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HOST-COLONIZATION BEHAVIORS
OF THE BEAN WEEVIL, ACANTHOSCELIDES OBTECTUS (SAY),
IN STORED BEANS

By

Martha Erica Quentin

A DISSERTATION

Submitted to
Michi an State University
in partial fu lment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Entomology

1991

ABSTRACT
HOST-COLONIZATION BEHAVOIRS
OF THE BEAN WEEVIL, ACANTHOSCELIDES OBTECTUS (SAY),
IN STORED BEANS
By

Martha Erica Quentin

In laboratory choice tests, adult Acanthoscelides obtectus (Say) reared for 14
generations on light red kidney beans laid twice as many eggs on kidney, navy and
pinto beans than on great northern, black beans or cowpea. Suitability to bean
weevil larvae was greater for some of the bean hosts less preferred for oviposition
than those receiving many eggs. Suitability of beans for A. obtectus development
appeared to be highly inﬂuenced by seed coats rather than cotyledons. Larval
survival was improved about ﬁve fold via pin holes through the testa.

Direct observation and close-up video cinematography revealed that, to effect
entry into a bean, larval A. obtectus must wedge their bodies into the cleft formed
where one bean abuts another or the surface of a container. Such a purchase
apparently enables a larva to develop an appreciable beanward force to its scraping
strokes, and prevents the larva from being displaced forward. Entry into a bean
was gained only after >19 h of nearly continuous scraping during which time there
was no evidence of larval feeding; or by using a hole already completed by another
larva, a pioneer. Ratio of pioneers to followers was 2:1.

In light of the above biological facts, it appeared that A. obtectus in stored
beans might be controlled by periodically tumbling beans so as to dislodge larvae

from their'digging sites and move partially dug holes away from abutting surfaces.

Indeed excellent (>96%) control of A. obtectus in red kidney beans held in jars,
buckets and gunny sacks was attained by rolling or tumbling the containers three
times per day. This control method should prove highly useful to smallholder
farm families in bean-producing areas of developing countries, and possibly to
large-scale producers where the technique could be mechanized for control of the
bean weevil. Moreover, tumbling might prove useful for other pests whose entry

into seeds is temporarily and spatially constrained.

To In husband,
Robert ent Lawrence
who was always there when I needed him
and for his love and belief in my efforts,

and
To my mother,

for her love and constant support
throughout my studies.

In memory of my father

iv

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ACKNOWLEDGEMENTS

I would like to express special thanks to my major professor, Dr. James R.
Miller, for his guidance in insect behavioral science, constructive criticism and
patience during my dissertation preparation.

I would also like to thank the other members of my guidance committee for
their contributions towards my doctoral program: Dr. Frederick W. Stehr for his
advice, encouragement and great support; Dr. William J. Mattson for enlightening
discussions and generously providing me with laboratory facilities, particularly, the
use of computers; Dr. Guy L. Bush for his advice and valuable suggestions; and Dr.
Robert F. Ruppel for sharing informative discussions on the management of the
bean weevil and serving as a member of my committee early in my program.

Special thanks go to Dr. Lawrence Copeland, Dr. Catherine Bristow, and Dr.
Edward Walker who served as members of my graduate committee following Dr.
Robert Ruppel’s retirement and while Drs. William J. Mattson and Guy L. Bush
were on sabbatical leave.

I am also grateful to Dr. Edward J. Graﬁus for allowing me to participate in
his research on onion maggot grth regulators as a partial fulﬁllment of my
doctoral enrichment program, and to Beth Bishop for valuable discussions and
friendship during that study.

Sincere thanks go to Sokoine University of Agriculture, Morogoro, Tanzania
for providing the Tanzania strain of the bean weevil. The Michigan strain of bean
weevil provided by the late Dr. Alfred Saettler was of great value to my research
and was deeply appreciated. I also thank Dr. Clarence D. Johnson, Dr. Richard

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Pratt, and Dr. John McLaughlin for materials and advice on establishment and
maintenance of common bean weevil cultures.

My special thanks go to Dr. Robert K. Lawrence for advising on computer
programming for statistical analysis and on graphics techniques. I also wish to
thank Dr. John Gill for his advice on statistical analysis.

I thank James E. Keller for his excellent technical assistance, and other
colleagues in Dr. J. R. Miller’s lab, especially Joseph L. Spencer, for assistance in
my ﬁnal dissertation experiment. The friendship and support extended by the crew
of Dr. W. J. Mattson’s lab was greatly appreciated.

I am very grateful to the Bean/Cowpea CRSP (Collaborative Research
Support Program) for providing ﬁnancial support during my Ph.D. program and to
the Sage Foundation for funding the ﬁnal preparation of my dissertation. I thank
Sokoine University of Agriculture for granting me leave from Morogoro, Tanzania,
and all colleagues there who have been supportive. I am sincerely grateful to Dr.
Pat Barnes-McConnell and Dr. Matt J. Silbernagel of the Bean/Cowpea CRSP, for
making my Ph.D. program at Michigan State University possible.

My deepest gratitude goes to my family in Tanzania and Mozambique, and to
my father- and mother-in-law, Dr. and Mrs. Robert G. Lawrence, for their constant

support during my dissertation preparation when things seemed most challenging.

LIST
LIST
GEN]

GENT

TABLE OF CONTENTS

LIST OF TABLES ............................................ xv
LIST OF FIGURES ........................................... xix
GENERAL INTRODUCTION .................................... 4
GENERAL LITERATURE REVIEW ................................ 4
A. Insect/Plant Interactions ................................. 4

1. Host-Colonization Behaviors ......................... 4

Host ﬁnding .................................. 4

Host examining and consuming .................... 6

2. Factors Inﬂuencing Host-Plant Suitability ................ 9

Allomones ........... - ........................ 9

Plant nutrients ............................... 12

Plant tissue toughness .......................... 14

Tissue thickening ............................. 15

Seed hardness ................................ 16

Seed coat integrity ............................ 17

Trichomes ................................... 17

B. Bruchid/Bean System .................................. 18

1 Beans ......................................... 18

2. Bean Weevil .................................... 19

Life cycle and damage .......................... 19
3. Adult/bean interaction ............................. 20
Oviposition .................................. 20
4. Larva/bean interaction ............................. 22
5. Control of the bean weevil .......................... 23
6. Limitations on current controls ....................... 28

CHAPTER 1 - Factors inﬂuencing host acceptance and suitability of
commercial bean classes for the bean weevil,

Acanthoscelides obtectus (Say) ........................ 3 1
INTRODUCTION ....................................... 32
GENERAL MATERIALS AND METHODS ................... 33

Bean weevil cultures ................................ 33
Bean classes ...................................... 34
Procedures ....................................... 34

EXPERIMENT 1 - Effects of Bean Classes, Photoperiods and
Petri dish type on Ovipositional Preference
and Larval Survival of the Bean Weevil in

a Two-choice Test. ....................... 35

Materials and Methods .............................. 35
Results and Discussion ............................... 38
Ovipositional acceptance ........................ 38
Larval survival ............................... 4O

EXPERIMENT 2 - Host-selection behavior and survival of
A. obtectus larvae when presented with
their rearing host vs. alternative host a
two-choice test. .......................... 40

viii

Materials and Methods .............................. 40

Effect of egg-placement site on larval survival ........ 40
Effects of bean seed coat on larval survival .......... 42
Results and Discussion ............................... 43
Inﬂuence of egg-placement site on larval survival ...... 43
Impact of bean seed coat factors on larval survival ..... 45

EXPERIMENT 3 - Larval survival of the bean weevil as affected
by seed coat perforation in three bean classes. . . . 47

Materials and Methods .............................. 47
Three-choice test .............................. 48
No-choice test ................................ 48

Results and Discussion ............................... 50
Three-choice test .............................. 50
No-choice test ................................ 53

EXPERIMENT 4 - Suitability of six bean classes as hosts of

the bean weevil. ......................... 53

Materials and Methods .............................. 53
Results and Discussion ............................... 57
Larval survival ............................... 57
Developmental period .......................... 59

Adult dry weights ............................. 59

EXPERIMENT 5 - Effects of bean seed stability on larval
performance of the bean weevil in six
bean classes. ............................ 64

Materials and Methods .............................. 64

Results and Discussion ............................... 66

larval survival ............................... 66
Developmental period .......................... 69
Adult dry weights ............................. 69

EXPERIMENT 6 - Effects of larval density on growth and
development of the bean weevil in six

bean classes. ............................ 75

Materials and Methods .............................. 75
Results and Discussion ............................... 75
Larval survival ............................... 75

Adult dry weights ............................. 77

GENERAL DISCUSSION ................................. 86
Experimental considerations ........................... 86
Practical considerations .............................. 87
CONCLUSIONS ........................................ 87

CHAPTER 2 - Ovipositional preference and larval performance of a
USA. vs. Tanzania strain of the bean weevil,
Acanthoscelides obtectus (Say) as inﬂuenced by bean class. . . 90

INTRODUCTION ....................................... 91
MATERIALS AND METHODS ............................ 92
Insects and Host materials ............................ 92
Oviposition: Six-choice test ........................... 93
Oviposition: No-choice test ........................... 95
Oviposition: Two-choice tests ......................... 95
Host suitability: Six-choice test ........................ 97

Host-suitability: No-choice test ........................ 97

RESULTS AND DISCUSSION ............................. 98
Oviposition: Six-choice test ........................... 98
Oviposition: No-choice test .......................... 101
Oviposition: Two-choice tests ......................... 104
Host Suitability: Larval survival ....................... 107

Six-choice test ............................... 107

No-choice test ............................... 109

Host Suitability: Adult dry weights ..................... 109
Six-choice test ............................... 109

No-choice test ............................... 114

Correlation between larval survival and oviposition ........ 117
Inter-strain differences .............................. 120
CONCLUSIONS ....................................... 121

CHAPTER 3 - Ovipositional behavior, larval growth, and survival of
the bean weevil, Accmthoscelides obtectus (Say):

Effects of bean seed damage. ....................... 123
INTRODUCTION ...................................... 124
MATERIALS AND METHODS ........................... 125

Insects .......................................... 125
Beans . ......................................... 125
Oviposition: Choice-test ............................. 125
Larval survival: Choice-test .......................... 126
Larval survival: No-choice test ........................ 127

RESULTS ............................................ 127

Oviposition: Choice test ............................. 127

Larval survival: Choice test .......................... 128

Larval survival: No-choice test ........................ 133
DISCUSSION ......................................... 137
CONCLUSIONS ....................................... 140

CHAPTER 4 - Bean attack behavior of neonate larvae of the bean

weevil, Acanthoscelides obtectus (Say). ................. 142
INTRODUCTION ...................................... 143

METHODS - Methods devised for exposure of bean parts and
establishing contact between bean parts and lid
of culture dish. .............................. 144

EXPERIMENT 1 - Effects of different bean seed areas and
zone of contact on the boring behavior

of the bean weevil larva. .................. 147
Materials and Methods ............................. 147
Results and Discussion .............................. 148

Effects of zone of contact, bean seed area and

bean weevil strain ............................ 148

Effects of bean moisture content ................. 152

Duration of larval seed attack activities ............ 154

larval behaviors ............................. 154

EXPERIMENT 2 - larval seed-boring behavior under

"natural" storage conditions. ............... 158

Materials and Methods ............................. 158
Results and Discussion .............................. 160

xii

Distribution of entry holes among different areas of
bean seed .................................. 160

Distribution of entry holes among beans ........... 164

EXPERIMENT 3 - Videotaping bean weevil seed entry behavior. . . 167

Materials and Methods ............................. 167
Results and Discussion .............................. 168
Pre-boring and settling of pioneer larvae ........... 171
Seed boring behavior of pioneer larva ............. 171

Pre-entry and entry into seed by the
follower larvae .............................. 175

Disturbance of pioneers by followers during

seed boring ................................. 178
Disturbance of seed entering follower by

other followers .............................. 178
GENERAL DISCUSSION ................................ 183

Factors that may affect boring success and the duration
of boring in intact bean seeds by the bean weevil larvae ..... 183
Future research areas and applicability of study ........... 185
CONCLUSIONS ....................................... 185

CHAPTER 5 - Tumbling of beans as a control measure for the

common bean weevil, Acanthoscelides obtectus (Say). ...... 188
INTRODUCTION ...................................... 189
MATERIALS AND METHODS ........................... 191
Experimental design ............................... 191
Insects and Rearing Conditions ....................... 191
Beans . ......................................... 192

xiii

 

APP

LII]

Containers and Tumbling Protocols .................... 192

RESULTS AND DISCUSSION ............................ 193

CONCLUSIONS ....................................... 197

APPENDIX ................................................ 198

LITERATURE CITED ........................................ 200
xiv

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6,

Table 7.

