RESPONSE OF THE WHITEFLY Bemisia tabaci B BIOTYPE TO REPEATED EXPOSURES
OF IMIDACLOPRID
By
Mamy L Rakotondravelo

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Entomology - Doctor of Philosophy
2013

ABSTRACT

RESPONSE OF THE WHITEFLY Bemisia tabaci B Biotype TO REPEATED EXPOSURES
OF IMIDACLOPRID

By

Mamy L Rakotondravelo

The whitefly, Bemisia tabaci (Gennadius), is among the most damaging of all pests in
cropping ecosystems. Among the different biotypes found in this complex species, B biotype is
one of the most widespread in agricultural systems. Within a few years of its initial introduction
imidacloprid became the standard product for control of whitefly. This study evaluates the effect
of frequent applications of imidacloprid to a population of whitefly. B biotype whitefly collected
from a commercial greenhouse in Michigan, was cultured and put under imidacloprid selection
to determine the rate of development of resistance to this insecticide. Before imidacloprid
treatments started this population already had a reduced susceptibility to imidacloprid with a
resistance ratio RR of 11-fold compared with that of a reference strain that we obtained from
University of California, Riverside. When adults of this resistant population were exposed to an
increasing concentration of imidacloprid, the resistance level increased to 218-fold in the F11
generation, indicating a rapid development of resistance after repeated exposure to high doses of
imidacloprid. The realized heritability h2 of the resistance was also determined. The overall
mean estimate of realized heritability for imidacloprid resistance in this B biotype population
was 0.10, suggesting that phenotypic variation in imidacloprid resistance in this population was
accounted for by an additive genetic variation. The response of the resistant population to
treatment with other neonicotinoids was then assessed. The imidacloprid resistant population

exhibited a low level of cross resistance to thiamethoxam (RR=9fold) and to acetamiprid (7fold). To have a complete picture of our assessment, the effect of exposure of whitefly to a very
low concentration of this insecticide was also determined. Three different populations were
used:a reference strain, a resistant and a rebuilt reference strain. The exposure of immatures of
the resistant strain to a low priming concentration of imidacloprid (0.0012 mg a.i. per pot),
followed by a subsequent exposure to imidacloprid as adults elicited an increase in fecundity
when compared with non-primed females (77.0 eggs/female compared with a 66.4 eggs/female).
Resistant females pre-exposed to low priming concentration (0.0012 mg a.i per pot) were also
found to exhibit greater egg production than resistant females primed with a high concentration
of imidacloprid (12 mg a.i. per pot ) (54.4 and 46.0 eggs per female). F1 female progeny of the
reference strain from parents primed with imidacloprid at 0.0012 mg a.i. per pot produced more
eggs than F1 female progeny from non-primed parents (19.6 and 13.6 eggs/female). The test was
repeated on the rebuilt reference strain using two priming concentrations (0.0012 and 0.00012
mg a.i. per pot). The test on the parental generation of this rebuilt reference strain showed that
females primed with the two low concentrations of imidacloprid 0.0012 and 0.00012 mg a.i. per
pot have greater egg production than the non-primed females for the rebuilt reference strain
(191.8, 186.7 and 169.3 eggs per female, respectively). However, no difference was found in the
fecundity of the F1 progeny of females for this rebuilt reference strain. All the results suggest
that treatment with a low amount of imidacloprid applied during an early immature stage of
development has an effect on adult whitefly fecundity. The result of this study has provided more
insight into the strategy for whitefly management and into the effects of residual amount of
insecticide in the organism.

Copyright by
MAMY L RAKOTONDRAVELO
2013

ACKNOWLEDGEMENTS

My deepest and continuing gratitude goes to my major Professor Dr. David Smitley who
has been most gracious to devote his time and energy to supervise my work with great care, and
without whom I would not have reached this point. He has been both professionally and
personally a real support for my growth as a researcher. I am grateful to my guidance
committee, namely Dr. Ke Dong, Dr. Rufus Isaacs and Dr. Cristi Palmer for their insightful
comments and fruitful discussions that have contributed meaningfully to the improvement of this
work. I am also indebted to Dr. David Mota-Sanchez, who has helped me with his guidance. I
would like to thank Terry Davis who not only provided all the materials that I needed but also
helped me with his advice. I am grateful to Dr. Steven Castle and Nilima Prabhaker Castle who
generously provided the reference strain of whitefly that I have been working on. I would like to
extend my gratitude to Gary Parsons for his valuable help when I needed to work with the
growth chambers. A special thanks to Haether Lenartson-Kluge for her assistance so that I could
pursue my degree program. I am thankful to my all friends that have made in my journey here in
Michigan State.

v

TABLE OF CONTENTS

LIST OF TABLES ....................................................................................................................... ix
LIST OF FIGURES ................................................................................................................... xiv
CHAPTER I. Introduction........................................................................................................... 1
I Whitefly Bemisia tabaci B biotype. ........................................................................................... 1
II Whitefly pest management. ...................................................................................................... 2
III Realized heritability h2 ........................................................................................................... 5
IV Resistance and cross resistance to neonicotinoids in whitefly ............................................. 6
V Hormesis. ................................................................................................................................... 9
VI Objectives ............................................................................................................................... 19
CHAPTER II. Selection and heritability of imidacloprid resistance in Bemisia tabaci B
biotype. ......................................................................................................................................... 20
I Introduction. ............................................................................................................................. 20
II Materials and methods. .......................................................................................................... 22
II-1 Plants. ................................................................................................................................... 22
II-2 Insecticide. ............................................................................................................................ 22
II-3 Insects. .................................................................................................................................. 23
II-3-1 Resistant strain. ............................................................................................................ 23
II-3-2 Reference strain. ........................................................................................................... 23
II-4 Selection. ............................................................................................................................... 24
II-4-1 History of selection and the resistant strain. .............................................................. 24
II-4-1-a) Exposure of adult whitefly................................................................................... 25
II-4-1-b) Exposure of both adults and immature whiteflies. ........................................... 28
II-5 Exposure of the resistant strain MI-r2a to soil-treated plants. ....................................... 30
II-6 Bioassay for determining LC50 .......................................................................................... 32
II-7 Data analysis. ....................................................................................................................... 32
II-7-1 Estimation of resistance ratio RR. ............................................................................. 32
II-7-2 Estimation of realized heritability (h2). ...................................................................... 33
III Results. ................................................................................................................................... 34
IV Discussion. .............................................................................................................................. 39
V Conclusion................................................................................................................................ 49
CHAPTER III. Response of imidacloprid-resistant whitefly B biotype to pesticides used for
whitefly control............................................................................................................................ 51
I Introduction. ............................................................................................................................. 51
II Material and Methods. ........................................................................................................... 53
II-1 Plants. ................................................................................................................................... 53
II-2 Insecticides. .......................................................................................................................... 53
II-3 Whitefly. ............................................................................................................................... 54
II-3-1 Reference strain. ........................................................................................................... 54
vi

II-3-2 Resistant strain. ............................................................................................................ 54
II-4 Systemic uptake bioassay for determining LC50. ............................................................. 55
II-5 Efficacy of insecticides to the resistant strain MI-r2a. ..................................................... 55
II-5-1 Test on adults. ............................................................................................................... 55
II-5-2 Test on immature. ......................................................................................................... 57
II-6 PBO synergism test.............................................................................................................. 58
II-7 Data analysis. ....................................................................................................................... 59
III Results. ................................................................................................................................... 60
IV Discussion ............................................................................................................................... 67
V Conclusion................................................................................................................................ 77
CHAPTER IV. Induction of hormesis in whitefly Bemisia tabaci B biotype by low priming
doses of imidacloprid. ................................................................................................................ 79
I Introduction. ............................................................................................................................. 79
II Materials and methods ........................................................................................................... 82
II-1 Plants .................................................................................................................................... 82
II-1-1 Plants used between 2009 and 2012......................................................................... 82
II-1-2 Plants used in 2013 .................................................................................................... 82
II-2 Insecticides. .......................................................................................................................... 83
II-3 Whitefly. ............................................................................................................................... 83
II -3-1 Whitefly used between 2009 and 2012. ...................................................................... 83
II -3-1 a) Reference strain. .................................................................................................. 83
II -3-1 b) Resistant strain. ................................................................................................... 83
II-3-2 Whitefly used in 2013. .................................................................................................. 84
II-4 Hormesis on the resistant and reference strains between 2009 and 2012. ..................... 84
II-4-1 Assay for the collection and priming of immature. ................................................... 85
II-4-1-a) Assay with priming of immature. ....................................................................... 85
II-4-1-b) Assay without priming of immature. ................................................................. 85
II-4-2 Assay for the collection and exposure of adults. ....................................................... 86
II-4-3 Test for hormesis in the parental generation. ............................................................ 87
II-4-3-a) Response of females of the reference and resistant colonies to imidacloprid.
............................................................................................................................................... 87
II-4-3-b) Response of resistant and reference females with different priming
concentrations. ..................................................................................................................... 88
II-4-3-c) Response of resistant and reference females primed with 0.0012 mg a.i. per
pot.......................................................................................................................................... 89
II-4-3-d) Response of resistant females with and without priming. .............................. 89
II-4-3-e) Response of resistant females with high and low priming concentrations ...... 90
II-4-4 Test for hormesis in the F1 generation of resistant and reference strains. ............. 90
II-5 Hormesis in the rebuilt reference strain in 2013. ............................................................ 92
II-5-1 Response of the rebuilt population from the reference strain without priming. .... 93
II-5-2 Response of the rebuilt population from the reference strain with priming. ......... 93
II-5-3 Test for hormesis on the parental generation of the rebuilt reference strain. ........ 95
II-5-4 Test for hormesis on the first generation of the rebuilt reference strain. ............... 96
II-6 Data analysis. ....................................................................................................................... 97
III- Results. .................................................................................................................................. 97
vii

III-1 Hormesis with the resistant strain. ................................................................................... 97
III-1-1 Study of the effect of priming exposure on the resistant adults. .......................... 97
III-1-2 Comparison of fecundity and longevity of primed and non-primed resistant
females. ................................................................................................................................... 105
III-1-3 Comparison of the fecundity and longevity between resistant females primed with
imidacloprid at 0.0012 and 12 mg a.i. per pot. ................................................................... 109
III-1-4 Study of the effect of priming of parents on F1 generation female offspring of the
resistant strain. ...................................................................................................................... 113
III-2 Hormesis in the reference strain (2009 - 2012).............................................................. 116
III-2-1 Study of the effect of priming on the reference strain. ......................................... 116
III-2-2 Study of the effect of priming of parents on the F1 generation female progeny of
the reference strain................................................................................................................ 123
III-3 Hormesis in the rebuilt reference strain (2013). ........................................................... 127
III-3-1 Study of the effect of the priming on females of the rebuilt reference strain. ..... 127
III-3-2 Study of the effect of priming on the parental generation of the rebuilt
population. ............................................................................................................................. 139
III-3-3 Study of the effect of priming of parents on the first generation of the rebuilt
reference strain. ..................................................................................................................... 145
IV Discussion ............................................................................................................................. 151
V Conclusion.............................................................................................................................. 159
Summary and future research direction................................................................................. 161
APPENDICES ........................................................................................................................... 165
APPENDIX A. Log probit dose plot. ................................................................................... 166
APPENDIX B. Exposure of the rebuilt reference strain to imidacloprid. ....................... 168
APPENDIX C. Record of deposition of voucher specimens ............................................. 175
LITERATURE CITED ............................................................................................................ 176

viii

LIST OF TABLES

Table 2.1. History of selections for the resistant whitefly culture…………………...…………26
Table 2.2. Summary of the selection for imidacloprid resistance in the resistant strain MI-R1 by
exposing the whitefly adults to imidacloprid…………………………………………………….35
Table 2.3. Summary of the selection for imidacloprid resistance in the resistance strain MI-R2
by exposing whitefly immatures and adults to imidacloprid………………………………….…37
Table 2.4. Resistance level of the resistant strain MI-r2a reared continuously on plants treated
with imidacloprid at a recommended dose (12 mg a.i. per pot) for whitefly control……………38
Table 2.5. Estimation of realized heritability of resistance to imidacloprid in Bemisia tabaci B
biotype collected from a commercial greenhouse in Michigan………………………………….44
Table 3.1. Synergistic effect of PBO on the toxicity of imidacloprid to F8 and F11 generation of
the resistant strain MI-R1 of Bemisia tabaci B biotype…………………………………………61
Table 3.2. Response of the reference strain (CA-s1) and the generation F5 of the imidaclopridresistant strain (MI-R1) of Bemisia tabaci B biotype to three neonicotinoid insecticides in a
systemic uptake bioassay………………………………………………………………………...62
Table 3.3. Efficacy ± SE of neonicotinoids against immatures of the resistant strain (MI-r2a) and
the reference strain (CA-s2) of Bemisia tabaci B biotype whitefly on cotton plants. *: Mean
number of immatures from four replicates…………………………………………………...….64
Table 3.4. Efficacy of buprofezin and pyriproxyfen against clip-caged immatures of the resistant
strain (MI-r2a) and the reference strain (CA-s2) of whitefly Bemisia tabaci B biotype on cotton
plants. *: mean number of immature whitefly from four replicates………………………………....65
Table 3.5. Efficacy ± SE of lambda- cyhalothrin and Beauveria bassiana against clip-caged
immatures of the resistant strain and the reference strain (CA-s2) of whitefly Bemisia tabaci B
biotype on cotton plants. *: Mean number of immatures from four replicates…………………..66
Table 3.6. Efficacy ± SE of flonicamid, fenpropathrin, and pymetrozine against clip-caged
immatures of the resistant strain (MI-r2a) and the reference strain (CA-s2) of whitefly Bemisia
tabaci B biotype on cotton plants. *: Mean number of imatures from four replicates………..…68
Table 3.7. Efficacy ± SE of neonicotinoids (imidacloprid, acetamiprid, thiamethoxam,
dinotefuran) against clip-caged resistant adults (MI-r2a) and reference adults (CA-s2) of whitefly
Bemisia tabaci B biotype exposed for 3 days to the treated cotton plants. *: Mean number of
adults from four replicates……………………………………………………………………….69
ix

Table 3.8. Efficacy ± SE of pyriproxyfen, Beauveria bassiana, buprofezin and lambdacyhalothrin against clip-caged resistant adults (MI-r2a) and reference adults (CA-s2) of whitefly
Bemisia tabaci B biotype exposed for 3 days to the treated cotton plants. *: Mean mean number
of adults from four replicates………………………………………………………………..……………70
Table 4.1a. Mean ± SE total egg production and longevity of females of Bemisia tabaci B
biotype collected from the resistant colony and exposed to different concentrations of
imidacloprid as adults…………………………………………………………………………....98
Table 4.1b. Summary of the generalized linear mixed model for the data in Table 4.1a….........99
Table 4.2a. Mean ± SE total egg production and longevity of females of the resistant strain
Bemisia tabaci B biotype primed with seven different concentrations of imidacloprid as
immatures, then exposed to the same concentrations of imidacloprid as adults……………….100
Table 4.2b. Summary of the generalized linear mixed model for the data in Table 4.2a……...101
Table 4.3a. Mean ± SE total egg production and longevity of females of the resistant strain
Bemisia tabaci B biotype primed with imidacloprid at 0.0012 mg a.i. per pot as immatures, and
then exposed to different concentrations of imidacloprid as adults………………………….....102
Table 4.3b. Summary of the generalized linear mixed model for the data in Table 4.3a……...103
Table 4.4a. Mean ± SE fecundity and longevity of the resistant strain primed with imidacloprid
at 0 and 0.0012 mg a.i. per pot as immatures, then exposed as adults to 9 concentrations of
imidacloprid centered about 0.0012 mg ai per pot……………………………………………..106
Table 4.4b. Summary of the generalized linear mixed model for the data on fecundity in Table 4.
4a……………………………………………………………………………………..…………107
Table 4.4c. Summary of the generalized linear mixed model for the data on longevity in Table 4.
4a………………………………………………………………………………………………..108
Table 4.5a. Mean ± SE egg production and longevity of resistant females primed with
imidacloprid at 0.0012 and 12 mg a.i. per pot as immatures, then exposed to different
concentrations of imidacloprid as adults………………………………………………….…….110
Table 4.5b. Summary of the generalized linear mixed model for the data on fecundity in Table 4.
5a…………………………………………………………………………………………..……111
Table 4.5c. Summary of the generalized linear mixed model for the data on longevity in Table
4. 5a……………………………………………………………………………………………..112

x

Table 4 6a. Mean ± SE egg production and longevity of the F1 generation females from
resistant parents primed with imidacloprid at 0.0012 mg a.i. per pot exposed to different
concentrations of imidacloprid as adults…………………………………………………..……114
Table 4.6b. Summary of the generalized linear mixed model for the data in Table 4. 6a…......115
Table 4.7a. Mean ± SE egg production and longevity of females of Bemisia tabaci B biotype
collected from the reference colony and exposed to different concentrations of imidacloprid as
adults………………………………………………………………………………………..…..117
Table 4.7b. Summary of the generalized linear mixed model for the data in Table 4. 7a…......118
Table 4.8a. Mean ± SE total egg production and longevity of females of the reference strain of
Bemisia tabaci B biotype primed with seven different concentrations of imidacloprid as
immatures, then exposed to the same concentrations of imidacloprid as adults……………….119
Table 4.8b. Summary of the generalized linear mixed model for the data in Table 4. 8a….….120
Table 4.9a. Mean ± SE total egg production and longevity of females of the resistant strain
Bemisia tabaci B biotype primed with imidacloprid at 0.0012 mg a.i. per pot as immatures, and
then exposed to different concentrations of imidacloprid as adults………………………….…121
Table 4.9b. Summary of the generalized linear mixed model for the data in Table 4. 9a……..122
Table 4.10a Mean ± SE egg production and longevity of the F1 generation of the reference
females from the parents with or without priming with imidacloprid at 0.0012 mg a.i. per pot,
then exposed to different concentrations of imidacloprid as adults………………………….....124
Table 4.10b. Summary of the generalized linear mixed model for the data on fecundity in Table
4.10a………………………………………………………………………………….…………125
Table 4.10c. Summary of the generalized linear mixed model for the data on longevity in Table
4.10a………………………………………………………………………………………….…126
Table 4.11a. Mean ± SE total egg production and longevity of female of the rebuilt reference
strain exposed as adults to different concentrations of imidacloprid up to 15 mg a.i. per pot…128
Table.4.11b. Summary of the generalized linear mixed model for the data in Table 4.11a. ….129
Table 4.12a. Mean ± SE total egg production and longevity of female of the rebuilt reference
strain exposed as adults to different concentrations of imidacloprid up to 60 mg mg a.i per
pot………………………………………………………………………………………………130
Table.4.12b. Summary of the generalized linear mixed model for the data in Table 4.12a…...131

xi

Table 4.13a. Mean ± SE egg production and fecundity of females of the rebuilt reference strain
when primed as immatures and then exposed as adults to imidacloprid at 0, 36 and 72 mg a.i per
pot………………………………………………………………………………………………133
Table 4.13b. Summary of the generalized linear mixed model for the data on fecundity in Table
4.13a……………………………………………………………………………………………134
Table 4.13c. Summary of the generalized linear mixed model for the data on longevity in Table
4.13a. …………………………………………………………………………………………...135
Table 4.14a. Mean ± SE total egg production and longevity of females of the rebuilt reference
strain exposed to three priming concentrations of imidacloprid (0, 0.00012 and 0.0012 mg a.i.
per pot) as immatures and then exposed to plants systemically treated for 36 hours with
imidacloprid at 0 and 12 mg a.i per pot. ……………………………………………………….136
Table 4.14b. Summary of the generalized linear mixed model for the data on fecundity in Table
4.14a…………………………………………………………………………………………….137
Table 4.14c. Summary of the generalized linear mixed model for the data on longevity in Table
4.14a……………………………………………………………………………………….…...138
Table 4.15a. Mean ± SE total egg production and longevity of the rebuilt reference females of
the parental generation P11 exposed to three priming concentrations of imidacloprid (0, 0.00012
and 0.0012 mg a.i. per pot) as immatures and then exposed to plants systemically treated for 72
hours with imidacloprid at 0, 6 and 12 mg a.i. per pot…………………………………………140
Table 4.15b Mean ± SE total egg production and longevity of the rebuilt reference females of
the parental generation P12 exposed to three priming concentrations of imidacloprid (0, 0.00012
and 0.0012 mg a.i. per pot) as immatures and then exposed to plants systemically treated for 72
hours with imidacloprid at 0 and 6 mg a.i. per pot…………………………………………..…141
Table 4.15c. Mean ± SE total egg production and longevity of the rebuilt reference females of
the parental generation P13 exposed to three priming concentrations of imidacloprid (0, 0.00012
and 0.0012 mg a.i. per pot) as immatures and then exposed to plants systemically treated for 72
hours with imidacloprid at 0, 6 and 12 mg a.i. per pot. ………………………………….….....142
Table 4.15d. Summary of the generalized linear mixed model for the data on fecundity in Table
4.15a, b, c……………………………………………………………………………………….143
Table 4.15e. Summary of the generalized linear mixed model for the data on longevity in Table
4.15 a, b, c……………………………………………………………...……………………….144
Table 4.16a. Mean ± SE total egg production and longevity of the F11 female progeny of the
rebuilt reference strain from parents with and without priming exposure to imidacloprid exposed
as adults to imidacloprid at 0, 6 and 12 mg a,i. per pot………………………………………...146
xii

Table 4.16b. Mean ± SE total egg production and longevity of the F12 female progeny of the
rebuilt reference strain from parents with and without priming exposure to imidacloprid exposed
as adults to imidacloprid at 0 and 6 mg a,i. per pot……………..……………………………...147
Table 4.16c. Mean ± SE total egg production and longevity of the F13 female progeny of the
rebuilt reference strain from parents with and without priming exposure to imidacloprid exposed
as adults to imidacloprid at 0, 6 and 12 mg a,i. per pot………………………………………...148
Table 4.16d. Summary of the generalized linear mixed model for the data on fecundity in Table
4.16a, b, c. ………………………………………………………………………………….….149
Table 4.16e. Summary of the generalized linear mixed model for the data on longevity in Table
4.16 a, b, c………………………………………………………………………………………150
Table A1. Proportion of affected female adults that exhibited various symptoms of toxicity after
imidacloprid application. Some females recovered after a few days but some was died within
five days. ……………………………………………………………………………………….168

xiii

LIST OF FIGURES
Figure 2.1. Selection of adults of the resistant strain MI-R1 from May 2007 until April 2008
using a systemic uptake bioassay…………………………………………………………...……27
Figure 2.2. Selection of the resistant strain MI-R2 from April 2009 to December 2009 using
systemic uptake bioassay and soil drench treatment……………………………………….…….29
Figure 2.3. Maintenance of the resistant whitefly MI-r2a on plants treated with imidacloprid
applied as soil drench from May 2009 until present time. This resistant strain MI-r2a was used
for the insecticide efficacy test and the study on sublethal effects of imidacloprid on whitefly...31
Figure 2.4. Effect of heritability (h2) on number of generations of Bemisia tabaci B biotype
required for a 10-fold increase in LC50 of imidacloprid at different selection intensities……….45
Figure A1. Log-probit dose plot for the toxicity of imidacloprid to the reference strain CA-s1
(dark blue triangles), the parental strain MI-R1 collected from a commercial greenhouse
(turquoise diamonds), and the F5 (green triangles) generation of Bemisia tabaci B biotype after
selection with imidacloprid……………………….…………………………………………….166
Figure A.2. Log-probit dose plot for the toxicity of imidacloprid to the reference strain CA-s2
(black diamonds), P: the resistant parental of the resistant strain MI-R2 (black triangles)
retrieved after the period of mite and thrips infestation, and the F3 (red triangles) and the F7
(blue diamonds) generation of the resistant strain MI-R2 after selection with
imidacloprid……………………………………………………………………………….....…167
Figure A3. Cumulative mean total egg production per female of the first parental generation P11
of the rebuilt reference strain primed with different concentrations of imidacloprid and then
subsequently exposed to imidacloprid at (a) 12 mg a.i. per pot, (b) 6 mg a.i per pot, and (c) 0
mg a.i. per pot as adults………………………………………………………………………...169
Figure A4. Cumulative mean total egg production per female of the second parental generation
P12 of the rebuilt reference strain primed with different concentrations of imidacloprid and then
subsequently exposed to imidacloprid at (a) 6 mg a.i. per pot, (b) 0 mg a.i. per pot as
adults……………………………………………………...…………………….........................170
Figure A5. Cumulative mean total egg production per female of the third parental generation P13
of the rebuilt reference strain primed with different concentrations of imidacloprid and then
subsequently exposed to imidacloprid at (a) 12 mg a.i per pot, (b) 6 mg a.i per pot, and (c) 0
mg a.i. per pot as adults…………………………………………………………………..….....171
Figure A6. Cumulative mean total egg production per F11 female offspring of the rebuilt
reference strain primed with different concentrations of imidacloprid and then subsequently
exposed to imidacloprid at (a) 12 mg a.i. per pot, (b) 6 mg a.i. per pot, and (c) 0 mg a.i. per pot
xiv

as adults……………………………………………………………………………………..…..172
Figure A7. Cumulative mean total egg production per F12 female offspring of the rebuilt
reference strain primed with different concentrations of imidacloprid and then subsequently
exposed to imidacloprid at (a) 6 mg a.i. per pot, (b) 0 mg a.i. per pot as adults……….……...173
Figure A8. Cumulative mean total egg production per F13 female offspring of the rebuilt
reference strain primed with different concentrations of imidacloprid and then subsequently
exposed to imidacloprid at (a) 12 mg a.i. per pot, (b) 6 mg a.i. per pot as adults and (c) 0 mg a.i
per pot……………………………………………………………………………………..……174

xv

CHAPTER I. Introduction.
I Whitefly Bemisia tabaci B biotype.
Bemisia tabaci (Gennadius) (Insecta: Hemiptera: Homoptera: Aleyrodidae) is a
polyphagous insect found on over 500 plants species in every continent except Antartica
(Oliveira et al., 2001). Whitefly Bemisia tabaci is a complex species that comprises up to 20
biotypes (Perring, 2001). Among the different biotypes, Q biotype and B biotype are the most
studied. Recently, it was reported that Bemisia tabaci is a cryptic species complex consisting of
at least 24 species which cannot be distinguished morphologically (DeBarro et al., 2011). Based
on the new classification, the B biotype whitefly is identified as Middle East- Asia Minor
whereas the Q biotype as Mediterranean (De barro et al., 2011). However, the term biotype is
still frequently used in published studies when addressing these whiteflies (Kontsedalov et al.,
2012). These two whitefly biotypes vary in fecundity, host range, and efficiency of virus
transmission (DeBarro et al., 2006; Gottlieb et al., 2010). The Q and B biotype are
reproductively incompatible, probably due to a prezygotic barrier (Sun et al., 2011). Both
biotypes may spread rapidly through the sale of ornamental plants (Debarro et al., 2011). B
biotype is the most widespread in agricultural ecosystems (Byrne et al., 2010). It is also known
as Bemisia argentifolii and may be referred to as silverleaf or sweetpotato whitefly (Debarro and
Driver, 2007). The B biotype whitefly may displace indigenous biotypes due to its high
fecundity, its mating behavior, and its higher fitness on several taxa of host plants (De Barro,
2011). However, the Q biotype whitefly tends to develop higher levels of resistance to
insecticides than the B biotype whitefly, so that Q biotype may be more widespread in
production systems where insecticides are used intensively (Horowitz et al., 2005; Kontsedalov
et al., 2012). For example, a monitoring study in Israel demonstrated that Q biotype was the
predominant biotype in greenhouses where chemical use was more frequent whereas B biotype
1

whitefly constitutes the majority of the whitefly found in open fields due to their higher fitness
under field conditions (Kontsedalov et al., 2012). This trend was also noticed with
neonicotinoids. For example, B biotype was the predominant biotype in China in 2006 (Chu et
al., 2007), and the subsequent monitoring study reported that Q biotype displaced B biotype due
to the increasing use of neonicotinoids (Chu et al., 2010).
Bemisia tabaci is among the most damaging of all pests in cropping ecosystems. It feeds
on plant phloem, excretes honeydew upon which sooty mold grows, and also transmits viruses
(Oliveria et al., 2001). The first reported damage caused by B biotype whitefly in the USA was
on poinsettia, Euphorbia pulcherrima in Florida in 1986 (Price and Schuster, 1991). It soon
spread to other southern states including California, Texas and Arizona (McAuslane, 2010). If
left uncontrolled, whitefly infestations can induce significant crop loss. The agricultural loss
associated with whitefly in Arizona, Florida, Texas and California was estimated at $500 million
in 1991 alone (Perring et al., 1993).

II Whitefly pest management.
Different strategies have been adopted to control whitefly. Among them is the use of
natural enemies, including fungi such as Ascherosnia, Beauveria, and Metarzhium, and predators
like Amblyseius swirskii, Macrophlophus calliginosus, or the parasitoids in the genera Encarsia
or Eretmocerus (Horowitz et al., 2011). However, despite many attempts to protect, augment or
release many different natural enemies, the use of chemicals is still the main strategy adopted for
whitefly control (Palumbo et al., 2001). Conventional insecticides such as carbamates,
organophosphates and pyrethroids were among the chemical classes most heavily used for
whitefly control before 2000 (Debarro, 2011). However, numerous studies reported the

2

development of resistance to these insecticides (Denholm et al., 1998). The use of mixtures of
pyrethroids with other classes of insecticides such as organophospahtes or carbamates was also
commonly adopted due to their efficacy for whitefly control especially in the southwestern
United States (Palumbo et al., 2001; Horowitz et al., 2011). Similar to the other conventional
pesticides, resistance to these pyrethroid mixtures was also reported after a few years of their use
(Dennehy and Wiliams, 1997). The diversity of chemicals for the whitefly control was increased
with the introduction of insect growth regulators IGR. Pyriproxyfen and buprofezin are among
the most heavily used IGR’s for whitefly control (Toscano et al., 2001). Buprofezin interferes
with chitin formation and affects the development of immature insects (Ishaaya et al., 1988),
whereas pyriproxyfen is a juvenile hormone analog that affects embryogenesis and
metamorphosis (Ishaaya and Horowitz, 1992).
In the early 1990s, imidacloprid, belonging to a different class of insecticide called the
neonicotinoids, was introduced into the market (Nauen and Denholm, 2005). This compound is
very effective in controlling sucking insects such as planthoppers, aphids and whiteflies due to
its systemic properties (Tomizawa and Casida, 2003). After imidacloprid, several additional
neonicotinoid products also became available to growers, including thiomethoxam, acetamiprid,
and dinotefuran. In 2006, neonicotinoids made up 17% of the global insecticide market with an
annual sale of 1.5 billion US dollars (Jeschke and Nauen, 2008). Imidacloprid is one of the most
successful neonicotinoid insecticides; it has been registered for use on 140 crops in more than
120 countries, and it was predicted to continue to be one of the leading insecticides in a few
years to come (Elbert et al., 2008). Imidacloprid acts on the central nervous system as an agonist
of the nicotinic acetylcholine receptors (nAChR) in insects (Liu and Casida, 1993). Since
imidacloprid had a new mode of at the time of the introduction, it was quickly adopted as the

3

best insecticide to use on resistant whiteflies (Rauch and Nauen, 2003). However, due to its
overuse high levels of resistance were reported in B biotype whitefly in various countries. For
example, a high resistance factor to imidacloprid (RR = 230 fold) was documented in B biotype
whitefly collected from the field in Mexico and in Guatemala in 2005 (Gorman et al., 2010).
Vassiliou et al. (2011) also found a wide range of imidacloprid resistance levels (RF= 67 to 392fold) in B biotype whitefly collected from different regions in Cyprus. Rauch and Nauen, (2003)
documented a high resistance level (874-fold) in a B biotype whitefly collected from roses in
greenhouses in Israel. In contrast to these results, no report of whitefly control failure was
documented for outdoor crops in Arizona and Florida despite the reliance on imidacloprid for
whitefly control in these regions in 1990s (Palumbo et al., 2001). Castle et al. (2010) suggested
that biological, ecological and operational factors could explain this modulation of the
development of insecticide resistance in whiteflies. For example, the authors reported that the
presence of untreated crops of alfalfa in Imperial Valley, California, which comprises 70% of the
area, could serve as refugia for susceptible whitefly and slowed down the development of
resistance to imidacloprid in this region despite its heavy use. This differential level of
imidacloprid resistance in whitefly has also been observed in different regions across the world
with different cropping systems, and the resistance level was found to be not necessarily
associated with the amount of use of the pesticides. For example, Vasillou et al. (2011) reported
resistance levels varying from moderate to high in B biotype whitefly populations collected from
different regions in Cyprus, and the resistance level was not correlated with the history of
neonicotinoid application. In addition, different biotypes in the same regions in China also
showed a different levels of resistance to imidacloprid, with B biotype whitefly being
susceptible to this compound whereas Q biotype whitefly being resistant (Luo et al., 2010). All

4

these observations seems to hint that in addition to environmental factors present in the field,
other factors such as biological, physiological or genetic factors may also contribute to the
development of different resistance levels in the field populations of whitefly.