Table 8,

Table 9‘

Table 10

Table 11

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

Table 10.

Table 11.

LIST OF TABLES

Eggs laid by A. obtectus exposed to six bean classes tested
against the black bean in a two-choice test ................ 39

Adult emergence of A. obtectus from six bean classes tested
against the black bean in a two-choice test ................ 41

Effects of egg site and bean class on seed infestation levels,
larval survival, larval mortality outside seeds, by A.
obtectus in a two-choice test ........................... 44

Effects of seed coat perforation and bean class on seed
infestation levels, larval survival, larval mortality outside
seeds, by A. obtectus in a two-choice test ................. 46

Effects of seed coat perforation and bean class on survival
of A. obtectus in a three-choice test ..................... 51

Effects of seed coat perforation on larval survival of A.
obtectus in a three-choice test .......................... 52

Effects of bean class on larval survival of A. obtectus in
a three-choice test ................................... 54

Effects of bean class and seed coat perforation on larval
survival of A. obtectus in a no-choice test ................. 55

Effects of seed coat perforation on larval survival of A.
obtectus in a no-choice test ............................ 56

Effects of bean class on developmental period of A.
obtectus larvae ...................................... 60

General linear models procedure (SAS) for analysis of

variance of adult dry weights of A. obtectus when larvae

were exposed to six bean classes with two seed coat

perforation treatments ................................ 62

Tabl-

Tab}

Table

Table

Table

Table

Table

Table

Table

Table ;
Table 1

Table 2

Table 12.

Table 13.

Table 14.

Table 15.

Table 16.

Table 17.

Table 18.

Table 19.

Table 20.

Table 21.

Table 22.

Table 23.

Table 24.

Table 25.

Effects of seed coat perforation and seed attachment on
larval survival (number and percentage of adults emerged)
of the bean weevil: a contingency table ................... 67

Effects of bean class and seed attachment on larval survival
(number and percentage of adults emerged) in the bean
weevil: a contingency table ............................ 68

Effects of bean class and seed coat perforation on larval
survival (number and percentage of adults emerged) in
the bean weevil: a contingency table ..................... 70

General linear models procedure (SAS) for analysis of
effects of seed attachment, bean class and seed coat
perforation on developmental time of A. obtectus larvae ...... 71

General linear models procedure (SAS) for analysis of
effects of seed attachment, bean class and seed coat
perforation on the resulting dry weight of the adult

A. obtectus ........................................ 73
Effects of seed coat perforation and larval density on larval

survival of A. obtectus ................................ 76
Effects of larval density on larval survival of A. obtectus ...... 78
Effects of larval density and bean class on adult dry weight

of A. obtectus ...................................... 80
Effects of larval density on adult weights of A. obtectus ....... 81
Effects of bean class and seed coat perforation on adult

weight of A. obtectus ................................ 83
Effects of bean classes on adult weights of A. obtectus ....... 85
Effects of bean class on oviposition of A. obtectus in a

six-choice test ...................................... 99
Numbers of eggs laid by two A. obtectus strains in a

six-choice test ..................................... 100
Effects of bean class on oviposition of A. obtectus in a

no-choice test ..................................... 102

Table 26.

Table 27.

Table 28.

Table 29.

Table 30.

Table 31.

Table 32.

Table 33.

Table 34.

Table 35.

Table 36.

Table 37.

Table 38.

Table 39.

Number of eggs laid among six bean classes by each strain

of A. obtectus in a no-choice test ...................... 103
Numbers of eggs laid by two strains of A. obtectus in a

two-choice test .................................... 105
Numbers of eggs laid by two strains of A. obtectus in a

two-choice test .................................... 106
Effects of bean class on larval survival of A. obtectus in

a six-choice test .................................... 108
Effects of bean class on larval survival of two strains of

A. obtectus in a six-choice test ......................... 110
Inﬂuence of bean classes on larval survival of A. obtectus

in a no-choice test .................................. 111
Effects of bean class on larval survival of the two strains

of A. obtectus in a no-choice test ...................... 112
Effects of A. obtectus strain and bean class on dry weights

of emerging adults in a six-choice test ................... 113
Dry weights of adult A. obtectus emerging from various

beans in a six-choice test ............................. 115
Dry weights of adult A. obtectus emerging from beans in

a no-choice test .................................... 116
Effects of A. obtectus strain and bean class on dry weights

of emerging adults in a no-choice test ................... 118
Adult emergence and bean infestation levels of two strains

of A. obtectus when data for seed damage treatments were

pooled .......................................... 134
Dry weights of A. obtectus adults emerging from different

seed damage treatments in a no-choice test ............... 134
Inﬂuence of different bean seed areas on sustenance of

larval boring by the 12th hour in the two strains of A.

obtectus: a contingency table of bean seed area vs. strain

of bean weevil ..................................... 150

Table 40.

Table 4

Table 4

Table .'

Table .

Table

Table

Table

Table 40. Inﬂuence of different bean seed areas on the ﬁnal number
of holes bored and entered by larvae of the two strains of
A. obtectus by the 48th hour: a contingency table of bean
seed area vs. strain of bean weevil ...................... 151

Table 41. Effects of bean seed moisture content on sustenance of
larval boring in the two strains of A. obtectus ............. 153

Table 42. Effects of bean seed moisture content on ﬁnal numbers of
holes bored and entered by the larvae of two strains of
A. obtectus ....................................... 153

Table 43. Timing of stages of A. obtectus entry into bean seeds as
determined by periodic examination under a dissecting
microscope ....................................... 155

Table 44. Positions and relative frequencies of A. obtectus entry
holes on various parts of bean seeds (N = 601 entry holes) . . . 161

Table 45. Number of A. obtectus entrance holes in seed parts with
normalization for differing surface areas of respective parts . . . 162

Table 46. Distribution of numbers of entry holes per bean (N =
2,076 beans) with fitted Poisson distribution ............... 166

Table 47. Timing of stages of A. obtectus entry holes into bean seeds
as determined by continuous videotaping ................. 177

Table 48. Type and frequency of disturbance of follower larvae by
other followers during entry of a hole previously made by
a pioneer ........................................ 181

Table 49. Inﬂuence of container rolling (tumbling) on population
increases of the common bean weevil, A. obtectus .......... 194

LIST OF FIGURES

Figure 1. Petri dish (60 x 15 mm) divided into two sections by two
wax ridges ......................................... 37

Figure 2. Bean seed coat conditions: (a) intact (no seed coat
perforation), (b) one perforation in each cheek, and
(c) ﬁve perforations in each cheek of the seed .............. 49

Figure 3. Distribution of Acanthoscelides obtectus adults emerging
from seeds of six bean classes with and without seed coat
perforation treatment. Bean class codes: K = light red
kidney; P = pinto; N = navy; G = great northern;
B = black; C = cowpea. Log-likelihood ratio for
contingency tables signiﬁed lack of independence between
bean class and seed coat perforation treatments (P<0.033;
df =5) ............................................ 58

Figure 4. Inﬂuence of seed coat perforation and bean class on the
developmental period (mean -_t SE) of Acanthoscelides
obtectus larvae. Bean class codes are given in Figure 3.
Developmental period = number of days from larval
introduction onto beans to adult emergence. Means were
not significantly different at P=0.01, Student—Newman-
Keuls’ test (SNK) ................................... 61

Figure 5. Inﬂuence of seed coat perforation and bean class on dry
weights (mean :t SE) of the emerging Acanthoscelides
obtectus adults. Bean class codes are given in Figure 3.
Means were not statistically different at P=0.01, SNK ........ 63

Figure 6. Bean seed attached to glass petri dish by melted wax.
Part of one check of the bean is in contact with the ﬂoor
of the dish ........................................ 65

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Inﬂuence of bean class on developmental period (mean

1 SE) of Acanthoscelides obtectus. Bean class codes are

given in Figure 3. Data were square-root transformed

prior to ANOVA. Seed attachment and seed coat data

were pooled before separation of means. Means

accompanied by the same letter are not signiﬁcantly

different (P=0.01, SNK). Numbers in parentheses =

numbers of beetles weighed ........................... 72

Adult dry weights (mean t SE) of emerging

Acanthoscelides obtectus as affected by different bean

classes. Dry weight data were log(x+ 1) transformed prior

to ANOVA. Seed attachment and seed coat perforation

effects were pooled prior to mean separation test. Means

were not signiﬁcantly different among the six bean classes

(P=0.01, SNK). Numbers in parentheses = numbers of

beetles weighed .................................... 74

Percent survival (mean t SE) of Acanthoscelides obtectus

when larvae were exposed to six bean classes with two seed

coat perforation treatments. Bean class codes are given

in Figure 3. Data were arcsine transformed for ANOVA.

Density effects were pooled. Means followed by the same

letter are not statistically different at P=0.01, SNK ......... 79

Inﬂuence of bean classes and seed coat perforation on dry

weights (mean t SE) of emerging adults of Acanthoscelides
obtectus. Bean class codes are given in Figure 3. Density

effects were pooled for mean separation. Means followed

by the same letter were not signiﬁcantly different at

P=0.01, SNK (also see Table 21) ....................... 84

Petri dish is divided into six sections by wax ridges.
B = bean placement area; I = insect introduction area ...... 94

Oviposition (mean t SE) by Acanthoscelides obtectus

among six bean seed damage treatments (choice test).

Seed damage treatments: IB = intact seed; CB = seed

with cracked seed coat at both ends; HB = seed with

one "exit" hole; SD = cotyledon with inner side facing

down; SU = cotyledon with inner side facing up;

NB = no bean. Data were square-root transformed

prior to ANOVA. Bars accompanied by the same letter

are not significantly different at P=0.05, Tukey’s

studentized range (HSD) test ......................... 129

mks.»

Figure 13.

Figure 14.

Figure 15.

Figure 16.

Figure 17.

Mean number of eggs laid (:SE) by two strains of
Acanthoscelides obtectus among six bean damage
treatments (choice test). Treatment codes are given in
Figure 12. Data were square-root transformed prior to
ANOVA. Bars within each strain accompanied by the
same letter are not signiﬁcantly different at P=0.05,

Tukey’s studentized range (HSD) test .................

Adult emergence (mean 1 SE) of Acanthoscelides obtectus
in ﬁve bean seed damage treatments (choice test).
Treatments were exposed to 54 larvae per dish. Treatment
codes are given in Figure 12. Data were log(x+ 1)
transformed for ANOVA Bars accompanied by the same
letter are not signiﬁcantly different at P=0.05, Tukey’s

studentized range (HSD) test ........................

Adult emergence (mean t SE) of two strains of
Acanthoscelides obtectus in ﬁve bean seed damage
treatments (choice test). Treatments were exposed to 54
larvae per dish. Treatment codes are given in Figure 12.
Data were log(x+ 1) transformed for AN OVA. Bars within
a strain accompanied by the same letter are not statistically

different at P=0.05, Tukey’s studentized range (HSD) test . . .

Adult emergence (mean t SE) of two strains of
Acanthoscelides obtectus in ﬁve bean seed damage
treatments (no-choice test). Each treatment was exposed
to 20 larvae. Treatment codes are given in Figure 12.
Bars within a strain accompanied by the same letter are
not signiﬁcantly different at P=0.05, Tukey’s studentized

range (HSD) test .................................

Mean number seeds infested (: SE) by each strain of
Acanthoscelides obtectus in ﬁve bean seed treatments
(no-choice test). Each treatment was exposed to 20
larvae. Treatment codes are given in Figure 12. Bars
within a strain accompanied by the same letter are not
statistically different at P =0.05, Tukey’s studentized range

(HSD) test .....................................

. 130

. 131

132

. 135

. 136

Ti:
h:

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.

Figure 24.

Figure 25.

Figure 26.

Figure 27.

Figure 28.

Dry weights (mean 1' SE) of two strains of Acanthoscelides
obtectus adults emerging from ﬁve bean seed treatments
(no-choice test). Treatment codes are given in Figure 12.
Bars within a strain accOmpanied by the same letter are
not signiﬁcantly different at P=0.05, Tukey’s studentized

range (HSD) test .................................

Side view of a bean seed showing various areas exposed

for colonization by Acanthoscelides obtectus larvae .......

Bean positioned with the cheek touching the lid to create

a zone of contact (ZOC) ...........................

Distribution of Acanthoscelides obtectus elapsed times

for settling to dig into beans (N = 63) .................

Distribution of Acanthoscelides obtectus elapsed times

for completing bean entry (N = 63) ...................

Side view of a bean seed showing various areas used to

. 138

. 145

. 146

. 156

. 157

estimate the surface area for each bean part ............. 159

Estimated surface area (mean t SE) for various bean
seed areas or zones of a light red kidney bean

(N = 20 beans). CHK = cheek; KLB = keel:back;

KLF = keelzfront; HLM = hilum + areas close to hilum;

End = end area .................................

Frequency histogram for Acanthoscelides obtectus entry
hole numbers per bean under normal storage conditions

(N = 2076 beans) ................................

Sequence of behaviors whereby pioneer Acanthoscelides
obtectus larvae colonize a bean contained in a petri dish

arena .........................................

Sequence of behaviors whereby primary follower
Acanthoscelides obtectus larvae colonize a bean contained

in a petri dish arena ..............................

Movements and positioning of ﬁrst-instar Acanthoscelides

obtecms larvae entering a bean. See text for explanation . . . .

. 165

. 170

172

T
lgure

17gb
re

F
is;
are

Figure 29.

Figure 30.

Figure 31.

Prothoracic plates of the ﬁrst instar larva of Acanthoscelides
obtectus: (a) Prothoracic plates with teeth on upper arms

and medians, (b) dorsal view of plates on larval prothorax,

and (c) position of the plates on larva (lateral view).

Head of larva is retracted within the thorax in (b) and (c) . . . 174

Behaviors and their timing for three Acanthoscelides
obtectus larvae (A,B,C) during bean colonization .......... 176

Frequency distribution of intervals without disturbance
during seed entry of the follower larvae ................. 182

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GENERAL INTRODUCTION

The common bean, Phaseolus vulgaris l.., is a subsistence crop and an
important component of diets for many families of tropical developing nations.
Beans are a source of dietary proteins, particularly where animal protein is not
readily available or affordable. Although common beans are grown in temperate,
sub-tropical and tropical regions of the world, great losses in the post-harvest crop
from attacks of the common bean weevil, Acanthoscelides obtectus (Say), are often
experienced in the latter two regions. Depending on the climatic conditions,
infestation by the bean weevil may appear as early as 30 days (approximately, the
duration of one generation) after harvest under tropical conditions. Because of the
unlimited bean supply in stores, bean weevil populations escalate rapidly, thus
reducing the quality and quantity of the crop for human consumption.

In many parts of the tropical developing world, particularly among small
farmers, facilities for long term storage and protection of dry beans from bean
weevil infestations are not easily available or affordable. In Tanzania, about 90%
of the total legumes produced come from small farms (Due, et al., 1985), however,
the crop cannot always be stored effectively for later use. Because of the
susceptibility of stored beans to damage by bean weevils, losses up to 40% are
common in Tanzania (Kiula and Karel, 1985). In some areas of Colombia, the
duration of dry bean storage on farms ranged from four to 38 days after harvest, and
about a month and a half in warehouses (Schoonhoven, 1976). Beans were stored
for very short periods to avoid losses caused by bean weevils. With the awareness

of the nutritional importance of beans, and the existing widespread food shortages

1

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in many parts of the tropics, efforts to minimize post-harvest losses, particularly
during storage should be emphasized. Research and development of better
storage systems to ﬁt local conditions are essential if investments in ongoing
development of high yielding bean varieties are to pay off.

Post-harvest losses from insect infestations are often as serious as those
sustained in the ﬁeld. This became apparent to me while investigating both ﬁeld
and stored insect pest problems with bean farmers in Mgeta (Morogoro, Tanzania).
Farmers were very concerned about losses in stored beans due to bean weevil
infestations. I, therefore, spent two and a half years trying to learn as much as I
could about the pest under the farmers’ ﬁelds and storage conditions, and also
tested the effectiveness of some control methods commonly available to subsistence
farmers in Mgeta. I found in ﬁeld trials, that the common bean weevil attacked
beans prior to harvest, and pre-harvest infestation by bean weevils varied with
growing seasons and bean cultivars. Some of the questions that arose were: 1)
how are beans attacked by the bean weevils, and 2) do different bean classes and
cultivars truly vary in their susceptibilities? Knowledge of insect/ host interactions
at a basic level appeared to be a necessity for eventually ﬁnding new control
techniques and effectively utilizing existing ones. I, therefore, chose to pursue
studies on the behavior of the bean weevil for my Ph.D. research. The main
objectives of my dissertation research were to: 1) understand some major host-
colonization behaviors of the bean weevil in stored beans, and 2) determine the
possible implications of these behaviors for control measures for this pest in beans.

The dissertation research is presented in ﬁve chapters. Chapter One
develops baseline information for further behavioral studies of the bean weevil.
The inﬂuence of different bean classes and cowpea on A. obtectus ovipositional
behavior, larval survival, and host-selection by neonate larvae was investigated.

Further, effects of: 1) seed coat perforations, seed stability, and larval density on

505

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3

some aspects of larval performance, and 2) effects of dish type and light on
oviposition and larval survival are also reported.

In Chapter Two, ovipositional preference of the bean weevil and suitability of
ﬁve bean classes and a cowpea were studied in Tanzania and Michigan strains of
bean weevils. This investigation was undertaken to isolate possible differences
between the strains in their host preference and acceptance behaviors and in larval
performance.

In Chapter Three, the inﬂuence of various bean seed damage types on
ovipositional behavior, larval growth, and larval survival for the Tanzania and
Michigan A. obtectus strains was examined.

Chapter Four reports the host-attacking behavior of the neonate bean weevil
larvae. The ﬁrst experiment attempts to explain the possible effects of different
parts of the bean and the zone of contact between a bean and another surface (e.g.,
storage container or another bean) on success of seed entry by larvae. In the
second experiment, bean attack behavior of larvae is assessed under normal storage
conditions in the laboratory. Frequency of seed entry holes among beans in the
container, and number of entry holes in different parts of a bean seed are reported.
Another experiment describes seed attack behavior of the neonate larvae as viewed
from a monitor of a close-up video system. larval behaviors outside the seed were
videotaped, described and quantiﬁed.

Chapter Five reports effects of disruption of larval boring activity by seed
tumbling as a control against the bean weevil. Tumbling of beans was achieved by
rolling storage containers. This study was based on ﬁndings from Chapter One and
Chapter Four regarding the importance of seed stability and zone of contact
between seed and container surface on the success of seed entry by A. obtectus

larvae.

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GENERAL LITERATURE REVIEW

A. Insect/Plant Interactions

1H - loni ithvir

Host plants provide insects with food and/ or shelter for their survival. Major
stages of host-colonization include ﬁnding, examining, and consuming of the host
plant by the insects (Miller and Strickler, 1984). At each stage, various sensory
interactions between insect and plant occur during the host-colonization process.
These interactions may vary with different insect/host-plant systems in given
environments, and possibly with individual insects (Papaj and Rausher, 1983) within

a population.

mainline

Prior to utilization of the host-plant, the insect must ﬁnd the appropriate or
potential host among other plants. The host-ﬁnding process may involve random
movements triggered by the physiological conditions of the insect (e.g., need to
feed, oviposit, or mate in some cases), or may involve use of non-contact cues (e.g.,
chemical volatiles or visual stimuli) from the potential host plant.

Plant volatiles are known to be involved in the orientation of many
phytophagous insect to possible host-plants (Schoonhoven 1972; Visser, 1986). In
host-ﬁnding behavior of a gravid female cabbage root ﬂy, Delia radicum (L.),

isothiocyanates (volatile products of glucosinolates) have been shown to guide this

5

insect to its host (Hawkes and Coaker, 1976). D. radicum females responded
anemotactically to brassica and allylisothiocyanate odor plumes in a wind tunnel in
the absence of visual stimuli (Hawkes et al., 1978; Nottingham and Coaker, 1985).
The insect, however integrates other sensory modalities in addition to olfaction
when approaching a potential host plant (Nottingham, 1988), and thus the use of
visual cues increases as the D. radicum nears the plant.

For Colorado potato beetle, (Leptinotarsa decemlineata (Say)) the process of
host-plant ﬁnding appears to be a chance event rather than the use of special cues
from the host such as volatiles (Jermy et al., 1988). However, the spatial
maneuvering in the insect appears to somehow depend on light direction (photo-
menotaxis?). Ramaswamy (1988) also observed that the highly polyphagous
Heliothis armigera (Hiibner) and H. zea (Boddie) may not be dependent on
chemical volatiles for host ﬁnding whereas many specialist insects (monophagous or
oligophagous) depend on chemical cues for host ﬁnding.

Synergistic effects of odor components of the complex blend rather than the
individual plant volatiles seem to elicit stronger attraction in insects towards their
host plants (Visser, 1986; Mustaparta, 1984). For example, Nordlander et a1.
(1986) found that adult pine weevils (Hylobius abietis L) were able to locate
suitable conifer roots for oviposition by perception of host-volatiles diffusing
through the soil. Furthermore, a complete bouquet of the pine condensate proved
to be more attractive to the weevils than individual fractions of host terpenes. A
similar situation was noted in the carrot ﬂy (Psila rosae (F.)), where the insect
responded to several individual compounds of carrot foliage odor, yet, the strongest
response was obtained with the mixture of these functionally different types of
carrot leaf volatiles (Guerin et al., 1983).

Visual host cues also play an important role in the host-ﬁnding process of

phytophagous insects. Behavioral responses such as attraction and landing to the

6

color yellow are known to occur in various insects (Moericke, 1969; Kennedy, Booth
and Kershaw, 1961; Fricke et al., 1954). Three species of the genus Dacus (Diptera:
Tephritidae) are more attracted to yellow hue sticky traps than other colored traps
(Hill and Hooper, 1984). Visual stimuli such as color, size, form and orientation of
host or model, have also been shown to elicit attraction to host trees in apple
maggot ﬂies Rhagoletis pomonella (Walsh) (Moericke et al., 1975). Red spheres of
model fruits (especially those closer to real fruit size) positioned within foliage of
the host tree were attractive to this insect (Prokopy, 1977). Red hue of the ripe
fruit may be responsible for its detection by the apple maggot ﬂy (Owens and
Prokopy, 1986). A study by Katsoyannos, et al. ( 1986) on ovipositional site
selection showed strong preference by wild Mediterranean fruit ﬂies, Ceratitis
capitata (Wiedemann) for black, blue, and red ceresin wax domes (18 mm in
diameter) over other colors. This effect was attributed to the color, hue, and
intensity of total reﬂected light (brightness) of surrogate hosts. Tuttle et al. (1988)
found that the most effective landing or host-ﬁnding cues for D. radicum were color
(and size of the area with such color), height of substrate, visual quality, and host
volatiles. Thus, host ﬁnding by adult insects seems to involve complex responses to

multiple stimuli rather than a single "key" stimulus.

'nin min
Often, insects need special physical and/or chemical adaptations to
successfully colonize host plants. Upon landing on the potential host plant, the
insect should be able to maintain contact with the host plant part during host-
examination or ensuing consumption activities. Possession of specialized
structures has made host-attachment by herbivorous insects possible (Southwood,
1973). Many insects possess pre-tarsal structures presumed to permit gripping of

smooth surfaces (Holway, 1935). In larvae of some herbivorous insects, the end of

.7

the abdomen may be used for firm attachment on the smooth surface of the host-
plant part during various events or when plant part is suddenly shaken. Some
examples of the use of the terminal segment of the abdomen for host-attachment
has been noted in some larvae of the Chrysomelidae (e.g., Galerucella); in larvae of
some Miridae (Southwood and Leston, 1959); and in young larvae of
Coniopteryidae which also secrete a sticky substance from terminal segments during
locomotion (Collyer, 1951).

Biochemical adaptations to host plants by insects are also necessary for
survival on a given host. Nutritional condition, secondary plant substances and
changes in composition or physiology of the host-plant, all affect colonization of the
host plant (Southwood; 1973; van Emden and Way, 1973), as do host phenology,
natural enemies and unfavorable environmental conditions.

Upon contact with the surface of the potential host plant, physical (e.g., tactile,
visual) and chemical (gustatory, olfactory) plant cues inﬂuence acceptance for
oviposition or feeding. Phytophagous insects have been seen to "inspect" plants
before feeding or oviposition. Palpation, dapping, drumming, and walking on
plants are behavioral adaptations to sample the surface of the possible host (Miller
and Strickler, 1984; Stiidler, 1986). Species-speciﬁc examining sequences are
usually followed by the ﬁrst bite or probe, and if accepted, continued feeding
(probing in aphids (Miles, 1972)), or oviposition on or the near the host plant
occurs. Female butterﬂies contacting leaf surfaces move their forelegs vigorously
back and forth ("drumming") when sampling a potential ovipositional site (Calvert
and Hanson, 1983; Feeny et al., 1983; Kolb and Scherer, 1982). On host leaves,
drumming is usually followed by oviposition; but, on non-hosts, ﬂight generally
ensues (Stadler, 1986). Cabbage, carrot, and onion ﬂies perform similar
ovipositional "runs" or "walks" on host foliage prior to laying eggs in the soil near the
plant (Stadler, 1977, 1978; Harris and Miller, 1983). Harris (1986) documented

var
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8

both stem and substrate runs being performed by onion ﬂy (Delia antiqua (Meigen))
before oviposition.