III Realized heritability h2
As a matter of fact, Tabashnik (1992) argued that the rate of development of the
resistance to insecticides could be predicted through the analysis of the heritability of resistance
to the insecticide in the species of interest. The phenotypic variation (VP) of a threshold trait
such as insecticide resistance is a combination of additive genetic variation (VA) and
environmental variation (VE) (Falconer, 1989, Firko and Hayes, 1990). Heritability in a narrow
sense is defined as the proportion of the total phenotypic variation attributable to the additive
genetic variation (Falconer, 1989; Tabashnik, 1992). Thus, heritability enables an estimation of
the relative contribution of the gene and the environment to the variation of the trait of interest
(Visscher et al., 2008). Heritability depends on different factors such as the allele frequency of
the resistant level but also to the environmental conditions under which the population is studied
(Falconer, 1989, Tabashnik, 1992). Indeed, laboratory populations which are likely to have less
environmental variation have a much higher heritability than field populations (Bull et al., 1982).
Thus, the heritability of laboratory populations may not necessarily reflect the situation in the
field (Falconer, 1989, Tabashnik and McGaughey 1994). However, several studies also
suggested that heritability in the laboratory could still predict the time of development of
resistance in field. For example, Li et al. (2012), starting with a field-collected population of
Musca domestica in China, selected for imidacloprid resistance for 21 generations, and estimated
a relatively low heritability (h2 = 0.10) in the houseflies. The authors noted that no reports of
5

resistance in housefly were yet documented in China. Wang et al. (2008) also estimated a very
low h2(0.08) in the planthopper, Nilaparvata lugens, collected in China and selected for
imidacloprid resistance for 27 generations. The authors also noted that the field population of
planthopper remained susceptible to imidacloprid from 1996 to 2003. Resistance was only
detected in 2005, some 13 years after the first use of imidacloprid in China. These studies are in
contrast to the results found by Sivasupramanian and Watson (2000) who estimated a high
heritability of resistance to the mixture of acephate and fenpropathrin on B biotype whitefly in
Arizona, and this high heritability estimate was consistent with the rapid observation of control
failure of field populations, which occurred within two years (Dennehy and Wiliams, 1997). A
relatively high heritability (h2 = 0.40) was also estimated in the greenhouse whiteflies,
Trialeurodes vaporarium, selected for dichlorovos resistance in the laboratory. This value was
also found to be in agreement with a rapid development of resistance in field (Omer et al., 1993).
However, it is also noteworthy that some other reported heritability values for insecticides in
other pests were found to be not consistent with the rate of resistance development in field
populations. For example, Firko and Hayes (1991) reported a very high heritability of 0.85 for
cypermethrin in the tobacco budworm, however no field control failure was documented until 10
years after the introduction of this insecticide. In brief, heritability could be one factor that may
partially explain the wide variation in how rapidly resistance develops among different
populations of one species or biotype.

IV Resistance and cross resistance to neonicotinoids in whitefly.
In addition to a pronounced difference in levels of resistance to imidacloprid among
different populations of B biotype whitefly, a wide variation in the observed levels of cross
6

resistance to other neonicotinoid has also been observed. For example, the level of crossresistance to neonicotinoids in populations of B biotype whitefly was found to vary from very
low (RRs of 5 and 2 for acetamiprid and thiamethoxam, respectively) to a high (RRs of 78 and
more than 900) (Prabhaker et al., 2005; Rauch and Nauen, 2003). Resistance to imidacloprid
was attributed to the P450 detoxification system in whiteflies (Rauch and Nauen, 2003). An
overexpression of one of the P450 genes, CYP6M1PvQ, was found in imidacloprid-resistant
populations of both B and Q biotypes (Karunker et al., 2008). In a subsequent study, Roditakis et
al. (2011) demonstrated that indeed CYP6M1PvQ has a potential to metabolize imidacloprid in
whiteflies. However, the same study showed that thiamethoxam was not metabolized by the
protein of this P450 gene. In fact, Nauen et al. (2003) showed that thiamethoxam was converted
to clothianidin in cotton plants and in the insects ingesting it, and now clothianidin is known to
be a byproduct of thiamethoxam. Interestingly, Roditakis et al. (2011) found that as opposed to
thiamethoxam but similar to imidacloprid, indeed clothianidin was metabolized by the gene
responsible of imidacloprid resistance in whiteflies, CYP6M1PvQ. In addition, Roditakis et al.
(2011) demonstrated that similar to thiamethoxam, the protein of the P450 gene CYP6M1PvQ
also did not metabolize acetamiprid in whiteflies. In agreement with the metabolism study, most
of the reports on cross resistance levels in B biotype whitefly resistant to imidacloprid showed a
high level of resistance to thiamethoxam but a moderate level of resistance to acetamiprid. For
example, Gorman et al., (2010) showed that population of B biotype whitefly from Guatemala
having a resistance ratio (RR) of 230 to imidacloprid exhibited a resistance ratio of 150-fold to
thiamethoxam whereas it was only 15-fold for acetamiprid. The same study also reported that the
B whitefly population collected from Mexico having a resistance ratio of 230-fold to
imidacloprid exhibited 73-fold resistance to thiamethoxam but only 15-fold resistance to

7

acetamiprid. Similarly, different B biotype whitefly populations collected from different regions
in Cyprus, and having a moderate to high resistance level to imidacloprid (RR of 77 to 392),
also showed a moderate level of resistance to thiamethoxam (RR = 50 to 164) and a low level of
resistance to acetamiprid (RR = 7 to 12) (Vasillou et al., 2011). The same observation of higher
resistance to thiamethoxam than to acetamiprid was even noted in several imidacloprid resistant
populations of Q biotype whitefly. For example, Rao et al., (2012) reported that a population of
Q biotype collected from China which has a similar high resistance level for thiamethoxam and
imidacloprid (244 and 273-fold respectively) showed only 68-fold of resistance to acetamiprid.
The selection history was already shown to influence the resistance level to other neonicotinoids
in B biotype whitefly. Horowitz et al. (2004) found that whitefly population selected for a
thiamethoxam resistance showed a very low resistance to acetamiprid whereas an acetamipridselected population was highly resistant to thiamethoxam. In contrast, Feng et al. (2009)
selected B biotype whitefly for thiamethoxam resistance (RR: 25.6) found a similar level of
resistance to acetamiprid (RR 35-fold) and imidacloprid (47.3-fold). Interestingly, Karunker et
al., (2008) found the CYP6CM1 as the P450 gene that was over-expressed in imidaclopridresistant whiteflies Q and B biotype. However, Xie et al. (2012) observed an overexpression of
three other P450 genes, CYP6a8, CYP4v2, and CYP6v5, in thiamethoxam-resistant whitefly.
These results seem to suggest that a different history of selection could also select a different
gene, causing a different level of resistance between neonicotinoids in whiteflies.
Dinotefuran is also another neonictoinoid used for growers for whitefly control.
However, this insecticide has a different structure as it does not have the pyridine or thiazole ring
similar to the previously introduced neonicotinoids such as imidacloprid, acetamiprid and
thiamethoxam (Wakita et al., 2005). Prabhaker et al. (1997) demonstrated that the population of

8

B biotype whitefly selected for imidacloprid resistance in laboratory did not exhibit an
appreciable cross resistance to this compound. Similarly, this non-significant cross resistance to
dinotefuran was also observed in some Q biotype whitefly. Three populations of Q biotype
collected from three different regions in China with a high resistance level to imidacloprid (201,
244, 219-fold) showed a resistance level of 2, 4 and 6- fold to dinotefuran, respectively (Rao et
al., 2012).
Besides the different metabolism of neonicotinoids, an increasing number of studies also
demonstrated that the bacterial symbionts hosted by both B and Q biotype Bemisia tabaci also
could influence the susceptibility of these whiteflies to neonicotinoids. Ghanim and Kontsedalov
(2009) showed that Q biotype was double-infected by the bacterial symbiont Ricketssia and
Arsenophonus on one hand, or Wolbachia and Arsenophonus on other hand, were more
susceptible to imidacloprid and thiamethoxam than the Q biotype whitefly infected by a single
symbiotic bacterium, Arsenophonus. Kontsedalov et al. (2008) showed that B biotype whitefly
harboring the symbiotic microorganism Rickesttsia was more susceptible to thiamethoxam and
acetamipird than individuals not infected by these microorganism, however the presence of these
Rickettsia did not affect susceptibility to imidacloprid. The compositions of these facultative
symbionts can vary between regions or biotypes (Xie et al., 2012). All of these studies suggest
that differential susceptibility to insecticides between strains or biotypes could also be partially
explained by the presence of different symbiotic microorganism in these whiteflies.

V Hormesis.
Bonmatin et al. (2003) reported that imidacloprid can still be found 12 months or longer
after its application in the field. The same authors detected the amount of imidacloprid to be as

9

low as 0.5-20 μg/kg in soils, plants, and pollens. Exposure of pests to a low amount of
imidacloprid is then expected for the entire crop production period. Some toxicants are known to
induce a stimulatory response at low doses, and this phenomenon is known as hormesis in the
toxicological literature (Calabrese and Blain, 2005). Hormesis is an adaptive response which is
characterized by biphasic dose response, with a stimulatory effect at the low doses of the toxicant
and an inhibitory effect at the high doses (Calabrese and Baldwin, 2002). The pioneer studies on
this biphasic dose response were conducted in the 1880s by a German pharmacologist, Hugo
Schluz, who observed a stimulation of yeast metabolism at low dose of various chemical agents
whereas the chemicals have inhibitory effects at high doses (Calabrese, 2002). As Schulz and
his colleague psychiatrist Rudolph Arndt developed the concept of this dose response, this
phenomenon was previously known as the Arndt-Schluz- law after the publication of Schluz’
work in 1912 (Calabrese, 2002). Several investigations on this phenomenon were then
conducted by different researchers in the 1920’s and 1930’s (Calabrese, 2002). After the 1930’s
the concept drew less attention from scientists due to some limitations related to the concepitself
and due to the fact some investigators associated the phenomenon with the unpopular
homeopathy concept (Calabrese, 2002, Calabrese, 2005, Calabrese et al., 2010, Rozman and
Dull, 2003). The term hormesis (from Greek word which means to excite) was used for the first
time by Chester Southam and John Ehlrich in 1943 (Calabrese, 2009). A resurgence of interest in
hormesis was noted again in late 1970s due to the needs in risk assessment and the availability of
improved methodology for detecting low doses of various chemicals (Calabrese et al., 2010).
Over the last two decades, Edward Calabrese, a toxicologist in Massachusetts University, has
been one of the leading researchers in the study of hormesis and has published several
documents on this concept. Through an exhaustive analysis of a huge volume of literature,

10

Calabrese and his colleagues set some criteria, principles and properties that characterize
hormesis in his several documents (Calabrese et al., 1999, Calabrese and Blain, 2005). For
example, hormesis was found to be induced by a wide variety of chemical and physical agents in
different organisms from bacteria to mammals (Calabrese and Blain, 2005). Calabrese and
Baldwin (2003) reported that in most cases hormetic stimulation has an amplitude of 130 to
160% of the untreated controls. The hormetic dose that induces stimulation above the control is
known as the hormetic zone, and is usually located just below the No Observed Adverse Effect
Level (NOAEL). The width of the zone is usually in the 10 to 100-fold dosage range , but 70%
of the studies are about in 20-fold (Calabrese and Baldwin, 2003). Calabrese and Baldwin
(2002) also defined two types of hormesis: first, the overcompensation stimulation hormesis
(OCSH) which is a result of an overcompensatory response following a disruption of
homeostasis, and second, the direct stimulation hormesis (DSH). In contrast to OCSH, the direct
hormesis is an adaptive response that is associated with the regular function of metabolism, thus
with no disruption of homeostasis to be repaired (Calabrese and Baldwin, 2002). The hormetic
effect may be detected with a single treatment, or sometimes it can only be determined after
challenging the cell or organism with a subsequent treatment. When a subsequent treatment is
required the effect is known as ‘conditioning hormesis’ (Calabrese et al., 2007). In conditioning
hormesis, the first treatment can protect the organism against the toxic effects of a subsequent
more severe treatment (Calabrese et al., 2007; Calabrese and Mattson, 2011). In fact, Stebbing,
(2002, 2009) viewed hormesis as an acquired tolerance. If the subsequent treatment does not
occur, the overcorrection response would be seen as stimulation above the control (Stebbing,
2009).
Hormesis has some characteristic properties. One of them is a lack of response of the

11

organism depending on the similarity or difference between the stress agent used in pre-exposure
and the subsequent exposures. For example, one inducing agent used for the pre-exposure could
protect the organism against the damaging effect caused by a subsequent higher dose of the same
stress agent only (Crysper and Johnson, 2002). However in some cases, one stress agent can
elicit an adaptive response to cope with the detrimental effects of the subsequent exposure to
more other stress agents (Chujo et al., 2012), a phenomenon that was also known as crossadaptation or cross-tolerance (Zhao and Wang, 2012; Chujo et al., 2012).
The dose used for the pre-exposure can also influence hormesis. For example, Chujo et
al., (2012) found that in C. elegans only the low but not the high dose of the stress agent such as
heat stress, osmotic stress and oxidative stress can elicit an adaptive response that protects the
worm against the damaging effect of the subsequent higher dose of these stress agents.
However, Wang and Xing (2010) demonstrated that pre-treatment of C. elegans with UV
irradiation at 15, 20 and 30 J/m2/min elicited an adaptive response, thus it protected the worm
against the reproductive toxicity of a subsequent exposure to cadmium. However the preexposure to a lower dose of this UV irradiation (5 J/m2/min) did not induce such a response.
These results suggest that the pre-exposure is only effective within a certain range of dose.
Stebbing (2009) already proposed in his model the absence of response of the organism if the
dose of the inducing factor is too low to trigger the phenomenon, but at too high of a dose the
damaging effect would prevail.
Another important characteristic of hormesis is that this phenomenon can be observed
when the inducing factors are applied at specific life stages. For example, Lebourg (2005)
demonstrated adaptive hormesis in Drosophila melanogaster by pre-exposing young males to
hypergravity, which induced a longer lifespan than the non-pre- exposed individuals when they

12

were later exposed to heat shock. Nascarella et al. (2003) also reported that the larva of the blow
fly, Phormia regina, fed a diet with a low dose of cadmium exhibited a high pupation rate.
However the treatment adversely affected the adult emergence of this species, indicating an
occurrence of hormesis at the larval stage only. But in some cases, the hormetic effect can occur
in adulthood when the mild stress was applied at an early stage of development of the individuals
(Costantini et al., 2010).
As far as insects are concerned, one remarkable study of stimulation caused by low doses
of insecticides was conducted by Luckey in 1968. Luckey (1968) demonstrated a stimulation of
growth of the house cricket Acheta domesticus to sublethal concentrations of 14 insecticides.
This stimulation was suggested to be associated with hormoligosis, a term used first time by
Luckey in 1955 to describe the biphasic dose response that the author found, thus a stimulatory
effect at low doses of the toxicants and an inhibitory effect at higher doses (Luckey, 2008).
Hormoligosis and hormesis indicate a stimulation induced by low doses of different stressors, but
until now there has not been any evidence that the two concepts describe the same phenomenon,
thus induced by the same mechanism, or that they are different from each other. Different
authors already attempted to differentiate the two effects. For example, Cutler (2012) and Cohen
(2006) pointed out the suboptimal conditions to be present for hormoligosis to occur to separate
the two concepts. In a different approach, Rozman and Doull (2003) proposed that hormesis
refers to a homeostatic overcompensation response triggered by an initial inhibition whereas
hormoligosis is associated with the overcompensation response induced by an initial stimulation.
Because hormesis induces a modest response (30-60% higher than the control) its
biological significance is sometimes overlooked (Calabrese, 2000). In agricultural ecosystems,
some investigators support the idea that hormesis may contribute to a pest resurgence or

13

secondary outbreak following an insecticide application (Guedes et al., 2010; Morse, 1998,
Cohen, 2006). Recently an increasing number of studies have investigated potential stimulatory
effects of sublethal doses of pesticides on populations of insects. For example, Guedes et al.
(2010) demonstrated that low doses of deltamethrin induced the maize weevil, Sitophilus
zeamais, to increase the intrinsic rate of population growth. A list of studies reporting an
occurrence of hormesis on different endpoints in arthropods was documented by Cohen (2006).
Recently, Cutler (2012) also published a more specific review on this concept by giving the main
focus of hormesis induced by insecticides in insects. Longevity and fertility were the biological
endpoints often reported in these studies (Cohen, 2006, Cutler, 2012). In some cases, the cause of
the stimulation was not well determined whether it was through a direct effect of the toxicants or
through an indirect effect such as through a change in physiology of the treated host plants or
other factors (Cohen, 2006, Cutler, 2012). For example, Van der Laan, (1961) reported an
increase in populations of whitefly after the application of DDT, and the author suggested that
this might have been related to a physiological change in the cotton host plants after the DDT
application, favoring the rate of viability of eggs. The authors excluded the reduction of natural
enemy as the main cause of the increase because others chemicals including endrin and toxaphan
which are as toxic to the natural enemy as DDT were studied simultaneously, however no
increase of whiteflies were observed in the plots treated with these chemicals.
The mechanism of hormesis is not yet elucidated, but Calabrese and Baldwin (2002)
argued that the occurrence of hormesis on different biological measures across different taxa
suggest that the induction of hormesis could not be explained by a single mechanism. These
authors argued that despite the unlikely occurrence of a single mechanism, it seems that the
hormesis results from a common strategy which consists of allocation of resources with the main

14

objective to conserve homeostasis in the organism (Calabrese and Baldwin, 2002). Several
studies also suggested that hormesis is a phenomenon that is related to an occurrence of stress
(Stebbing, 2009; Chujo et al., 2012). Calabrese et al., (2010) also reported several stress
responsive genes that were involved in the hormesis phenomenon in mammals. Zhao and Wang
(2012) reported that hormesis in the nematode Caenorhabditis elegans was also often associated
with a regulation of various metabolic and signaling pathways in addition to the involvement
of the antioxidant system triggered by various stress agents. An increasing number of authors
working on mammals suggested that hormesis might also be associated with an epigenetic
mechanism (Scott et al., 2009; Vaiserman, 2010, 2011).
An epigenetic phenomenon is suggested by the observation of hormesis on adult life
when treatment was applied at an early stage of development (Costantini et al., 2010). In fact, an
increasing body of evidence shows that an exposure of mammals to various chemical factors
during early stages of development can influence the phenotype of individuals when they
become adults, and this phenomenon is believed to be associated with an epigenetic mechanism
(He et al., 2006; Zawia and Basha, 2011). Epigenetics are the processes causing a heritable change
of gene expression without an alteration in the DNA sequence. Instead, the heritable changes are
caused by changes in DNA methylation, histone modification, and remodeling of chromatin which
consequently causes activation or repression of the genes (Jaenisch and Bird, 2003; Decluve et al.,
2009). In fact, the epigenetic status of a cell or an organism is vulnerable to modification

triggered by environmental factors during critical periods of development (Janish and Bird, 2003,
Reik et al., 1993). Once established, the epigenetic modification can be inherited by the daughter
cells after cell division (Ng and Gurdon, 2008), and the associated phenotype can therefore be
observed during the adult life of the individuals. In mammals, for example, this sensitivity to
modification may occur during embryo development or during the post natal period (Decluve et
15

al., 2009). The epigenetic modification induced by environmental factors can affect the somatic
cells or the germ cells as well. In mammals, one of the periods of sensitivity of the germ cell is
during the period of gonadal sex determination of the embryo when there is an epigenetic
reprogramming event (demethylation and then remethylation of the DNA) in the germ cells
(Skinner, 2008). It was noticed that the timing of the epigenetic reprogramming differs between
sexes and species. For example, remethylation of germ cells occurs so late in female mice that it
doesn’t happen until after birth (Reik et al., 2001). In rats, the demethylation of primordial germ
cells (PCG) occurs when they migrate to the precursor embryonic gonads, and the remethyaltion
of the germ cells starts in the gonads (Anway et al., 2005). Any treatment after this short period
of time did not have any effect on the germ cell (Anway et al., 2005). An epigenetic modification
that affects the germ cell can be inherited by the subsequent generation (Jabalonka and Raz,
2009). Skinner (2008) proposed two definitions to categorize inheritance of the traits by the
subsequent generation: multigenerational and transgenerational effects. In the multigenerational
effect, the F1 offspring are exposed directly to the modifying agent when they are inside the
treated pregnant mother. In the transgenerational effect, the effect was inherited by individuals of
the subsequent generation that were not directly exposed to the modifying agent, like the F3
generation in some studies with mammals. Pesticides are among the stress agents that can induce
transgenerational effects in various organisms. An increasing number of studies have shown a
transgenerational inheritance of the effect of pesticides on the reproductive system of mammals.
For example, the pyrethroid insecticide permethrin and the insect repellent N,N-Diethylmetatoluamide (DEET) were shown to induce a decrease of the primordial follicle pool size in the
ovary of the F3 generation in rats (Manikkam et al., 2012). Anway et al. (2005) also showed
that the fungicide vinclozolin and the pesticide methoxychlor reduced the number and motility of

16

sperm in rats and the effects persist until the F4 generation of the males.
Little is known about the sensitivity to the environmental factors or epigenetic
reprogramming and its timing in insects. However some studies on honeybees help elucidate this
phenomenon in insects. Changing the DNA methylation of honeybees during larval stage was
found to elicit a phenotypic plasticity in the adults; silencing of methyltransferases dnmt3
(enzyme responsible for the de novo DNA methylation) by treatment with small interfering RNA
of new hatched larva generated a high proportion of queens which have a well-developed ovaries
whereas the control individuals gave workers with rudimentary ovaries (Kukcharski et al., 2008).
Similarly, feeding honeybee larva with a royalactin, a protein of the royal jelly, during larval
stage can also induce a queen phenotype in honeybee and housefly adults, thus exhibiting an
increased fecundity and longevity (Kamakura, 2001). All of these studies seem to suggest that
the larval stage in insects might be vulnerable to change triggered by different environmental
factors. In addition, the effect of treatment applied during the larval stage of insects can even
persist in a few subsequent generations. For example, a change of the length of the antennae
from a short phenotype to a long phenotype in the Mediterranean flour moth Ephestia kuehniella
was observed when the fifth larval instar or pupae of this species was treated with a temperature
at 25 C or lithium, and the change was inherited by the offspring and persist until the F4
generation (Pavelka and Koudelova, 2001). Similarly, treatment of the third instar larva of moth
Spodoptera exigua with fenitrothion also caused a malformation of eggs in this species, and the
effect was observed in three unexposed generations (Adamski et al., 2005).
Reproduction is a key component of fitness of an organism, thus a study of effects of
insecticides on reproduction is important. It was already known that treatment of some insect and
mite species with insecticides could enhance their fecundity. For example, imidacloprid was found

to enhance the fecundity of the spider mites and this effect was suggested to be mediated by
17

some change in the treated plants (Szczepaniec et al., 2011). However, an elevated juvenile
hormone (JH) was also reported in species of insect exhibiting an increased fecundity after
treatment with imidacloprid during immature stage. For example, Yu et al. (2007) showed that
Chilo suppresalis treated with imidacloprid since neonate exhibited an elevated JH and an
increased fecundity of this species. Fecundity and JH titer of Tryporyza incertulas were also
reported to increase when this pest was treated with imidacloprid since egg stage (Wang et al.,
2005). These results seem to suggest that the effect of imidacloprid might be mediated by a
physiological change in the insect rather than in the treated host plants. The effect of low dose of
imidacloprid on fecundity in insects seems to support this hypothesis. Indeed, several studies
indicated that a low dose of imidacloprid applied during adult stage elicited an inhibitory effect
on the reproduction in various insect species. For example, He et al., (2012) found that low doses
of imidacloprid reduced the fertility of the B. tabaci whitefly treated with this insecticide as
adults. Boina et al. (2011) also reported a decrease in fecundity of the citrus psyllid, Diaphorina
citrius treated with imidacloprid as adults. However, in studies on some other insects, a
variability in results was noticed in the same species treated with the same insecticide depending
on the stage treated. For example, Bao et al., (2009) found a decrease in fecundity of brown
planthopper, Nilaparvata lugens exposed to sublethal dose of imidacloprid applied as topical
treatment during the adult stage. In contrast, Yin et al., (2008) reported an increase in fecundity
of females of the same species that grew on plant rice treated with imidacloprid since thirdinstar. This discrepancy in result was also observed with aphids Myzus persicae. For example,
Devine et al., (1996) showed that the number of nymphs produced by M. persicae treated with a
low dose of imidacloprid as adults declined when comparing with these of the untreated aphids.
In contrast, Ayyanhath et al. (2013) found an increase in fecundity of the same species of aphids

18

when the immature stages was treated. All these results across different species of insects seem
to suggest that the increase of fecundity induced by low amount of imidacloprid happens only
under specific conditions, which is when the treatment is applied during immature stage of the
insects, and the inhibitory effect was observed when the treatment is carried out on the adult
stage. However, Yu et al., (2010) also demonstrated an increase in reproduction of M. persicae
when the newly emerged adults of this species were treated with imidacloprid, thus even without
treating the immature stage. Therefore, it might be that the effect of imidacloprid can also be
inherited by the following generation; so that an increase in fecundity might be observed even
the individual offspring was not exposed during larval stage as long as the parents was already
exposed during their larval stage. A study on hormesis in fecundity, thus the study of effect of
low dose, will be then an adequate approach to help elucidate this phenomenon.

VI Objectives
In order to develop a more complete picture of the effect of imidacloprid on the population of
silver leaf whitefly repeatedly exposed to this compound, we initiated experiments to meet the
following objectives:

1. Determine the rate of development and heritability of imidacloprid resistance in whiteflies
heavily exposed to high doses of this compound.
2. Screen the response of other pesticides that are registered for control of whiteflies in
commercial greenhouses to determine levels of cross resistance.
3. Determine whether or not a continuous exposure to a low amount of imidacloprid can induce
hormesis in whitefly that might contribute to maintaining and promoting whitefly population.

19

CHAPTER II. Selection and heritability of imidacloprid resistance in Bemisia tabaci B
biotype.
I Introduction.
The whitefly Bemisia tabaci (Gennadius) (Insecta: Hemiptera: Homoptera: Aleyrodidae) is
an important pest of greenhouse plants, vegetables, legumes, grains and cotton, and may cause
significant crop losses (Oliveria et al., 2001, De Barro, et al., 2011). Chemical sprays are the
primary method used for its control in various cropping system (Horowitz et al., 2011). After a
series of control failures on many crops in the 1980’s when organochlorines, pyrethroids, and
organophosphate insecticides were heavily used, imidacloprid became the most widely used
insecticide for whitefly control (Palumbo et al., 2001). Due to extensive use of this insecticide,
high levels of resistance to this insecticide were documented in many different countries,
including Guatemala, Mexico, and Israel (Raunch and Nauen, 2003; Gorman et al., 2010). In
contrast to these results, and despite the reliance on imidacloprid for whitefly control on cotton in
Arizona in the 1990s, no report of whitefly control failure was documented there or in Florida
(Palumbo et al., 2001). However, selection of imidacloprid resistance in a laboratory in
California suggested a potential development of resistance in the B biotype whitefly (Prabhaker
et al., 1997). Moreover, in previous research where the level of imidacloprid resistance was
monitored in field populations, susceptibility varied considerably over time. Schuster et al.
(2010) reported reduced susceptibility to imidacloprid in some field populations collected in
Florida in 2001, 2002 and 2003, followed by increased susceptibility in the populations of the
same regions in 2004 and 2005. Dennehy et al. (2004) also found a high susceptibility of
whitefly to imidacloprid on cotton in Arizona in 2000 and 2003 after reduced susceptibility was
reported in previous years. This variation suggested an occurrence of reduction of susceptibility

20

to the imidacloprid in B biotype whitefly followed by a reversion of the insecticide resistance.
These observations from field suggest that several factors could influence the
development and loss of imidacloprid resistance in field populations. Biological, ecological and
operational factors in field production were suggested to modulate or accelerate insecticide
resistance in whitefly and in arthropods in general (Castle et al., 2010; Rosenheim and
Tabashnik, 1992). However, determining the most important factors that can cause change in the
susceptibility of natural populations of whitefly to insecticides is not easy due to the complexity
of the environmental factors involved. Alternatively, selection for resistance in a laboratory is an
effective method to study insecticide resistance to provide information such as the potential rate
for the development of the resistance and stability of the resistance (Brown and Payne, 1988).
Another advantage of selection experiments in the laboratory is that they enable researchers to
estimate the heritability (h2) of the resistance trait, thus the proportion of total phenotypic
resistance variation that is associated with the additive genetic variation (Falconer, 1989), and to
evaluate the risk of resistance to a pesticide (Tabashnik, 1992). Because populations of B biotype
whitefly are known to vary considerably among different geographic regions and cropping
systems (Tabashnik, 1992; De Barrow et al., 2011), it is also important for the greenhouse
floriculture industry to determine the heritability of imidacloprid resistance from B biotype
whitefly collected from a commercial greenhouse in the United States.
The initial objective of this study was to determine the rate of development of imidacloprid
resistance in a population of B biotype whitefly collected from a commercial greenhouse in
Michigan, by putting the population under imidacloprid selection pressure. Initially adults were
used in the selection regime. However, cultures in large cages failed to thrive due to infestations
of thrips and mites. This selection process was then replaced by a more controlled selection
21

approach using adults and immatures. The study enabled us to determine the heritability of
resistance as a tool for understanding the risk of development of imidacloprid resistance in
B. tabaci B biotype.

II Materials and methods.
II-1 Plants.
Cotton was used as a host plant for the whitefly culture and the experiments in this study.
The variety ST5599B was used initially; and a second variety, FM958LL, was used after
February 2009. The cotton seeds were provided by Cotton Incorporated, Cary, NC. Seeds were
planted first in a 48-cell tray with standard potting medium soil in the laboratory or in the
greenhouse with a 16:8 h (light/dark) photoperiod and grown until they reached the two true-leaf
stage. After, the cotton seedlings were planted in a 6’ pot with sterilized potting soil in the
greenhouse where they were grown. The young cotton plants at the three to four true-leaf stage
were introduced into the caged culture to maintain the whitefly population. Cotton seedlings at
the two-true leaf stage were also used for the bioassays.

II-2 Insecticide.
The formulated product Merit 75 WP was used as the source of imidacloprid for our tests,
the insecticide used was less than two years old. Tap water was used to dilute the formulation to
the concentrations needed in this study no physicochemical of the water was used.

22

II-3 Insects.
II-3-1 Resistant strain.
We obtained a few poinsettia plants infested with silverleaf whitefly from a commercial
greenhouse in Michigan in September 2006. The poinsettia plants were put in a cage at the MSU
greenhouse. The whiteflies were allowed to grow on these plants. Cotton plants were put in the
culture cage for the emerging adults to colonize. Colonies were then maintained on cotton plants
from January 2007 to April 2007. Cotton plants were systemically treated with a soil drench of
imidacloprid at the rate of 6 mg a.i. per 6’ pot before placing them into the culture cage (Table
2.1). The resistant strain collected from poinsettia was reared and selected for resistance with
increasing concentrations of imidacloprid from May 2007 until April 2008. This resistant strain
is referred to as MI-R1. Details of the selection procedure are described below in Table 2.1 and
Figure 2.1. Between April 2008 and March 2009 the culture became infested with mites and
thrips and was nearly lost. In April 2009 the resistant culture was split into two resistant cultures.
In one culture resistance was selected for from April 2009 to December 2009 with increasing
concentrations of imidacloprid. This resistant strain is referred to as MI-R2. Details of the
selection procedure are in Table 2.1 and Figure 2.2. A second resistant strain, referred to as MIr2a, was maintained on cotton plants treated with a constant concentration of imidacloprid (12
mg a.i. per pot). This resistant strain MI- r2a has been maintained from May 2009 until the
present time.
II-3-2 Reference strain.
We also maintained a population provided to us by Dr. Nilima Prabhaker, at the
University of California Riverside, CA. The population was started from a few cotton leaves
infested with whitefly pupae. The infested leaves were placed in a cage containing young cotton

23

plants, so that emerging adults would oviposit on them. This culture was maintained as a
reference strain from April 2007 to January 2009. This reference strain is referred to as CA-s1.
The reference population also became heavily infested with thrips and mites between September
and December 2008, and failed to thrive. The culture was replaced in February 2009 by a second
set of susceptible whitefly provided to us by Dr. Nilima Prabhaker and reared on a new variety of
cotton plants (FM958LL). This second reference strain, referred to as CA-s2, was also started
from a few cotton leaves infested with whitefly pupae. This reference strain was used from
February 2009 until present time. All whiteflies were reared in double-walled walk-in screencage with double-doors in a section of the Plant Sciences greenhouses at Michigan State
University. High intensity lamps are positioned above the cages to provide adequate lighting for
the plants.