Chemicals on plant surfaces have been shown to affect various insect
behaviors. For example, the initiation of feeding in grasshoppers and locusts
generally involves palpation of leaf surface followed by biting (Woodhead and
Chapman, 1986), but rejection can occur at either stage (Blaney et al., 1985).
Lacuna migraton'a L. reject sorghum (Sorghum bicolor (L.) Moench) seedling leaves
at palpation, but feed readily on mature sorghum of the same cultivar and on
seedlings with surface wax removed (Woodhead, 1983). Major alkane and ester
fractions of the seedling sorghum wax were unpalatable and deterrent. On the
other hand, extracts from the surface of Poa foliage stimulated biting in L.
migraton'a (Blaney and Chapman, 1970).

Mechanical cues such as physical structure and texture of the surface of host-
plant (Stadler, 1976) or host-plant part are also known to inﬂuence host-selection in
various insect herbivores. For example, perception of shape and texture of the
host surface as well as the internal consistency of substrate for oviposition in R.
pomonella is viewed as mechanical in nature (Boller and Prokopy, 1976). In the
cowpea weevil (Callosobruchus maculatus (F.)), Nwanze and Horber (1976)
reported that tactile stimuli are used when selecting ovipositional sites, thus the
beetle avoids rough surfaces and chooses smoother-coated cowpea seeds for laying
eggs.

Furthermore, many herbivorous insects are known to avoid ovipositing on host
plants or host-plant parts bearing conspeciﬁc eggs (Prokopy, 1981; Prokopy et al.,
1984). Gravid females may utilize various cues to detect eggs on potential
oviposition sites. Some butterﬂies appear to rely mainly on vision to detect the
brightly colored eggs of conspeciﬁcs (Shapiro, 1981; Williams and Gilbert, 1981),

while other insects may rely on chemical cues (Kozlowski et al., 1983), or may use

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more than one in some cases. Pien's brassicae L. utilizes multiple cues such as
visual, olfactory and contact chemoreception in detecting previously laid eggs on
host plants (Rothschild and Schoonhoven, 1977). Euura lasiolepis Smith, a stem-
galling sawﬂy, uses plant wound compounds from oviposition scars as cues in
avoiding resources already occupied by eggs of conspeciﬁcs (Craig et al., 1988).
The cowpea weevil, C. maculatus, has been observed to use both chemical and
physical (tactile) stimuli in egg-recognition on host seeds (Messina and Renwick,
1985).

On the other hand, egg load assessment in insects is not limited only to
conspeciﬁc eggs. Interspeciﬁc egg recognition leading to avoidance of such
oviposition sites has been seen in Pieris rapae L. females in the presence of P.
brassicae eggs on host plants. The deterrent factor sensed by olfaction was
suspected to be an oviposition deterring pheromone (ODP) from the P. brassicae
egg loads (see Klijnstra, 1982; Rothschild and Schoonhoven, 1977). When the
insect was presented with cabbage leaves bearing P. brassicae ODP and control
leaves, P. rapae highly preferred depositing eggs on the control leaves over treated
leaves even when control leaves became laden with conspeciﬁc eggs (Klijnstra,

1985).

2 n' -In

Allomones

Phytophagous insects are affected by a wide range of secondary plant
substances (Bell, 1981) as they attempt colonization of plant species. Secondary
plant chemicals such as alkaloids, non-protein amino acids, amines, saponins, simple
phenols, ﬂavonoids (including tannins), cyanogenic glycosides, sesquiterpenes,

lactones, and many others are abundant in various plants (Matthews and Matthews,

10

1978), and may act as allomones (Whittaker and Feeny, 1971) against herbivores.
The compounds may be toxic to some insects or they may modify insect behaviors
(Blaney and Simmonds, 1983; Simmonds and Blaney, 1984; Simmonds et al., 1985;
Blaney et al., 1987). For example, Bemays (1981) found that the inﬂuence of
tannins (plant polyphenols) on insects varied from having almost no effect on some
insects to affecting adversely growth and survival of other insects. In C. maculatus,
the cowpea weevil, condensed tannins from seed coats of Vicia faba L. var. ‘Gaente
Portugaise’ were shown to affect larval survival and prolong development of
surviving larvae fed diets containing four fractions of tannins (Boughdad et al.,
1986). Mortality was highest for fourth instar larvae and males of the cowpea
weevil.

Several other secondary plant compounds have also been tested on C.
maculatus. Castanospermine, an alkaloid from the legume Castanospennum
australe A. Cunn. (Hohenschultz et al., 1981), when incorporated in larval diets,
proved toxic to C. maculatus (Nash et al., 1986). The compound inhibited activities
of both alpha- and beta-D-glucosidases in the alimentary tract of the larvae.
Castanospermine is also a feeding deterrent to Acyrthosiphon pisum (Harris), the
pea aphid (Dreyer et al., 1985). Seed lectins (phytohaemagglutinins) found in many
plants particularly in legumes, are known to be toxic and contain haemagglutining
activity. For example, toxic effects were noted in C. maculatus larvae that fed on
artiﬁcial diets containing lectin fractions from Phaseolus vulgaris L. seeds
(Gatehouse et al., 1984). Lectins bind to the midgut epithelium and disrupt the
epithelial cells, thereby affecting normal nutrient movement into cells, while
enhancing absorption of toxic subtances (Gatehouse et al., 1984). A non-protein
amino acid from seeds of the legume, Dioclea megacarpa Rolfe, called canavanine is
tolerated by its seed specialist bruchid, Caryedes bras'ilienszlr, but has been shown to

be toxic to larvae of C. maculatus at lower concentrations than those naturally

11

found in seeds of the legume (Rosenthal et al., 1976; Bell, 1981).

Plant allomones may also deter insect herbivores. The deterrent effects of
secondary substances may in some cases be overriden by feeding stimulants in a
given host plant (Schoonhoven and Derksen-Koppers, 1976). Furthermore,
allomones may be generally toxic, yet some insects appear to be adapted to them.
For instance, the Colorado potato beetle, L. decemlineata, that feeds on potato
foliage, is adapted to solanine, a potato alkaloid toxic to some other insect
herbivores (Harborne, 1977). In some specialist insects, adaptation to host plants
may go beyond tolerance of defensive substances. Phytophagous insects that
tolerate toxic substances in their host-plants are presumed to possess physiological
mechanisms for: a) avoiding harmful effects, or b) detoxifying toxins, and in some
cases c) sequestering these substances for protection against predators
(Schoonhoven, 1981). larvae of Catyedes brasilienszls (Bruchidae) feeding on seeds
of legume, Dioclea megacarpa, appear to have adapted biochemically to the
presence of L-canavanine in the seed, and can metabolically sequester and
metabolize the compound (Rosenthal, 1983). Some Diabrotica species are known
to feed on the Curcubitacae which contain cucurbitacins as secondary plant
compounds. Cucurbitacins act as allomones in protecting plants from insect
herbivores, yet they also behave as kairomones for host-selection in the Diabrotica
beetles. In addition, these beetles further concentrate and sequester relatively
large amounts of cucurbitacins in free and derived forms after feeding on
Curcurbitacae, perhaps for deterring some of their predators (Metcalf, 1986).
Similarly, aposematic larvae of the pyralid, Uresiphita reversalis (Guenée), (Bernays
and Montllor, 1989) feed on Genisteae legumes, which bear various quinolizidine
alkaloids (Kinghom and Balandrin, 1984). The larvae preferred feeding on the
youngest leaves of the plant which contain four to ﬁve times the amount of alkaloids

found in older leaves. Although much of the alkaloids were excreted, the

cOmb
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12

sequestered alkaloids generally appeared on or near the surface of the larvae
(Montllor et al., 1990).

Induced changes in defences and nutritional quality of the host plant have also
been associated with herbivory (Schultz and Baldwin, 1982; Karban and Carey,
1984). For example, wounded solanaceous plants were observed to contain
proteinase inhibitors of trypsin and chymotrypsin, and the proteinase-inhibitor
inducing factor was produced at the site of damage on the plant (Green and Ryan,
1972; Ryan, 1983). Risch (1985) reported that induced chemical changes may
occur in many damaged plants or plant parts and that feeding preferences may be
affected in some insects, particularly the more generalists. In addition to induced
chemical changes, gummosis, a physical plant defensive reaction may also result
from initial tissue damage by insects on some plants. The plant secretes a gummy
substance that may immobilize or drown the insect, hence preventing further
wounding. The mechanism is particularly common in conifers (Harris, 1960).

Aging of the plant or plant parts is also known to induce changes in plant
chemistry. Nutritive value of plants is generally considered to decline with aging.
Scriber and Slansky (1981) and Rausher (1981) observed decrease in nitrogen and
water levels as the season progressed. Further, levels of secondary plant
compounds also vary with age of host plant. Tannins and other phenols occur in
higher amounts in mature foliage of some tree species (Feeny, 1970; Haukioja et al.,

1978), thus affecting colonization of host in some insects.

Plantautdems

Apart from secondary substances, host-plant nutrient types, levels, or different
combinations of these, also contribute to the host-selection process in insects (van
Emden and Way, 1973). Even when requisite nutrient types are present, their

levels and ratios must also be "correct" to assure success in colonization of hosts by

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13

their herbivores.

Nutritional status may vary among different parts of plants. The ability of
insects to respond to different nutrient concentrations in their hosts, thus enabling
them to locate the most suitable part of plant for feeding and oviposition, has been
demonstrated in the larvae of European corn borer, Ostrinia nubilalis (Hiibner) and
in the larvae of H. zea in selecting parts of corn on the basis of sucrose levels
(Schoonhoven, 1973).

Insects may also respond to different nutrient ratios in host plants while
selecting those on which they survive best. For example, the ratio of stimulants to
inhibitants appeared to be essential in eliciting feeding by Phomzia regina (Meigen);
it is presumed that the feeding preference of the blowﬂy is regulated by several host
chemicals which are excitatory, additive, neutral, inhibitory or synergistic in
behavior (Mukwaya, 1986).

Ability to adapt to changes in diet quality, or to compensate for inadequate
nutrient levels in host plants has been reported in some insects. Simpson et al.
( 1988) noted dietary selection behavior in nymphs of the oligophagous acridid,
Locusta migraton’a and in polyphagous larvae of the noctuid, Spodoptera littoralis
(Boisduval). Insects conditioned on four artiﬁcial diets, and then presented with a
choice of diets, selected for nutrients deﬁcient in the conditioning diet. L.
migratoria selected carbohydrate (C) over protein (P) (54:46) whereas S. littoralzls'
selected protein over carbohydrate (33:67, C:P). Further, a polyphagous acridid,
Melanoplus bivittatus (Say) also performed better when provided with several
species of plants than when fed on a single species (MacFarlane and Thorsteinson,
1980). A similar selection for higher carbohydrate-to-protein ratio in diets has also
been recorded in an omnivorous cockroach, Supella longipalpa (F .) (Cohen et al.,
1987). As for S. littoralis, the selection for higher protein-to-carbohydarte ratio

was also demonstrated earlier in Heliothis zea (Waldbauer et al., 1984) where the

14

larvae selected caseinzsucrose ratio (79:21) when offered two diets, each lacking one
of the nutrients. Moreover, when the last instar larvae of H. zea were offered two
diets, each lacking either lipid or certain vitamins essential for completing their
development, the larvae fed from both diets, obtaining a mixture superior to either
diet (Schiff et al., 1988).

Interactions between chemical stimuli may inhibit or synergize feeding
behavior in insects (Schoonhoven, 1973). For example, salt (NaCl) at low
concentrations (below 0.175 M in salt/sucrose mixtures) enhanced sugar intake in
Phormia regina (Mukwaya, 1986), and this synergistic effect was a conﬁrmation of
earlier observations by Dethier (1968), Kuwabara (1961), and Morita et a1. (1965).

Strength of response to precise nutrient information may generally vary with
the degree of specialization of the herbivore, whether with regard to plant species or
just part of the host plant. The pea aphid, Acrythosiphon pisum, has a relatively
narrower host range and stronger preference for amino acids and sucrose
combinations than does the polyphagous potato aphid, Macrosiphum euphorbiae

(Thomas) (Cartier, 1968).

W

Of many mechanisms in plant defence against insects, hardness of plant tissue
is the most common (Van Emden and Way, 1973). Hardening and thickening of
plant tissues are known to interfere with feeding and oviposition of insects (Norris
and Kogan, 1980). Tough plant parts may resist action of mandibles during
feeding, penetration of the insect into host, or insertion of the ovipositor into plant
tissue during egg laying.

Raupp (1985) found that tough leaves not only eroded the mandibular cutting
edges of the leaf beetle (Plagiodera versicolora laich.), but also slowed consumption

and reduced oviposition. Leaf toughness has been associated with seasonal

15

changes in a number of cases. Increase in hardness of leaves as well as tannin
levels during a season affected normal feeding behavior in the winter moth
(Operophtera brumata L.) larvae on oak leaves (Feeny, 1970). The toughness of
mature foliage in combination with decreasing availability of nitrogen (tannins are
known to reduce availability of nitrogen) may have caused such results. Lowman
and Box (1983) also found that leaf toughness and chemical toxicity of ﬁve species
of Australian rain forest trees increased with leaf age.

Mineral deposits in plant cuticle may also harden the foliage and render it
resistant to insects. Pubescence of many plants is fortiﬁed either by calciﬁcation or
siliciﬁcation (Uphof, 1962). Some plant species, especially those of Grarninae,
Cyperaceae and Palmae, have silica deposits in their epidermal cell walls (Norris
and Kogan, 1980); and a silicated epidermis confers resistance against some pest
species. Mandibles of Chilo suppressalis (Walker), the rice stem borer, feeding on
stems of a silicated variety of rice were highly worn when compared to those of stem

borers feeding on normal rice (preferred host) (Sasamoto, 1958).

I. l i l .

Thickening of plant tissues may also interfere or restrict utilization of host
plants by insects. Thickened pod walls of some cowpea (Vigna unguiculata (L.))
varieties resist entry of cowpea curculio (Chalcodermus aeneus Boheman) into pods
(Cuthbert and Davis, 1972). In shoot ﬂy (Athen’gona soccata Rondani) resistant
sorghum varieties, lignin-rich thickened cell walls covering sheaths of vascular
bundles of the central whorl of young leaf blades provide resistance against the pest
(Blum, 1968). The tightly packed, tough vascular bundles of hard, woody stems of
Cucurbita species also act as a barrier against penetration and normal feeding of the

squash borer (Melittia cucurbita (Harris)) larvae (Howe, 1949).

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Madness

Hardening of seed coat as well as cotyledons occurs in some seeds, including
beans. Successful entry of ﬁrst instar bruchid larvae into host seeds is hindered by
hard seed coats (Thiery, 1984).

Storage conditions can play an important part in the hardening of dry bean
seed. For example, storage of beans at high temperatures and humidities induced
changes in both structure and biochemistry of the seeds (Varriano-Marston and
Jackson, 1981; Jackson and Varriano-Marston, 1981; Hincks and Stanley, 1986).
Structural changes in seed coat and in cotyledon components (cell walls, middle
lamella, starch granules and membranes) were more highly implicated in inducing
hardness than changes in the proteins, carbohydrates, phytate, polyphenols, and
lignin of the stored bean seeds (Stanley and Aguilera, 1985). Black beans exposed
to storage conditions of 41 0C and 75% and 100% RH for one or two weeks showed
detachment of the plasmalemma from the cell wall in seeds and loss of granularity
of cytoplasm (Varriano-Marston and Jackson, 1981).

Hincks and Stanley (1986) observed hardening of beans by the fourth month
of storage at 30 0C and 85% RH. They concluded that phytate loss during the
earlier part of storage period and phenol metabolism in the extended storage time
were the major contributors to seed hardness in beans, and that phenol metabolism
may act as a ligniﬁcation-like mechanism. Seed resistance to entry by bean weevil
larvae has been mainly attributed to lignin in seed coats (Stamopoulos, 1987).
Furthermore, in some sunﬂowers, resistance of the pericarp to puncture or damage
by larvae of sunﬂower moth (Homoeosoma electellum (Hulst.)) was reported in

achenes having a phytomelanin layer in their pericarps (Stafford et al., 1984).

17

S 1 . .

Integrity of seed coat is also essential in prevention of seed infestation by seed
feeders particularly in stored grains. Integrity of husks of stored rice confers
resistance against certain coleopterous pests. Poor closing of glumes in grains of
some rice varieties allows larvae to enter seeds easily (Link and Rossetto, 1972;
Sauphanor, 1988). Mechanical damage as well as shattering of rice husks also
provide entry of storage pests such as Sitotroga cereallela (Olivier) into the grain

(Sauphanor, 1988).

Trichomes

Trichomes are unicellular or multicellular structures originating from the
epidermal layer of leaves, stems, and roots (Uphof, 1962), and they may act as a
mechanical barrier against insect attachment, movement, feeding or oviposition on
the plant. The effectiveness of trichomes in plant resistance against insect
herbivores appears to depend on density, length, erectness, and shape of the hairs
(Norris and Kogan, 1980). More erect and hook-shaped pubescence on foliage of
the bean cultivar, California light red kidney, effectively captured nymph and adult
leafhoppers, Empoasca kraemeri Rose & Moore (Pillemer and Tingey, 1976). A
Medicago glandulosa David clone with seed pods densely covered with long,
glandular trichomes prevented alfalfa seed chalcid (Bruchophagus roddi
(Gussakovsky)) seed infestation completely, while other alfalfa species were
infested (Brewer, et al., 1983). Removal of trichomes from foliage of resistant wild
tomato and a suceptible tomato cultivar signiﬁcantly increased the suitability of the
two plants to larvae of H. zea (Farrar and Kennedy, 1987).

Other than their role as a physical barrier or exuding gummy materials when
fractured, some trichomes also contain chemical substances toxic to insects

(Stipanovic, 1983; Duffey, 1986). For example, in wild tomatoes (Lycopersicon

18

species), resistance to attack from larvae of Keiferia ch0persicella (Walsingham)
(tomato pinworrn) was attributed not only to the sticky exudate from glandular
trichomes which trapped the larvae, but also to the presence of non-alkaloid

antibiotic, 2-tridecanone in the plant foliage as well (Lin and Trumble, 1986).

B. Bruchid/Bean System

1, Beans

Food legumes are among the major storable and commercial foods of the
world (Davies, 1983). Phaseolus vulgaris, the common bean, is a widely cultivated
species of legume in the temperate, subtropical and tropical regions of the world.
P. vulgaris is a native of the new world; its native range probably includes Mexico,
Guatemala, and the Andean region of of South America (Duke, 1981; Kaplan,
1981)

P. vulgaris is an annual polymorphic dicotyledonous plant. Depending on the
cultivar type, it may be either erect, bushy or twinning in growth habit. The plant
self-pollinates and bears seeds in ﬂat to cylindrical pods (Duke, 1981).

Beans are an important crop in many countries of the world, especially to
those where they serve as a major source of dietary protein. Beans are eaten as
immature green pods (snap beans), mature ripe beans and mature dry beans. The
crop is generally stored as dry bean seeds.

In Tanzania, most of the bean crop for local consumption is grown by small
farmers at a subsistence level (Due et al., 1985). Although yields up to 2,000 kg/ ha
have been attained by researchers in farmers’ ﬁelds, the usual yield of dry beans is
about 600. kg/ha (Sibernagel and Teri, 1990). Storage of dry beans for later

consumption is not always possible for many farmer families and other consumers.

19

Damage during storage by bean weevils is one of the major constraints limiting long
term storage of dry beans. This problem in turn leads to shortages of beans

between the growing seasons, particularly in the non-producing areas of the country.

2, Bean Weevil

14mm

The common bean weevil, Acanthoscelides obtectus (Say), belongs to the
subfamily Bruchinae in the family Bruchidae. The Bruchidae are generally known
as seed beetles and the family consists of approximately 1300 species (Southgate,
1979). The bean weevil is a major pest of dry beans. It has been reported in
Central and South Arnerica, in Asia and Paciﬁc islands and West Indies (Southgate,
1978), in Africa (Decelle, 1981) and in the USA.

During storage, dry beans are particularly susceptible to damage by the bean
weevils. A. obtectus constitutes a principal pest of beans wherever this crop is
grown or stored (Williams, 1980), particularly in the higher altitudes or cooler areas.
Initial infestation begins in the ﬁeld but continues and increases rapidly in storage.
A few generations of A. obtectus in stored beans will produce signiﬁcant damage to
the crop. In the ﬁeld, female bean weevils lay their eggs in a hole bitten into the
maturing pod or inside a dry dehiscent pod (Skaife, 1926). In stores, the beetles
scatter their eggs loosely amongst dry bean seeds (Howe and Currie, 1964;
Pankanin-Franczyk, 1980). Their whitish eggs are elongate and about 0.65 mm
long (Whitman and Southgate, 1982). Eggs hatch in about 3 to 5 days and ﬁrst
instar larvae penetrate seeds where they feed and develop into adults (Williams,
1980; Pankanin-Franczyk, 1980). larvae molt 4 times prior to pupation, and the
last instar prepares a circular "exit window" under the seed coat through which the

adult will emerge eventually by pushing or cutting open the cover (Howe and

20

Currie, 1964; Southgate, 1979; Williams, 1980). The pupal stage lasts from 7 to 28
days (Southgate, 1979).

The life cycle (from eg to adult) is completed in about 29 days at 30 OC and
70% RH. Up to 10 generations can occur within a year (Williams, 1980) in
favorable storage conditions. Adult bean weevils are greyish-brown, measure
about 3 mm long, and have moniliform antennae. They generally mate soon after
emergence and oviposit within 48 hours. The pre-ovipostional period may last less
than a day; eggs are laid at maximum rates during earlier life. A female may lay up
to 60 eggs during an average lifetime of 15 days (Howe and Currie, 1964).

According to Howe and Currie (1964) and Pankanin-Franczyk (1980), adults
do not seem to feed in storage, but they have been seen to take water, sugar solution
or nectar when provided. In the ﬁeld, adult A. obtectus has been observed to feed
on nectar and pollen (Levinson and Levinson, 1978); and in some cases, they feed
on grass pollen that is wind blown and collects on plants (Jarry, 1987). Though
adults feed on pollen and larvae feed on bean cotyledons, the carbohydrate

composition of these two diets is similar (Leroi et al., 1984).

Q . i'

Site selection for oviposition by the bean weevils has been suggested to
operate in a "two-way specialization" of the stimuli receptors of the insect
(Muschinek et al., 1976), e.g., the stimulatory and inhibitory stimuli that inﬂuence
the egg-laying behavior in female phytophagous insects (Matthews and Matthews,
1978; Muschinek et al., 1976). In A. obtectus, the host-plant or plant parts
stimulate oviposition and induce fecundity (Szentesi, 1975). Contact with host-

plant or bean seeds by females induces oogenesis and oviposition (Pouzat, 1978).

ovo;

srrai

21

Gustatory perception of host surface (Monge, 1983; Pouzat, 1978) and probably
tactile interactions may also stimulate oviposition in bruchids (Pouzat, 1976). The
labial and maxillary palps of the bean weevil appear to be the major organs involved
in sensing stimuli for oviposition (Pouzat, 1978; Messina et al., 1987).

Mating, in addition to host-plant presence, provides further stimulus for
ovogenesis and oviposition (Szentesi, 1975), but its effect may vary with different
strains of the bean weevil. Huignard (1976) found that in one strain of A. obtectus,
mating alone induced oviposition in absence of beans, while in the other strain,
mating and presence of host were necessary for egg-laying. Butare and Biemont
(1987) also worked with a strain of bean weevil that produced mature oocytes in the
absence of hosts. Females reared in isolation, however, produced more oocytes
than those in groups. Inhibition of oogenesis in the grouped females was
attributed to direct contact stimulus exchanges between the group members via their
antennae.

In other bruchids such as Callosobruchus maculatus and C. chinensis L., seed
physical factors play an important role in host-selection. Smooth-coated seeds and
checks of the seed are preferred sites for oviposition in C. maculatus (Nwanze et al.,
1975). In C. chinensis, seed curvature and size stimulate oviposition (Avidov, et
al., 1965). Plant developmental stages also vary in their stimulatory effects upon
bruchids. Preference for oviposition may range from full-size green pods to mature
dry pods, but exposed seeds are normally most preferred (Messina, 1984).

Availability of preferred host plants usually leads to rejection of other possible
hosts or non-hosts for oviposition in the bean weevil (J ermy and Szentesi, 1978).
However, in the absence of host plants, non-hosts, even if unsatisfactory for larval
survival, may be utilized. For example, A. obtectus oviposited some eggs on dry
pea (Pisum sativum L.) seeds in the absence of beans. Dry peas are thought not to

contain stimulatory or inhibitory substances affecting the bean weevil oviposition

22

(Jermy and Szentesi, 1978). Therefore, non-preference of the beetle for some
ovipositional hosts may be due to lack of stimulants or presence of inhibitory
factors, or both.