II-4 Selection.
II-4-1 History of selection and the resistant strain.
The resistant strain and reference strain of whitefly were identified as B biotype by Dr.
Frank Byrne at the University of California, Riverside, CA. The timetable of the selection is
summarized in Table 2.1. Two sequences of selection were made to build-up resistance levels.
The first set of selections was made by exposing the resistant whitefly adults to imidacloprid
using systemic uptake bioassay method, and moving survivors to new non-treated plants. The
second selection procedure was conducted by exposing both adults and immatures to
imidacloprid. Immatures were exposed by rearing resistant whitefly on plants systemically
treated with imidacloprid. The emerged adults were collected and treated with successively
higher rates of imidacloprid using the systemic uptake bioassay, and moving survivors to new

24

plants that were systemically treated with imidacloprid.
II-4-1-a) Exposure of adult whitefly.
The first set of selections was made by exposing whitefly adults (MI-R1) from May 2007
to April 2008 to increasing concentrations of imidacloprid (Figure 2.1, Table 2.1). Whitefly
adults were selected for imidacloprid resistance by using a systemic uptake bioassay as described
by Cahill et al. (1996) and Li et al. (2000) with a slight modification. Cotton seedlings at the two
true-leaf stage were cut at the main stem, and the stems immersed in a 20ml-glass vial containing
imidacloprid (Merit 75 WP) at different selecting concentrations (from 30 to 500 mg/L) (Table 2.
1) for 24 h. After 24 h of imidacloprid uptake by the cotton seedlings, leaf discs were cut and put
in scintillation vials containing 2 ml of agar (1.3%) in the bottom of the vial. The leaf was put
upside down on the agar in the bottom of the vial so that the underside of the leaf faces up.
Fifteen to thirty six seedlings were used for each selection. Whitefly adults from the resistant
strain MI-R1 were collected into vials containing imidacloprid-treated leaf discs; the collection
was conducted during the adult emergence peak. For the collection, the mouth of the vial was put
onto the cotton leaf in a way that surrounds the adults to be collected, and then the cotton leaf
was shaken so that the whiteflies flew into the vial. Then the vial was capped with parafilm
which was punctured several times with an insect pin to allow some aeration. Fifty to 126 vials
were used for each selection and each vial contained 30 to 224 whitefly adults, the total of
number of adults collected and then treated was shown in Table 2.2 for each selection. The adults
were then placed on treated leaf discs for 48h at room temperature, after which the surviving
adults were collected and released on 18-30 new non-treated cotton plants in a clean cage. Eggs
produced by these females were allowed to grow until adulthood, and a new selection was made

25

Table 2.1. History of selections for the resistant whitefly culture.
Date

From September
2006 to April 2007
MI-R1

MI-R2

8 May 2007
4 June 2007
1 July 2007
2 August 2007
26 August 2007
27 September 2007
30 October 2007
17 December 2007
17 January 2008
18 February 2008
19 March 2008
23 April 2008
From May 2008 to
August 2008
From September
2008 to March 2009
April 2009
May 2009
June 2009
July 2009
August 2009
September 2009
October 2009
November 2009
December 2009

Generation

Selecting
concentration for
adult (mg/L)

Soil drench
concentration
(mg
a.i./6”pot)
Population
6
build up
(one treatment
per month)
Parental P
30
F1
30
F2
100
F3
200
F4
200
F5
350
F6
500
F7
500
F8
500
F9
500
F10
500
F11
500
6
(one treatment
per month)
Population near extinction
Parental P’
F’1
F’2
F’3
F’4
F’5
F’6
F’7
F’8

100
no adult selection
no adult selection
no adult selection
100
150
250
400
500

26

6
12
12
12
12
12
12
12

whitefly adults
collected and put in
leaf discs from
seedlings treated
systemically with
imidacloprid

egg hatch grow
trough larval stages
to become adults

Surviving adults
released to nontreated plants in a
clean cage to lay
eggs

Surviving adults
released to nontreated plants in a
clean cage to lay
eggs

egg hatch grow
through larval stages
to become adults

whitefly adults
collected and put in
leaf discs from
seedlings
systemically treated
with imidacloprid

Figure 2.1. Selection of adults of the resistant strain MI-R1 from May 2007 until April 2008
using a systemic uptake bioassay

27

(Figure 2.1). The selection procedure was repeated for 11 generations of the cultured whitefly
from May 2007 to April 2008.
The selection process was interrupted from May 2008 to March 2009 as the population
was heavily infested by mites and thrips, and the resistant culture was nearly lost. From May to
August 2008 the culture was maintained on cotton plants treated with imidacloprid at half of the
label rate (6 mg a.i. per pot), and from September 2008 to March 2009 the culture was
maintained on non-treated plants to allow the population to recover. Because there was an
interruption of selection during this period, the reversion of resistance was documented during
that time period.
II-4-1-b) Exposure of both adults and immature whiteflies.
A new variety of cotton (FM958LL) was used after the first set of selections. This second
selection procedure was conducted on the resistant strain MI-R2 from April 2009 to December
2009. Both adults and immatures of MI-R2 were exposed to imidacloprid (Figure. 2.2, Table
2.1). Whitefly adults were selected by using the same systemic uptake bioassays as described
above. Adults of the resistant strain MI-R2 were collected into 20 ml- scintillation vials and
exposed for 48 h to the leaf discs systemically treated with imidacloprid at different selecting
concentrations (100, 100, 150, 250, 400, 500 mg/ L) for 24 h (Table 2.1, 2.3). The collection was
performed when adult emergence reached a generation peak. Thirty two to 66 vials were used for
the selection and each vial contained 36 to 127 adults. The total numbers of collected adults were
shown in Table 2.3. Meanwhile, 12-18 new young cotton plants were treated with the selecting
concentration of imidacloprid (6 and 12 mg a.i per pot). Imidacloprid was applied as a soil
drench by pouring the solution of imidacloprid into the soil around the base of the plants. After
the collection of the emerged adults, the cotton plants were removed from the cage and the new

28

whitefly adults
collected and put in
leaf discs from
seedlings treated
systemically with
imidacloprid

Surviving adults
released to new
treated plants to lay
eggs

egg hatch grow
trough larval stages
to become adults
Plants removed from the
cage after adult collection

New clean systematically
treated plants introduced in
the cage
Surviving adults
released to new
treated plants to
lay eggs

egg hatch grow
through larval stages
to become adults

whitefly adults
collected and put in
leaf discs from
seedlings
systemically treated
with imidacloprid

Figure 2.2. Selection of the resistant strain MI-R2 from April 2009 to December 2009 using
systemic uptake bioassay and soil drench treatment

29

treated young cotton plants were introduced in the same cage. The surviving adults after the 48 h
exposure to imidacloprid were released on these new treated plants to lay eggs and this new
generation was allowed to become adults for a new selection. This selection procedure was
repeated for 8 generations of this resistant strain from April 2009 until December 2009, the
population was lost afterwards . Also, the number of surviving adults of F’4 after the adult
selection was very low; therefore the host cotton plants were not treated in order to obtain more
emerged individuals in the next generation.

II-5 Exposure of the resistant strain MI-r2a to soil-treated plants.
A second resistant strain, referred to as MI-r2a, has been reared from May 2009 until the
present time and studied at the same time with the selected resistant strain MI-R2. However as
opposed to the first resistant strain (MI-R2), this population (MI-r2a) was not selected for
resistance with increasing concentrations of imidacloprid but was maintained on cotton plants
treated with imidacloprid at 12 mg a.i. per pot. This second resistant strain MI-r2a was initiated
from eggs deposited on cotton plants by the new parental generation of the first resistant strain
MI-R2 recovered in April 2009. These eggs and few remaining resistant adults were allowed to
grow on these plants. Clean cotton plants treated with imidacloprid at 12 mg a.i per pot were
introduced monthly in the same cage to maintain the population. The imidacloprid was applied
as soil drench by pouring the solution of imidacloprid at the base of the plants. This rate was
adopted with this product in this study as it is the label rate recommended for whitefly control
with the imidacloprid Marathon 60WP. Cotton plants were only removed from the cage when
they are died (Figure 2.3). For this resistant strain M-r2a, generations of whitefly were not
separated and individuals of all ages could be found at any time, thus no exact number of

30

new plants added to
cage every month after
drenching soil with
imidacloprid at 12 mg
a.i. per pot

cage

dead or dying plants
removed and destroyed

Figure 2.3. Maintenance of the resistant whitefly MI-r2a on plants treated with imidacloprid
applied as soil drench from May 2009 until present time. This resistant strain MI-r2a was used
for the insecticide efficacy test and the study on sublethal effects of imidacloprid on whitefly.

31

generations could be determined in such a continuous culture.

II-6 Bioassay for determining LC50.
The susceptibility of the whitefly (resistant strain and reference strain) to imidacloprid
was evaluated by using the systemic uptake bioassay method as described above. Cotton
seedlings at the two true-leaf stage were cut at the main stem, and the stems were immersed in 20
ml of imidacloprid (Merit 75 WP) at different concentrations for 24 h. Six to ten different
concentrations of imidacloprid giving more than 0% and less than 100% mortality were used for
each test, and three to four cotton seedlings per concentration were used. Whitefly adults from
the resistant strains were collected into the vials as described above. Three to four replicates were
used for each concentration and each replicate contained 30-80 individuals. Mortality was
checked after the 48h- exposure. The same systemic uptake bioassay was also conducted on the
reference strain.

II-7 Data analysis.
II-7-1 Estimation of resistance ratio RR.
Data from the resistance bioassay were used to determine the slope and LC50 (SAS
version 9.1 software) for the reference strain and the resistant strain. The resistance ratio was
determined as the LC50 of the resistant strain divided by the LC50 of the reference strain. The
LC50 for the reference strain for the first regime of selection was the average of the two LC50
determined during this selection procedure. The LC50 for the reference strain for the second
regime of selection was also the average of the two new LC50 determined during this selection
32

procedure.
II-7-2 Estimation of realized heritability (h2).
The realized heritability (h2) for this resistant strain of Bemisia tabaci B biotype was
estimated based on the method used by Tabashnik (1992) according to the following formula:

h2 = R/S
where R is the response to selection ( Falconer, 1989) which is estimated as :

R = [log (final LC50) – log (initial LC50)] x 1/n
In this formula final LC50 is the LC50 of offspring after n generations of selection, and initial
LC50 is the LC50 of the parental generation before n generations of selection. Returning to the
original formula, S is the selection differential (Falconer, 1989) which is estimated as:

S = iσp
where i is the intensity of selection estimated from p, the percentage of population with values
above the selection threshold (percentage surviving selection) using Appendix A of Falconer
(1989). The phenotypic standard deviation (σp) is estimated as:

σp = [1/2(initial slope + final slope)] -1
To determine a change of the rate of resistance development during the course of selection, the
heritability of first half (F0 to F5) and second half (F5 to F11) of the selection regime by treating
adults of the resistant strain MI-R1 were determined (Tabashnik, 1992). The heritability in the
33

second set of selection by treating adults and immatures of the resistant strain MI-R2 was not
estimated as no adult selection (thus no mortality data) could be carried out in a few generations
(F’1, F’2, and F’3) during the selection due to the low number of emerged adults.
The number of generations G required for a 10-fold increase in LC50 is also estimated. For that,
the response to selection R was estimated with the mean slope obtained from F0 to F11 by
using the following formula:

R = h 2S
Then, the prediction of number of generation required for an increase of LC50 was estimated
according to the Tabashnik’ s formula (1992) :

G = R-1 = (h2S)-1

III Results.
The population of silverleaf whitefly (B biotype) MI-R1 collected from a commercial
greenhouse in Michigan showed a reduced susceptibility to imidacloprid (LC50 = 23.42
compared to a LC50 of 1.97 for our reference strain CA-s1 (Table 2.2), indicating a development
of resistance to imidacloprid under greenhouse conditions. When adults of this population were
exposed to an increasing concentration of imidacloprid, the LC50 level of resistance increased
from 23.42 mg /L in the parental generation to 284.47 mg /L in the F5 generation, and to 431.08
mg /L in the F11 generation (Table 2.2). However, the LC50 of the untreated reference strain
34

Table 2.2. Summary of the selection for imidacloprid resistance in the resistant strain MI-R1 by exposing the whitefly adults to
imidacloprid.

Strain

Generation

CA-s1
MI-R1
MI-R1

RFc
Parental
F1

MI-R1
MI-R1
MI-R1
MI-R1
MI-R1
CA-s1
MI-R1
MI-R1
MI-R1
MI-R1
MI-R1

F2
F3
F4
F5
F6
RF
F7
F8
F9
F10
F11

Selecting
dose
(ppm)
-

No. adults
treated
-

%
Mortality
-

Slope
± SE
1.03 ± 0.16

LC50
(ppm)
1.77

95% FLa

RRb

0.80-3.11

-

30
30

7471
7981

63
50

23.42
nd

16.68-33.51
nd

11
nd

100
200
200
350
500
500
500
500
500
500

11244
21756
5820
7179
5288
4885
3975
5966
8343
10328

57
87
62
57
45
47
46
46
47
51

1.15 ± 0.10
ndd
0.80 ± 0.06
nd
nd
0.71 ± 0.07
nd
0.60 ± 0.08
nd
1.22 ± 0.20
nd
nd
0.62 ± 0.04

57.87
nd
nd
284.87
nd
2.18
nd
411.07
nd
nd
431.08

38.20-85.22
nd
nd
197.62- 418.50
nd
1.16-3.87
nd
240.97-786.11
nd
nd
274.26-692.53

29
nd
nd
144
nd
nd
208
nd
nd
218

a) FL: fiducial limits
b) RR: resistance ratio =LC50 of resistant strain / LC50 of reference strain (where LC50 of reference strain is the average of the
two LC50 values of RF (1.97)).
c) RF: reference strain CA-s1
d) nd: not conducted

35

CA-s1 remained similar to the parental population when checked after six months (LC50 from
1.77 to 2.18 mg /L) (Table 2.2, Figure A1). When compared with the average LC50 (1.97 mg /L)
of our reference strain CA-s1, the level of resistance increased from 11-fold in the parental
generation to 29-fold in the F3 generation, 144-fold in the F5 generation, and to 218-fold in the
F11 generation (Table 2.2).After the F11 generation, the selection was interrupted for one year
due to a heavy infestation of the culture by thrips and mites. The culture was not exposed to
imidacloprid for 7 months to allow the population to recover. After this 7 month period, the
average LC50 of the new reference strain C-s2 was 4.16 mg/L whereas the LC50 of the resistant
strain MI-R2 was 48.48 mg/L (Table 2.3). This indicated a resistance ratio (LC50 resistant/LC50
reference strain) of 11- fold. This suggests that resistance can be significantly reduced (from
218–fold to 11-fold) in a short period of time (7 months) when selection pressure is not applied,
but not lost. Apparently imidacloprid resistance is not very stable in B. tabaci B biotype.
After this time, when selection was initiated again by exposing both immatures and adults
of MI-R2, the LC50 increased slightly from 48.48mg /L to 58.1 mg/L in F’3 generation, then to
193.75 mg/L in the F7 generation (Table 2.3, Figure A2). During the same period of time the
LC50 reference strain CA-s2 remained stable (4.12 and mg/L and 4.21 mg/L) (Table 2.3).
Comparing the LC50 of the resistant strain MI-R2 to the reference strain CA-s2, the resistance
ratio of the resistant strain MI-R2 increased from 11-fold in the first generation after the 7-month
hiatus in selection to 14-fold in the F3 generation, and to 46-fold in the F7 generation.
The response to imidacloprid exposure of resistant strain MI-r2a that has been reared
continuously on plants treated with imidacloprid at 12 mg a.i. per pot was shown in Table 2.4.
36

Table 2.3. Summary of the selection for imidacloprid resistance in the resistance strain MI-R2 by exposing whitefly immatures and
adults to imidacloprid.

Strain

Generation

CA-s2
MI-R2
MI-R2

RFc
P’
F’1

MI-R2
MI-R2
CA-s2
MI-R2
MI-R2
MI-R2
MI-R2
MI-R2

F’2
F’3
RF
F’4
F’5
F’6
F’7
F’8

Soil applied
dose
a.i.( mg)
-

Adultselecting
dose
(ppm)
-

6
12

100
ndd
nd
nd
100
150
250
400
500

12
12
12
12
12
12

No.
adults
treated
-

%
Mortality

Slope
± SE

LC50
(ppm)

95% FLa

RRb

-

1.15 ± 0.16

4.12

1.58-11.87

-

5187
nd

63
nd

0.94 ±0.20
nd

48.48
nd

8.90-386.88
nd

11
nd

nd
nd
2851
2027
2552
3113
4421

nd
nd
74
42
53
62
62

nd
1.01 ± 0.13
0.96 ± 0.11
nd
nd
nd
0.82 ± 0.10
nd

nd
58.1
4.21
nd
nd
nd
193.75
nd

nd
28.55-111.87
2.13 - 7.92
nd
nd
nd
112.39- 87.25
nd

nd
14
nd
nd
nd
46
nd

a) FL: fiducial limits
b) RR: resistance ratio =LC50 of resistant strain / LC50 of reference strain (where LC50 of reference strain is the average of the two
LC50 values of RF (4.16).
c) RF: reference strain CA-s2
d) nd: not conducted

37

Table 2.4. Resistance level of the resistant strain MI-r2a reared continuously on plants treated
with imidacloprid at a recommended dose (12 mg a.i. per pot) for whitefly control.

95% FLa

RRb

0.96 ± 0.11
0.95 ± 0.07

LC50
(ppm)
4.21
30.54

2.13 - 7.92
22.62 - 40.39

7

834
783

1.09 ± 0.06
1.03 ± 0.01

4.08
37.47

3.31 - 5.02
23.62 - 56.12

9

841
944

1.28 ± 0.15
1.18 ± 0.07

6.17
59.41

3.90 - 10.63
49.84 - 70.74

9

Date

Strain

Number
of adults

Slope ± SE

July 2009

Reference CA-s2
Resistant MI-r2a

632
563

Reference CA-s2
Resistant MI-r2a

April 2011 Reference CA-s2
Resistant MI-r2a

June 2010

a) FL: fiducial limits
b) RR resistance ratio (LC50 of resistant strain / LC50 of reference strain)

38

The resistance level in this resistant strain MI-r2a was relatively stable over two years (Table
2.4). The resistance ratio RR was slightly increased from 7-fold on July 2009 to 9-fold two years
later, indicating that the level of resistance increase much slower with this type of imidacloprid
pressure.
The overall mean estimate of realized heritability for imidacloprid resistant in this B biotype
whitefly population was 0.10, with a mean response of 0.10 and a mean selection differential (S)
of 0.99 (Table 2.5). This suggests that phenotypic variation for imidacloprid resistance in this
population of B biotype whitefly was accounted for by an additive genetic variation. The realized
heritability in the first half of the selection was higher (0.16) comparing with the second half of
the selection (0.02) (Table 2.5). This suggests that the additive genetic variance declines over the
course of selection. The projected rate of resistance evolution is proportional to the value of
heritability h2 and the selection intensity. For example, a 10-fold increase of LC50 would be
obtained in 3 generations with an intensity of selection causing 90 % mortality and h2 of 0.16
whereas the same rate of increase would require 10 generations with an intensity of selection
causing 50% mortality and with a h2 of 0.10 (Figure 2.4).

IV Discussion.
The population of whitefly (B. tabaci B biotype) collected from a commercial greenhouse
in Michigan showed a reduced susceptibility to imidacloprid compared to our reference strain,
indicating that resistance to imidacloprid developed under commercial greenhouse conditions.
By putting the population under selection pressure, the resistance level increase considerably.
Prabakher et al. (1997) also demonstrated development of imidacloprid resistance in a B biotype

39

whitefly population initially collected from the field in California and put under selection
pressure in a laboratory. Similarly, Wang et al. (2009) reported a high level of resistance in a B
biotype whitefly collected from a greenhouse in China after selection for imidacloprid resistance
for 30 generations in the laboratory. All of these results suggest that B. tabaci B biotype have a
potential to develop imidacloprid resistance when exposed to selection pressure.
After a 7-month hiatus from imidacloprid treatments, the resistance ratio RR of our
imidacloprid-resistant strain decreased 95%. This drop could be explained by the loss of resistant
alleles and an increase in susceptible alleles during this time. The rapid decline of resistance
levels suggests that imidacloprid resistance is not stable in this strain B biotype whitefly. Other
authors also reported the instability of imidacloprid resistance in B. tabaci B biotype. For
example, Schuster et al. (2010) reported a reduction by 80% of imidacloprid resistance in a lab
population of B biotype whitefly that was not exposed to the insecticide for four to six
generations. Gutierrez-Olivares et al. (2007) also reported a significant decrease of resistance
level in the B biotype whitefly without exposure to imidacloprid. Raunch and Nauen (2003)
reported a significant drop of the resistance level of the population of B biotype whitefly from
870-fold to 361-fold within six months. These relatively fast declines in resistance in unexposed
B biotype whitefly is in contrast to what has been observed for the Q biotype whitefly where an
elevated level of resistance is maintained even without exposure to imidacloprid for 2 years
(Nauen et al., 2002). Theoretical models indicate that the stability of insecticide resistance
depends on different factors including the level of the allele frequency in the population, the size
of untreated refuges and the fitness cost associated with the resistance (Carriere and Tabashnik,
2001). For example, Wilson et al. (2007) demonstrated that a reduction of pyriproxyfen
resistance in Q biotype whitefly was caused by the fitness cost associated with the resistance.

40

Whatever is the cause of decline, this instability of the imidacloprid resistance associated with
this temporal refuge when there was no exposure might partly contribute along with other factors
to the considerable variation of susceptibility of field populations of B biotype whitefly to
imidacloprid.
When imidacloprid treatments were initiated again, and a new regime of selection was
performed, the resistance ratio (RR) of our resistant strain MI-R2 increased again from 11-fold in
the parental generation P’ to 46-fold in the F’7 generation. This suggests that resistant alleles
were still present in the population despite the temporal refuge without exposure, and the
population could still respond to the new selection. Wilson et al. (2007) demonstrated that the
pyriproxyfen resistance in Q biotype whitefly was also found to be unstable but was not lost
completely even without the insecticide exposure, and the simulation study projected that the
pyriproxyfen resistant allele would remain in the unexposed Q biotype population for 55
generations, thus for 5 years (Crowder et al., 2005).
The size of the resistant population used in the first set of selections of MI-R1 (May
2007 to April 2008) was much larger than the population used in the second set of selections of
MI-R2 (April 2009 to December 2009, Tables 2.2, 2.3). The low number of emerged adults
prevented us from performing a selection in the first three generations, F’1, F’2 and F’3 (April
2009 to June 2009), of the resistant strain MI-R2 (Table 2.3). The small size of the population
may be due to prolonged exposure of sessile immatures, which are also more susceptible to
imidacloprid than the adults. Indeed, the resistance in the current study is likely associated with
the P450 detoxification system ( Chapter 3 Table 3.1 ). Nauen et al. (2008) demonstrated that
the expression of the P450 gene depends on the age in whitefly B and Q biotypes, and these

41

authors stated that the level of expression in the immature stage was so low that whitefly was
vulnerable at this early stage. Thus, high mortality in the immature was not surprising.
The second resistant strain, MI-r2a, has been maintained on plants treated with
imidacloprid at 12 mg a.i. per pot, a label rate for whitefly control when using imidacloprid
Marathon 60WP. Generations overlapped in this culture as plants were only removed when they
died. Resistant levels remained relatively stable for two years (Table 2.4). This suggests that the
regular use of imidacloprid as a soil drench will maintain resistance in the population but that the
level of resistance increases slowly when compared with the resistant strain MI-R2 selected with
an increasing concentration of imidacloprid (Table 2.3, 2.4).
The study of realized heritability is commonly used to summarize the results of selection
for insecticide resistance (Tabashnik, 1992). Rosenheim (1991) did a thorough analysis of
different factors that were not controlled in the current study and that may cause an
underestimation or overestimation of the realized heritability value. He reported that the h2
estimate was underestimated if there is a differential uptake of the toxicants between all
individuals in the population studied, especially between the more tolerant and less tolerant
individuals. A difference of tolerance to the toxicant between male and female could also induce
an underestimation of the realized heritably, especially if the selection is carried out in adults that
have been already mated (Rosenheim, 1991; Bloch and Wool, 1994). No investigation of the
effect of imidacloprid on male and females were performed in this study. Conversely, an
overestimation of realized heritability can occur if the sublethal effect of the toxicant induces
difference in the reproduction of the surviving individuals with less fecundity in the less tolerant
individuals. Thus, the interpretation of the realized heritability h2 estimate needs caution due to

42

these biases and limits that could be associated with the different factors mentioned above (Bloch
and Wool, 1994).
The overall realized heritability h2 estimate in this study was 0.10. Heritability of
a population can change over time, and the heritability value during the time when the most
significant change in resistance occurs was suggested to represent better the heritability of the
population (Huang et al., 1999). Thus by using the heritability in the first half of the selection,
the heritability estimate in this population was 0.16 under the conditions used in this study, this
estimate was similar to that found by Sethi et al. (2008) with a different population in an
independent study (h2 = 0.16). The phenotypic variation (VP) of a threshold trait such as
insecticide resistance is a combination of the additive genetic variation (VA) and environmental
variation (VE) (Falconer1989, Firko and Hayes, 1990). And heritability (in a narrow sense) is
defined as the proportion of the total phenotypic variation attributable to the additive genetic
variation (Falconer, 1989; Tabashnik, 1992). Thus, the heritability analysis in this study
indicated that 10% of the total imidacloprid tolerance variation in this population of B biotype
whitefly is accounted for by an additive genetic variation.
An analysis of h2over a course of selection was also performed. Additive genetic
variation, thus the heritability, has a maximal value when the allele of the trait of interest
is at intermediate frequency and is zero when the alleles of interest is absent or fixed in the
population (Falconer, 1989). Similar to the result found by Sethi et al. (2008) who also selected
for imidacloprid resistance in B biotype whitefly, the h2 estimates in the current study declined
from the first half to the second half of the first set of selections, suggesting a decrease in
additive genetic variation, thus an increase of the resistant alleles as the selection proceeds.
43

Table 2.5. Estimation of realized heritability of resistance to imidacloprid in Bemisia tabaci B biotype collected from a commercial
greenhouse in Michigan.

Generation

Estimation of response to selection

Estimation of selection differential

h2e

Initial LC50 (Log)

Final LC50 (Log)

Ra

ib

Mean Slope

σpc

Sd

F0-F5

1.369

2.454

0.180

1.020

0.930

1.075

1.096

0.164

F5-F11

2.454

2.634

0.025

0.766

0.665

1.503

1.152

0.022

F0-F11

1.369

2.634

0.105

0.880

0.885

1.129

0.994

0.106

a) R: Response to selection
b) i: intensity of selection
c) σp: phenotypic standard deviation
d) S: selection differential
e) h2: realized heritability estimate

44

12

h2 = 0.16

Generation

10

h2 = 0.10

8
6
4
2
0
50

60

70

80

90

Mortality (%)

Figure 2.4. Effect of heritability (h2) on number of generations of Bemisia tabaci B biotype
required for a 10-fold increase in LC50 of imidacloprid at different selection intensities.

*** For interpretation of references to color in this and other figures, the reader is referred to
electronic version of this dissertation.

45

The main purpose of heritability study is to be able to forecast the rate of resistance
development, thus to assess the risk of resistance to an insecticide (Tabashnik, 1992). The
projected rate of the resistance development in this study predicted that a 10-fold increase of
LC50 would be reached in 7 generations with a heritability h2 of 0.16 and a mortality of 50% per
generation (Figure 2.4). Interestingly, the result of selection in laboratory carried out by
Prabhaker et al. (1997) in California falls within this range. These authors selected for
imidacloprid resistance in B biotype whitefly by using 40 to 50% mortality per generation and an
increase of 10-fold of LC50 was observed between 5 and 7 generations of selection. Our results
also predicted that as few as 3 generations may be needed to develop a 10-fold increase of LC50
under higher selection intensity (90% mortality per generation) and a heritability h2 of 0.16
(Figure 2.4). This high mortality of 90% per generation was used by Wang et al. (2009) for
imidacloprid resistance selection in B biotype whitefly collected from cotton greenhouse in
China. Wang et al. (2009) did not show all of their results; however they reported a 254-fold
increase in LC50 after fifteen generations of selection. Thus, the results in this study seem to be
in agreement with those found from other independent laboratory experiments for imidacloprid
selection on B biotype whitefly. The laboratory results suggest that B biotype whitefly has a
potential to develop imidacloprid resistance and the resistance could be detected after a few
generations depending of the intensity of selection.
Despite this consistency of results among laboratory experiments, it may not necessarily
reflect the situation in field as the value of heritability h2 is specific to one particular population
under the specific environmental conditions to which they are subjected (Falconer, 1989;

46

Tabashnik and McGaughey, 1994). Moreover, heritability in laboratory is likely to have less
environmental variation than in the field (Bull et al., 1982). In addition, the h2 estimate may have
limitations and biases as explained previously; therefore caution should be used when using it as
a reference for field populations.
Consistent with many other reports studying quantitative genetic of insecticide resistance
in different pests (Lai and Su, 2011; Tabashnik, 1992), the current study found that the response
to selection, thus the rate of development of resistance in this whitefly population, depends on
the heritability of resistance and the intensity of selection (Figure 2.4). From this viewpoint,
assuming field population having even lower heritability found in the present study, the whitefly
population showing high level of resistance in field might have been experienced a very high
intensity of selection for several reasons such as the cropping system and pest management
practice. That would be probably the case for the whitefly population collected from the
greenhouse in Israel reported by Nauen et al. (2008).
The low realized heritability (0.10) observed in this study also suggests an importance of
the environmental variation in the imidacloprid tolerance phenotype variation in this population.
It was emphasized that the heritability parameter does not provide information on the exact
magnitude of the contribution of the environmental components and the genetic components in
the trait of interest; however environment can have some influence on the expression of the
phenotype (Visscher et al., 2008). Consistent with the low heritability value in the current study,
several studies have demonstrated previously that different environmental factors could influence
the susceptibility of whitefly to imidacloprid. For example, Castle et al. (2010) reported that the
quality of the host plant could influence the expression of imidacloprid resistance in B biotype
whitefly. These authors showed that the population of B. tabaci collected from cotton plants in
47

Arizona at the end of the season, thus when the host plant cottons were already in a low quality,
was found to be susceptible to imidacloprid; however a resistance to this insecticide was revealed
when the same population was reared on untreated cotton plants with a higher quality in the
laboratory. Huang et al. (2011) also reported that the susceptibility of B biotype whitefly to
imidacloprid increased with the temperature. Several studies also demonstrated that the
susceptibility of B biotype whitefly to several insecticides varies depending on the host plants on
which they were reared (Castle et al., 2009, Liang et al., 2006; Xie et al., 2010). A wide
variability of the level of imidacloprid resistance depending on the location where the whitefly
was collected was already reported previously. For example, Vasillou et al. (2011) reported some
resistance level varying from moderate to high in B biotype whitefly populations collected from
different regions in Cyprus, however the authors did not found a significant correlation between
the history of neonicotinoid application and the resistance level. This is in contrast to the results
found on Q biotype whitefly as demonstrated by Roditakis et al. (2008) where the variability of
the level of resistance in Q biotype populations collected from different locations in Crete was
well correlated with the number of imidacloprid applications. Besides the identified
environmental factors, some source of variations that have effect on the phenotypic variation are
unobserved. Recently, increasing number of studies working on heritability of quantitative traits
suggested that epigenetics might be the mechanism behind the observed or non-observed
environmental variation and genetic variation, thus on the heritability of the trait (Feninberg and
Irizarry, 2010, Petronis et al., 2010; Wong et al., 2005). All of these studies suggested that many
other factors might influence the evolution of resistance to insecticides in field population.
Therefore, in concurrence with several previous reports, this study suggest to take into account

48

the environmental situation in field, along with the results generated from laboratory, when
assessing and managing the risk of pesticide resistance in B biotype whitefly .