Apart from insect and plant-host factors, some components of the
environment are also known to affect the colonization success of A. obtectus.
Changes in temperature and light intensity affect ﬂight initiation rates of the
migrating or dispersing adults. No insects ﬂew at 20 0C, however, take-off rates
were considerable at 25 0C, peaked at 30 0C, but fell at 40 0C (Perttunen and
Hayrinen, 1969). The authors further observed that at a constant temperature of
25 0C, take-off rates increased with increasing light intensity from 0.5 lux to the
highest rate at 10001ux (Perttunen and Hayrinen, 1970).

Bean weevils are also sensitive to the relative humidity and temperature in
their ovipositional environments. A relative humidity of 70% and temperature of

25-30 0C (Howe and Currie, 1964) yields maximal A. obtectus oviposition.

g, Qwaﬂzeen interaetien

First instar bean weevil larvae must usually penetrate the seed coat of the seed
to feed on the cotyledon. Once inside, the developing larva does not change seeds
(Stamopoulos and Desroches, 1981). Some mortality of the ﬁrst instar larvae is
common during the boring process, but this varies depending on seed and larval
characteristics. Seed coat hardness enhances resistance against larval penetration
into bean seeds. This trait can be regulated by varying moisture content of the
testa (Thiery, 1984). Higher bean moisture content reduced seed coat hardness
and improved larval penetration into seed.

Seed age is thought to inﬂuence seed coat hardness. Hardness increases with

storage time even when seeds are kept at constant relative humidity and moisture

23

content (Thiery, 1982a). Srisuma et al. (1989) observed that adverse storage
conditions such as high temperatures combined with high relative humidities over
time induced seed hardness (Srisuma et al., 1989). Hardness has been correlated
with increased free phenolic acids in cotyledons and testa of bean seeds.

In addition to acting as a mechanical barrier, the testa of some ripening P.
vulgan's seeds has been shown to contain toxins not degradeable by all larvae of A.
obtectus (Stamopoulos and Desroches, 1981). Stamopoulos and Huignard (1980)
evaluated the role of seed coat as a chemical barrier by mixing ﬁnely ground bean
testa with bean cotyledon meal as a larval diet. This diet was lethal to bean weevil
larvae, particularly the third and fourth instars. It appears that non-ingestion by
the ﬁrst instar larvae while boring through the seed coat may be a toxin-avoidance
mechanism. This has also been suggested for ﬁrst instar larvae of the cowpea
weevil (Nwanze and Horber, 1976). Moreover, perforation of seed coats improved
survival of the bean weevil larvae more than 2 to 3 times that of non-perforated
seeds, and mortality of the insect before seed penetration was generally higher than
after seed entry (Stamopoulos and Desroches, 1981).

Upon successful penetration into the bean seed, larvae tunnel, feed and
develop within the cotyledons and consequently damage the seed. , More than one
bean weevil larva may enter a hole previously bored by another larva (Iayberie,

1960).

rlfh nwevil

Losses in both quality and quantity of stored beans in developing nations can
be severe, particularly for subsistence farmers. Vulnerability to bean weevil attack
directly affects the bean supply that will be available to the farmer and consumers

throughout the non-producing period of the year. Emphasis on increasing yield

We
afz
(Be

[lav

Ei'aj

24

alone will not alleviate shortages, if reserves cannot be stored because of insect
damage. The ability to store beans effectively for relatively long periods would
ensure sufﬁcient beans for the consumer and enough seed for planting by the
farmer. However, many subsistence farmers cannot store beans for longer than
three to six months unless control is exercised for the bean weevils.

Various control techniques for the bean weevil at the small farmer level
include use of synthetic insecticides, cultural, and traditional methods (e.g., mixing
beans with ashes). Lindane (gamma-HCH or gamma-BBC), synergised pyrethrin
dusts (Taylor and Evans, 1980) and malathion (Prevett, 1975) are some of the
commonly used insecticides in dry bean storage, where they are available and
affordable. The effectiveness of malathion dusts used in bean storage rarely lasts
for more than six weeks after application (Golob and Kilminster, 1982). Bean
weevil damage has been known to exceed the economic threshold in ﬁve months
after ﬁeld-infested beans were treated with malathion (3 ppm) soon after harvest
(Baier and Webster, 1990). Pirimiphos methyl and permethrin dust formulations
have been shown to be effective for the control of the bean weevil (Taylor and
Evans, 1980), however, their action did not extend beyond 24 weeks.

The limited effectiveness and irregular availability of insecticides in various
developing countries limit their use in control of the bean weevil. In addition,
ignorance of the hazards of misused insecticides may result in health and
environmental problems. Further, insecticide resistance in stored product insect
pests is not uncommon. Resistance to lindane by some stored product beetles
(e.g., Tribolium castaneum (Herbst), the red ﬂour beetle) is worldwide (Champ and
Dyte, 1977; Beeman and Stuart, 1990). T. castaneum is also reported to be
resistant to malathion (Beeman, 1983; Beeman and Nanis, 1986). Resistance to
phosphine, a fumigant, has also been detected in a number of stored grain pest

species, including Rhyzopertha dominica (F .) and Sitophilus oryzae (L.). (White and

25

Lambkin, 1990).

Small farmers and consumers in many developing countries have used
traditional, non-synthetic insecticide control techniques for years. However, these
methods are particularly feasible for storage of relatively small quantities of dry
beans. For example, stored pulses are covered with ashes from ﬁrewood stoves or
vegetable oils in some parts of Africa and Asia, respectively. In Malawi, some
small farmers use ash from the kitchen ﬁre, and tobacco leaf dust (among tobacco
growers) for control of insect pests in stored pulses and grains (Golob et al., 1982).

Various workers have tested the effectiveness of some locally available
materials in the control of the stored product pests. Golob et a1. (1982) tested
effects of dolomite, wood ash, tobacco dust, sawdust and sand, and concluded that
even though the treatments did not totally control infestation, almost all restricted
infestation levels of insect pests for 4 months in stored maize grain. For example,
the number of adult insects emerging from maize was reduced by about 91% when
maize was mixed with wood ash at 30% by weight; activity rankings in descending
order for other materials were: dolomite (88%), tobacco dust (87%), sawdust (69%)
and sand (66% at 1:1 maize to sand ratio). Mixing ashes (Schwartz and Galvez,
1980) or sand (CIAT, 1980) with dry beans was shown to be an effective method
where the physical barriers reduced infestation of bean weevils. Ashes at 20% by
weight that ﬁlled all spaces between beans gave good control of Acanthoscelides
obtectus for more than nine months in naturally infested beans, but for four months
in artiﬁcially infested beans (Baier and Webster, 1990). Inert dusts (e.g.,
crystalline silica, bentonite and magnesium carbonate) have also been known to kill
A. obtectus (Schwartz and Galvez, 1980). Activated kaolinitic clay caused 100%
mortality of another bruchid, Callosobruchus chinensis, up to 225 days when adult
insects were exposed to cowpea seeds coated with activated kaolin at 10 g/kg

(Swamiappan et al., 1976). Other tests on the effectiveness of inert dusts have also

CODIrol .

Obtained

C “Menu

26

been conducted by Parkin and Bills (1955) on control of bruchids in stored peas and
beans.

Mixing edible vegetable oils with legume seeds for protection against bruchid
infestation has long been used in India. In C. maculatus, the cowpea weevil,
groundnut oil at the rate of 5 nil/kg cowpeas provided 100% control of the insect
up to 180 days in stored cowpeas (Singh et al., 1978). Schoonhoven (1978) found
that a high level of control of Zabrotes subfasciatus (Boheman), the Mexican bean
weevil, was achieved by using various vegetable oils at the rate of 1 ml/kg of bean
seeds; however, for complete control lasting more than 75 days, the dosage of 5 or
10 ml/kg beans was necessary. Treatment of beans with oil increased adult
mortality, reduced oviposition, egg-hatch and larval survival in Z subfasciaau.
Oils appear to suffocate various insect life stages. Further, a triglyceride
component of African palm oil and oleic acid of the free fatty acids was also found
to be insecticidal to Z. subfasciams (Hill and Schoonhoven, 1981). In A. obtectus,
mixing soybean oil with dry beans at 5 ml oil/kg beans kept the infestation level
below the economic threshold (4% damage) for eight months after harvest (Baier
and Webster, 1990). Although this method appears to be promising in its
effectiveness against bruchids, the use of edible oils may not be highly practical for
different situations in dry bean storage. Such oils may not always be available in
amounts that can be allocated for both food and storage.

Other oils besides common edible oils have also been tested for their
insecticidal activities. Neem (Azadirachta indica A. Juss) kernel oil gave better
control of Z. subfasciatus than other vegetable oils after one month following the
treatment (Kiula and Karel, 1985). Vapors of Acorns calamus L. (sweetﬂag) oil
obtained from rhizomes of A. calamus (Araceae) were toxic to adults of several
stored-product insect pests, particularly C. chinensis (El-Nahal et al., 1989). All

C. chinensis contained in ﬁve test tubes within a 400 ml glass jar exposed to vapors

 

TC.
fra.

nan

27

from 1 and 10 u! of A. calamus oil for various periods in excess of 48 hours were
killed. However, these oils are rarely available for applied use presently.

The use of resistant varieties against the bean weevil attack has been
considered a promising alternative control (Schoonhoven and Cardona, 1982),
especially for small farmers in the developing nations. Screening for resistance
among various bean accessions and developing resistant varieties safe for human
consumption has been ongoing in many parts of the world in the recent years.
Phaseolus vulgaris seeds are known to contain two major proteins, the lectin
phytohaemagglutinin (PHA) and arcelin (Lioi and Bollini, 1989), which impart
some resistance to bruchid attack (Janzen et al., 1976; Osborn et al., 1988). Four
arcelin genes have been transferred from wild to cultivated bean lines (Harmsen et
al., 1988). Arcelin-containing lines of P. vulgan’s gave high resistance against Z.
subfasciatus but not against A. obtectus (CIAT, 1987; 1988). Resistance to both
A. obtectus and Z. subfasciatus has been found in one of the wild lines of P.
vulgarr's (G 12953) (Schoonhoven et al., 1983). This resistance was conﬁrmed by
Gatehouse et a1. (1987), who isolated a heteropolysaccharide fraction from the
resistant line and incorporated it into artiﬁcial beans. The heteropolysaccharide
fraction was toxic to the bean weevil at a concentration of 4% (its approximate
natural concentration within seed). It resulted in 80-85% larval mortality in A.
obtectus. Some resistance to A. obtectus has also been reported in some tepary
bean (Phaseolus acutifolius A. Gray) accessions (Shade et al., 1987). larval
survival and adult emergence were reduced while developmental period of the bean
weevil was lengthened in tepary beans when compared to P. vulgwr's cultivars.
Furthermore, incorporation of thiol proteinase inhibitors in artiﬁcial seed diet of A.
obtectus impeded larval development. When a speciﬁc thiol proteinase inhibitor,
E-64, wasincorporated into larval diet at 0.10 and 0.25%, all feeding bean weevils

died (Nielsen et al., 1988). The authors suggested transfer of thiol proteinase

and
[be
Ola]
Sboul
reusu
ﬁlSyr

ﬂoweri

28

inhibitors from their natural sources into beans to develop lines resistant to bean
weevils.

Ecological aspects of the bruchid biology in the context of developing
techniques for bruchid control have been suggested by a number of authors.
labeyrie (1977) found that in mixed cropping situations of beans and maize, the
maize crop shielded the beans and limited A. obtectus infestation to the border
area of the ﬁeld. On the other hand, presence of alternative hosts in the
neighboring area or within the ﬁeld may perpetuate bruchid populations around the
ﬁelds until the bean crop is available for infestation. When dry beans are not
available, the Mexican bean weevil is known to reproduce on wild beans of
Phaseolus lunatus L. vines growing in the periphery of the bean ﬁelds (Pimbert,
1985). However, around the harvest period, Z. subfasciams moves from P. lunatus
to dehiscing pods of P. vulgaris to oviposit, and consequently multiply extensively in
the stable environment of bean stores. Early harvest (prior to dehiscing of pods)
and destruction of alternative wild host plants has been suggested (Pimbert, 1985).
Use of non-dehiscent cultivars may also avoid ﬁeld infestation of Z. subfasciatus
(Pimbert and Pierre, 1983). In addition, phenological resistance to bean weevils
should also be considered. Tahhan and van Emden (1989) reported phenological
resistance to a bruchid, Bruchus dentipes Baudi, a pest of faba bean (Vicia faba L.)
in Syria. A faba bean accession (BPL33) escaped serious infestation by late

ﬂowering and pod-setting.
'mi i n n n r l
The use of ashes and other inert materials for control of the bean weevil may

not always be practical, particularly for storage of large quantities of dry beans.