V Conclusion.
The study of the realized heritability suggests that the imidacloprid resistance was
genetically based and can be inherited by the upcoming generations. The B biotype whitefly can
develop a high level of resistance to imidacloprid with an intensive use of this compound. This
could explain the observation of high resistance levels in some field populations. Similar to the
results found of a previous study, the realized heritability was found to be relatively low
suggesting that a small proportion of imidacloprid tolerance variation only was attributable to the
genetic variation in this population under the conditions used in the current study. This might
also explain partially the wide variability of the susceptibility of resistance in different fields.
However, due to different limitations and biases that can be associated with the realized
heritability estimate; this explanation remains only suggestive but not conclusive. Another
important characteristic of resistance is its stability after an arrest of the insecticide exposure.
Consistent with other previous findings, this study showed that the imidacloprid resistance was
not stable in B biotype whitefly as it was significantly reduced within a few months without
imidacloprid exposure. This instability of the resistance might also play an important role to
moderate the development of resistance. That could be another additional cause of the observed
variability of imidacloprid susceptibility in field population over time. However, despite the
increase in susceptibility after a temporal refuge, the resistance was not completely lost and the
population was able to respond with a new set of selections after seven months. Therefore, it
would be advisable to give enough time for the population to regain susceptibility before using
49

imidacloprid again. In brief, this experiment in laboratory suggests that in addition to the
ecological, operational factors which are already known to influence the evolution of resistance
in field, some other factor such as heritability, instability of resistance can influence evolution of
resistance. Thus, a combination of results from both the laboratory and field assessment would
be the better strategy to manage the imidacloprid resistance in field population.

50

CHAPTER III. Response of imidacloprid-resistant whitefly B biotype to pesticides used for
whitefly control.
I Introduction.
Bemisia tabaci is a complex species of whitefly that comprises up to 20 biotypes (Perring
et al., 2001). Among them, biotype B and biotype Q are the most invasive and most damaging.
These two biotypes vary in their biology such as fecundity, host range, efficiency to virus
transmission (DeBarro et al., 2006, Gottlieb et al., 2010) and also insecticide resistance where Q
biotype is prone to develop higher resistance levels than B biotype (Horowitz et al., 2005,
Kontsedalov et al., 2012). Bemisia tabaci B biotype was first found to cause economic damage
on poinsettia in Florida in 1986, followed by reports from Arizona, California and other states
(McAuslane, 2010). The impact of whitefly feeding, virus transmission and sooty mold on
cultivated plants has caused enormous annual crop losses worldwide (Oliveria et al., 2001). As a
result of widespread use of chemicals for its control, whitefly has become resistant to many
conventional insecticides including many organophosphates and pyrethroids (Cahill et al., 1995;
Dennehy and William, 1997). In the early 1990s, imidacloprid, belonging to a different class of
insecticide called the neonicotinoids, was introduced to growers as one option of chemicals to
control whitefly (Nauen and Denholm, 2005; Horowitz et al., 2011). Imidacloprid acts on the
central nerve system as an agonist of the nicotinic acetylcholine receptors (nAChR) in insects
(Liu and Casida, 1993). Because imidacloprid had a mode of action that was different from
insecticides being used at the time, it gave excellent control of resistant populations of whitefly
(Rauch and Nauen, 2002). Imidacloprid is very effective in controlling sucking insects due to its
systemic properties (Tomizawa and Casida, 2003). Within a few years imidacloprid became the
standard product for control of whitefly in North American greenhouses, and by 2004 resistance

51

to imidacloprid had been well documented in the Q biotype of Bemisia tabaci, which originated
from overseas. Between 1995 and 2011 several additional neonicotinoid products also became
available to greenhouse growers, including thiamethoxam, acetamiprid, and dinotefuran. Since
these insecticides are in the same chemical class and share a common target sites as imidacloprid
(Honda et al., 2006), entomologists working with the greenhouse industry were wary of crossresistance problems. Most of the research on whitefly resistance to imidacloprid was done with
the Bemisia tabaci Q biotype (Nauen and Denholm, 2005), and recommendations for growers to
use insecticides in other chemical classes became widespread, despite the fact that most growers
had problems with the B biotype, not the Q biotype (Byrne et al., 2010).
Research projects focusing on whitefly resistance to imidacloprid demonstrated that
populations of both Q and B biotype could develop resistance to imidacloprid. However, a wide
variation in the response of imidacloprid resistant whitefly to other neonicotinoid insecticides
was noticed. For example, different populations of B biotype were found to vary from having a
resistance ratio (RR) of 2 and 5 in the most susceptible populations to a ratio of 78 and > 900 in
the most resistant populations, for acetamiprid and thiamethoxam, respectively (Prabhaker et al.,
2005; Rauch and Nauen, 2003). Elucidating the amount of cross resistance in an imidaclopridresistant population of B biotype whitefly in the Midwestern USA would be very helpful to
greenhouse growers considering the use of neonicotinoid insecticides to control whitefly.
Because the level of cross-resistance in imidacloprid-resistant populations of B biotype is
uncertain, we conducted our research with a B biotype culture that was started from infested
plants in a commercial greenhouse in Michigan. The susceptibility of this population to other
insecticides of different classes registered for whitefly control was also compared with a
reference strain.

52

II Material and Methods.
II-1 Plants.
Cotton plants were used in the study (FM958LL). Cotton seeds were planted in a 48-cell
tray with potting soil and the trays held in a laboratory or in the greenhouse until plants reached
the two true-leaf stage. Seedlings in the two true-leaf stage were used in a systemic uptake
bioassay or they were planted in 6” pots with potting soil in the greenhouse as described
previously. Cotton plants in the four to six true-leaf stage were used to test the susceptibility of
the whitefly to different insecticides. Seedlings were treated with Pylon (chlorfenapyr, 0.98
ml/L) when they were in the cotyledon stage to prevent infestations of thrips and mites.

II-2 Insecticides.
Formulated products registered for control of whitefly on greenhouse or other crops were
used in the present study. Ten insecticides belonging to six different chemical classes and one
microbial insecticide were tested. The insecticides tested, their product name and the chemical
class and group according to Insecticide Resistance Action Committee (IRAC) are:
imidacloprid (Merit 75WP, neonicotinoid 4A); acetamiprid (Tristar 30SG, neonicotinoid, 4A);
dinotefuran (Safari 20G, neonicotinoid 4A); thiamethoxam (Meridian 25WG, neonicotinoid,
4A); lambda-cyhalothrin (Scimitar GC, pyrethroid, 3A); fenpropathrin (Tame 2.4 EC,
pyrethroid, 3A); flonicamid (Aria 50WG, novel chemistry, 9C); pyriproxyfen (Distance, insect
growth regulator [IGR], 7C); buprofezin (Talus 40SC, buprofezin inhibitors of chitin
biothesythisis, 17); pymetrozine (Endeavor 50WG, pymetrozine, 9B); and Beauveria bassiana
(Botanigard 22WP, live fungal spores). All insecticides used were less than two years old. All
formulated products were diluted with tap water to the needed dose, no physicochemical

53

properties of the water were tested.

II-3 Whitefly.
II-3-1 Reference strain.
We maintained a reference strain, CA-s1, of the B biotype whitefly in the greenhouse
(with an LC50 of 1.97 mg /L for imidacloprid) from April 2007 to January 2009 (see Chapter I).
This reference strain was used for testing for cross-resistance among neonicotinoid insecticides
using systemic uptake bioassay. The CA-s1 population was replaced in February 2009 by a new
reference strain, CA-s2, (with an LC50 of 4.26 mg /L for imidacloprid) from the same source as
the first one (from Dr. Prabhaker, University of California Riverside, CA.) due to mites and
thrips infestation (see chapter I). This second reference strain (CA-s2) was used in the insecticide
efficacy testing under greenhouse conditions from June 2009 until October 2009.
II-3-2 Resistant strain.
The resistant strain, MI-R1, was initially obtained from a commercial greenhouse in
Michigan. This population was subjected to selection for imidacloprid resistance from May
2007 to April 2008 (Table 2.2 of chapter I). It was maintained and split into two cultures in
April 2009, as described in Chapter I. The two resistant strains MI-R1 and MI-r2a were used in
this study. Tests on cross resistance to neonicotinoid insecticides using a systemic uptake
bioassay method in the laboratory were conducted on generation F5 of the selected resistant
strain MI-R1. In September 2007, this population was 144-fold more resistant compared with
the reference strain CA-s1, with an LC50 for imidacloprid being 284.87 mg/l (Table 2.2 of
chapter I). The test of the synergistic effect of pyperonyl butoxide (PBO) which is an inhibitor of

54

P450 detoxification enzyme was also performed on the F8 generation (in January 2008) and on
F11 generation (in April 2008) of this selected resistant strain MI-R1.
Tests on insecticide efficacy and cross resistance under greenhouse conditions were made
with the resistant strain, MI-r2a, between June 2009 and October 2009. This population was 7fold more resistant to imidacloprid when compared with the reference strain CA-s2. The LC50
value for imidacloprid of this resistant strain MI-r2a was 30.54 mg/L (Table 1.4 of chapter I).

II-4 Systemic uptake bioassay for determining LC50.
A systemic uptake bioassay method as described in the previous section (II-6 of Chapter
I) was used for the study of cross-resistance of our imidacloprid-resistant whitefly colony to
thiamethoxam, acetamiprid and dinotefuran. Briefly, cotton seedlings at the two true-leaf stage
were systemically treated with different concentrations of imidacloprid for 24h. Six to nine
concentrations giving more than 0% and less than 100% mortality were used. Whitefly adults
from the generation F5 of the resistant strain MI-R1 were collected as described previously into
the scintillation vials. Four replicates were used for each concentration and each replicate had
thirty to fifty adults. Mortality was checked after 48h. The same experiment was conducted with
the reference strain.

II-5 Efficacy of insecticides to the resistant strain MI-r2a.
II-5-1 Test on adults.
The experiment was conducted in the greenhouse. Cotton plants in the four to six trueleaf stage were planted in 6’ pots in the greenhouse for this test. Cotton plants were treated with

55

insecticides at the highest labeled rate. If products are labeled for use as a soil drench, they were
applied that way. However, if they are only labeled as a foliar spray, they were sprayed. Four
neonicotinoid insecticides were tested: imidacloprid (Merit 75WP) applied as a soil drench at a
rate of 12 mg a.i./pot; acetamiprid (Tristar 30SG) applied as a foliar spray at 5.3 oz /100 gal;
dinotefuran (Safari 20SG) applied as a soil drench at a rate of 24 oz /100 gal; and thiamethoxam
(Meridian 25WG) applied as foliar spray at 8.5 oz/100 gal, buprofezin (Talus 40SC) applied as a
foliar spray at a rate of 9 fl. oz /100gal, pyriproxyfen (Distance IGR) applied as a foliar spray at
8 fl. oz. /100 gal; Beauveria bassiana (Botanigard 22WP) applied as a foliar application at 1 lb
/100 gal; and lambda-cyhalothrin (Scimitar GC) applied as a foliar spray at 148 ml /100 gal.
For soil drench treatments, insecticide was applied by pouring 100 ml of solution around the base
of each plant. The treatment was performed 24 h before the beginning of the test to allow cotton
plants time to uptake the insecticide. For the foliar treatment, one liter of solution of insecticide
was prepared and the insecticide was applied with a garden plant sprayer. The upper side and
lower side of the leaf were sprayed until first runoff to ensure good coverage. Plants were
sprayed three hours before caging whiteflies on the plants to allow time for the spray to dry on
the leaves. Water was sprayed for the control treatment.
Unsexed whitefly adults from the resistant strain MI-r2a and reference strain CA-s2 were
collected in 20 ml scintillation vials by filling them with CO2, and keeping them capped with a
thumb until the vial’s mouth was placed over whiteflies on a leaf. The leaf was shaken to
encourage the whiteflies to move into the vial. Anesthetized whitefly adults were counted and
transferred into a clip-cage by pouring them from the vial into the clip-cage.
The clip cage was attached to a wooden stake as a support and was placed on the
underside of well-developed leaves of treated plants. Each plant supported two clip-cages, one

56

on each of its two opposite leaves. On each plant one of the clip cages contained reference adults
and the other one resistant adults. Each treatment was replicated four times and each replicate
contained between thirty and ninety adults. Adult mortality was checked after 3 days.
II-5-2 Test on immature.
Adults from the resistant strain MI-r2a and the reference strain CA-s2 were collected in
vials as previously described. Thirty unsexed adults were transferred with a thin brush into one
clip-cage for oviposition. Clip-cages containing the whiteflies were then put on plants so that
each plant supported one cage for susceptible adults and one cage for resistant adults. Adults
were released after two days, and the clip-cages re-established to cover the eggs which had been
deposited. The number of larvae emerging from the deposited eggs was counted under a
dissecting microscope after eight days. Clip-cages were re-established after counting and
treatment. Cotton plants with four to six true-leaves, grown in 6’ pots in the greenhouse were
used. Plants were treated with insecticide at the highest labeled rate. The same insecticide
treatments used in the adult assay were used again for this test on immatures. Three additional
insecticides were also tested: flonicamid (Aria 50WG) applied as a soil drench at a rate of 120
g/100 gal, fenpropathrin (Tame 2.4 EC) applied as a foliar spray at 16 fl. oz./gal, and
pymetrozine (Endeavor 50WG) applied as a foliar spray at 20 g/100 L. The clip cage was
removed for the foliar application and then re-established after the treatment. Treatments were
made on 20 and 21 June 2009 for the four neonicotinoids (imidacloprid, dinotefuran,
thiamethoxam and acetamiprid); on 30 June 2009 for buprofezin and pyriproxyfen; on 24 July
2009 for lambda-cyhalothrin and Beauveria bassiana and on 20 September 2009 for flonicamid,
fenpropathrin and pymetrozine. Water was used for control.
Mortality was checked under the microscope one week after treatment. Immature mortality at

57

the end of the test was also determined by subtracting the number of empty pupal cases left by
emerged adults from the number of treated first instars; this count was performed from 17 to 20
days after the treatment. Cotton plants used in the test for cross resistance to flonicamid,
fenpropathrin and pymetrozine became so heavily infested with mites and thrips that the test was
ended one week after treatment. Each treatment was replicated four times; each replicate
contained 92 to 418 larvae.

II-6 PBO synergism test.
A piperonyl butoxide (PB0) synergism test was also conducted for determining the
possible involvement of P450 monooxygenases in the expression of resistance. Cotton seedlings
at the two true-leaf stage were collected from the laboratory. Discs from the cotton leaves were
cut and immersed for 10 s in 10 ml of 500 ppm PBO solution containing 10 µl of Triton X-100.
Then, the leaf discs were air-dried at room temperature. After drying, leaf discs were transferred
into scintillation vials containing 2 ml of agar (1.3%) in the bottom of the vial. The leaf disc was
put upside down so that the lower side of the leaf faces up. Whiteflies from the generation F11 of
the reference strain MI-R1 were collected into the vial as described previously (II 4-1a of chapter
I). Then the adults were exposed to these PBO-treated leaf discs for 7 hours for F11. After, the
PBO pre-exposed adults were transferred to new vials with imidacloprid-treated leaf discs for the
systemic uptake bioassay described previously. For the transfer, the mouth of two vials was
connected to each other so that the vial with whitefly adults was placed below the other vial.
After shaking the vials gently the whitefly adults moved upward into the vial above. Six
concentrations giving more than 0% and less than 100% mortality were used for the assay. Four
replicates were used for each concentration and each replicate contained thirty to fifty adults.

58

The mortality was checked after 48h. The same procedure was performed with adults preexposed for 7 h to leaf disc immersed for 10 s in 10 ml of water containing with 10 µl of Xtriton. Then, the same systemic uptake bioassay for the exposure of the whiteflies to
imidacloprid for 48h was performed. The same test was also conducted on generation F8 of this
resistant strain MI-R1 but with a shorter exposure time of 2 hours to a higher concentration of
the PBO solution (1000 ppm). This test was conducted during the trial for determination of the
right dose to be used for PBO test.

II-7 Data analysis.
SAS (version 9.1) software was used to determine the LC50 value of the neonicotinoids.
The ratio of LC50 of the resistant strain and the reference strain was calculated to determine the
cross resistance level. Two LC50 are considered significantly different when their 95 % fiducial
values do not overlap.
The number of individuals tested were different between treatments, thus the efficacy of
insecticides on immatures and adults was determined by using Henderson -Tilton formula
(1955):

Efficacy (%) = [1 - (Ta x Cb / Tb x Ca)] x 100

in which Ta = live individuals in the treatment after treatment
Tb = live individuals in the treatment before treatment
Ca = live individuals in the control after treatment
Cb = live individuals in the control before treatment
59

In order to determine whether imidacloprid resistant whiteflies exhibit resistance to other
insecticides registered for whitefly control, the efficacy of the resistant strain MI-r2a and
reference strain CA-s2 was compared with t-student test by using SPSS 17.5.

III Results.
In fall of 2007 the LC50 of imidacloprid for the resistant strain CA-s1 was found to be
1.97 mg/l when building a high level of resistance in the imidacloprid-resistant strain MI-R1.
Two years later in April 2009 after the population decline due to mite and thrips infestations,
new cotton variety and new reference strain CA-s2 were used in the study. The LC50 value of
imidacloprid for the reference strain CA-s2 was 4.16 mg/L when we tested for insecticide
efficacy under greenhouses conditions.
When the generation F8 of the resistant strain MI-R1 were pre-exposed to PBO, the LC50 value
decreased from 411.07 mg/L (no PBO exposure) to 81.59 mg/L (pre-exposed to PBO at 1000
ppm) (Table 3.1). Similarly, the LC50 of imidacloprid to generation F11 of this resistant strain
dropped from 431.08 mg/L to 32.47 mg/l when they were pre-exposed to PBO at 500 ppm for 7
hours (Table 3.1). These results suggest a synergistic effect of PBO and suggest that cytochrome
P450 monooxygenases could be involved in the imidacloprid resistance mechanism for this
population.
Cross-resistance of whitefly in the imidacloprid-resistant strain MI-R1 to other
neonicotinoids was also studied. When the resistant strain MI-R1 and reference strain CA-s1
were treated with thiamethoxam, the LC50 for resistant strain was 29.20 mg/l, compared with

60

Table 3.1. Synergistic effect of PBO on the toxicity of imidacloprid to F8 and F11 generation of
the resistant strain MI-R1 of Bemisia tabaci B biotype.
95% FLa

RRb

1.22 ± 0.20

LC50
(ppm)
411.07

240.87-86.11

208

773

1.02 ± 0.11

81.59

54.17-124.84

45

1602

0.62 ± 0.04

431.08

274.26-92.53

218

1204

0.79 ± 0.14

32.47

4.17-142.57

16

Generation

Treatment

Number
of adults

Slope ± SE

F8

Imidacloprid
without PBO
Imidacloprid
with PBO

959

Imidacloprid
without PBO
Imidacloprid
with PBO

F8 c
F11
F11d

a) FL fiducial limits
b) RR resistance ratio (LC50 of resistant strain / LC50 of reference strain (1.97)). Resistance
strain MI-R1 and reference strain CA-s1 were used in the test
c) whiteflies were pre-exposed to PBO at 1000 ppm for 2 hours
d) whiteflies were pre-exposed to PBO at 500 ppm for 7 hour

61

Table 3.2. Response of the reference strain (CA-s1) and the generation F5 of the imidaclopridresistant strain (MI-R1) of Bemisia tabaci B biotype to three neonicotinoid insecticides in a
systemic uptake bioassay.

Number
of
adults

Slope ± SE

LC50 (ppm)

95% FLb

RRc

Acetamiprid
Reference
Im-Resistanta

1252
674

0.81 ± 0.11
1.06 ± 0.09

1.21
8.65

(0.79 – 1.78)
(5.60 – 12.68)

7

Thiamethoxam
Reference
Im-Resistant

960
561

0.93 ± 0.15
1.51 ± 0.42

3.18
29.20

(2.19 – 4.61)
(6.10 – 60.36)

9

Dinotefuran
Reference
Im-Resistant

816
739

0.72 ± 0.06
0.74 ± 0.10

0.16
0.61

(0.09 - 0.26)
(0.20 – 1.35)

4

Insecticide
/Strain

a) Im-Resistant: F5 generation of the imidacloprid resistant strain MI-R1
b) FL: fiducial limits
c) RR: resistance ratio (LC50 of imidacloprid resistant strain / LC50 of reference strain).

62

3.18 mg/L for the reference strain, indicating a low level of cross-resistance (9-fold, Table 3.2).A
similar level of resistance (7-fold) was also observed with acetamiprid as indicated by the
difference in LC50 of the resistant strain MI-R1 and reference strain CA-s1 (8.65 and 1.21 mg/l,
respectively) (Table 3.2). No significant difference in the LC50 values was found among the
resistant MI-R1 and reference strain CA-s1 for dinotefuran as their 95% fiducial limits
overlapped (Table 3.2).
Results of a standard efficacy test on the whitefly populations for imidacloprid at the
labeled rate indicate that mortality of whitefly from the resistant strain MI-r2a was significantly
lower than that of the reference strain CA-s2 at one week (79.1% and 21.9% mortality,
respectively, p < 0.0001) and 18 days (80.1% and 27.2% mortality, p = 0.002) after the
treatment (Table 3.3). A second efficacy test gave similar results for adults (83.4% and 3.7%
mortality, respectively, p < 0.0001) (Table 3.7). For thiamethoxam, no difference in mortality
was observed for the immature stages one week and two weeks after treatment (Table 3.3).
However, a significant difference in mortality was observed with the adults between the two
strains (92.8 and 62.3%, p = 0.004), indicating a reduced susceptibility of the resistant whitefly
adults MI-r2a to thiamethoxam (Table 3.7). No difference of efficacy of the two other
neonicotinoids (acetamiprid and dinotefuran) was observed for both immature and adult
whiteflies of MI-r2a and CA-s2 (Table 3.3, 3.7). Results of efficacy tests of the nonneonicotinoid insecticides buprofezin, pyriproxyfen and Beauveria bassiana indicate no
significant differences in efficacy for immatures and adults between the resistant strain MI-r2a
and reference strain CA-s2 (Table 3.4, 3.5, 3.8). The only observed difference in efficacy for any
non-neonicotinoid insecticide was for lambda-cyhalothrin, where a slight but non-significant
difference in mortality of immatures and adults was observed (Table 3.8, 3.5). No differences in
63

Table 3.3. Efficacy ± SE of neonicotinoids against immatures of the resistant strain (MI-r2a)
and the reference strain (CA-s2) of Bemisia tabaci B biotype whitefly on cotton plants. *: Mean
number of immatures from four replicates.
Insecticide
/strain

Imidaclopridd
Reference
Resistant
Acetamipride
Reference
Resistant
Thiamethoxamf
Reference
Resistant
Dinotefurang
Reference
Resistant
Untreated control
Reference
Resistant

Immature
alive *
Before
treatment

Efficacy
Immature
(%)
alive*
7 days
after treatment
(6/28/09)

Efficacy
Immature
(%)
alive*
18 days
after treatment
(7/9/09)

35.7

80.1 ± 5.6a

31.7

204.2

79.1 ± 5.7ac
21.9 ± 5.2b

75.5

27.2 ± 8.6b

71.2

212.5
227.5

98.8 ± 0.8a
97.8 ± 0.8a

1.2
2.1

99.3 ± 0.6a
97.9 ± 0.8a

0.5
1.7

249.5
187.0

98.8 ± 0.5a
94.5 ± 2.6a

1.5
4.5

99.2 ± 0.4a
94.5 ± 2.3a

1.0
4.5

233.7
235.2

99.8 ± 0.2a
97.7 ± 1.2a

0.3
1.7

99.7 ± 0.2a
97.9 ± 0.8a

0.2
0.5

255.2
233.7

-

157
126

-

146.7
119.2

312.2

c) Same letter within the same insecticide in the same column indicates lack of statistical
difference in insecticide efficacy between the resistant strain and the reference strain according
to Student t test at 0.05 level
d) Imidacloprid (Merit 75WP) applied as a drench treatment at 12 mg a.i. per pot
e) Acetamiprid (Tristar 30SG) applied as a foliar spray at 5.3 oz/100gal
f) Thiamethoxam (Meridian 25WG) applied as foliar spray at 8.5 oz/100 gal
g) Dinotefuran (Safari 20G) applied as a soil drench at a rate of 24 oz/100gal
All treatments were conducted on June 21, 2009, thus eight days after eggs deposition when all
eggs hatched.

64

Table 3.4. Efficacy of buprofezin and pyriproxyfen against clip-caged immatures of the resistant
strain (MI-r2a) and the reference strain (CA-s2) of whitefly Bemisia tabaci B biotype on cotton
plants. *: mean number of immature whitefly from four replicates.
Insecticide
/strain

Buprofezind
Reference
Resistant
Pyriproxyfene
Reference
Resistant
Untreated control
Reference
Resistant

Immature
alive *
Before
treatment

Efficacy
Immature
(%)
alive*
7 days
after treatment
(7/7/09)

Efficacy
(%)

Immature
alive*
20 days
after treatment
(7/20/09)

3.5

100 ± 0.0a

0

294.7

97.9 ± 0.9ac
98. 2 ± 0.4a

4.0

100 ± 0.0a

0

186.2
162.0

75.4 ± 3.5a
72.6 ± 2.9a

33.0
33.7

99.8 ± 0.2a
99.7 ± 0.2a

0.2
0.2

168.0
172.2

-

121.2
130.0

-

115.2
125.7

237.5

c) Same letter within the same column indicates statistically different between the insecticide
efficacy against the resistant strain and the reference strain within the same insecticide according
to Student t test at 0.05 level
d) buprofezin (Talus 40SC) applied as a foliar spray at a rate of 9 fl. oz/100gal
e) pyriproxyfen (Distance IGR) applied as a foliar spray at 8 fl. oz./100gal
All treatments were conducted on June 30, 2009, thus eight days after eggs deposition when all
eggs hatched.

65

Table 3.5. Efficacy ± SE of lambda- cyhalothrin and Beauveria bassiana against clip-caged
immatures of the resistant strain and the reference strain (CA-s2) of whitefly Bemisia tabaci B
biotype on cotton plants. *: Mean number of immatures from four replicates.
Insecticide
/strain

Beauveria
bassianac
Reference
Resistant
Lambdacyhalothrind
Reference
Resistant
Untreated control
Reference
Resistant

Immature
alive *
Before
treatment

Efficacy
Immature
(%)
alive*
7 days
after treatment
(7/31/09)

Efficacy
Immature
(%)
alive*
17 days
after treatment
(8/10/09)

23.7

90.8 ± 0.5a

8.0

193.2

73.2 ± 3.7ab
82.0 ± 4.0a

19.7

90.9 ± 0.8a

9.0

184.5
204.5

88.6 ± 3.9a
82.2 ± 2.1a

10.7
19.7

90.1 ± 4.2a
82.8 ± 2.2a

8.7
17.5

176.0
149.7

-

89.5
82.0

-

84
76

174.0

b) Same letter within the same column indicates statistically different between the insecticide
efficacy against the resistant strain and the reference strain within the same insecticide according
to Student t test at 0.05 level
c) Beauveria bassiana (Botanigard 22 WP) applied as a foliar spray at 1 pound/100gal
d) lambda-cyhalothrin (Scimitar GC) applied as a foliar spray at 148ml /100 gal
All treatments were conducted on July 7, 2009, thus eight days after eggs deposition when all
eggs hatched.

66

mortality of immatures were also observed between the two strains MI-r2a and CA-s2 with the
other insecticides tested (flonicamid, fenpropathrin and pymetrozine), suggesting an absence of
resistance to these insecticides in this resistant strain MI-r2a (Table 3.6).

IV Discussion
Resistance to insecticides can be caused by different mechanism in insects. Several
published studies reported that cytochrome P450 enzymes were involved in imidacloprid
resistance in Q and B biotype of Bemisia tabaci (Rauch and Nauen 2003, Karunker et al., 2008
). The results of this study also suggest that cytochrome P450 monooxygenases are an important
part of the resistance mechanism of whitefly in our resistant strain because pre-treatment with the
P450 inhibitor, PBO, reduced the level of imidacloprid resistance from an RR of 218 to 16.
However, it is also noteworthy that resistance was not completely eliminated by the PBO pretreatment, suggesting an additional mechanism is involved (Table 3.1). In a previous study a
mutation of the target site nAChR and an overexpression of P450 gene have been shown to cause
imidacloprid resistance in a population of aphids, Myzus persicae, from southern France (Bass et
al., 2011). A mutation on the target site nAChR was also observed in imidacloprid-resistant
brown planthoppers Nilaparvata lugens (Liu et al., 2005).
When imidacloprid was applied to potted plants in an efficacy test designed to determine
what level of control would be obtained by greenhouse growers using products at a rate of 12 mg
a.i. per pot, which is the label rate recommended for the imidacloprid product Marathon 60WP,
it was effective (83.4 % mortality) on whitefly from the reference strain CA-s2 but had little
effect on immature or adult whiteflies (27.2 or 3.7% mortality) from the resistant strain (MI-r2a)
with a level of resistance RR of 7-fold (LC50 = 30.54 mg/L) (Table 3.3, 3.7). These results are

67

Table 3.6. Efficacy ± SE of flonicamid, fenpropathrin, and pymetrozine against clipcaged immatures of the resistant strain (MI-r2a) and the reference strain (CA-s2) of
whitefly Bemisia tabaci B biotype on cotton plants. *: Mean number of imatures from
four replicates.
Insecticide/
Strain

Flonicamidc
Reference
Resistant
Fenpropathrind
Reference
Resistant
Pymetrozinee
Reference
Resistant
Untreated control
Reference
Resistant

Immatures*
alive
Before
treatment

Efficacy ± SE
(%)

Immatures*
alive

240.2

94.2

240.5

42.6 ± 5.7ab
41.9 ± 6.0a

252.7
214.5

71.4 ± 6.8a
66.6 ± 7.3a

48.7
47.2

184.5
204.5

48.6 ± 7.0a
42.6 ± 12.8a

10.7
19.7

195.0
139.2

-

66.7
51.5

7 days
after treatment
(9/27/09)

96.5

a) Same letter within the same insecticide in the same column indicates lack of statistical
difference in insecticide efficacy between the resistant strain and the reference strain
according to Student t test at 0.05 level
b) flonicamid (Aria 50WG) applied as a soil drench at a rate of 120g/100gal
c) fenpropathrin (Tame 2.4 EC) applied as a foliar spray at 16 fl. oz./gal
d) pymetrozine (Endeavor 50WG) applied as a foliar spray at 20g/100l
All treatments were conducted on September 20, 2009, thus eight days after eggs
deposition when all eggs hatched.

68

Table 3.7. Efficacy ± SE of neonicotinoids (imidacloprid, acetamiprid, thiamethoxam,
dinotefuran) against clip-caged resistant adults (MI-r2a) and reference adults (CA-s2) of whitefly
Bemisia tabaci B biotype exposed for 3 days to the treated cotton plants. *: Mean number of
adults from four replicates.

Insecticide/
Strain

Imidaclopridd
Reference
Resistant
Acetamipride
Reference
Resistant
Thiamethoxamf
Reference
Resistant
Dinotefurang
Reference
Resistant
Untreated control
Reference
Resistant

Adult*
alive
Before
exposure

Efficacy ± SE
Adult*
(%)
alive
3 days after
insecticide exposure
(6/11/09)

48.0

83.4 ± 3.5ac
3.7 ± 0.9b

45.5

63.0
53.5

100 ± 0.0a
100 ± 0.0a

0.0
0.0

41.7
49.0

92.8 ± 3.4 a
62.3 ± 5.9 b

3.2
18.0

50.2
50.0

100.0 ±0.0 a
97.8 ±1.2a

0.0
1.0

77.7
66.7

-

76.5
67.5

54.2

8.7

c) Different letter within the same insecticide in the same column indicates statistical difference
in insecticide efficacy between the resistant strain and the reference strain according to Student t
test at 0.05 level
d) Imidacloprid (Merit 75WP) applied as a soil drench at a rate of 12 mg a.i. per pot
e) Acetamiprid (Tristar 30SG) applied as a foliar spray at 5.3oz/100gal
f) Thiamethoxam (Meridian 25WG) applied as foliar spray at 8.5 oz/100 gal
g) Dinotefuran (Safari 20G) applied as a soil drench at a rate of 24oz/100gal
All treatments for the drench application (imidacloprid and dinotefuran) were conducted on
June7, 2009 and on 8 June, 2009 for foliar application (thiamethoxam and acetamiprid).
69

Table 3.8. Efficacy ± SE of pyriproxyfen, Beauveria bassiana, buprofezin and lambdacyhalothrin against clip-caged resistant adults (MI-r2a) and reference adults (CA-s2) of whitefly
Bemisia tabaci B biotype exposed for 3 days to the treated cotton plants. *: Mean mean number
of adults from four replicates.
Insecticide/
Strain

Pyriproxyfenc
Reference
Resistant
Beauveria bassianad
Reference
Resistant
Buprofezine
Reference
Resistant
lambda-cyhalothrinf
Reference
Resistant
Untreated control
Reference
Resistant

Adult*
alive
Before
exposure
(7/6/09 and 7/7/10)

Efficacy ± SE
Adult*
(%)
alive
3 days after
insecticide exposure
(7/9/09 and 7/10/09)
32.2

50.2

29.2 ± 9.1ab
15.1 ± 4.0a

44.7
47.7

19.1 ± 5.2a
20.0 ± 7.7a

34.0
36.2

47.5
44.0

10.7 ± 3.4a
11.9 ± 4.8a

39.7
36.5

45.7
42.0

63.5 ± 11.6a
55.4 ± 8.8a

15.7
17.5

37.5
42.7

-

35.2
40.5

48.5

40.5

b) Different letter within the same insecticide in the same column indicates statistical difference
in insecticide efficacy between the resistant strain and the reference strain according to Student t
test at 0.05 level
c) pyriproxyfen (Distance IGR) applied as a foliar spray at 8 fl.oz./100gal
d) Beauveria bassiana (Botanigard 22 WP) applied as a foliar spray at 1 pound/100gal
e) buprofezin (Talus 40SC) applied as a foliar spray at a rate of 9 fl.oz/100gal
f) lambda-cyhalothrin (Scimitar GC) applied as a foliar spray at 148 ml/100 gal
All treatments were conducted on July, 6 2009 except for lambda- cyhalothrin applied on July 7,
2009.