The amount of materials needed to sufﬁciently cover beans may not be easily

5/76
ace:
Fara
1H nu
leafs
resista
deployi
other L‘
durablljr
Since P12
developm
e'lPlOred Cl

29

available. Dusts or ashes are also messy to handle and necessitate more water for
cleaning beans prior to cooking. Where water supplies are limited, these methods
are not likely to be used. In addition, such materials occupy a large proportion of
storage area (e.g., if ashes or sand were used to completely cover the beans) that
could otherwise be available for beans. The use of sand causes excessive weight to
storage containers, making the methodology less attractive. However, where these
methods are applicable, they may provide practical storage protection.

Use of edible oils for protection of dry beans in storage also has its limitations.
Such oils may not be available in quantities adequate for both food and storage or
the effective oils may not be available at all in some localities. Similarly, the use of
plant allelochemicals is currently not widely used at the small farmer level. More
research is needed in the use of plant compounds that are locally available.

Breeding for plant resistance against insect herbivores may the best alternative
to almost all methods discussed. However, the process of screening, developing,
and producing resistant varieties is generally long. In addition, varieties developed
should not only be resistant to the bean weevil, but should also be non-toxic and
acceptable (e.g., in color, cookability, palatability) to farmers and consumers.
Furthermore, there is always a possibility of insect adaptation to resistant cultivars.
In numerous cases reported, adaptation of insect genotypes occurred in less than 7
years (Gallun, 1977; Sosa, 1981; Pathak and Heinrichs, 1982). The durability of
resistance in the varieties produced depends on the strategies developed for
deploying the resistant germ plasm (Gould, 1986a). For example, integration of
other tactics with a resistant wheat cultivar has been shown to enhance the
durability of Hessian ﬂy-resistance in winter wheat (Gould, 1986a; Gould, 1986b).
Since plant breeding is expensive and generally requires a long time for the
development of resistant cultivars (Kennedy et al., 1987), other methods need to be

explored concurrently that could: I) be integrated with resistant varieties in a given

30

IPM system; and 2) be used when insects become adapted to the resistant cultivars.

In conclusion, there is still a strong need for better methods for control of the
bean weevils. Integration of more than one control technique rather than total
dependence on a single method may be the best approach in dealing with bean
weevil control. For example, appropriate cultural practices are always beneﬁcial
when used in conjunction with various conventional or traditional methods.
Furthermore, when developing and introducing pest control methods for the small
farmer in developing nations, the control "packages" should be readily available,

safe, affordable, easy to operate, and not too time consuming.

CHAPTER 1

Factors influencing host acceptance and suitability of
commercial bean classes for the bean weevil,

Acanthoscelides obtectus (Say).

31

5U.
lb
bea
10291
High
QXam

COlOIL

INTRODUCTION

The bean weevil, Acanthoscelides obtectus (Say), is a widely distributed insect
pest of stored beans (Phaseolus vulgaris L) wherever the crop is cultivated or stored
(Williams, 1980), particularly in higher altitudes or cooler areas. Infestation of
beans by A. obtectus generally begins in the ﬁeld and proliferates during storage.
Females deposit their eggs individually or in clusters near, around, or between bean
seeds. Eggs of A. obtectus are not attached to the host-seed surface as are those
of cowpea weevil, Callosobruchus maculatus (F.) and Mexican bean weevil,
Zabrotes subfasciams (Bohemann). Neonate larvae bore through the testa into the
seed, where they feed and develop into adults (Williams, 1989; Pankanin-Franczyk,
1980).

In this chapter, patterns of ovipositional preference of A. obtectus and host
suitability were determined for various bean classes readily available in Michigan.
These tests were conducted to determine if the bean weevil discriminated among
bean classes within P. vulgan's or between P. vulgaris and the cowpea, Vigna
unguiculata (L.) Walp. Some bean characteristics and environmental factors that
might inﬂuence A. obtectus behavioral patterns in the laboratory were also
examined to establish valid methodology for behavioral experiments on host

colonization by the bean weevil.

32

Phi
Mo
coll
colt
bar.
and

lildi

3V0ll

33

GENERAL MATERIALS AND METHODS

Bean weevil cultures

Two colonies of Acanthoscelides obtectus were established from ﬁeld-infested
Phaseolus vulgaris in 1985; one was obtained from Mgeta (on the Uluguru
Mountains), a bean-growing region in Morogoro, Tanzania, and the other strain was
collected from a bean-growing area in Saginaw county, Michigan, USA. The
colonies were maintained ﬁrst on red kidney beans for 4 generations, then
transfered to black beans for 8 generations (due to abundance of the bean then),
and ﬁnally on light red kidney beans. Transfer of the bean weevil cultures to a red
kidney bean was done to conform to the rearing conditions of other researchers in
the USA. All colonies were kept in a walk-in environmental chamber at 25 0C,
60 i 5% RH, and 16L:8D photoperiod.

The bean weevils were reared in half-liter glass jars one-third ﬁlled with beans.
Development of the two strains was synchronized to permit comparative tests. To
avoid contamination of strains, the two colonies were kept in separate 77 x 47 x 32
cm glass cages within the environmental chamber. Further, colonies were also
kept free of the "active" morphs (Caswell, 1960; Sano, 1967; Utida, 1972; Taylor,
1974; Sano-Fujii, 1986; Messina, 1987) of the bean weevils by maintaining low
densities of insects in jars, as well as using clean jars and beans at every generation.
Eggs collected from each generation were used to propagate the bean weevil
cultures. The egg stage was used as colony propagation material so as to avoid

unintentional selection for certain adult characteristics.

light :
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comp.
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34

Bean classes

Five U.S.A. commercial P. vulgaris classes used in these experiments were:
light red kidney bean, pinto bean, great northern bean, navy bean, and black bean.
In addition, a cowpea (V. unguiculata), the black eye pea, was included for
comparative studies of ovipositional host preference / acceptance and host suitability.
Beans and cowpeas (Jack Rabbit Brand, Berger and Company) were purchased at a
lansing-area food store in 16 oz. (454 g) plastic packages. Beans in sealed plastic
bags were also stored within the environmental chamber.

In starting cultures, intact and/or slightly cracked beans were used. These
beans were visually sorted. Split beans, broken beans, beans without testa, or those
exposing at most a third of their cotyledon, were eliminated to prevent the
possibility that later intars might fall out of seeds and die. Damaged seeds were
also avoided because the frass of excavating older larval instar falling out of large
cracks in seeds would accumulate in jars, making it difﬁcult to collect eggs after
adult emergence and oviposition. Excessive frass also enhanced development of

molds in jars.

Procedures

In every experiment, beans were sorted according to the requirements of the
tests. For example, beans selected were either intact, slightly cracked at each end,
or split. In regular ovipositional tests, seeds with intact seed coats were preferred
to damaged seeds because it was easier to ﬁnd eggs. Cracked seed coats provided
bean weevil females with cracks to insert and "hide" their eggs under the testa.
Experiments were generally placed within glass cages (aquaria) similar to those

holding culture jars in the same walk-in environmental chamber.

CD5

(3011

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inla

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HUI]

Cha

35

In oviposition experiments, generally, 1-7 day-old adults were used. To
ensure mating, both male and female adults were released together into dishes
containing beans. Beetles were sexed according to Halstead ( 1964) and Horacio
(1977). Eggs and larvae used in comparative tests were also age-synchronized.
Insect introductions to experimental petri dishes were randomized within blocks and
dishes were likewise positioned within cages in a randomized block design. Light
intensity in the cages housing cultures and experiments was about 740 lux. The
source was cool and warm-white ﬂuorescent lights. All cultures and experiments

were maintained within the environmental chamber unless otherwise stated.

EXPERIMENT 1

Effects of Bean Classes, Photoperiods and Petri dish type on Ovipositional
Preference and Larval Survival of the Bean Weevil in a Two-choice Test.

Mri th

Five bean (P. vulgaris) classes used in this experiment were: black bean (B),
light red kidney bean (K), navy bean (N), pinto bean (P), great northern bean (G),
and a cowpea (V. unguiculata), black eye pea (C). For convenience, the cowpea
will in this report sometimes be referred to as a bean class. All seeds used had
intact seed coats and were from the same respective stock.

The black bean was used because it had been the host of the bean weevil for
six generations by the time this experiment was conducted, and it received high
numbers of eggs in a no-choice situation. Bean weevils used in all experiments in
Chapter 1 were obtained from the Tanzania stock colony. Beetles used for

Experiment 1 were from the tenth generation.

tom
the t

103

photo,
on the

méSb/c

36

Glass and plastic (Corning Brand) petri dishes (60 mm diam. x 15 mm high)
were each divided into two equal sections by two parafﬁn wax (Walnut Hill
Company) ridges about 2m high (Figure 1). Black beans were spread in a
monolayer on one half of the dish. An equal weight of test or variable bean was
spread on the other half. Ten pairs of adult bean weevils (1-7 day olds) were
introduced at the center of each dish, for all treatment combinations. The six bean
combinations were replicated six times for each of the two petri dish types. Half of
the replicates from each dish type were exposed to total darkness and the other half
to a 16L:8D photoperiodic regime.

Two glass cages, each measuring 77 x 47 x 32 cm were used to establish the
photoperiod conditions. For total darkness, one cage was painted completely black
on the outside and its access ports were covered by two layers of black cloth (28
mesh/cm) to exclude light but permit ventilation within the cage. The other cage
was not painted but had a similar cloth covering the access openings to ensure
uniformity. All dishes were placed inside their appropriate cages held in a walk-in
environmental chamber maintained at 25 0C, 65 t 5% RH, and 16L:8D
photoperiod.

After 72 h, adult beetles and beans were removed from one section of the dish
at a time, and eggs were counted. The adults were then discarded. All beans
were returned carefully to the appropriate dish section, and the dishes were
returned to their original cages where they remained during egg hatch and larval
penetration into seed. After two weeks, beans from each section of a dish were
transferred to separate dishes for monitoring adult emergence. Adults were
counted and removed from dishes daily for 21 days.

This .6 x 2 x 2 factorial experiment was analyzed by a three-way ANOVA.

Data for number of eggs laid by the bean weevil were square root transformed and

37

 

Petri dish

 

 

 

Wax ridges

Figure 1. Petri dish (60 x 15 mm) divided into two sections by two wax ridges.

 

 

da
de

38

that for adult emergence was log(x+ 1) transformed for homogeneity of variances

prior to analysis of variance.

Reselge end Dieeuesien

1 n It

When beans were presented in a two-choice test, differences in numbers of
eggs laid by the bean weevil among black beans and variable beans were signiﬁcant
(P<0.001, ANOVA) (Table 1). A. obtectus females preferred black bean over
other beans for oviposition. The descending rank order of preference was cowpeas
(C), great northern beans (G), navy beans (N), light red kidney beans (K), and
lastly, pinto beans (P). Probably, the use of black bean as a rearing substrate for
the bean weevil for six generations prior to testing selected for high acceptance of
this bean.

Environmental factors including light and petri dish types evaluated in this
experiment had considerable inﬂuence on the ovipositional behavior of A. obtectus.
Signiﬁcantly more eggs per dish were laid in total darkness (116.7 t. 9.4, mean:SE)
than under the 16L:8D light regime (91.6 :t 8.4) (P = 0.001, ANOVA). These
data conﬁrm the report of Howe and Currie (1964) that bright light intensity
decreases oviposition by A. obtectus.

Difference in ovipositional responses between dishes of different material
types was also evident. Mean eggs laid in glass petri dishes were signiﬁcantly
higher (120.6 3 9.7) than for plastic petri dishes (87.7 i 7.8) (P <0.001, ANOVA).
Perhaps, the beetles may have somehow adapted to glass containers (glass rearing
jars) or, more likely, plastic dishes might emit signiﬁcant levels of inhibitory
chemicals.

Tabl
clas

combi

39

Table 1. Eggs laid by A. obtectus exposed to six bean
classes tested against the black bean in a two-choice test.

 

Number eggs laid (Mean i SE)

 

 

 

Bean Black Variable Total
combination1 bean bean
2

B-B 109.5 i 15.1 b 133.4 i 15.4 a 242.9 i 27.3 ab
B-C 188.7 i 20.1 ab 75.0 i 12.0 b 263.7 i 30.0 a
B-G 115.6 i 16.4 ab 52.8 i 9.5 b 168.4 : 23.1 ab
B-N 192.4 1 22.3 a 46.8 i 10.3 b 239.2 i 23.3 ab
B-K 140.0 1 21.0 ab 36.7 i 4.3 bc 176.7 i 22.1 ab
B-P 143.1 1 21.6 ab 15.9 i 3.6 c 159.0 i 20.6 b
1

Bean combination = black bean vs. variable bean. B =

black bean, C = cowpea, K = light red kidney bean, N = navy
bean, G = great northern bean, P = pinto bean.