70

consistent with many reports by greenhouse growers over the last 15 years, that imidacloprid
failed to provide adequate control of whitefly. Our colony of resistant B-biotype was started
from whitefly collected from a production greenhouse in southeast Michigan. The initial test of
the whitefly population before selecting for higher levels of imidacloprid resistance indicated an
LC50 of 23.42 mg/L (RR=11). Although this is a low level of imidacloprid resistance, our
standard efficacy tests with products at labeled rates indicate that at a similar resistance level
(LC50 = 30.54 mg/L, RR = 7) efficacy is already affected. Control in production greenhouses
may be even more affected because growers are expecting at least 6 weeks of protection after a
single soil drench of imidacloprid or other neonicotinoid insecticides.
We observed high mortality of immature whitefly from the resistant strain MI-r2a (27% at 18
days after imidacloprid treatment) (Table 3.3), indicating that this population is not
homogenously resistant and that susceptible alleles still exist at a certain frequency in the
population, which is consistent with its low level of resistance (RR = 7). In addition, whitefly
immatures were suggested to be more susceptible to imidacloprid than whitefly adults (Jones et
al., 2011; Nauen et al., 2008). All individuals in the reference strain CA-s2 was also not
susceptible to imidacloprid at the time of the test. Instead of seeing 90-100% mortality after
treatment with imidacloprid, only 80% of immature whiteflies died after treatment, suggesting
that some individuals from the resistant strain may have found their way into the reference strain.
The level of cross-resistance of imidacloprid resistant populations to other
neonicotinoids has been a subject of controversy due to variation in the results obtained from
several studies. In the current study, the resistant strain MI-R1 with a relatively high level of
resistance (LC50 = 284.47 mg/L, RR = 144) was used to study the cross resistance to these

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neonicotinoids using a systemic bioassay. In addition, the response of the resistant strain MI-r2a
with a lower resistance level (LC50 = 30.54 mg/L, RR =7) to three neonicotinoids was also used
and compared to the response of the reference strain CA-s2 under the greenhouse conditions.
The results of cross resistance in these studies are consistent with those of Prabhaker et al.
(2005) who found a low level or no cross resistance to neonicotinoids with a B biotype whitefly
population. In our tests, three neonicotinoids (acetamiprid, thiamethoxam and dinotefuran)
caused as much mortality of immatures of the resistant whitefly MI-r2 a as they did for
immatures of the reference strain CA-s2 (Table 3.3). This is not surprising given the fact that
P450 monooxygenases might play an important role in the resistance in this population as
described above, and previous study demonstrated an age specific expression of the P450 gene
responsible of imidacloprid resistance in whitefly, with a low level of expression in immatures,
leaving this stage more vulnerable to the neonicotinoids than the whitefly adults (Jones et al.,
2011; Nauen et al., 2008).
When the adult whiteflies were tested for insecticide efficacy, the resistant whitefly
showed some cross-resistance to one of three neonicotinoids tested. Resistant adults of MI-r2a
with a low level of resistance (RR =7) were less susceptible to thiamethoxam than reference
adults CA-s2 (Table 3.7). This is consistent with results obtained with the resistant strain MI-R1
with a high resistance level (RR =144) by using the systemic bioassay method, where a low level
of cross resistance (9-fold) was found (Table 3.2). Recently, an overexpression of one P450
gene, CYP6M1PvQ, was observed in an imidacloprid-resistant population of both B and Q
biotypes (Karunker et al., 2008). In a subsequent study, Roditakis et al. (2011) demonstrated that
indeed CYP6M1PvQ has a potential to metabolize imidacloprid in whitefly. However, the same
study showed that thiamethoxam was not metabolized by the protein of this P450 gene. In fact,
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Nauen et al. (2003) showed that thiamethoxam was converted to clothianidin in cotton plants and
in the insects after its application, clothianidin is a byproduct of thiamethoxam. Interestingly,
Roditakis et al. (2010) found that as opposed to thiamethoxam but similar to imidacloprid,
clothianidin was metabolized by the protein of the P450 gene, CYP6M1PvQ, responsible for
imidacloprid resistance in whitefly. All these studies seem to support the findings in this study
showing a resistance to thiamethoxam; thus the thiamethoxam could be first converted to
clothianidin in the plants and whitefly, and then the protein of the P450 gene of the whitefly
could degrade the metabolite clothianidin that was generated, which consequently makes the
mother compound thiamethoxam less toxic to the whitefly. A low level of resistance (7-fold) was
also observed with acetamiprid using the systemic bioassay with the resistant strain MI-R1
having a high level of resistance to imidacloprid (144-fold) (Table 3.2). However, the result of
the efficacy test shows no difference in response of the adults of the resistance strain MI-r2a with
low RR= 7 and the reference strain C-S2 to this compound (Table 3.7). This discrepancy could
be explained by the fact that acetamiprid has a high level of insecticidal activity when applied as
foliar treatment (Horowitz et al., 1988). In addition, Roditakis et al. (2010) demonstrated that
similar to thiamethoxam, the protein of the P450 gene CYP6M1PvQ also did not metabolize
acetamiprid in whitefly. These authors also suggested that this could be another explanation of
the observation of low level of cross-resistance to neonicotinoids in some whitefly populations.
Apparently in agreement with the results on the metabolism of these two compounds
(acetamiprid and thiamethoxam) by the protein of P450 gene, several published studies on cross
resistance to neonicotinoids using a leaf dip assay found that the level of resistance was much
higher for thiamethoxam than for acetamiprid in imidacloprid-resistant whiteflies. For example,
the B biotype whitefly having a resistance ratio (RR) of 230 to imidacloprid exhibited a

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resistance ratio of 73-fold to thiamethoxam whereas it was only 15-fold for acetamiprid (Gorman
et al., 2010). Different B biotype whitefly populations collected from different regions in
Cyprus having a moderate to high resistance level to imidacloprid (RR of 77 to 392) also
showed a moderate level of resistance to thiamethoxam (RR = 50 to 164) but a low level of
resistance to acetamiprid (RR = 7 to 12) (Vasillou et al., 2012). The same observation of higher
resistance to thiamethoxam than to acetamiprid was also noticed in several imidacloprid resistant
populations of Q biotype whitefly (Rao et al., 2012). The LC50 for thiamethoxam in the resistant
strain MI-R1 (LC50 =29.20 mg/L) was higher than that for acetamiprid (LC50 = 8.65 mg/L) in
the current study, however, these values are not significantly different based on their 95 %
fiducial limit and the resistance ratio was similar for both compounds (9-fold and 7-fold for
thiamethoxam and acetamiprid respectively). Prabhaker et al. (2005) also observed this. It is
noteworthy that systemic bioassays were used in these two studies (this study and Prabhaker et
al., 2005) and that might partially explain the difference in the results. It is also likely that
difference in level of resistance could be caused by the predominant gene or mechanism that was
selected in different populations. For example, Horowitz et al. (2004) found that a whitefly
population selected with thiamethoxam showed a very low resistance to acetamiprid whereas
acetamiprid-selected population was highly resistant to thiamethoxam. In contrast, Feng et al.
(2009) selected B biotype whitefly for thiamethoxam resistance (RR: 25.6) found a similar level
of resistance to acetamiprid (RR 35-fold) and imidacloprid (47.3-fold). Interestingly, Xi et al.
(2012) observed an overexpression of three other P450 genes (CYP6a8, CYP4v2, and CYP6v5)
in thiamethoxam-resistant whitefly B. tabaci but not the P450 gene CYP6CM1 that was found
previously in imidacloprid-resistant whiteflies Q and B biotype (Karunker et al., 2008). Thus, a
different history of insecticide treatments can generate a difference in the predominant genes or
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mechanism responsible for resistance in different whitefly populations. All these studies suggest
that variation in cross resistance level between the three neonicotinoids (imidacloprid,
thiamethoxam and acetamiprid) is not unexpected. In addition, the bacterial symbionts hosted by
both B and Q biotype B. tabaci were recently found to influence the susceptibility of these
whiteflies to acetamiprid, thiamethoxam and imidacloprid and other insecticides as well (Ghanim
and Kontsedalov, 2009; Kontsedalov et al., 2008). The compositions of these facultative
symbionts can vary between regions or biotypes (Xie et al., 2012). That could be one more
possible cause that might partially explain the variation in level of resistance to these compounds
in different populations.
Results from a systemic bioassay test and from a standard greenhouse efficacy test
indicate that imidacloprid-resistant whitefly from our cultures did not exhibit any crossresistance to dinotefuran. Prabhaker et al. (2005) also detected no cross resistance to dinotefuran
in B-biotype whitefly selected for imidacloprid resistance. Similarly, different Q biotype
whitefly populations collected from fields in China having high resistance level to imidacloprid
also showed very low or no resistance to dinotefuran (Rao et al., 2012). Dinotefuran has a
different structure compared with the other neonicotinoids. Thus, it would not be unlikely that
the same protein of the P450 gene responsible for imidacloprid resistance in whitefly may
metabolize dinotefuran in a different way than for other neonicotinoids. Studies of binding
target sites demonstrate that even dinotefuran shares the same target site with other
neonicotinoids such as imidacloprid, thiamethoxam and acetamiprid in several different insects
including whitefly, dinotefuran may have a different interaction with the target site as shown in
the assay with a Homalodisca leafhopper (Honda et al., 2006). Liu et al. (2006) studied effects of
the mutation Y151S on the nicotinoic acetylcholine receptor, which causes imidacloprid

75

resistance in brown planthoppers, on the agonist potency of a few neonicotinoid compounds.
These authors found that the impact of this mutation Y151S on the agonist potency of
dinotefuran was the smallest among all the neonicotinoids in the study. Considering the results of
all these studies, a low or non-observation of cross resistance to dinotefuran is not unexpected.
The standard efficacy test also did not show a difference between the resistant strain MIr2a and reference strain CA-s2 for the other non-neonicotinoid insecticides tested. It is
noteworthy that the efficacy testing was conducted with a population having a low resistance
level (7-fold). It is very likely that a low or a moderate resistance to other insecticides might not
be detected with this level of resistance in the population used for the test. Thus, the results
observed with these insecticides are not conclusive. Indeed, no difference was observed between
resistant MI-r2a and reference strain CA-s2 with pymetrozine in the current study (Table 3.6).
However, a good correlation of level of resistance between pymetrozine and the neonicotinoids
imidacloprid and thiamethoxam was already observed in whitefly having a resistance ratio of
10,000 fold (Gorman et al., 2010). A slight difference in efficacy between the resistant strain MIr2 and the reference strain CA-s2 (82% and 90% respectively) was observed with lambdacyhalothrin (Table 3.5), however, this small difference is not significantly different. The two
insect growth regulator compounds, pyriproxyfen and buprofezin caused the lowest efficacy in
adults among the insecticides tested in the present study (Table 3.8). In fact, these compounds
which act mainly on the immature stages were known to have less activity to the adults (Palumbo
et al., 2001). Buprofezin interferes with chitin formation in the development of immature insects
(Ishaaya et al., 1988), whereas pyriproxyfen which is a juvenile hormone analog affects
embryogenesis, metamorphosis and formation of adults in insects (Ishaaya and Horowitz, 1992).
Consistent with their mode action, these two IGR compounds caused a high level of mortality to

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the immature stages of both strains (Table 3.4). Elbert and Nauen (2000) even suggested the two
IGR compounds as alternative insecticides in a pesticide rotation with neonicotinoids. Indeed,
pyriproxyfen was found to cause 100% mortality of eggs of Q biotype whitefly population which
has highly resistance to imidacloprid, whereas buprofezin caused an appreciable mortality rate
(68%) to the immature of the same population (Elbert and Nauen, 2000).
Similar to the result found by Oetting (2007) conducted on B biotype whitefly,
Beauveria bassiana was found to cause high mortality (90%) to immatures two weeks after
treatment in the current study (Table 3.5). No difference in efficacy for immatures or adults was
observed among the two strains MI-r2a and CA-s2. Efficacy of flonicamid against immatures
was similar to that observed by Parella (2006), with a moderate effect of this insecticide on
whitefly immatures one week after treatment, and no appreciable differences among the
reference strain and CA-s2 and resistant strain MI-r2a was observed in the current study (Table
3.6).

V Conclusion.
The current study found that resistance in this B biotype of Bemisia tabaci population
was mainly mediated by P450 monooxygenases as suggested by the significant synergistic effect
of imidacloprid in the resistant whitefly pre-treated with the P450 inhibitor, PBO. The
population showed a low level of cross resistance to thiamethoxam. This is consistent with the
recent study that found protein of the P450 gene responsible for imidacloprid resistance in
whitefly also metabolizes clothianidin, a direct metabolite of thiamethoxam (Roditakis et al.,
2010, Nauen et al., 2003).
In contrast no appreciable cross resistance was observed with dinotefuran in this

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population. A low level of cross-resistance to acetamiprid was also observed (Table 7). However
the product of the same P450 gene did not metabolize this compound in a recent study Roditakis
et al. (2010). This suggests that a different P450 gene or even an additional mechanism might
have been involved in the imidacloprid resistance in this population. In fact, as in previous
studies, the imidacloprid resistance was not completely removed by PBO in the current study, a
result that supports the occurrence of other mechanisms of resistance. Thus, the wide variation in
the level of resistance to imidacloprid observed in different populations of whitefly might be
attributed to differences in the mechanisms of resistance that were selected for, and that might be
related to the history of insecticide treatments. Regardless of the mechanism of resistance, the
results of this study suggest that greenhouse growers should avoid the use of thiamethoxam and
acetamiprid in their rotation of insecticides for whitefly control because of the history of
widespread use of imidacloprid over the past 15 years, and because of the documented cross
resistance to acetamiprid, and especially thiamethoxam. The response of the resistant whitefly to
insecticides with different modes of action was also studied and no appreciable resistance to
these insecticides was observed. However, the results were not conclusive as the level of
resistance of the imidacloprid-resistant population used in the study was too low to detect a low
level of cross resistance to these compounds. Still, the result is informative because the level of
resistance (7-fold) was similar to an imidacloprid resistant population of B biotype found in a
commercial greenhouse in southeast Michigan (LC50 = 30.54 mg/L), and therefore may be
representative of resistance levels that greenhouse flower growers are likely to have.

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CHAPTER IV. Induction of hormesis in whitefly Bemisia tabaci B biotype by low priming
doses of imidacloprid.
I Introduction.
Numerous studies have assessed the effects of sublethal doses of insecticides on insects.
Most of the investigations were made with adult insects and most of the results demonstrate an
inhibitory effect (Desneux et al. 2007, Tan et al., 2012; Boina et al., 2009). However, toxicants
may also cause different responses at very low doses that do not cause an observable adverse
effect, and this phenomenon is known as hormesis in toxicology (Calabrese and Blain, 2005). In
hormesis, the low dose of the stressors including insecticides may elicit a stimulatory effect
whereas higher doses of the same stressors are toxic or inhibitory (Calabrese and Baldwin,
2003a; Calabrese and Blain, 2005). There are two types of hormesis: first, an overcompensation
stimulation hormesis (OCSH) which is a result of an overcorrection response following a
disruption of homeostasis, and second, a direct stimulation hormesis (DSH) (Calabrese and
Baldwin, 2002). The stimulation can be observed at a low dose where the stressor does not elicit
any observable toxicity (Calabrese and Blain (2003a), and it may also be observed over a wide
range of doses, thus even at the dose where the stressors elicit adverse effects (Stebbing, 2009;
Chujo et al. 2012). The hormetic effect may be detected with a single treatment, but sometimes
it can only be determined after a pre-exposure or priming of the cell or organism. When a preexposure is needed the effect is known as ‘conditioning hormesis’ (Calabrese et al., 2007). In
conditioning hormesis, the first treatment can protect the organism against the toxic effects of a
subsequent and more severe treatment (Calabrese et al., 2007, Calabrese and Mattson, 2011).
Therefore, hormesis can be viewed as an acquired tolerance (Stebbing, 2002, 2009). The
underlying mechanism of hormesis is not fully understood. However, several investigators

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reported that different biological and physiological processes including stress response systems,
signaling pathways and metabolic mechanisms could be involved in hormesis (Zhao and Wang,
2012; Calabrese et al., 2010). Other authors suggest that hormesis might be associated with
epigenetic modification in the organism (Vaiserman, 2011; Costantini et al., 2011). Indeed,
increasing number of studies have shown that epigenetics are involved in hormesis and the
phenomenon is mediated by oxidative stress (Schroeder et al., 2013).
Hormesis can be induced by different toxicants across a wide range of taxa on several
endpoints. Recently, hormesis has gained interest among pest management researchers. Some
investigators support the idea that hormesis may contribute to a pest resurgence or secondary
outbreak following an insecticide application (Guedes et al., 2010; Morse, 1998). In insects, the
hormetic effect can happen at different stages of development including early adulthood
(Kristensen et al., 2003; Lebourg, 2005) or during the larval stage (Nascarella et al., 2003).
When conditioning hormesis is applied to management of insect pests of cultivated crops some
important questions arise: if insects in immature stages are exposed to a trace amount of
insecticide, will this stimulate the fecundity of the same individuals when they encounter the
same insecticide during their adult stage, and will it protect the individuals from a subsequent
exposure to higher doses of the same insecticide?. In addition, previous studies demonstrated that
side effects of a single exposure to pesticides applied at an early stage of the development can
persist in a few subsequent generations of mammals and insects (Manikkam et al., 2012,
Adamski, et al., 2005, Anway et al., 2005). The question could be then asked whether a
stimulation induced by a single exposure of insects to pesticides during immature stage can
reappear in their offspring.
Whitefly is a major pest in both greenhouses and agricultural fields. The life cycle of a

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whitefly starts with the egg which has a pedicel used to obtain water from the host leaf to which
it is attached (Byrne et al., 1990). After egg hatch, whitefly immatures go through four instars.
The last stage of the fourth instar is a pharate adult which is often mistakenly called a pupa
(Gelman et al., 2007). Then as adults, whitefly can reproduce parthenogenetically. Being
arrhenotokous, the virgin females can lay eggs, which become male if unfertilized or become
female if fertilized (McAuslane, 2007).
The neonicotinoid insecticide imidacloprid has been commonly used for control of sucking
insects such as whiteflies, aphids, and planthoppers since its introduction to the market in the early
1990s (Elbert et al., 2008). Imidacloprid may persist up to 12 months or longer after its application to
soil (Bonmatin et al., 2003). Therefore, pests on plants in fields where a soil application of
imidacloprid is made can be exposed to low doses of imidacloprid for the entire crop production
period. Because of this long exposure period it is important to know if trace amounts of imidacloprid
in plants have any effect on the fitness of the pest population. In previous research with the green

peach aphid some studies showed that a low dose of imidacloprid can inhibit fecundity (Devine
et al., 1996), while in other studies fecundity was increased (Yu et al., 2010; Cutler et al., 2009).
Other authors reported no effect of a low dose of imidacloprid on the fertility of green peach
aphid (Janmaat et al., 2011). This variability of results was also observed with other pest species
such as brown planthopper, Nialaparvata lugens, treated with imidacloprid (Bao et al., 2009;
Yin et al., 2008). This discrepancy in results suggests that imidacloprid can induce a stimulation
or hormesis in the fecundity of insects under specific circumstances.
This study investigates the potential of low dosages of imidacloprid to induce an increase
in whitefly fecundity, and the heritability of the hormetic effect. Because the long-term
persistence of resistant populations of whitefly is of critical importance to flower production,
vegetable production and cotton production, the current study was conducted on B. tabaci
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biotype, the most widespread resistant-biotype on these crops. This study focuses mainly on an
imidacloprid-induced increases in fecundity, because fecundity is a key component of fitness.

II Materials and methods
The study on hormesis was conducted at two stages: the first stage was conducted
between 2009 and 2012 with two cultures: a resistant whitefly strain and a reference strain. The
second stage was conducted in 2013 with a rebuilt population from the original reference strain.

II-1 Plants
II-1-1 Plants used between 2009 and 2012
Cotton seeds were planted in 48-cell trays filled with a soil-less medium (Baccto,
Michigan peat company, Texas), and grown in the greenhouse until they reached the two trueleaf stage. Cotton seedlings were then planted in 6” pots filled with the same growing medium.
Seedlings were treated with Pylon (chlorfenapyr, 0.98 ml/L) when they were in the cotyledon
stage to prevent thrips and mites infestation. Cotton plants at the four to six true-leaf stage were
used for the experiment.
II-1-2 Plants used in 2013
The same cotton seeds used in previous study (2009-2012) were used. However, cotton
plants were grown with a different type of soil medium, Supermix perlite, (Michigan grower
products, Inc, Michigan) provided by the greenhouses at Michigan State University. In addition,
in previous experiments no fertilizer was used for growing cotton plants, but because unfertilized
cotton plants grow very slowly, in 2013 soluble fertilizer (Everris, NA Inc., Ohio) was applied so
that more plants could be grown for the final set of experiments. One hundred ml of a fertilizer

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solution at 500 ppm N was applied twice per week to each plant.

II-2 Insecticides.
The formulated product Merit 75 WP (imidacloprid) was used in this study. Tap water
was used to dilute the imidacloprid into the needed concentrations. The physicochemical
properties of the water were not tested. Solutions were prepared immediately before each
experiment.

II-3 Whitefly.
II -3-1 Whitefly used between 2009 and 2012.
II -3-1 a) Reference strain.
We have maintained a reference strain of the B biotype in the greenhouse (Chapter II
section II-3). The second susceptible culture, initiated on February 2009, was used as a reference
population for this study. Its LC50 value for imidacloprid determined between April 2009 and
April 2011 ranged from 4.08 to 6.17 mg/L (Chapter II Table 2.2, 2.4).
II -3-1 b) Resistant strain.
The resistant strain was initially obtained from a commercial greenhouse as described in
an earlier section (Chapter II section II-3). The recovered resistant population has been
continuously reared on plants treated with imidacloprid at 12 mg a.i. per pot since May 2009.
This population was 9-fold more resistant to imidacloprid when compared with the reference
strain in June 2010 (LC50 was 37.47 mg/L) and April 2011 (LC50 was 54.19 mg/L, (Chapter II
Table 2.4).

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II-3-2 Whitefly used in 2013.
After July of 2012, when the initial research project was coming to an end the two
whitefly cultures used for this research were reduced to the whiteflies on few plants in each cage.
As the study was re-initiated in 2013, the resistant culture and susceptible culture used in
previous years were re-built from January to March of 2013 for the next round of experiments. It
is noteworthy that some mixing of these two populations occurred before and during the period
of rebuilding because of holes in the screen cages. The population used for experiments in 2013
is from the original reference culture, after some immigration from the resistant culture between
September 2012 and March of 2013. The LC50 to imidacloprid was not determined with a full
range of test doses, but results from the first preliminary experiments in 2013, described below,
indicate that this population now appears to have a low level of resistance to imidacloprid. It is
also noteworthy that a different type of soil medium provided by the greenhouses in Michigan
State University was used in the study. In addition, in previous experiments no fertilizer was
used for growing cotton plants, but because unfertilized cotton plants grew very slowly, in 2013
soluble fertilizer was applied at the standard rate as a drench so that more plants could be grown
for the experiments. Thus, the experiments in 2013 were conducted under conditions different
from that of the previous years, and this required some preliminary experiments to determine the
appropriate treatment concentrations.

II-4 Hormesis on the resistant and reference strains between 2009 and 2012.
Priming of an organism with a low dose of a toxicant can induce a stimulation of various
processes (Stebbing, 2002, Chujo et al., 2012). In addition, a mild stress applied at early stage of
an organism can have effect on the adult stage (Costantini et al., 2010). Therefore, in the current
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study whitefly immatures were primed with imidacloprid, followed by a subsequent exposure of
the same individuals to imidacloprid after they became adults.
II-4-1 Assay for the collection and priming of immature.
II-4-1-a) Assay with priming of immature.
Four to six new cotton plants in the four to five true-leaf stage were placed in the
reference culture or the resistant culture to allow adults to lay eggs on them. Plants were
removed after three days when they had enough eggs and were transferred into two separate
cages. These plants were then treated with imidacloprid (Merit 75WP) applied as a soil drench
by pouring 100 ml of the imidacloprid solution at the needed priming concentration (see each
experiment below) around the base of the plants. The eggs were allowed to grow on the treated
plants until the end of fourth stage, which occurs about 20 days after treatment, then pupae were
collected from mature leaves. When collecting, the largest pupae of any group were selected as
they usually turned out to be females. Pupae were collected by cutting a small piece (35-50 mm2)
of the cotton leaf under each pupa. Most of the pupae were collected individually on separate
leaf discs, but when they were very close together, 2 or 3 pupae were collected on the same leaf
disc. The small leaf disc with the pupa was then transferred into a 1.7 ml microcentrifuge tube
and held at room temperature until adult emergence (one to two days).
II-4-1-b) Assay without priming of immature.
The procedure for the experiment without priming of immature was the same as the one
with priming of immature as described above. The only difference is that the cotton plants
containing eggs laid by the adult females were not treated with imidacloprid in this assay so that
the whitefly nymphs grew and fed on untreated plants from egg hatch to end of the fourth instar.
Then females were exposed to imidacloprid after pupation and emergence as described on the

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assay for the adult exposure section below (II-4-2).
II-4-2 Assay for the collection and exposure of adults.
Upon emergence, the sex of the adults from the collected pupae was determined by
checking the tip of their abdomen under a microscope to ensure they were females. The few
males that were collected by mistake were discarded. Emerging females were transferred to
treated leaf discs cut from cotton plants in the four to five true-leaf stage after they were treated
systemically with imidacloprid (Merit 75WP) for three days at different concentrations ( see
each experiment) as a soil drench. Three plants were used for each treatment so that leaf discs
used for each treatment could be cut from the same leaf of the same plant to minimize the effect
of variation in the amount of imidacloprid in different plants. Each of the three plants provided
replicate leaf discs for all treatments. Young upper leaves in the same stage of development were
always used because the imidacloprid concentration in leaves can vary depending on their age
and location on a plant (Olson et al, 2004). The leaf discs were placed in a 1.7 ml
microcentrifuge tube containing a 1.25 ml layer of agar (1.3%) on the bottom. The
microcentrifuge tubes were punctured several times with an insect pin for aeration before the
experiment. Females were transferred as previously described (Chapter II) from the tube where
they emerged to the new tube containing the treated leaf disc. The number of eggs laid by these
females was monitored daily until the female’s death. The longevity of the female was also
recorded. Initially, each treatment consisted of fifteen female adults. Females which died due to
the transfer, fungi or because of a decayed leaf disc were eliminated from the test, thus each
treatment consisted of 8 to 15 replicates with each adult female on a separate leaf disc in a
microcentrifuge tube being a replicate. The death of females was considered to be due to the
imidacloprid when the females were seen to exhibit symptoms of toxicity including paralysis or

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long lasting inertia before death, or if the death occurred with no injury in the microcentrifuge
tube with a very clean leaf disc (no fungi, decay or excessive moisture that traps the female).
II-4-3 Test for hormesis in the parental generation.
II-4-3-a) Response of females of the reference and resistant colonies to
imidacloprid.
For this experiment, pupae were either collected from the reference culture where
immatures were not exposed to imidacloprid or from the resistant culture, where immatures grew
on plants that were treated with imidacloprid (Merit 75 WP) at 12 mg a.i. per pot. The assay for
the collection of pupae for adult emergence described above was followed (see II- 4-1-b). Then,
the emerged adults were collected and exposed to leaf discs taken from plants treated with
imidacloprid at different concentrations.
In hormesis, stimulation can be seen as a stimulation above the level of the control at
doses where the agent does not elicit an observable adverse effect (Calabrese and Baldwin,
2003b) but the stimulation can also be seen over a wide range of dosage even at high dose that
causes an adverse effects (Wang and Xing 2010, Crysper and Johnson, 2002, Stebbing, 2002).
Thus in the current study, a wide range of concentrations of imidacloprid were used, from a
very low dosage that would not be expected to show adverse effects in whitefly to relatively
high doses.
The lowest concentrations used were estimated based on the published studies. Indeed, a
decrease in fecundity induced by imidacloprid was often associated with reduction of excretion
of honeydew in insects, thus through the feeding behavior (Devine et al., 1996, Boina et al.,
2009). The EC50 for the suppression of honeydew is the concentration that causes a 50%
reduction of honeydew excretion in the insects. The EC50 for the suppression of honeydew
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excretion by whitefly adults in a systemic assay was estimated at 0.037 mg/L with a 95%
fidicual limit (FL) of 0.0059-0.11 (Nauen et al., 1998). The concentration of imidacloprid that
did not elicit any observable depression of honeydew excretion in another sucking insect, the
aphid species, using a systemic assay was shown to be at 0.0001 mg/L (Nauen, 1995). Based on
these published results, the lowest concentration of imidacloprid chosen was 0.00012 mg a.i per
pot in the current study. The concentration of 6 mg a.i per pot was selected as the highest
concentration, which is half of the rate of 12 mg a.i. per pot, a rate recommended for whitefly
control in greenhouses. Four more concentrations of imidacloprid between these two
concentrations were first used including 0.012, 0.012, 0.12, 1.2 mg a.i. per pot. These six
concentrations were then used to study the response in fecundity of whitefly adults to
imidacloprid exposures. The imidacloprid was applied according to the aforementioned method
(see II- 4- 2). Water was used on control plants.
II-4-3-b) Response of resistant and reference females with different priming
concentrations.
As no stimulation in fecundity was observed in the test described above, the effect of
priming was tested by applying imidacloprid at an early stage of development of the whitefly.
The test was then conducted on whiteflies from the reference strain and resistant strain that were
exposed to imidacloprid during their immature stage and adult stage.
The dosage for the priming exposure is a factor that can affect the occurrence of hormesis
(Stebbing, 2002). Perhaps because the mechanism causing hormesis is not well understood, little
information is available on how to select a priming rate. The study of Stebbing et al. (2002)
showed that doses below the threshold for an observable adverse effect could be used for
priming exposure to induce stimulation. Similarly, the result of the study of Davies et al. (1995)

88

on yeast suggested that the low concentration of a stressor that induces a stimulation above the
untreated control level could be used as a priming dose to study a conditioning hormesis. In the
current study, whitefly individuals were first exposed to a single dosage through their whole life.
For that, immatures were primed with the wide range of concentrations of imidacloprid used in
the previous experiment (0.00012, 0.0012, 0.012, 0.12, 1.2, 6 mg a.i. per pot) by using the
method described above (II-4-1-a ). Then, the emerged adults were subsequently exposed to the
same concentration used during their immature stage (0.00012, 0.0012, 0.012, 0.12, 1.2, 6 mg a.i.
per pot) following the method described above (II-4-2) and their fecundity was assessed. Water
was used for the control (0 mg a.i. per pot).
II-4-3-c) Response of resistant and reference females primed with 0.0012 mg
a.i. per pot.
The concentration of 0.0012 mg a.i. per pot was selected as priming concentration based
on the results of the previous experiment. The effect of priming of whitefly immatures with this
concentration of 0.0012 mg a.i. per pot was further investigated by expanding the range of
concentrations of the subsequent exposure by adding one lower concentration of imidacloprid
and one higher concentration. The experiment followed the assay described above (II-4-1-a and
4-2). Therefore, emerged adults were subsequently exposed to eight imidacloprid concentrations
(0.000012, 0.00012, 0.0012, 0.012, 0.12, 1.2, 6, and 12 mg a.i. per pot) as adults. Water was used
for the control (0 mg a.i. per pot).
II-4-3-d) Response of resistant females with and without priming.
To determine if priming is needed for resistant whitefly to exhibit hormesis on fecundity,
a comparison was made between the responses of resistant adults that were pre-exposed to the
priming concentration of imidacloprid at 0.0012 mg a.i. per pot and those that were not primed

89

with imidacloprid (no priming: 0 mg a.i per pot) during their immature stage. The experiment
was conducted as described above (II -4-1 and 2). Immatures of the resistant strain were either
reared on plants treated with imidacloprid at 0.0012 m a.i. g per pot from eggs to pupae or they
were reared on plants that were not treated with imidacloprid. Emerged adults from the collected
pupae were then subsequently exposed to different concentrations of imidacloprid. Three
additional concentrations were added to determine if resistant females exhibit a biphasic dose
response in fecundity: two concentrations 0.0009 and 0.003 a.i per pot, thus near the
concentration 0.0012 a.i per pot where stimulation was found, and one very high concentration
48 mg a.i per pot. Thus, the concentrations used were: 0.00012, 0.0009, 0.0012, 0.003, 0.012, 6,
12 and 48 mg a.i per pot. Water was used for the control (0 mg a.i. per pot).
II-4-3-e) Response of resistant females with high and low priming
concentrations.
This test aimed to determine the effect of priming concentration of imidacloprid on
hormesis. Immature resistant whiteflies were primed with a concentration of 0.0012 mg a.i per
pot or 12 mg a.i per pot (a 1,000-fold greater concentration). The experiment was conducted by
using the assay on the immature and adult exposure as described above (II -4 -1-a and 4-2). More
concentrations for the subsequent exposure were added in this test to determine if resistant
females exhibit a biphasic dose response in fecundity: 0.00012, 0.0003, 0.0009, 0.0012, 0.003,
0.006, 0.009, 0.012, 12 and 48 mg a.i per pot. Water was used for the control (0 mg a.i. per pot).
II-4-4 Test for hormesis in the F1 generation of resistant and reference strains.
A test on the F1 generation was conducted to determine whether hormesis can occur in
the first generation progeny of the primed immatures. A new parental cohort was used in this
study. Ten plants in the four to six true-leaf stage were placed in the reference culture and five

90

cotton plants were placed in the resistant culture for adults to lay eggs on. Plants were removed
from the cultures after three days and transferred in two different cages. These plants with
whitefly eggs were then treated with the priming concentration of imidacloprid (0.0012 mg a.i.
mg per pot), so that the immatures of this parental generation grew and fed on treated plants. The
other five plants introduced in the reference culture were not treated with imidacloprid (no
priming) and were used as negative controls. Eggs were allowed to grow to pupae on all the
treatments. When most of the eggs reached the pupal stage, at twenty days after eggs were
deposited, five new untreated plants were introduced in each three cages (for primed and non primed reference strain, and primed resistant strain). The newly emerged adults (parental
generation) were allowed to fly to the untreated plants where they could lay eggs (F1
generation). The F1 immatures grew on untreated plants (no priming). Then, the old plants on
which the immatures of parental generation grew were removed from the cage after one week,
after almost all the adults had emerged. At this point the experiment followed the procedure
described in section II 4-2. Pupae (F1) were collected for adult emergence, and upon emergence,
the female adults (F1) were subsequently exposed to imidacloprid at different concentrations (0,
0.0012, 0.12, 0.12, 12, 24, 48 mg a.i mg per pot). Fecundity of females from three treatments
groups was determined: F1 females of the resistant strain generated by primed parents (denoted
as F1RP), F1 females of the reference strain generated by primed parents (denoted as F1SP), and
F1 females of the reference strain generated from parents without priming exposure (denoted as
F1SN).

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II-5 Hormesis in the rebuilt reference strain in 2013.
Results from experiments conducted between 2009 and 2012 suggest that the fecundity of
female whiteflies can be stimulated by a very low priming dose given to the immature stage. The
experiments gave some information about the range of priming doses of imidacloprid and the
range of doses for subsequent exposures that result in the increased fecundity of primed
individuals. The priming effect was observed consistently throughout experiments conducted
from 2009 to 2012, but the experiments were not repeated to determine the reproducibility of the
results. Therefore, a set of experiments was conducted in 2013 where a comparison could be
made of the fecundity of primed and non-primed females. Instead of testing a wide range of
subsequent exposure concentrations, more replications were added to a few selected subsequent
exposure concentrations. Hormesis experiments from 2009 to 2012 also gave some interesting
results about the F1 offspring of primed whiteflies. In the experiment the F1 offspring of adults
that were primed as larvae also showed an increase in fecundity when they were given an
additional exposure as the F1 adults. These F1 experiments also need to be repeated with more
replicates per treatment. The goal for 2013 was to repeat the original priming experiment and
the F1 experiment, each three times, for a total of six new experiments, after appropriate
treatment doses were determined for the re-built culture.
Experiments in 2013 were conducted on the rebuilt population from the reference strain
to determine whether priming of whitefly females with a low concentration of imidacloprid
could protect them against the adverse effect of a subsequent and more severe imidacloprid
exposure. Because the culture of whitefly used in 2013 is a re-constituted reference culture, I
first tested subsequent exposure doses at concentrations expected to be just below levels that

92

cause mortality. However, because a new growing medium was used in 2013, and a fertilizer was
used for the first time (no fertilizer was used in to grow plants between 2009 and 2012), the
appropriate range of concentrations for the subsequent exposure still needed to be determined.
Similarly, the effect of the priming concentration used in the previous years was assessed again
under these new conditions.
II-5-1 Response of the rebuilt population from the reference strain without priming.
The experiments in 2013 were conducted under conditions different from that of the
previous years, and this required some preliminary experiments to determine the relevant
subsequent concentrations, thus first determining the concentrations that elicit reductions in
fecundity due to a mild effect of imidacloprid but not a toxic effect causing mortality. Therefore,
the response in fecundity of the rebuilt population of whitefly adults to a wide range of
imidacloprid concentration was assessed first. The assay described in section II -4-1b and II-4-2
was followed. Initially seven concentrations (0, 0.00012, 0.0012, 3, 6, 9, 12, 15 mg a.i. per pot)
were used based on results from experiments conducted in 2012. Because no significant decrease
in fecundity was observed, another experiment was conducted with higher concentrations (12,
24, 48, and 60 mg a.i. per pot). Water was used as control.
Initially, each treatment consisted of twenty female adults with each adult female on a separate
leaf disc in a microcentrifuge tube being a replicate. Females which died due to the transfer,
fungi or decay of the leaf disc were eliminated from the test.
II-5-2 Response of the rebuilt population from the reference strain with priming.
Priming concentrations used in the 2013 experiments were selected from the priming
concentrations that gave the greatest and most consistent hormesis effect when individuals were
given a subsequent exposure as adults: 0.0012 imidacloprid per pot. However due to the new

93

conditions in 2013, the assessment of the same priming concentration was conducted. Another
priming concentration close to 0.0012 mg a.i. per pot was added (0.00012 mg a.i. per pot)
because this priming concentration was also shown to elicit a non-significant increase in
fecundity in the 2009-2012 study.
A test on the fecundity of the rebuilt population was then conducted. Immature
whiteflies were pre-exposed to the priming concentration 0.00012 and 0.0012 mg a.i per pot.
Then, the emerged adults were collected and subsequently exposed to leaf discs treated with
imidacloprid at subsequent concentrations as following the method described above (see II- 4-2).
Subsequent exposure concentrations of imidacloprid of 36 and 72 mg a.i. per pot were used due
to the results of the experiment above. To make sure that the imidacloprid drench did not leak
through the pots the solution was applied uniformly over the surface of the soil.
The two concentrations were very toxic. However, the experiment was still carried out to
provide information on the effect of a very severe subsequent exposure. Since the experiment
was primarily intended to assess the effect of priming on the sublethal effect of imidacloprid on
fecundity, another experiment with less toxic effect was also conducted. Therefore, as soon as it
was observed that the two subsequent concentrations were highly toxic to the collected female
adults, the extra primed and non-primed female pupae (primed as larvae, but not used in the
experiment) were used for another experiment. Females were emerging rapidly from pupae, so
drenches (12 mg a.i. per pot) made as a second exposure had to be limited to a 36 hour period
before leaf discs were cut for the experiment instead of the standard 72 hour drench and uptake
period. A lower subsequent exposure concentration (12 mg a.i. per pot) was used to avoid female
mortality. Initially, each treatment consisted of twenty five female adults with each adult female
on a separate leaf disc in a microcentrifuge tube being a replicate. Females which died due to the

94

transfer, fungi or a decayed leaf disc were eliminated from the test.
II-5-3 Test for hormesis on the parental generation of the rebuilt reference strain.
After the aforementioned experiments on the response of fecundity under the new cotton
growing conditions, a test on hormesis was carried out. The effect of the priming exposure
during the immature stage on the fecundity of the same individuals subsequently exposed as
adults to the imidacloprid was then tested. Immatures were exposed to the two priming
concentrations of imidacloprid (0.00012 and 0.0012 mg a.i per pot) and water was used for the
non- primed females. The assay for the collection of pupae for adult emergence described above
was followed (see II- 4-2) with a slight modification. Twelve plants in the four to six true-leaf
stage were placed in the rebuilt reference culture for adults to lay eggs on. Plants were removed
from the cultures after three to five days and transferred into three different rearing cages (24 X
24 X 72") made of polyester/nylon netting, mesh size 48 x 48. These plants with parental eggs
were then treated with the priming concentration of imidacloprid (0.0012, 0.00012 or 0 mg a.i.
mg per pot). Emerging adults were exposed to the subsequent exposure concentrations. Two
subsequent exposure concentrations (6 and 12 mg a.i. per pot) were used due to the results of the
previous experiment and the same volume of water was used in the drench for the control.
Fifteen plants were used for each treatment. The imidacloprid solution was applied by pouring
imidacloprid uniformly over the surface of the soil for both the priming exposure and the
subsequent exposure. The experiment was placed in growth chamber set at 22 °C. The eggs
were counted every other day except for the two first experiments during which eggs were
counted daily. Eggs were removed from each tube every week to facilitate the counting. For that,
the females were transferred into an empty microcentrifuge, the eggs on the leaf discs were
removed with a white tissue and forceps, and then the female was transferred back to the tube

95

after the cleaning. Initially, each treatment consisted of thirty female adults with each adult
female on a separate leaf disc in a microcentrifuge tube being a replicate. Females which died
due to the transfer, fungi or decayed leaf disc were eliminated from the test. This experiment was
repeated three times with three different cohorts. The first parental generation is referred to as
P11. The second parental generation is referred to as P12. A third a parental generation is
referred to as P13.
II-5-4 Test for hormesis on the first generation of the rebuilt reference strain.
A test on generation F1 was conducted to determine whether the stimulatory effect can be
transferred to the first generation of offspring of the primed immatures. The assays followed the
procedure described in section II-4-4 with a slight modification. When eggs of the primed
(0.00012, 0.0012 a.i. per pot) or non- primed parental generation reached the pupal stage, four
new untreated plants in the four to six true-leaf stage were introduced in each of the three rearing
cages. The old plants on which the immatures of parental generation grew were removed from
the cage after one week. The experiment followed the procedure described in section II 4-2.
Female adults of the first generation were exposed to two subsequent exposure concentrations (6
and 12 mg a.i mg per pot) or a water drench.
This experiment on the F1 generation was repeated three times. The first generation progeny of
the parental generation P11 is referred to as F11. Likewise, progeny of P12 is referred to as F12,
and progeny of P13 as F13. Initially, each treatment consisted of thirty female adults with each
female being placed on a separate leaf disc in a microcentrifuge tube.

96

II-6 Data analysis.
The analysis of effect of exposure to imidacloprid on fecundity and longevity of females
was determined by using a generalized linear mixed model, PROC GLIMMIX with a log
link function (SAS 9.3). As treatment with imidacloprid can affect the longevity of females,
longevity was not included in the model for the analysis of the fecundity and the fecundity was
expressed as number of total eggs laid by females. In these experiments the effect of exposure
(priming exposure or subsequent exposure) was considered as a fixed effect, with the other
exposure and the female replicates being considered as random effects.
For experiments with the rebuilt reference strain that were repeated three times, each experiment
was treated as an experimental block. The degrees of freedom in the analysis were adjusted by
using Kenward- Roger method. The priming exposure and subsequent exposure are fixed
effects, and the experimental run and whitefly replicate are random effects. The interaction
between the effects of priming and subsequent exposures was determined as well. The post-hoc
Tukey-Kramer was used following detection of a significant difference among treatments.

III- Results.
III-1 Hormesis with the resistant strain.
III-1-1 Study of the effect of priming exposure on the resistant adults.
The response in fecundity of the resistant strain to an exposure of imidacloprid was
assessed first. Since the resistant colony was continuously maintained on cotton plants treated
with imidacloprid at 12 mg a.i. per pot, the immature stages of the tested females had been
exposed to imidacloprid. The mean fecundity and longevity of these females are shown in the
Table 4.1a. No significant difference was observed among the total egg production of females

97

Table 4.1a. Mean ± SE total egg production and longevity of females of Bemisia tabaci B
biotype collected from the resistant colony and exposed to different concentrations of
imidacloprid as adults.

Imidacloprid treatment
(mg a.i. per pot)
Subsequent
Priming
exposure (single Adult female
exposure
drench, leaf disc
count
(Single drench , collected 72 h
continuous leaf
after
exposure)
application)
12
12
12
12
12
12
12

0
0.00012
0.0012
0.012
0.12
1.2
6

13
12
13
15
13
14
12

98

Resistant strain

Eggs per
female

Longevity
(days until mortality)

113.0 ± 5.2
101.9 ± 9.9
104.0 ± 7.6
109.5 ± 8.6
114.3 ± 6.9
113.7 ± 8.3
102.0 ± 10.5

22.0 ± 1.3
21.2 ±1 .3
20.4 ± 1.2
21.9 ± 1.2
22.0 ± 1.3
21.6 ± 1.2
21.5 ± 1.3

Table 4.1b. Summary of the generalized linear mixed model for the data in Table 4.1a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Imidacloprid
exposure

6

85

0.52

0.79

Imidacloprid
exposure

6

85

0.20

0.97

Fecundity

Longevity

Differences of means

Fecundity

Longevity

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.00012
0.0012
0.012
0.12
1.2
6

111.7a
96.5a
100.5a
104.8a
112.0a
109.4a
96.9a

9.8
8.9
8.9
8.6
9.8
9.3
8.9

0
0.00012
0.0012
0.012
0.12
1.2
6

22.0a
21.2a
20.4a
21.9a
22.0a
21.6a
21.4a

1.3
1.3
1.2
1.2
1.3
1.2
1.3

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer .

99

Table 4.2a. Mean ± SE total egg production and longevity of females of the resistant strain
Bemisia tabaci B biotype primed with seven different concentrations of imidacloprid as
immatures, then exposed to the same concentrations of imidacloprid as adults.
Imidacloprid treatment
(mg a.i. per pot)
Priming
exposure
(single drench,
continuous leaf
exposure)
0
0.00012
0.0012
0.012
0.12
1.2
6

Subsequent
exposure (single
drench, leaf disc
collected 72 h
after application)
0
0.00012
0.0012
0.012
0.12
1.2
6

Resistant strain

Adult female
count

Eggs per
female

Longevity
(days until
mortality)

15
14
14
14
11
13
15

70.6 ± 4.3
78.0 ± 4.9
92.7 ± 5.7
81.0 ± 5.0
71.0 ± 5.1
66.9 ± 4.4
82.1 ± 4.9

18.6 ± 1.3
19.2 ±1.3
20.9 ± 1.4
20.5 ± 1.4
19.6 ± 1.5
19.9 ± 1.4
18.8 ± 1.3

100

Table 4.2b. Summary of the generalized linear mixed model for the data in Table 4.2a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Imidacloprid
exposure

6

89

2.81

0.01

Imidacloprid
exposure

6

89

0.54

0.77

Fecundity

Longevity

Differences of means

Fecundity

Longevity

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.00012
0.0012
0.012
0.12
1.2
6

68.7b
76.4ab
91.7a
79.4ab
70.4ab
65.6b
80.8a

4.5
5.1
6.0
5.3
5.4
4.6
5.2

0
0.00012
0.0012
0.012
0.12
1.2
6

18.6a
19.2a
20.9a
20.5a
19.6a
19.9a
18.8a

1.1
1.1
1.2
1.2
1.3
1.2
1.1

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

101

Table 4.3a. Mean ± SE total egg production and longevity of females of the resistant strain
Bemisia tabaci B biotype primed with imidacloprid at 0.0012 mg a.i. per pot as immatures, and
then exposed to different concentrations of imidacloprid as adults.
Imidacloprid treatment
(mg a.i. per pot)

Resistant strain

Priming
exposure
(single drench,
continuous leaf
exposure)

Subsequent
exposure
(single drench,
leaf disc collected
72 h after
application)

Adult
female
count

Eggs
per female

Longevity
(days until
mortality)

0.0012
0.0012
0.0012
0.0012
0.0012
0.0012
0.0012
0.0012
0.0012

0
0.000012
0.00012
0.0012
0.012
0.12
1.2
6
12

10
15
15
15
14
8
15
15
14

47.1 ± 4.6
58.6 ± 4.6
54.9 ± 4.4
64.9 ± 5.1
57.9 ± 4.7
51.2 ± 5.5
40.0 ± 3.3
53.6 ± 4.3
48.8 ± 4.1

14.9 ± 1.2
16.8 ± 1.0
16.6 ±1.0
16.8 ± 1.0
16.2 ± 1.0
17.5 ± 1.4
15.8 ± 1.0
15.9 ± 1.0
16.8 ± 1.1

102

Table 4.3b. Summary of the generalized linear mixed model for the data in Table 4.3a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Imidacloprid
exposure

8

112

3.26

0.002

Imidacloprid
exposure

8

112

0.92

0.92

Fecundity

Longevity

Differences of means

Fecundity
Imidacloprid
(mg a.i. per pot)
0
0.00012
0.00012
0.0012
0.012
0.12
1.2
6
12

Mean*
fecundity
47.0ab
58.5a
54.93ab
64.9a
57.2a
51.2ab
39.9b
53.5ab
48.8ab

Longevity
SE

Imidacloprid
(mg a.i. per pot)
0
0.00012
0.00012
0.0012
0.012
0.12
1.2
6
12

4.4
4.3
4.1
4.8
4.4
5.3
3.1
4.0
3.8

Mean
Longevity
14.9a
16.8a
16.6a
16.8a
16.2a
17.5a
15.8a
15.9a
16.8a

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

103

SE
1.2
1.0
1.0
1.0
1.0
1.4
1.0
1.0
1.0

treated with a wide range of concentration of imidacloprid from 0.00012 to 6 mg a.i. per pot (p =
0.79) (Table 4.1b). Similarly, imidacloprid treatments had no effect on the longevity of these
resistant females (p = 0.97) (Table 4.1b).
After that test, the resistant females were treated with various concentrations of
imidacloprid as immatures and then subsequently exposed to the same concentration as adults.
The mean fecundity and longevity of these females are shown in the Table 4.2a. The analysis
indicates a significant difference in the egg production of these resistant females (p = 0.01)
(Table 4. 2b). A follow-up Tukey-Kramer test indicates that females primed with the
imidacloprid concentration of 0.0012 mg a.i. per pot as immatures and then subsequently
exposed to the same concentration as adults produced more eggs than the control females (91.7
eggs/female and 68.7 eggs/female respectively, p = 0.04) and that of the treatment at 1.2 mg a.i.
per pot (p = 0.01) (Table 4.2b). No significant difference was observed in the longevity of these
resistant females (p = 0.77) (Table 4. 2b).
Stimulation of egg production above the control level was found following a priming
concentration of 0.0012 mg a.i. per pot. This concentration of imidacloprid 0.0012 mg a.i. per
pot) was selected and studied further in the next round of experiments. Females primed with
0.0012 mg a.i. per pot were subsequently exposed to a wide range of imidacloprid
concentrations. The mean egg production and longevity of these females are shown in Table 4.
3a. The test on the fecundity indicates a significant difference (p = 0.002) among treatments
(Table 4.3b). The mean total number of eggs laid by females that were treated as adults at 1.2 mg
a.i was lower than that of females treated with low concentrations from 0.00012 to 0.012 mg a.i
per pot (Table 4.3b). The fecundity was found to peak at the subsequent exposure concentration
of 0.0012 mg a.i. per pot (64.93 eggs/female) (Table 4.3a, b). The longevity of adult females

104

treated with all subsequent exposure concentrations of imidacloprid was slightly higher than the
untreated adults , however, the analysis indicates no significant difference among treatments (p
= 0.92) (Table 4.3b).
III-1-2 Comparison of fecundity and longevity of primed and non-primed resistant
females.
The egg production and longevity of resistant females primed with imidacloprid at 0.0012 and 0
mg a.i per pot and then subsequently exposed to different concentrations of imidacloprid are
shown in the Table 4.4a. The test on the effect of priming exposure on fecundity of these
whitefly females indicates a significant difference (p < 0.001) (Table 4.4b). The mean number of
eggs laid by females primed with imidacloprid at 0.0012 a.i. mg per pot was higher than that of
the non-primed females (77.0 and 66.4 eggs/female, p < 0.001) (Table 4.4b). Thus, the result
indicates an overall significant increase of 15% in egg production of the primed females. The
results also indicate a significant difference in fecundity among the subsequent concentrations (p
< 0.001) (Table 4. 4b). The follow-up Tukey Kramer test indicates that the number of eggs laid
by females subsequently exposed to imidacloprid at 48 mg a.i per pot was lower than that of all
other treatments (Table 4.4b). The test also indicates that the effect of priming on longevity of
these female adults is not significant (p = 0.65) (Table 4.4 c). Subsequent exposure of females to
imidacloprid also did not affect the longevity of females (p = 0.20) (Table 4.4c). This result also
suggests that a low uptake of imidacloprid might happen with the highest treatment at 48 mg a.i
per pot so that no decrease in longevity for the two groups of females (primed and non-primed)
was seen at this high concentration 48 mg a.i per pot.

105

Table 4.4a. Mean ± SE fecundity and longevity of the resistant strain primed with imidacloprid at 0 and 0.0012 mg a.i. per pot as
immatures, then exposed as adults to 9 concentrations of imidacloprid centered about 0.0012 mg ai per pot

Subsequent
exposure
concentration (single
drench, leaf disc
collected 72 h after
application
(mg a.i. per pot)
0
0.00012
0.0009
0.0012
0.003
0.012
6
12
48

Resistant strain
with no priming (0 mg a.i. per pot)

Adult
female
count
14
12
13
14
12
13
14
13
15

Eggs per
female
72.3. ± 3.9
68.7 ± 3.8
82.9 ± 6.1
84.4 ± 5.3
69.0 ± 5.1
68.8 ± 3.6
63.9 ± 5.3
69.0 ± 4.8
41.7 ± 3.9

Resistant strain
primed with 0.0012 mg a.i. per pot (single
drench, continuous leaf exposure)

Longevity
(days until
mortality)
18.5 ± 4.9
18.8 ± 5.4
20.6 ± 5.7
20.2 ± 5.4
19.6 ± 5.6
19.3 ± 5.3
17.4 ± 4.6
19.5 ± 5.4
18.5 ± 4.9

106

Adult
female
count
15
15
14
12
13
14
12
15
15

Eggs per
female
74.8 ± 4.4
78.4 ± 5.4
92.6 ± 5.0
100.0 ± 3.0
80.23 ± 6.0
76.21 ± 3.0
79.4 ± 5.4
81.6 ± 3.9
52.5 ± 5.0

Longevity
(days until
mortality)
18.4 ± 0.8
17.4 ±0.8
20.5 ± 0.7
20.9 ± 0.7
19.3 ± 0.7
19.0 ± 1.0
18.8 ± 1.0
21.0 ± 0.9
20.2 ± 1.5

Table 4.4b. Summary of the generalized linear mixed model for the data on fecundity in Table 4.
4a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

1

244

19.68

<0.0001

Subsequent exposure

8

125

11.98

< 0.0001

Differences of means
Priming exposure

Subsequent exposure

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

0
0.0012

66.4a
77.0b

4.5
5.2

0
0.00012
0.0009
0.0012
0.003
0.012
6
12
48

73.1ab
72.3ab
84.3ab
90.2a
72.1ab
71.4ab
70.3b
74.7ab
45.2c

6.3
6.3
7.3
7.9
6.3
6.2
6.1
6.5
4.0

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

107

Table 4.4c. Summary of the generalized linear mixed model for the data on longevity in Table 4.
4a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

1

244

0.25

0.61

Subsequent exposure

8

112

1.39

0.20

Differences of means
Priming exposure
Imidacloprid
(mg a.i. per pot)
0
0.0012

Mean*
longevity
18.4a
18.0a

Subsequent exposure
SE

Imidacloprid
(mg a.i. per pot)
0
0.00012
0.0009
0.0012
0.003
0.012
6
12
48

0.7
0.8

Mean*
longevity
18.4a
18.0a
20.5a
20.5a
19.5a
19.2a
18.1a
20.3a
19.6a

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer .

108

SE
0.7
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8

III-1-3 Comparison of the fecundity and longevity between resistant females primed
with imidacloprid at 0.0012 and 12 mg a.i. per pot.
It was found in the previous experiment that a priming of the resistant females with
0.0012 mg a.i. imidacloprid per pot could stimulate their fecundity when they become adults.
An experiment was designed to determine if a high priming concentration can also induce a
stimulation of fecundity of the female whitefly. In this regard, a comparison between the effect
of priming concentrations of imidacloprid at 0.0012 and 12 mg a.i. per pot on fecundity was
performed. The mean fecundity and longevity of these females are shown in the Table 4.5a. The
analysis indicates a significant effect of the priming concentration on the fecundity of the
resistant females (p = 0.01) (Table 4.5b). The overall egg production by females primed with
imidacloprid at 0.0012 mg.a.i per pot was significantly higher than that produced by females
primed with imidacloprid at 12 mg a.i per pot (54.49 and 46.09 eggs/female, p = 0.001) (Table
4.5 b). The priming exposure of females with a low level of imidacloprid (0.0012 mg a.i. per pot)
elicits an 18 % increase in egg production when compared to the females primed with a high
concentration of imidacloprid (12 mg a.i. per pot). The effect of subsequent exposure also shows
a significant difference between treatments (p < 0.0001) (Table 4.5 b). The fecundity of females
exposed as adults to imidacloprid at 48 mg (32.37 eggs/female) was lower than that of the other
treatments (Table 4. 5b). The fecundity of females treated with 12 mg a.i per pot was also lower
than these of the females treated with low level of concentrations from 0.0003 to 0.009 mg a.i
per pot (Table 4. 5b).
The priming exposure did not have a significant effect on the longevity of female adults (p =
0.62) (Table 4.5c). However the subsequent exposure influenced the longevity of females (p <
0.001) (Table 4. 5c). The fecundity of females subsequently treated with imidacloprid at 48 mg

109

Table 4.5a. Mean ± SE egg production and longevity of resistant females primed with imidacloprid at 0.0012 and 12 mg a.i. per pot
as immatures, then exposed to different concentrations of imidacloprid as adults.
Subsequent
exposure
concentration (single
drench, continuous leaf
exposure)
(mg a.i. per pot)
0
0.00012
0.0003
0.0009
0.0012
0.003
0.006
0.009
0.012
12
48

Resistant strain
primed with 12 mg a.i per pot (single drench,
leaf disc collected 72 h after application)
Adult
female
count
13
13
13
11
13
11
8
12
9
14
12

Eggs per
female
58.0 ± 7.3
57.3 ± 7.6
62.7 ± 8.2
61.7 ± 7.5
72.8 ± 7.5
58.9 ± 7.8
70.6 ± 8.3
58.0 ± 9.4
38.4 ± 7.3
28.8 ± 2.1
19.9 ± 3.8

Longevity
(days until
mortality)
22.3 ± 2.4
23.5 ± 2.5
24.6 ± 2.8
23.3 ± 2.7
24.4 ± 2.1
25.1 ± 1.6
23.8 ± 1.8
24.8 ± 2.7
17.0 ± 2.1
14.0 ± 0.5
12.7 ± 1.4

110

Resistant strain
primed with 0.0012 mg a.i. per pot (single drench,
leaf disc collected 72 h after application)
Adult
female
count
15
12
15
13
15
14
10
14
9
12
12

Eggs
per female
56.2 ± 9.0
63.5 ± 9.8
73.6 ± 8.0
72.2 ± 6.5
94.1 ± 5.5
78.1 ± 8.3
90.5 ± 8.1
81.4 ± 8.1
53.3 ± 9.9
36.3 ± 4.6
17.4 ± 3.7

Longevity
(days until
mortality)
20.2 ± 2.3
21.9 ±2.1
22.2 ± 2.1
25.0 ± 2.3
26.8 ± 1.3
26.1 ± 2.3
25.0 ± 1.7
27.0 ± 2.5
20.1 ± 2.2
16.2 ± 1.6
9.9 ± 1.2

Table 4.5b. Summary of the generalized linear mixed model for the data on fecundity in Table
4.5a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

1

245

6.59

0.01

Subsequent exposure

10

144

16.48

< 0.0001

Differences of means
Priming exposure
Imidacloprid
(mg a.i. per pot)
0
0.0012

Mean
fecundity
54.4a
46.0b

Subsequent exposure
SE

Imidacloprid
(mg a.i. per pot)
0
0.00012
0.0003
0.0009
0.0012
0.003
0.006
0.009
0.012
12
48

7.9
6.7

Mean
fecundity
51.2abc
52.8abc
63.5ab
65.6ab
79.3a
65.4ab
80.1a
64.5ab
40.0bc
31.9c
15.3d

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer .

111

SE
7.4
7.7
9.2
9.7
11.4
9.6
12.4
9.3
6.3
4.7
2.4

Table 4.5c. Summary of the generalized linear mixed model for the data on longevity in Table
4.5a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

1

245

0.24

0.62

Subsequent exposure

10

144

10.97

<0.0001

Differences of means
Priming exposure

Subsequent exposure

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.0012

21.0a
20.4a

0.7
0.7

0
0.00012
0.0003
0.0009
0.0012
0.003
0.006
0.009
0.012
12
48

21.0ab
22.1a
22.6a
24.4a
25.3a
25.7a
24.6a
25.6a
18.1ab
14.9bc
11.1c

1.4
1.5
1.5
1.7
1.7
1.8
1.9
1.7
1.5
1.1
0.9

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

112

a.i. per pot was also lower than that of females treated with low concentrations of imidacloprid
from 0 to 0.009 mg a,i per pot (Table 4.5c). The longevity of females treated with imidacloprid
at 12 mg a.i. per pot was also lower than that of the females treated with low concentrations from
0.00012 to 0.009 mg a.i per pot (Table 4.5c).
III-1-4 Study of the effect of priming of parents on F1 generation female offspring of
the resistant strain.
The fecundity of resistant females of the F1 generation from primed parents was
investigated (Table 4.6a). F1 females were not primed during their immature stage, only their
parents were primed with imidacloprid. The analysis of their egg production indicates a
significant difference among treatments (p <0.0001) (Table 4.6 b). The follow-up Tukey-Kramer
test indicates that egg production by F1RP females treated as adults with imidacloprid at 48 mg
a.i. per pot (7.34 eggs/female) are significantly decreased compared with that of the untreated
F1RP females as adults (42.6 eggs/female) (p = 0.02 and < 0.0001) (Table 4.6 c).The inhibitory
effect was associated with the lethal effect of imidacloprid as suggested by the decrease in
longevity at these high concentrations. Egg production peaked at a concentration of 0.0012 mg
a.i. per pot (44.2.2 eggs/female), but the fecundity at this concentration is not significantly
different from that of the untreated female adults (Table 4. 6b). The leaf disc used for the adult
exposure was found to decay earlier in this treatment due to the low quality of the cotton leaves
available. The analysis on longevity of female indicates a significant difference (p < 0.0001)
(Table 4. 6c). The longevity of females treated as adults with imidacloprid at 24 and 48 mg a.i.
per pot was lower than these of the other treatments (Table 4.6c).
.

113

Table 4.6a. Mean ± SE egg production and longevity of the F1 generation females from
resistant parents primed with imidacloprid at 0.0012 mg a.i. per pot exposed to different
concentrations of imidacloprid as adults.
F1 resistant strain
Imidacloprid
concentration for
adult exposure
(mg a.i. per pot)
0
0.00012
0.0012
0.012
12
24
48

Adult
Female
count
12
14
12
14
14
15
15

Eggs
per female

Longevity
(days until mortality)

42.6 ± 2.0
40.4 ± 3.5
46.2 ± 4.7
37.5 ±2.7
40.7 ± 4.1
25.6. ± 5.3
10.8 ± 3.4

13.7 ± 0.4
13.2 ± 0.7
10.9 ± 0.8
13.7 ±0.2
13.0 ± 1.0
10.4 ± 1.3
6.8 ± 1.4

114

Table 4.6b. Summary of the generalized linear mixed model for the data in Table 4.6a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Imidacloprid
exposure

1

89

11.57

<0.0001

Imidacloprid
exposure

6

89

6.45

<0.0001

Fecundity

Longevity

Differences of means
Fecundity

Longevity

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.00012
0.0012
0.012
12
24
48

42.2ab
39.0ab
44.3ab
36.6a
38.5ab
19.2b
7.3c

8.2
7.0
8.6
6.6
6.9
3.4
1.4

0
0.00012
0.0012
0.012
12
24
48

13.7a
13.2a
10.9a
13.7a
13.0a
10.4a
6.8b

1.7
1.6
1.4
1.7
1.6
1.3
0.9

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

115

III-2 Hormesis in the reference strain (2009 - 2012).
The study on hormesis due to imidacloprid exposure as measured by fecundity was also
conducted with the reference strain between 2009 and 2012. No fertilizer was used during this
study.
III-2-1 Study of the effect of priming on the reference strain.
The response of the reference strain to exposure to different concentrations of
imidacloprid was first determined when these female adults were not primed with imidacloprid
as immatures (0 mg a.i per pot) and the results was shown in the Table 4.7a. The analysis of the
results indicates a significant difference in the fecundity among treatments (p = 0.01) (Table
4.7b). The follow up pairwise comparison test indicates that egg production of females at 6 mg
a.i per pot was significantly lower than that of untreated females (76.7 and 124.2 eggs/female, p
= 0.015) (Table 4.7b). This indicates that a sublethal concentration of imidacloprid affects the
fecundity of females of the reference strain. Imidacloprid exposure had no effect on the longevity
of these reference females (p = 0.64) (Table 4.7b).
After observation of this adverse effect of imidacloprid on fecundity, the reference females were
pre-exposed to various concentrations of imidacloprid as immatures, then exposed to the same
concentration as adults (Table 4.8a). As opposed to the results without priming, no statistical
difference was observed among treatments with these primed females of the reference strain (p =
0.15) (Table 4.8 b). The significant reduction in fecundity observed at higher concentration of
imidacloprid (6 mg a.i per pot) in reference females without priming was not seen anymore.
However, it was noteworthy that the effect of priming with a relatively high concentration of
imidacloprid (6 mg a.i. per pot) on survivorship of the immatures was not assessed in this
experiment. The egg production was observed to peak at the concentration of 0.00012 mg a.i. per

116

Table 4.7a. Mean ± SE egg production and longevity of females of Bemisia tabaci B biotype
collected from the reference colony and exposed to different concentrations of imidacloprid as
adults.
Imidacloprid treatment
(mg a.i. per pot)
Subsequent
Adult
Priming
(single drench, female count
(single drench,
leaf disc
continuous leaf
collected 72 h
exposure)
after application)
0
0
0
0
0
0
0

0
0.00012
0.0012
0.012
0.12
1.2
6

10
14
15
14
15
15
14

117

reference strain
Eggs per
female

Longevity
(days until mortality)

128.2 ± 10.3
115.9 ± 5.5
116.1 ± 8.0
102.3 ± 8.7
93.2 ±7.1
97.6 ± 7.2
81.7 ± 8.0

23.9 ± 0.8
26.3 ± 1.0
25.9 ± 1.2
24.5 ± 1.5
23.2 ± 1.2
25.2 ± 1.7
23.5 ± 1.5

Table 4.7b. Summary of the generalized linear mixed model for the data in Table 4.7a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Imidacloprid
exposure

6

90

2.80

0.015

Imidacloprid
exposure

6

90

0.70

0.64

Fecundity

Longevity

Differences of means
Fecundity

Longevity

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.00012
0.0012
0.012
0.12
1.2
6

124.3a
114.1ab
112.0ab
96.3ab
89.5ab
93.1ab
76.7b

13.9
10.8
10.3
9.2
8.3
8.6
7.4

0
0.00012
0.0012
0.012
0.12
1.2
6

23.8a
26.1a
25.9a
24.6a
23.2a
25.3a
23.5a

1.6
1.4
1.3
1.3
1.3
1.3
1.3

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

118

Table 4.8a. Mean ± SE total egg production and longevity of females of the reference strain of
Bemisia tabaci B biotype primed with seven different concentrations of imidacloprid as
immatures, then exposed to the same concentrations of imidacloprid as adults.
Imidacloprid exposure
(mg a.i. per pot)
Priming
(single drench,
continuous leaf
exposure)

Subsequent
(single drench,
leaf disc collected
72 h after
application)

0
0.00012
0.0012
0.012
0.12
1.2
6

0
0.00012
0.0012
0.012
0.12
1.2
6

Reference strain

Adult female
count

Eggs per
female

Longevity
(days until
mortality)

14
14
14
12
10
13
14

72.1 ± 6.1
90.4 ± 7.5
84.4 ± 7.0
77.7 ± 7.0
67.0 ± 6.7
66.6 ± 5.9
71.1 ± 6.0

17.0 ± 1.0
20.0 ± 1.4
19.7 ± 1.2
18.9 ± 1.0
17.9 ± 1.2
18.3 ± 1.4
16.9 ± 1.2

119

Table 4.8b. Summary of the generalized linear mixed model for the data in Table 4.8a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Imidacloprid
exposure

6

84

1.61

0.15

Imidacloprid
exposure

6

84

1.05

0.40

Fecundity

Longevity

Differences of means
Fecundity

Longevity

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.00012
0.0012
0.012
0.12
1.2
6

67.9a
88.3a
81.7a
75.2a
65.7a
61.9a
66.2a

6.8
8.7
8.1
8.1
7.8
6.4
6.6

0
0.00012
0.0012
0.012
0.12
1.2
6

16.9a
20.0a
19.7a
18.9a
17.8a
18.3a
16.9a

1.1
1.2
1.2
1.3
1.4
1.2
1.1

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

120

Table 4.9a. Mean ± SE total egg production and longevity of females of the resistant strain
Bemisia tabaci B biotype primed with imidacloprid at 0.0012 mg a.i. per pot as immatures, and
then exposed to different concentrations of imidacloprid as adults.
Imidacloprid treatment
(mg a.i. per pot)

Reference strain

Priming
(single drench,
continuous leaf
exposure)

Subsequent
(single drench,
leaf disc collected
72 h after
application)

Adult
female
count

Eggs
per female

Longevity
(days until
mortality)

0.0012
0.0012
0.0012
0.0012
0.0012
0.0012
0.0012
0.0012
0.0012

0
0.00012
0.00012
0.0012
0.012
0.12
1.2
6
12

10
15
14
15
14
8
13
13
15

36.7 ± 4.3
44.3 ± 4.2
51.3 ± 5.0
46.2 ± 4.4
45.5 ± 4.5
35.3 ± 4.7
34.7 ± 3.6
40.5 ± 4.2
36.0 ± 3.5

15.5 ± 1.2
16.6 ± 1.0
16.4 ± 1.0
16.7 ± 1.0
15.0 ± 1.0
13.1 ± 1.2
16.1 ± 1.1
17.0 ± 1.1
15.9 ± 1.0

121

Table 4.9b. Summary of the generalized linear mixed model for the data in Table 4.9a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Imidacloprid
exposure

8

108

1.84

0.07

Imidacloprid
exposure

8

108

0.88

0.53

Fecundity

Longevity

Differences of means
Fecundity

Longevity

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.00012
0.00012
0.0012
0.012
0.12
1.2
6
12

36.6a
44.3a
51.3a
46.2a
45.5a
35.3a
34.7a
40.5a
35.9a

4.3
4.2
5.0
4.4
4.5
4.7
3.6
4.2
3.5

0
0.00012
0.00012
0.0012
0.012
0.12
1.2
6
12

15.5a
16.6a
16.4a
16.7a
15.0a
13.1a
16.1a
17.0a
15.9a

1.2
1.0
1.0
1.0
1.0
1.2
1.1
1.1
1.0

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

122

pot (90.43 eggs/female) (Table 4.8a, b). No difference in longevity was observed among
treatments (p = 0.40) (Table 4.8b).
The results of the previous experiments shows a non-significant increase of egg
production when the reference strain was primed with concentrations of imidacloprid of 0.00012
and 0.0012 mg a.i. per pot. Also, the priming with imidacloprid at 0.0012 mg a.i. per pot causes
a significant increase in the total egg production in the resistant strain (Table 4.2a). Thus this low
concentration of imidacloprid (0.0012 mg a.i. per pot) was selected as the priming concentration
for the next round of experiments. The females primed with this concentration 0.0012 mg a.i.
perpot were subsequently exposed to a wide range of imidacloprid concentrations up to 12 mg
a.i. per pot and the results are shown in the (Table 4.9a). The analysis of the results indicates an
absence of significant difference in the fecundity of reference females (p = 0.07) (Table 4.9b).
Thus, no significant difference was observed between the number of eggs deposited by the
untreated female adults and the females subsequently exposed to imidacloprid at 12 mg a.i. per
pot (36.70 and 36.00 eggs/female) (Table 4.9b). No difference in longevity was observed among
treatments (p = 0.53) (Table 4.9 b).
III-2-2 Study of the effect of priming of parents on the F1 generation female
progeny of the reference strain.
The fecundity and longevity of F1 reference female offspring of non-primed parents
(F1SN) and primed parents (F1SP) was analyzed and the results are shown in the Table 4.10a.
The effect of priming exposure of the parental generation on egg production of the F1 female
adults indicates a significant difference (p = 0.0002) (Table 4.10b). The number of eggs laid by
F1SP females from primed parents was higher than that of F1SN females from non-primed

123

Table 4.10a Mean ± SE egg production and longevity of the F1 generation of the reference females from the parents with or without
priming with imidacloprid at 0.0012 mg a.i. per pot, then exposed to different concentrations of imidacloprid as adults.
Subsequent
exposure
concentration (single
drench, continuous leaf
exposure)
(mg a.i. per pot)
0
0.00012
0.0012
0.012
12
24
48

Reference strain from
non-primed parents

Female
adult
count
13
14
13
14
15
15
15

Eggs per
female
34.9 ± 3.2
28.2 ± 1.9
24.6 ± 2.2
28.6 ± 2.9
19.8 ±2.8
7.5 ± 1.7
1.4 ± 0.6

Reference strain from
parents primed with 0.0012 mg a.i. per pot (single
drench, leaf disc collected 72 h after application)

Longevity
(days until
mortality)
12.0 ± 0.5
11.8 ± 0.7
9.6 ± 0.5
12.0 ± 0.6
10.3 ± 1.1
7.6 ± 1.2
2.8± 0.4

124

Female
adult
count
14
13
14
11
13
15
15

Eggs
per female
33.9 ± 3.2
39.0 ± 2.9
31.7 ± 3.1
37.0 ± 2.3
36.7 ±4.3
13.8 ± 3.2
4.7 ± 2.5

Longevity
(days until
mortality)
11.5 ± 0.6
11.9 ± 0.9
10.8 ± 0.8
13.9 ± 0.5
11.9 ± 0.8
8.6 ± 0.1.2
5.1 ± 1.2

Table 4.10b. Summary of the generalized linear mixed model for the data on fecundity in Table
4.10a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

1

192

14.73

0.0002

Subsequent exposure

6

97

38.84

<0.0001

Differences of means
Priming exposure

Subsequent exposure

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

0
0.0012

13.6a
19.6b

5.6
8.0

0
0.00012
0.0012
0.012
12
24
48

32.4a
31.8a
26.7a
30.6a
26.1a
9.1b
1.9c

6.5
6.4
5.4
6.3
5.3
1.9
0.4

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

125

Table 4.10c. Summary of the generalized linear mixed model for the data on longevity in Table
4.10a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

1

192

1.96

0.16

Subsequent exposure

6

97

22.69

<0.0001

Differences of means
Priming exposure

Subsequent exposure

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.0012

11.7a
11.8a

0.8
0.8

0
0.00012
0.0012
0.012
12
24
48

11.7a
11.8a
10.2ab
12.5a
11.0a
8.1b
3.9c

0.8
0.8
0.8
0.9
0.8
0.6
0.4

*: means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

126

parents (19.6 and 13.6 eggs/female, p = 0.0002) (Table 4.10b), indicating an overall increase of
44 % in egg production of females that were generated from parents primed with imidacloprid
during their immature stages. The subsequent exposure of female adults was also shown to
induce a significant difference in fecundity of these females (p < 0.0001) (Table 4.10b). The
fecundity of F1 reference females treated as adults with imidacloprid at 24 and 48 mg a.i. per pot
was lower than that for females exposed to the lower treatment doses (Table 4.10b). The
priming of the parental generation does not cause a statistical difference in the longevity of the
F1 reference female offspring (p = 0.16) (Table 4.10c). However, the effect of subsequent
exposure of F1 female adults to imidacloprid had a significant effect on longevity (p < 0.0001)
(Table 4.10c). The F1 reference females treated as adults with imidacloprid at 48 mg a.i. pot had
lower longevity than the F1 females treated with the other concentrations (Table 4.10c).
Longevity of females treated with 24 mg a.i. per pot was also lower than that the other treatment
except that of the treatment with imidacloprid at 0.0012 mg a.i. per pot (Table 4.10c).

III-3 Hormesis in the rebuilt reference strain (2013).
III-3-1 Study of the effect of the priming on females of the rebuilt reference strain.
The females of the rebuilt reference strain were exposed to various concentrations of
imidacloprid from 0.00012 to 15 mg a.i. per pot. The results are shown in the Table 4.11a.
Analysis of the data indicates no difference in fecundity among the treatments (p = 0.80) (Table
4.11b). There is also no significant difference in longevity among treatments (p = 0.57) (Table
4.11b, c).
As no significant decrease in fecundity was noticed, a second experiment was then
conducted by using higher concentrations (up to 60 mg) as shown in Table 4.12a. The analysis of

127

Table 4.11a. Mean ± SE total egg production and longevity of female of the rebuilt reference
strain exposed as adults to different concentrations of imidacloprid up to 15 mg a.i. per pot.
Imidacloprid treatment
(mg a.i. per pot)
Subsequent
Priming
(single drench,
(single drench,
leaf disc
continuous leaf
collected 72 h
exposure)
after application)
0
0
0
0
0
0
0
0

0
0.00012
0.0012
3
6
9
12
15

Rebuilt reference strain
Adult female
count

Eggs per
female

Longevity
(days until mortality)

19
18
17
18
18
19
19
19

59.3 ± 2.5
62.0 ± 4.5
65.2 ± 3.5
60.2 ± 2.5
58.3 ± 4.1
65.7 ± 3.0
63.0 ± 2.3
63.2 ± 4.3

16.0 ± 1.2
16.3 ± 1.2
16.9 ± 1.3
18.1 ± 1.3
16.5 ± 1.2
16.7 ± 1.2
18.5 ± 1.3
17.2 ± 1.2

128

Table.4.11b. Summary of the generalized linear mixed model for the data in Table 4.11a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Imidacloprid
exposure

7

139

0.54

0.80

Imidacloprid
exposure

7

139

0.81

0.57

Fecundity

Longevity

Differences of means
Fecundity

Longevity

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.00012
0.0012
3
6
9
12
15

58.7a
60.2a
64.2a
59.6a
56.8a
65.0a
62.5a
61.4a

3.5
3.7
4.0
3.7
3.5
3.8
3.7
3.7

0
0.00012
0.0012
3
6
9
12
15

16.0a
16.3a
16.9a
18.1a
16.5a
16.7a
18.5a
17.2a

0.9
0.9
0.9
1.0
0.9
0.9
0.9
0.9

*: means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

129

Table 4.12a. Mean ± SE total egg production and longevity of female of the rebuilt reference
strain exposed as adults to different concentrations of imidacloprid up to 60 mg mg a.i. per pot.
Imidacloprid treatment
(mg a.i. per pot)
Subsequent
Priming
(single drench,
(single drench,
leaf disc
continuous leaf
collected 72 h
exposure)
after application)
0
0
0
0
0
0

0
12
24
36
48
60

Rebuilt reference strain
Adult female
count

Eggs per
female

Longevity
(days until mortality)

20
19
20
19
20
20

92.3 ± 5.9
84.6 ± 4.3
85.9 ± 5.7
79.6 ± 5.4
79.7 ± 3.9
77.2 ± 7.2

15.6 ± 1.1
17.4 ± 1.2
16.3 ± 1.1
18.7± 1.2
18.2 ± 1.2
16.7 ± 1.1

130

Table 4.12b. Summary of the generalized linear mixed model for the data in Table 4.12a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Imidacloprid
exposure

5

112

1.06

0.38

Imidacloprid
exposure

5

112

1.61

0.16

Fecundity

Longevity

Differences of means
Fecundity

Longevity

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
12
24
36
48
60

92.3a
87.6a
85.9a
79.6a
79.7a
77.2a

6.1
5.9
5.7
5.4
5.3
5.1

0
12
24
36
48
60

15.6a
17.4
16.3a
18.7a
18.2a
16.7a

0.8
0.9
0.9
0.9
0.9
0.9

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

131

results indicates no significant difference in fecundity among treatments (p = 0.38) (Table
4.12b). However, a non-significantt decrease in egg production was observed for the relatively
high concentrations of 36, 48 and 60 mg a.i. per pot when compared with the controls (Table
4.12b). It is noteworthy that the imidacloprid solution was poured at the base of the plants for
these two experiments. The effect of priming of immatures with an exposure of 0, 0.000012 or
0.0012 mg a.i. imidacloprid per pot, on the fecundity of females of the rebuilt reference strain
was tested by a subsequent exposure of females to imidacloprid at 36 or 72 mg imidacloprid a.i
per pot. The results were shown in Table 4.13a. Results from this experiment indicates that the
two subsequent exposure concentrations of 36 and 72 mg a.i per pot applied as drench over the
entire soil surface were highly toxic. Many females showed symptoms of toxicity or died after
the subsequent exposure for the three groups of females (non-primed and primed with 0.00012
and 0.0012 mg a.i. per pot) (Table A1). Apparently application of the drench over the entire soil
surface increased uptake of imidacloprid. The effect of priming exposure on the fecundity of
these females was not significantly different (p = 0.31) (4.13b). Thus, there is no difference
between egg productions of non-primed females and females primed with 0.00012 and 0.0012
mg ai per pot. However, subsequent exposure was found to influence the fecundity of the
females (p = 0.0005) (4.13b). The egg production of females treated as adults with imidacloprid
at 72 mg a.i per pot was lower than that for females treated with 36 mg a.i per pot and for
untreated female adults (38.6, 97.6, and 74.3, p = 0.0005, 0.01) (Table 4.13b). The priming
exposure did not have a significant effect on the longevity of females (p = 0.42) (Table 4.13c).
However, the subsequent exposure effected the longevity of females (p = 0.008) (Table 4.13c).
The females treated as adults with imidacloprid at 72 mg a.i per pot had reduced longevity
compared with females treated with imidacloprid at 36 mg a.i per pot and untreated female

132

Table 4.13a. Mean ± SE egg production and fecundity of females of the rebuilt reference strain when primed as immatures and then
exposed as adults to imidacloprid at 0, 36 and 72 mg a.i. per pot.
Imidacloprid
(mg a.i. per
pot)

Priming exposure
(single drench, continuous leaf exposure)
0

0.00012

0.0012

Subsequent
exposure
(single drench,
leaf disc
collected 72 h
after
application)

Adult
female
count

Eggs
per female

Longevity
(until
mortality)

Adult
female
count

Eggs
per female

Longevity
(until
mortality)

Adult
female
count

Eggs
per female

Longevity
(until
mortality

0
36
72

25
25
25

101.2 ± 4.8
88.5 ± 8.0
58.8 ± 10.0

14.8 ± 0.6
15.2 ± 1.1
12.6 ± 1.6

25
25
25

91.0 ± 5.0
75.0 ± 10.2
62.0 ± 10.5

14.0 ± 0.8
12.9 ± 1.4
11.9 ± 1.4

25
25
25

107.2 ± 7.0
95.2 ± 9.9
47.0 ± 11.4

14.6 ± 0.7
15.1 ± 1.3
10.0 ± 1.6

133

Table 4.13b. Summary of the generalized linear mixed model for the data on fecundity in Table
4.13a.
Type III tests of Effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

2

222

1.15

0.31

Subsequent exposure

2

72

8.40

0.0005

Differences of means
Priming exposure
Imidacloprid
(mg a.i. per pot)
0

Mean*
fecundity
54.0a

0.00012
0.0012

Subsequent exposure
SE
26.8

Imidacloprid
(mg a.i. per pot)
0

Mean*
fecundity
97.6a

16.1

37.7a

18.7

36

74.3a

12.3

40.1a

19.9

72

38.6b

6.4

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

134

SE

Table 4.13c. Summary of the generalized linear mixed model for the data on longevity in Table
4.13a.
Type III tests of fixed effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

2

222

5.87

0.42

Subsequent exposure

2

72

5.07

0.008

Differences of means
Priming exposure

Subsequent exposure

Imidacloprid
(mg a.i. per pot))

Mean*
longevity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0

13.3a

0.8

0

14.4a

1.0

0.00012

11.9a

0.7

36

14.0a

1.0

0.0012

12.1a

0.7

72

10.8b

0.8

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

135

Table 4.14a. Mean ± SE total egg production and longevity of females of the rebuilt reference strain exposed to three priming
concentrations of imidacloprid (0, 0.00012 and 0.0012 mg a.i. per pot) as immatures and then exposed to plants systemically treated
for 36 hours with imidacloprid at 0 and 12 mg a.i per pot.
Priming exposure
(single drench, continuous leaf exposure)

Imidacloprid
treatment
(mg a.i per pot)

Subsequent
exposure
(single drench,
leaf disc
collected 36 h
after
application)
0
12

0

0.00012

0.0012

Adult
female
count

Eggs
per female

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female

Longevity
(days until
mortality)

29
29

148.7 ± 8.0
147.3 ± 9.5

16.3 ± 0.9
16.0 ± 0.8

30
29

149.1 ± 8.3
176.0 ± 7.8

15.8 ± 0.6
17.1 ± 0.5

29
29

162.5 ± 8.3
164.1 ± 8.7

16.5 ± 0.6
16.5 ± 0.7

136

Table 4.14b. Summary of the generalized linear mixed model for the data on fecundity in Table
4.14a.
Type III tests of Effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

2

172

2.05

0.13

Subsequent exposure

1

58

1.03

0.31

Differences of means
Priming exposure

Subsequent exposure

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
fecundity

SE

0
0.00012
0.0012

140.2a
156.3a
156.0a

6.2
6.8
6.9

0
12

150.1a
158.7a

5.8
6.1

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

137

Table 4.14c. Summary of the generalized linear mixed model for the data on longevity in Table
4.14a.
Type III tests of Effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure

2

172

0.23

0.79

Subsequent exposure

1

58

1.03

0.31

Differences of means
Priming exposure

Subsequent exposure

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
longevity

SE

0
0.00012
0.0012

15.7a
16.2a
16.1a

0.5
0.5
0.5

0
12

15.9a
16.1a

0.4
0.4

*: Means followed by different letters within the same column are statistically different
according to Tukey-Kramer.

138

adults (10.8, 14.4, 14.0 days, p = 0.01, 0.02) (Table 4.13c).
Another test was also conducted by using a shorter exposure time (36 hours) with a lower
subsequent exposure concentration (12 mg a.i. per pot) as shown in the Table 4.14a. Analysis of
the data shows that the effect of priming exposure on the fecundity of the female adults was not
statistically significant (p= 0.13) (Table 4.14b). The overall egg production of females primed
with imidacloprid at 0.00012 and 0.0012 mg a.i per pot was higher than that of non-primed
females but not statistically different (p = 0.18, 0.19) (Table 4.15b). The effect of subsequent
exposure on fecundity among the treatments was also not statistically significant (p = 0.31)
(Table 4.14b) as well as the effect of priming exposure on longevity (p =0.79) (Table 4.14c).
Similarly, the subsequent exposure did not affect the longevity of the rebuilt reference female (p
=0.63) (Table 4.15c).
III-3-2 Study of the effect of priming on the parental generation of the rebuilt
population.
At this point there was enough information to run an experiment on parental generation
that could be repeated several times. The three parental generations are referred to as P11, P12
and P13. The fecundity and longevity of the females of these parental generation are shown in
Table 4. 15 a,b,c. Analysis of the data indicates no interaction of the effects of priming exposure
and subsequent exposure on the egg production of the females (p = 0.34) (Table 4. 15d).
However, the effect of priming exposure on egg production of females shows a significant effect
(p = 0.004) (Table 4.15d). The follow-up Tukey-Kramer test indicates that egg production of
females primed with imidacloprid at 0.00012 and 0.0012 mg per pot was higher than that of the
non-primed females (186.7, 191.1 and 169.9, eggs/female, p = 0.03, 0.004) (Table 4.15d). This
suggests an increase in fecundity of the rebuilt reference females primed with low concentrations

139

Table 4. 15a. Mean ± SE total egg production and longevity of the rebuilt reference females of the parental generation P11 exposed to
three priming concentrations of imidacloprid (0, 0.00012 and 0.0012 mg a.i. per pot) as immatures and then exposed to plants
systemically treated for 72 hours with imidacloprid at 0, 6 and 12 mg a.i. per pot.
Priming exposure (single drench, continuous leaf exposure)
Imidacloprid
concentration
(mg a.i. per pot

0

0.00012

0.0012

subsequent
exposure
(single drench,
leaf disc
collected 72 h
after
application)

Adult
female
count

Eggs
per female
(P11)

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female
(P11)

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female
(P11)

Longevity
(days until
mortality)

0
6
12

27
30
30

114.0 ± 7.9
126.7 ± 11.5
116.6 ± 10.0

20.8 ± 1.2
23.4 ± 1.8
17.7 ± 1.4

30
30
30

101.1 ± 8.9
163.2 ± 15.1
140.4 ± 13.2

17.7 ± 1.4
25.5± 1.8
18.8 ± 1.6

29
28
28

129.0 ± 11.4
163.4 ± 14.0
121.9 ± 0.6

21.7 ± 1.4
25.7 ± 1.8
17.7 ± 1.4

140

Table 4.15b Mean ± SE total egg production and longevity of the rebuilt reference females of the parental generation P12 exposed to
three priming concentrations of imidacloprid (0, 0.00012 and 0.0012 mg a.i. per pot) as immatures and then exposed to plants
systemically treated for 72 hours with imidacloprid at 0 and 6 mg a.i. per pot.

Imidacloprid
concentration
(mg a.i. per pot

Priming exposure (single drench, continuous leaf exposure)
0

0.00012

0.0012

subsequent
exposure
(single drench,
leaf disc
collected 72 h
after
application)

Adult
female
count

eggs
per female
(P12)

Longevity
(days until
mortality)

Adult
female
count

eggs
per female
(P12)

Longevity
(days until
mortality)

Adult
female
count

eggs
per female
(P12)

Longevity
(days until
mortality)

0
6

27
29

119.6 ± 9.9
133.9 ± 9.8

17.6 ± 1.0
20.1 ± 1.6

30
27

127.3 ± 8.8
153.6 ± 14.4

18.1 ± 1.2
19.6± 1.4

29
28

140.3 ± 10.4
166.6 ± 11.8

20.1 ± 1.0
20.3 ± 1.4

141

Table 4. 15c. Mean ± SE total egg production and longevity of the rebuilt reference females of the parental generation P13 exposed to
three priming concentrations of imidacloprid (0, 0.00012 and 0.0012 mg a.i. per pot) as immatures and then exposed to plants
systemically treated for 72 hours with imidacloprid at 0, 6 and 12 mg a.i. per pot.
Priming exposure
(single drench, continuous leaf exposure)

Imidacloprid
(mg a.i. per
pot)

0

0.00012

0.0012

subsequent
exposure
(single drench,
leaf disc
collected 72 h
after
application)

Adult
female
count

Eggs
per female
(P13)

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female
(P13)

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female
(P13)

Longevity
(days until
mortality)

0
6
12

28
29
30

312.7 ± 18.3
304.7 ± 17.0
344.0 ± 19.5

29.7 ± 1.8
28.5 ± 1.8
32.6 ± 1.7

28
28
29

342.2 ± 20.8
325.6 ± 25.1
349.0 ± 17.7

32.3 ± 2.0
29.8 ± 2.2
29.4 ± 1.5

29
30
29

316.0 ± 17.5
351.3 ± 21.9
365.5 ± 20.6

28.0 ± 1.9
30.4 ± 2.1
31.7 ± 1.7

142

Table 4.15d. Summary of the generalized linear mixed model for the data on fecundity in Table 4.15a, b, c.
Type III tests of Effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure (P)

2

674.3

5.53

0.004

Subsequent exposure (S)

2

674.2

8.45

0.0002

PXS

4

674.6

1.13

0.34

Differences of means
Priming exposure (P)
Imidacloprid
(mg a.i. per pot)
0

Mean *
fecundity
169.3a

0.00012
0.0012

Subsequent exposure (S)
SE
51.7

Imidacloprid
(mg a.i. per pot)
0

Mean*
fecundity
168.7a

51.4

186.7b

56.9

6

195.2b

59.4

191.8b

58.4

12

184.7ab

56.4

*Mean followed by the same letter are not statistically different according to Tukey-Kramer.

143

SE

Table 4.15e. Summary of the generalized linear mixed model for the data on longevity in Table 4.15 a, b, c.
Type III tests of Effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure (P)

2

672.1

0.31

0.73

Subsequent exposure (S)

2

667.6

6.25

0.002

PXS

4

672.5

0.20

0.93

Differences of means
Priming exposure (P)
Imidacloprid
(mg a.i. per pot)
0

Mean *
longevity
22.8a

0.00012
0.0012

Subsequent exposure (S)
SE
3.3

Imidacloprid
(mg a.i. per pot)
0

Mean*
longevity
22.4a

3.2

22.7a

3.3

6

24.6b

3.6

23.3a

3.4

12

21.8a

3.2

*: Mean followed by the same letter are not statistically different according to Tukey-Kramer.

144

SE

of imidacloprid during immature stages. The effect of the subsequent exposure on the egg
production of the females also indicates a significant difference among treatments (p = 0.0002)
(Table 4.15d). The egg production of females treated as adults with imidacloprid at 6 mg a.i per
pot was higher than that for untreated female adults (195.2 and 168.7 eggs/female, p = 0.0001)
(Table 4.14d). The fecundity of the females of these three parental generations of the rebuilt
reference strain was illustrated in the cumulative mean egg production graphs (Figure A3, A4,
and A5). The graph of the cumulative fecundity of primed females was seen to be above of that
of the non-primed females at the concentration of 6 mg a.i per pot. The analysis also indicates
that there is no interaction of the effects of priming exposure and subsequent exposure on
longevity of the females (p = 0.93) (Table 4.15e). Similarly, the effect of priming exposure on
longevity was also not statistically significant (p = 0.73) (Table 4.15e). However, the effect of
the subsequent exposure on longevity was statistically significant among the treatments (p =
0.002) (Table 4. 15e). Females treated as adults with imidacloprid at 6 mg a.i per pot have an
increased longevity compared with females treated with imidacloprid at 0 and 12 mg a.i. per pot
( 24.6, 22.4 and 21.8 days, p = 0.01, 0.005) (Table 4.15e).
III- 3-3 Study of the effect of priming of parents on the first generation of the rebuilt
reference strain.
The effect of priming was found to be transferred to the offspring of the first generation
of the reference strain in the study of 2009-2012. Therefore, a test on the first generation of
offspring was also conducted on the rebuilt reference strain to determine if a priming effect can
be transferred to the offspring. The fecundity and longevity of the first generation of female
offspring from three experiments (F11, F12, F13) are shown in the Table 4.16a, b, c. Data
analysis indicates that the priming exposure of the parental generation did not cause a significant

145

Table 4.16a. Mean ± SE total egg production and longevity of the F11 female progeny of the rebuilt reference strain from parents
with and without priming exposure to imidacloprid exposed as adults to imidacloprid at 0, 6 and 12 mg a,i. per pot.
Priming exposure of parents
(single drench, continuous leaf exposure)

Imidacloprid
(mg a.i. per
pot)

0

0.00012

0.0012

subsequent
exposure
(single drench,
leaf disc
collected 72 h
after
application)

Adult
female
count

Eggs
per female
(F11)

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female
(F11)

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female
(F11)

Longevity
(days until
mortality)

0
6
12

27
28
25

222.5 ± 21.4
273.3 ± 24.5
263.4 ± 22.7

29.7 ± 3.1
36.2 ± 2.8
31.6 ± 2.5

29
28
26

255.1 ± 21.5
258.7 ± 16.7
244.2 ± 26.3

31.5 ± 2.6
31.7± 2.0
27.1 ± 2.6

27
26
26

259.8 ± 19.7
295.5 ± 21.1
304.3 ± 22.9

36.0 ± 2.8
37.7 ± 2.7
34.3 ± 2.5

146

Table 4.16b. Mean ± SE total egg production and longevity of the F12 female progeny of the rebuilt reference strain from parents with
and without priming exposure to imidacloprid exposed as adults to imidacloprid at 0 and 6 mg a,i. per pot.

Priming exposure of parents
(single drench, continuous leaf exposure)

Imidacloprid
(mg a.i. per
pot)

0

0.00012

0.0012

subsequent
exposure
(single drench,
leaf disc
collected 72 h
after
application)

Adult
female
count

Eggs
per female
(F12)

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female
(F12)

Longevity
(days until
mortality)

Adult
female
count

Eggs
per female
(F12)

Longevity
(days until
mortality)

0
6

27
28

214.0 ± 16.6
216.2 ± 20.6

33.3 ± 1.9
32.9 ± 1.9

29
28

212.6 ± 16.4
232.5 ± 16.4

31.0 ± 1.7
32.0 ± 1.5

27
26

179.2 ± 21.2
223.0 ± 19.6

27.5 ± 2.2
30.3 ± 2.1

147

Table 4.16c. Mean ± SE total egg production and longevity of the F13 female progeny of the rebuilt reference strain from parents with
and without priming exposure to imidacloprid exposed as adults to imidacloprid at 0, 6 and 12 mg a,i. per pot.

Priming exposure of parents
(single drench, continuous leaf exposure)

Imidacloprid
(mg a.i. per
pot)

0

subsequent
exposure
(single
drench, leaf
disc collected
72 h after
application)

Adult
female
count

0
6
12

28
29
30

Eggs
per
female
(F13)

324.7 ± 18.7
332.2 ± 16.6
299.0 ± 19.6

0.00012
Longevity
(days until
mortality)

37.2 ± 1.5
37.8 ± 1.5
36.8 ± 2.1

Adult
female
count

28
28
29

Eggs
per female
(F13)

0.0012
Longevity
(days until
mortality)

277.9 ± 21.1 34.6 ± 2.0
347.8 ± 20.4 42.1± 21.0
294.1 ± 27.4 34.3 ± 2.0

148

Adult
female
count

Eggs
per female
(F13)

Longevity
(days until
mortality)

29
30
29

303.2 ± 22.4
351.0 ± 26.8
364.8 ± 19.1

34.8 ± 2.0
38.7 ± 1.6
38.9 ± 1.6

Table 4.16d. Summary of the generalized linear mixed model for the data on fecundity in Table 4.16a, b, c. .
Type III tests of Effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure (P)

2

643

2.05

0.125

Subsequent exposure (S)

2

643.9

5.01

0.006

PXS

4

643.2

1.89

0.11

Differences of means
Priming exposure (P)

Subsequent exposure (S)

Imidacloprid
(mg a.i. per pot)

Mean *
fecundity

SE

Imidacloprid
(mg a.i. per pot)

Mean*
Fecundity

SE

0

257.3a

31.4

0

248.0a

30.1

0.00012

257.0a

31.4

6

278.6b

33.9

0.0012

275.7a

33.7

12

263.8ab

32.6

*: Mean followed by the same letter are not statistically different according to Tukey-Kramer.

149

Table 4.16e. Summary of the generalized linear mixed model for the data on longevity in Table 4.16 a, b, c.
Type III tests of Effects
Effect

Numerator df

Denominator df

F

P value

Priming exposure (P)

2

3.93

0.64

0.57

Subsequent exposure (S)

2

647

18.04

<0.0001

PXS

4

647

2.93

0.02

Differences of means
Priming exposure (P)
Imidacloprid
(mg a.i. per pot)
0

Mean *
longevity
34.1a

0.00012
0.0012

Subsequent exposure (S)
SE
2.2

Imidacloprid
(mg a.i. per pot)
0

Mean*
longevity
32.8a

1.8

32.2a

2.1

6

35.4b

2.0

34.0a

2.2

12

32.2a

1.8

*: Mean followed by the same letter are not statistically different according to Tukey-Kramer.

150

SE

difference on fecundity of F1 female offspring among the treatments (p = 0.12) (Table 4.16d).
The fecundity of the F1 progeny from primed parents was higher than that of the F1 progeny
from the non-primed parents (175 and 157 eggs/female), however this increase is not statistically
different. The cumulative mean egg production of these F1 females offspring was also shown in
Figure A6, A7 and A8. The graph of cumulative fecundity of F1 females from primed parents at
0.0012 mg a.i. per pot was seen to be above of that of the two others groups of females at 12 mg
a.i. per pot. The effect of the subsequent exposure of the F1 progeny on their fecundity was
statistically significant (p = 0.006). The fecundity of F1 rebuilt reference females treated as
adults with imidacloprid at 6 mg a.i. per pot was higher than the untreated F1 female adults (0
mg a.i. per pot) (278. 6 and 248.0 eggs/female , p = 0.004) (Table 4.16d). The effect of the
priming exposure of the parental generation on longevity of the F1 female progeny was not
significantly different among the treatments (p =0.57) (Table 4.16e). However, the effect of the
subsequent exposure on longevity was statistically significant (p < 0.0001). The longevity of
rebuilt reference females treated as adults with imidacloprid at 6 mg a.i per pot was higher than
for females treated with 0 and 12 mg a.i. per pot (35.4, 32.8 and 32.2 days, p <0.0001, <0.001).
Analysis of longevity data for the F1 progeny females also shows an interaction of the effects of
priming of the parent generation and the subsequent exposure of the F1 female progeny (p =
0.02).

IV Discussion
Low concentrations of toxicants can induce a stimulation of many biological processes in a
variety of organisms including insects, and this phenomenon is known as hormesis (Calabrese
and Blain, 2005). In hormesis, the stimulation can be revealed as an increased positive response

151

above the level of the control, or it can also be expressed as an adaptive response of the organism
to a second and more severe exposure to the stressors (Calabrese and Baldwin, 2002; Stebbing,
2009). Several important factors can determine the occurrence of the stimulation, among them
are the dose of the toxicants and the stage of the development of the organism. This study set a
hypothesis that treatment of whitefly females with low doses of imidacloprid applied at an early
stage in their development will induce an increase in fecundity of the same females when they
become adults.
A wide range of concentrations was first tested to determine the priming concentration
used to treat the immature stage. A stimulatory response in fecundity of adult females in the
resistant strain was observed for individuals that were primed with imidacloprid at 0.0012 mg a.i.
per pot during the immature stage then treated with the same concentration as adults (Table 4.
2b, c). This result suggests that priming of females at this concentration during immature stage
might increase the fecundity of adult females. Indeed, in a following test, an overall increase in
fecundity of 15% was noticed in resistant females primed with imidacloprid at 0.0012 mg a.i per
pot when compared to females without priming and using a wide range of the subsequent
exposure concentrations (Table 4.4b). An overall 18% increase in egg production was also
observed for these resistant females primed with 0.0012 mg a.i per pot compared to that of
females primed with 12 mg a.i per pot (Table 4.5b). This also suggests that only a low
concentration of imidacloprid may elicit a stimulation in fecundity. In a related experiment,
Ayyanath et al. (2013) exposed immatures of the aphid M. persicae to imidacloprid for several
generations, and these authors found that only a continuous exposure to low concentrations of
imidacloprid of 0.025 µg/l caused an increase in fecundity of the aphids whereas the continuous
exposure to the higher concentrations (0.1 to 25 µg/L) did not cause a stimulation.

152

Preliminary tests were also conducted with the reference strain to determine appropriate
subsequent and priming concentrations. A desirable subsequent exposure concentration is one
that decreases fecundity without causing significant mortality. In an initial test, the fecundity of
the non-primed reference females decreased after they were treated with imidacloprid at 6 mg a.i
per pot, without causing significant mortality (Table 4.7b). This is consistent with a report on
reduction of egg production in whitefly females exposed to sublethal dose of imidacloprid. He et.
al. (2013) showed that females of B. tabaci treated with imidacloprid at a sublethal concentration
of 5 ppm for 24h exhibited a reduction of egg production. Then a test was conducted to
determine an appropriate priming concentration of imidacloprid for treating immatures. Two low
concentrations, 0.0012 and 0.00012 mg a.i. per pot, were found to elicit a non-significant
increase of the fecundity of adults (4.8a,b). The reference females were then primed with the
concentration of 0.0012 mg a.i per pot in the following experiment; and as opposed to the
previous results without priming, no decrease of fecundity was seen with the reference females
even at a relatively high subsequent exposure concentration of 12 mg a.i. per pot (4.9b).
However, since no females without priming were used as controls in that experiment, it is not
known whether this disappearance of inhibition of egg production might be due to reduced
uptake of imidacloprid by the plants. A test on the F1 generation was also conducted to assess
the inheritance of a priming effect with the reference strain. The reference female progeny from
primed parents exhibited 44% higher fecundity than the female progeny from non-primed
parents when they were exposed to different concentrations of imidacloprid as adults (Table 4.
10b).
After observing a stimulation in fecundity of whitefly that were primed as immatures
with a low dose of imidacloprid, the study was re-initiated with the rebuilt reference strain for a

153

set of experiments to confirm these results. It is noteworthy that for this new set of experiments
fertilizer was used to grow cotton plants more rapidly, and that the MSU greenhouses had
switched to a new growing medium that we used to grow our plants. The initial objective was to
investigate if priming of whitefly females with a low concentration of imidacloprid could protect
them against the adverse effect of a subsequent and more severe imidacloprid exposure, a
phenomenon known as conditioning hormesis (Calabrese and Mattson, 2011). However, no
decrease in fecundity was observed for females exposed to concentrations up to 15 mg a.i. per
pot (Table 4.11b). Only a slight but not significant decrease of egg production was observed
when the imidacloprid concentration was increased up to 60 mg a.i. per pot (4.12 a, b).
A test was conducted using different priming concentrations (0, 0.0012, 0.00012 mg a.i.
per pot) and relatively high subsequent exposure concentrations (36 and 72 mg a.i per pot). In
contrast to the results of the aforementioned experiment, these high concentrations exhibit
toxicity in the females (Table 4.13b). It is noteworthy that in this experiment, the imidacloprid
solution was applied uniformly over the soil surface instead of pouring the solution around the
base of the plants, to ensure that the solution did not leak through the new potting soil. The
uptake of imidacloprid by the plants may have been more efficient when the imidacloprid drench
was applied in this way; however no experiment was conducted to confirm this. In brief, no
difference in fecundity was observed between primed and non-primed females and the exposure
of females to concentrations 72 mg a.i per pot caused a significant reduction in fecundity of all
females in the experiment (Table 4.13b, c). A significant decrease in fecundity was also observed
when primed females of the resistant and reference strain were exposed to concentrations of 24
and 48 mg a.i. per pot (Table 4. 4b, c). These results suggest that above a certain level of severity
of subsequent exposures, the effect of priming on the stimulation of fecundity may no longer be

154

efficient. The treatment with the higher concentration of imidacloprid (72 mg a.i per pot) was
then found to cause a decrease in fecundity and high mortality in this experiment whereas the
treatment with 36 mg a.i per pot elicits various symptoms of toxicity in the females but did not
cause a significant decrease of their fecundity (Table 4.13b, c). This lack of an adverse effect of
high concentration of imidacloprid on fecundity on this rebuilt reference strain may be associated
with some increase in resistance due to movement of the whiteflies between cages. But some
differences in these results compared with earlier experiments may also be associated with the
use of fertilizer. In fact, Bi et al. (2001) already showed that fertilizer can modulate the
metabolism of sugar and amino acid in cottons, which consequently enhance the growth of adults
and immatures of B. tabaci B biotype. He et al. (2013) showed that the adverse effect of
sublethal dose of imidacloprid on reproduction of whitefly B. tabaci was associated with the
antifeedant effect of this compound. Therefore, the use of fertilizer on the cotton plants might
increase the nutrients available to the whitefly, which may consequently soften the antifeedant
effect. This may partially explain the difficulty of finding a sublethal concentration that
negatively affects fecundity without causing mortality of the females under the new conditions.
Thus, the lower concentrations of 6 and 12 mg a.i per pot that did not elicit symptoms of toxicity
and did not affect mortality under the conditions of this study were used in the next set of
experiments to test the effect of priming on fecundity. The stimulatory effect of priming on
fecundity was evaluated in these experiments but the protective effect of the priming exposure,
thus the conditioning hormesis, could not be evaluated. In this new set of experiments with the
rebuilt reference strain it was found that the fecundity of females primed as immatures with
imidacloprid at 0.00012 and 0.0012 mg a.i. per pot, and then given a subsequent exposure as
adults at concentrations of 0, 6 or 12 mg a.i per pot, was 9% and 12% greater than that of non-

155

primed females (Table 4.15d). It was also noticed that fecundity of females given subsequent
exposures of 6 mg a.i. per pot as adults was higher than that for females given a subsequent
exposure of 0 or 12 mg a.i. per pot (Table 4.15d). The test was also conducted on the F1
progeny. However, no difference in fecundity was observed between the F1 female offspring
from primed and non-primed parents.

Hormesis is a phenomenon characterized by a stimulation of various biological processes
by a low dose of toxicant and inhibition of these processes by high doses of the toxicant
(Calabrese and Baldwin, 2002). Numerous studies have reported a stimulation of different
biological endpoints by sublethal doses of insecticides in insects as cited in the review paper of
Cutler (2013). The results of our study suggest that application of imidacloprid at an early stage
elicits an increase in the fecundity of whitefly adults. Such hormesis in reproduction has already
been observed in some insects such as aphids (M. persicae) exposed to a low dose of
imidacloprid (Annyath et al, 2013), a parasitoid wasp (Pimpla turionellae) exposed to a low dose
of malathion (Büyükgüzel, 2006), and in a predatory stinkbug, Podisus distinctus, exposed to a
low dose of permethrin (Zanuncio et al., 2013). These studies are similar to ours when
considering the fact that the insecticide exposure was carried out during immature stages of these
species. The study of a parasitoid wasp and of a stink bug show that reproduction in these two
species were stimulated by a single treatment of the immature stage (Büyükgüzel, 2006,
Zanuncio et al., 2013). In the parasitoid study, the wax moth host, Galleria mellonella, was fed a
diet with a very small amount of malathion (0.01 and 0.1 ppm). The parasitoid, P. turionellae,
grew in the pupal stage of the moth, and after adult emergence from the host, exhibited an
increase in fecundity (Büyükgüzel, 2006). Similarly, the third instar of a stinkbug was treated

156

once with low dose of permethrin applied as topical treatment, and the emerging female adult
exhibited a hormesis, without a subsequent exposure of the adults. In our research with
whiteflies, fecundity was also observed with the priming of the immature stage. However, it is
noteworthy that a consistent trend throughout all sets of our hormesis experiments is that a
subsequent exposure of adults seems to affect the observed level of increase in fecundity, while
the fecundity of primed and non-primed females that were not treated as adults with imidacloprid
are not different. The results of Ayyanath et al. (2013) also showed that fecundity increased
when aphids were continuously exposed to a low concentration of imidacloprid (0.025 µg/L) and
reproduction was not increased if the aphids were treated only once in the first instar with the
same concentration 0.025 µg/L. However, these authors found an increase in fecundity of aphids
when females were treated with imidacloprid once during the immature stage with a higher
concentration of imidacloprid (0.1 and 10 µg/L) (Ayyanath et al. 2013). In fact, numerous
authors already suggested that time is an important factor in the study of hormesis, and that the
occurrence of hormesis might be determined by the pharmacodynamic and pharmacokinetic of
the chemicals, thus the level of the dose of the chemicals and its related effect, their rate of
absorption, degradation, and elimination in the organism over a course of time (Rozman and
Doull, 2003). That suggests that a repeated exposure may be required for some chemicals or for
some dose of the chemical depending of chemical’s rate of absorption, distribution and
excretion, thus numerous factors that were not investigated in the current study. A use of a wide
range of priming doses at different times and with a different number of exposures would
provide a better explanation of this phenomenon.
The occurrence of a wide variation in the fecundity of whiteflies used in our tests was
also a limiting factor in our research. This variation was mainly associated with variation in the

157

nutritional quality of the cotton plants. Although our cotton plants were grown as uniformly as
possible and leaves of the same age were used in each test, average fecundity levels still varied
considerable from one experiment to another. Variation in the uptake of imidacloprid by cotton
plants from one experiment to another is also suspected. However, variation of fecundity within
each experiments was small, as indicated by the similarity of the fecundity of the untreated
controls between the groups of females to be compared (Table 4.4a, 4.5a). In our experiments
variation of fecundity caused by a plant effect was minimized by cutting the leaf discs of
replicates of treatments to be compared (primed and non-primed for example) from the same
plant. Therefore the leaf discs used in replicate of a primed and non-primed comparison were cut
from the same leaves, and so on. This approach enabled us to detect even a 10% difference in
fecundity of the female whiteflies.
Low concentrations of imidacloprid have been reported in soil and plants up to one year
after its application in some cropping systems, and residual amounts have been detected in
untreated plants the following crop season (Bonmatin et al, 2003). Thus the application of
imidacloprid to one crop may contaminate the following crop that whitefly may use as a host.
The effect of a residual amount of imidacloprid on the population growth of whitefly has not
been determined for a cropping system, but results of recent hormesis studies, mostly conducted
in the laboratory raise some interesting questions. Ayyanath et al. (2013) did report a significant
increase of the population size of the aphids exposed to low doses of imidacloprid in their
greenhouse experiments.
Longevity of the reference strain and the resistant strain was also analyzed in this study.
The priming of the females with low concentration of imidacloprid during immature stage seems
to not affect the longevity of these females. Ayyanath et al. (2013) also reported to not observing

158

a significant increase of longevity in green peach aphids that were treated with a low
concentration of imidacloprid in the immature stage. Cutler et al. (2009) also found a significant
increase in fecundity but not on longevity of the green peach aphids that they exposed to low
dose of imidacloprid. However, this study showed that the treatment of females of the rebuilt
reference strain with imidacloprid at 6 mg a.i. as adults increased their longevity (Table 4.15e,
4.16 d). An increase in fecundity was also noticed along with the increase in longevity at this
concentration. This effect might be associated with the effects of imidacloprid on cotton plants.
Indeed, imidacloprid has already been reported to have some effect on the physiology of treated
plants. For example, Szczepaniec et al. (2011) found that elm trees treated with imidacloprid
had larger leaves than the untreated trees, and the spider mites Tetranychus schoenei on these
treated trees exhibited higher fecundity than on untreated controls. It is also a possibility that
treatment with imidacloprid at a relatively high but not lethal doses might induce a stimulatory
effect in the insects but with different mechanism.

V Conclusion
The present study shows that a low concentration of imidacloprid applied at an early stage of
development of female whiteflies can induce a stimulation of their fecundity when they become
adults. The priming of whitefly during their immature stage is then necessary for the stimulation
to occur. Although the current study did not identify the range of doses that is most efficient for
this priming, the results suggest that the effect can occur with a very low priming dose, while
priming of immature whitefly with a high dose does not appear to induce a stimulation. It also
appears that there is a certain level of subsequent exposure above which the stimulation induced
by the priming effect could not be observed. Overall, the rate of increase of the fecundity due to

159

priming was modest, 10 to 20 % greater than the untreated control. This modest increase could
be easily masked by variation of the endpoints. It also appears that the stimulatory effect induced
by a single priming exposure can be transferred to the first generation of female offspring. A
higher fecundity was observed in reference females generated from the parents primed with low
concentration of imidacloprid in the current study. However, the stimulation was less consistent
and less obvious. A significant increase in fecundity of the female progeny of the rebuilt
reference strain was not observed. Overall, the current study suggests that treatment with a low
dose of imidacloprid applied during an early immature stage of development has an effect on
adult whitefly fecundity. Further molecular and biochemical studies are needed to elucidate this
phenomenon.

160

Summary and future research direction.
Among the neonicotinoid insecticides, imidacloprid is one of the most successful as
indicated by its volume of sales worldwide (Elbert et al., 2008 ). Because imidacloprid is still
widely used for many key pests, including whiteflies, it is important to determine the effects of
this insecticide at range of concentrations to provide insight into long-term resistant management
strategies.
In the research presented in this dissertation it was found that heavy use of imidacloprid
could lead to the rapid development of resistance in populations of B biotype whitefly. However
the resistance appears to be unstable as it was lost with a temporal refuge without exposure to
imidacloprid. Also, heritability of the resistance was found to be relatively small (0.10)
suggesting that only a small proportion of the resistance phenotype variation could be attributed
to the additive genetic variation. However, resistance can rise again rapidly with a new selection
regime. All of these findings could partially explain the wide variation in the level of
imidacloprid resistance in B biotype whiteflies in space and time as reported in the literature.
Thus, the frequency of use of imidacloprid is one of the key factors to be considered to modulate
the development of the resistance in whitefly. Allowing enough time for resistance to decline
before the re-use of imidacloprid would be then a good approach to manage resistance to this
insecticide.
After the introduction of imidacloprid, more neonicotinoid products became available to
growers to control whitefly including thiamethoxam, acetamiprid and dinotefuran. Therefore, the
development of cross resistance between these neonicotinoids is a concern for growers in
greenhouse industry. In previous research on cross resistance in B biotype whitefly, variability in
the level of cross resistance was found among several published studies. In this research the

161

heavy use of imidacloprid caused a cross-resistance to thiamethoxam. The P450 detoxification
system may be involved in the resistance as suggested by the synergistic effect of PBO, an
inhibitor of P450. A low level of cross-resistance to acetamiprid was also found in the same
resistant population. These results seem to be consistent with published studies on metabolism of
neonicotinoids in whitefly and in plants. No significant cross resistance to dinotefuran was
observed in the present study, however an occurrence of cross resistance to this neonicotinoid
insecticide in a population having much higher resistance level to imidacloprid is not ruled out.
This research also increases our understanding of the causes of variation in the levels of cross
resistance to neonicotinoids found in populations of B biotype Bemisia tabaci in different
regions. The results suggest that in addition to P450 detoxification, another mechanism of
resistance is very likely involved in whitefly resistance to neonicotinoids, and that the level of
the cross resistance among neonicotinoids depends on the mechanisms of how resistance
developed in each population. Therefore, the results of this study do not support the use of
neonicotinoids as alternative insecticides for control of imidacloprid-resistant populations of
whitefly.
Determining the effect of low doses of imidacloprid on populations of whitefly may be
important because of the persistence of imidacloprid after its application. Several studies have
reported that sublethal doses of insecticide are either neutral or even helpful for pest management
because low doses may reduce the rate of reproduction of the pests. Bonmatin et al. (2003)
detected a residual amount of imidacloprid as low as 0.1−1 μg/kg in soils 1 to 2 years after
application of this insecticide as seed-dressing. The imidacloprid concentration used in the
current study was 1.2 μg a.i per pot, thus a concentration that may be encountered in field 2 years
after imidacloprid application. The results of this study suggest that this residual amount of

162

imidacloprid can cause an increase in fecundity of whitely when applied at the immature. The
current study provides evidence that exposure to a low dose of imidacloprid applied during an
early stage of development has an effect on adult whitefly fecundity. .
Hormesis has drawn the attention of researchers studying aging and senescence, and
more recently from those studying the effects of various pollutants on health. One contribution
of the research in this dissertation is to alert other researchers that hormetic effects can be subtle
and are easily overlooked. The underlying mechanism of the hormesis is not well determined yet.
Calabrese and Baldwin (2002) argued that no single mechanism could explain hormesis given
that this phenomenon was observed on many endpoints across several taxa with various hormesis
-inducing agents. However, it was also often reported that conditioning hormesis involves the
antioxidant defense system and reactive oxygen species (ROS) in various organisms such as
paramecia, yeast, nematodes, insect and mammals (Smith et al., 1996; Davies et al. 1995; Zhao
and Wang, 2012; Lebourg, 2007; Ramakrishnan et al., 2013). ROS such as peroxide hydrogen
H2O2 and the antioxidant system was already found to play a role in mammalian reproduction
through the egfr signaling pathway in mammal species (Shkolnik et al., 2012). In insect, an
increased activity of the superoxide dismustase SOD, one of the defense enzymes of the
antioxidant system that protects organism against reactive oxygen species (ROS), was already
found to be positively correlated with an increased fecundity. For example, Büyükgüzel (2006)
reported a positive correlation between SOD and fecundity and longevity in the female of the
parasitoid wasps Pimpla turionellae that emerged from the host wax moth treated with a low
dose of malathion. It is noteworthy that exposure to imidacloprid was already known to induce
an increase of SOD in whitefly Bemisia tabaci. (Gao et al., 2013). In addition, in the present
study the pre-exposure to low amount of imidacloprid was conducted when the whitefly

163

individuals were at immature stage, thus at an early stage of development when organism was
susceptible to epigenetic change induced by environmental stimuli (Janish and Bird, 2003,
Decluve et al., 2009 ). Recent studies in mammals and yeast showed that hormesis was
associated with epigenetic modification in these organism and this link was mediated by ROS
and antioxidant system (Schroeder et al.; 2013, Bernal, 2013). In addition, the current study
found that the stimulation in fecundity appear in the F1 generation of female offspring of the
reference strain when fertilizer was used, This suggests an occurrence of transfer of the hormetic
effect to the offspring females. Previous studies in mammals have already shown that the side
effects of pesticides applied at an early stage of the individuals on reproductive system in
mammals could be transferred to few generations of their offspring (Manikkam et al. 2012,
Adamski, et al., 2005; Anway et al., 2005). A transgeneration inheritance of the extension of
longevity was also reported in the yeast and this inheritance was mediated by epigenetic
phenomenon (Greer et al., 2011). This inheritance of the effect was not seen with F1 female
progeny of the rebuilt population of the reference strain.

164

APPENDICES

165

APPENDIX A. Log probit dose plot.

98

7

95

6

80
70
60
50
40
30
20

5

Probit

Percent mortality

90

4

10
5
2

0.01

0.1

1

10

100

1000

3

10000 100000

ppm

Figure A1. Log-probit dose plot for the toxicity of imidacloprid to the reference strain CA-s1
(dark blue triangles), the parental strain MI-R1 collected from a commercial greenhouse
(turquoise diamonds), and the F5 (green triangles) generation of Bemisia tabaci B biotype after
selection with imidacloprid.

166

98

S

95

F7

7

6

80
70
60

5

50
40
30
20

probits

percent of mortality

90

4

10
5

P F3

2

0.1

1

3

100

10

1000

10000

Figure A2. Log-probit dose plot for the toxicity of imidacloprid to the reference strain CA-s2
(black diamonds), P: the resistant parental of the resistant strain MI-R2 (black triangles)
retrieved after the period of mite and thrips infestation, and the F’3 (red triangles) and the F’7
(blue diamonds) generation of the resistant strain MI-R2 after selection with imidacloprid.

167

APPENDIX B. Exposure of the rebuilt reference strain to imidacloprid.
Table A1. Proportion of affected female adults that exhibited various symptoms of toxicity
after imidacloprid application. Some females recovered after a few days but some was died
within five days.
0

Priming
concentration

0.00012

0.0012

(mg a.i. per pot)

Subsequent
exposure
concentration

female

female

female

female

female

female

affected

dead

affected

dead

affected

dead

36

14/25

4/25

20/25

7/9

16/25

4/25

72

16/25

4/25

21/25

9/25

18/25

13/25

(mg a.i. per pot)
0

168

P11 12 mg (a)

eggs/female

150
100
50
0
0

7

0 mg

14
0.00012 mg

200

eggs/female

21

28

35

0.0012 mg

42
day

P11 6 mg (b)

100

0
0
0 mg

7

28
35
42
0.0012 mg
day

P11 0 mg (c)

150
eggs/female

14
21
0.00012 mg

100

50
0

0
0 mg

7

14

21

0.0012 mg

28

35

0.0012 mg

42
day

Figure A3. Cumulative mean total egg production per female of the first parental generation P11
of the rebuilt reference strain primed with different concentrations of imidacloprid (0, 0.0012,
0.0012 mg a.i per pot) and then subsequently exposed to imidacloprid at (a) 12 mg a.i per pot,
(b) 6 mg a.i per pot, and (c) 0 mg a.i. per pot as adults.

169

P12 6 mg (a)

eggs/female

200
150
100
50
0
0

7

0 mg

14

21

0.00012 mg

28
0.0012 mg

35
day

P12 0 mg (b)

eggs/female

150
100
50
0
0
0 mg

7

14
0.00012 mg

21

28
0.0012 mg

35
day

Figure A4.Cumulative men total egg production per female of the second parental generation
P12 of the rebuilt reference strain primed with different concentrations of imidacloprid and then
subsequently exposed to imidacloprid at (a) 6 mg a.i per pot, (b) 0 mg a.i. per pot as adults.

170

400

P13 12 mg (a)

eggs/female

300
200
100
0
0

7

0 mg

14

28

0.00012 mg

400
eggs/female

21

35

42

0.0012 mg

49
day

P13 6 mg

300

200
100
0
0

7

0 mg

21

28

0.00012 mg

P13

400

eggs/female

14

35

42

0.0012 mg

49
day

0 mg (b)

300
200
100
0
0

7
0 mg

14

21

0.00012 mg

28

35

42

0.0012 mg

49

day

Figure A5. Cumulative mean total egg production per female of the third parental generation P13
of the rebuilt reference strain primed with different concentrations of imidacloprid and then
subsequently exposed to imidacloprid at (a) 12 mg a.i per pot, (b) 6 mg a.i per pot, and (c) 0
mg a.i. per pot as adults.

171

eggs/female

400

F11 12 mg (a)

300
200
100
0
0

7

0 mg

14

21

28

35

0.00012 mg

400

42

49

0.0012 mg

56
day

F11 6 mg (b)

eggs/female

300
200
100
0
0

7

0 mg

14

21

28

35

0.00012 mg

42

49

0.0012 mg

56
day

F11 0 mg (c)

eggs/female

300
200
100
0

1
0 mg

7

14

21

28

0.00012 mg

35

42

49

0.0012 mg

56
day

Figure A6. Cumulative mean ± SE total egg production per F11 female offspring of the rebuilt
reference strain primed with different concentrations of imidacloprid and then subsequently
exposed to imidacloprid at (a) 12 mg a.i per pot, (b) 6 mg a.i per pot, and (c) 0 mg a.i. per pot as
adults

172

F12 6 mg (a)

eggs/female

250
200
150
100
50
0
0

7

0 mg

21

28

0.00012 mg

35

42

0.0012 mg

49
day

F12 0 mg (b)

250

eggs/female

14

200

150
100
50
0
0
0 mg

7

14

21

0.00012 mg

28

35

42

0.0012 mg

49
day

Figure A7. Cumulative mean total egg production per F12 female offspring of the rebuilt
reference strain primed with different concentrations of imidacloprid and then subsequently
exposed to imidacloprid at (a) 6 mg a.i per pot, (b) 0 mg a.i. per pot as adults

173

F13 12 mg (a)

eggs/female

400
300
200
100
0
0

7

0 mg

21

28

0.00012 mg

35

42

0.0012 mg

49
day

F13 6 mg (b)

400
eggs/female

14

300
200
100
0

0
7
0 mg

14
21
28
0.00012 mg

35
42
0.0012 mg

49

35
42
0.0012 mg

49
day

day

F13 0 mg (c)

eggs/female

400
300
200
100
0
0
7
0 mg

14
21
28
0.00012 mg

Figure A8. Cumulative mean total egg production per F13 female offspring of the rebuilt
reference strain primed with different concentrations of imidacloprid and then subsequently
exposed to imidacloprid at (a) 12 mg a.i per pot, (b) 6 mg a.i. per pot as adults and (c) 0 mg a.i
per pot.

174

APPENDIX C. Record of deposition of voucher specimens
The specimens listed below have been deposited in the named museum as samples of those
species or other taxa, which were used in this research. Voucher recognition labels bearing the
voucher number have been attached or included in fluid preserved specimens.

Voucher Number: ________#_2013-13__________

Author and Title of thesis: Response of the whitefly Bemisia tabaci B biotype to repeated
exposures of imidacloprid
By Mamy L. Rakotondravelo

Museum(s) where deposited:
Albert J. Cook Arthropod Research Collection, Michigan State University (MSU)

Specimens:
Family

Genus-Species

Life Stage

Quantity

Preservation

Aleyrodidae

Bemisia tabaci (B biotype)

adult

10

alcohol

Aleyrodidae

Bemisia tabaci (B biotype)

larva

10

alcohol

Aleyrodidae

Bemisia tabaci

pupae

10

alcohol

(B biotype)

175

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