2

Means followed by the same letter within a column are not
significantly different at P = 0.05, Tukey's studentiiEg
range (HSD) test. Data were transformed to (x+0.5)
prior to factorial ANOVA. (N = 12).

nean
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Larval survival was signiﬁcantly higher for cowpea (Table 2) than for the other
bean types which followed in this rank order: light red kidney, pinto, black, navy,
and great northern bean. Obviously, the pattern of larval survival did not agree
with that of ovipositional host acceptance by the adult A. obtectus (Table 1).

Bean weevil adult emergence was not signiﬁcantly affected by the two light
treatments in this test (P=O.464, ANOVA). On the other hand, container type
appeared to have a substantial inﬂuence on the resulting adult emergence.
Signiﬁcantly more adults emerged from glass (8.8 :t 1.4) than plastic petri dishes
(5.1 .4: 1.0) (P = 0.002, ANOVA). High adult emergence from glass dishes can be
explained by the greater oviposition in glass dishes. There is also a possibility that
plastic dishes provided less favorable conditions (e.g. volatiles, texture) for seed
boring by neonate larvae. Some larvae attempted boring or bored through the
plastic dishes instead of entering beans. Glass containers were therefore employed

thereafter.

EXPERIMENT 2

Host-selection behavior and survival ofA. obtectus larvae
when presented with their rearing host vs. alternative
host a two-choice test.

Mri Mh

- l i rviv
A two-choice test was conducted using black bean and cowpea to determine
whether larvae move from the site of egg deposition to bean seeds at an adjacent

site. A60 x 15 mm glass petri dish was divided into two equal sections by an

Tabl
clas

last

41

Table 2. Adult emergence of A. obtectus from six bean
classes tested against the black bean in a two-choice test.

 

Number adults emerging (Mean i SE)

 

 

Bean Black Variable Total
combination1 bean bean
2
B-C 5.7 i 1.5 a 31.8 i 3.1 a 37.5 i 3.0 a
B-K 2.5 i 1.4 a 12.9 i 2.5 b 15.4 i 2.6 ab
B-P 2.9 i 1.1 a 6.5 i 2.2 be 9.4 i 2.7 bc
B-B 4.7 i 2.9 a 5.2 i 1.7 cd 10.0 i. 3.7 bc
B-N 5.5 i 2.0 a 1.3 i 0.7 de 6.8 i 2.0 bc
B-G 3.7 i 1.9 a 0.4 i 0.2 e 4.2 i 1.9 c

 

1

Bean combination = black bean vs. variable bean per dish.
B =b1ack bean, C = cowpea, K = light red kidney bean, N =
navy bean, G = great northern bean, P = pinto bean.

2

Means followed by the same letter in a column are not
significantly at P = 0.05, Tukey's studentized range (HSD)
test. Data were log(x+1) transformed for a valid
factorial ANOVA. (N = 12).

in

the

42

approximately 2 mm high parafﬁn wax ridge. Approximately ten black bean seeds
were spread on one side of the dish, and same number of cowpea seeds were spread
on the opposite side. Visually intact bean seeds were used in each case. The
cowpea seed size was similar to that of the black bean (means = 220 and 250 mg,
respectively). Twenty eggs (1-2 days old) from a bean weevil culture (13th
laboratory generation) were placed among beans of one type at the center of one
half of the dish. A block, therefore, consisted of one dish with eggs in the cowpea
section and another dish with eggs in the black bean section. Eggs were introduced
into the two dishes randomly via a ﬁne paint brush. There were 20 replications for
the bean class x egg-placement site (2 x 2) factorial experiment.

Unhatched eggs were counted two weeks after eggs were introduced into the
dishes. One week later, beans were transferred from each section of a dish to
separate dishes for assessing adult emergence. Emerging adults were counted and
removed from dishes daily. Numbers of seeds damaged by the bean weevil and
numbers of larvae found dead outside the bean seeds were also recorded for each
treatment combination. A two-way ANOVA was used to analyze the data, and
treatment means were compared using Tukey’s Studentized Range Test (SAS

Institute 1985).

Eﬂmiheanseedrnatonlmm

A two-choice experiment was conducted to determine the effect of
unpunctured and punctured seed coats on larval survival of the bean weevil. Seed
coat condition was considered a possible factor contributing to the differences in
larval survival observed between the cowpea and black bean. Eight seeds of black
bean were placed on one side of a dish (Figure 1) and eight of cowpea on the
opposite side. In an experimental block, one dish had intact seeds (seed coat

perforation = 0), and the other dish had seeds with one perforation in each cheek

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(P<0.001, AV

43

(seed coat perforation = 1). The testa of the perforated seed treatment was
artiﬁcially punctured with a dissecting needle tip so that the cotyledon was exposed
through an approximately 0.22 mm diam x 0.2 mm deep hole.

Twenty eggs (1-2 days old) from the 13th generation bean weevil stock were
randomly introduced at the center of each dish to ensure equal chances of larval
dispersal to each of the two bean sections. The 2 (bean class) x 2 (seed coat
perforation) factorial experiment was replicated 20 times. The numbers of
unhatched eggs were counted two weeks after eggs were introduced into the dishes.
Three weeks after the initiation of the experiment, beans from each section of the
dish were transferred to a separate dish for monitoring adult emergence.
Beginning with the ﬁrst day of adult emergence, the number of beetles eclosing was
recorded daily and the beetles were removed from dishes at each count. Numbers
of bean seeds damaged by the bean weevil were recorded for assessment of
infestation levels in each bean class. Numbers of larvae found dead outside of

beans for each dish section were also recorded. The experiment was analyzed

using a two-way AN OVA.
Besultsanﬂlimsion
f - l n i l rviv

Infestation levels of the bean weevil were signiﬁcantly affected by asymmetric
egg-placement (P = 0.046, ANOVA) in the two-choice tests (Table 3). In general,
more seeds were infested (2.3 i 0.2, mean 1 SE) when eggs were placed among
cowpeas than black bean (1.9 1- 0.2). Furthermore, infestation levels were
signiﬁcantly higher for the cowpea (3.0 i 0.2) than black bean seeds (1.2 1 0.1)
(P< 0.001, AN OVA) irrespective of egg-placement site. Apparently, neonate

larvae move about and examine various beans before penetrating a seed.

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A signiﬁcant interaction was found between egg-placement site and bean class
(P=0.003, AN OVA). As shown in Table 3, signiﬁcantly more adults emerged from
cowpea than black bean for either egg placement site (P = 0.01, Student-Newman-
Keuls (SNK)). The bean class effect was also highly signiﬁcant (P<0.001,
ANOVA). Mean numbers of adults emerging from cowpea seeds were 10.9 i 0.7
compared to 3.4 1 0.4, for black beans. Egg-placement site, by itself, was not a
signiﬁcant (P = 0.23, ANOVA) factor.

Mean overall larval mortality outside the bean seeds was signiﬁcantly higher
for cowpea (1.6 i 0.3) than black bean (0.7 i 0.2) (P = 0.03, ANOVA) (Table 3).
It is probable that some larvae moved to cowpeas after failing to bore through the
seed coats of black beans. Such larvae may have exhausted their energy reserves
for boring and died or become dependent on ﬁnding entry holes already bored by
other larvae.

In summary, some emerging larvae moved from the site of egg deposition to
the other side of a dish as evidenced by adult emergence from both bean hosts
separated by the wax ridge. Movement of larvae from one section to another was
also directly evidenced by dead larvae found (two weeks after egg introduction) near

seeds in both sections of a dish.

119W

Seed infestation by bean weevil larvae in each bean class with and without
perforation is presented in Table 4. When bean weevil eggs were placed between
the two bean types in a dish, numbers of seeds damaged were signiﬁcantly higher for
intact cowpea than intact black bean. However, among seeds with one perforation
per cheek, signiﬁcantly more black bean seeds were infested than cowpea, hence the
signiﬁcant interaction (P<0.001, ANOVA). Cowpea infestation was similar with

or without seed coat perforation; however, infestation of black bean seeds increased

46

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47

about three fold with perforation. Moderate resistance to A. obtectus was
therefore imparted by the seed coat of the black bean. When the seed coat was no
longer a barrier, the black bean became even more suitable than the cowpea to A.
obtectus. The overall number of bean seeds infested (mean 1- SE) was 2.6 t 0.2
for intact bean seeds, and 4.7 t 0.3 for seeds with one perforation in each cheek of
the seed.

Adult emergence from intact seeds was slightly higher for cowpea than black
bean (Table 4), whereas with seed coat perforation, adult emergence differed
signiﬁcantly between bean classes. This was conﬁrmed by a signiﬁcant interaction
between seed coat perforation and bean class (P<0.001, ANOVA). Bean class
effect was signiﬁcant (P=0.001, ANOVA); mean adult emergence from black bean
was 9.6 t 0.7, and 7.3 i 0.5 from cowpea. In addition, no dead larvae were found
in either section of the dish with perforated seeds, however, mean number of dead
larvae found outside intact seeds was 0.6 i 0.1. This clearly indicates the
signiﬁcant (P<0.001, ANOVA) effect of the seed coat upon bean suitability to A.
obtectus larvae. I conclude that the black bean is less suitable than the cowpea for

larval survival because of seed coat factors.

EXPERIMENT 3

Larval survival of the bean weevil as affected by seed coat
perforation in three bean classes.

W905

To determine if increasing the number of seed coat perforations on a seed
would increase larval survival signiﬁcantly, and if this would also vary among

different classes of Phaseon vulgaris, three-choice and no-choice tests were carried

 

It—l

48

out using: 1) three bean classes (light red kidney bean, black bean, and great
northern bean) with, 2) three different bean seed coat conditions (seed with intact
seed coat, seed with one perforation per cheek, and seed with 5 perforations per
cheek). Eggs from the 15th generation of the bean weevil stock were used for both
tests. The last two generations were reared on light red kidney bean. The change
to kidney bean as host for rearing was done to provide a similar host for the bruchid

as those used by other bean weevil researchers in the USA.

Three-eheiee 1§§l

One seed of each of the three bean classes, was placed in a 60 x 15 mm glass
petri dish. The three beans contained in a dish were either: 1) intact, 2) with one
seed coat perforation per cheek, or 3) with 5 seed coat perforations per cheek
(Figure 2). The 3 x 3 factorial experiment was replicated 20 times. To each dish,
21 one-two day-old eggs were introduced in random fashion. Unhatched eggs were
counted 14 days later. During the third week, each bean was transferred to a
separate dish for monitoring adult emergence. Emerging adults were counted and
removed from dishes over three weeks. The percentage of bean weevil survival
was calculated for each bean class on the basis of total eggs hatched in a given dish.
The data were arcsine transformed prior to subjecting them to analysis by a general

linear models procedure (GLM) (SAS Institute 1985).

bin-Mm

In another 3 x 3 (bean class x seed coat perforation) factorial experiment, a 60
x 15 mm glass petri dish was provided with: 1) only one seed of either light red
kidney bean, black bean, or great northern bean, at a time, and 2) the seed had
either an intact seed coat, one seed coat perforation on each cheek, or had ﬁve

perforations on each cheek. Thus, a block consisted of nine dishes with one bean

49

(a)

(b) ‘

E

(C)

Figure 2. Bean seed coat conditions: (a) intact (no seed coat perforation), (b) one
percfloration in each cheek, and (c) ﬁve perforations in each cheek of the
see .

seed
W69)
not l
remc

data

pOOle

50

seed per dish and was replicated 20 times. Each dish received ten 1-2 day-old bean
weevil eggs. Two weeks after eggs were introduced into dishes, the eggs that did
not hatch were counted. Adults emerging from each seed were counted and
removed from the dishes daily for a period of three weeks. Percentage survival

data were arcsine transformed and analyzed by AN OVA (SAS Institute 1985).

Results and Diseussien

W

When emerging larvae were exposed to three seeds of three bean classes in a
dish, their survival differed signiﬁcantly (P<0.001, GLM) with bean classes and seed
coat perforation (Table 5). With non-perforated seed coat, larval survival was
signiﬁcantly higher for light red kidney bean than the other two bean classes. The
trend in adult emergence among the three bean classes remained constant
throughout treatments (Light red kidney bean > Great northern bean > Black
bean). However, for bean seeds with one perforation in each cheek, the
differences in percentage larval survival among bean classes were reduced. These
differences were no longer statistically signiﬁcant among beans with 5 perforations
per cheek. Differences among bean classes decreased with increasing perforations
per seed, hence the signiﬁcant interaction between bean class and seed coat
perforation (P<0.001, GLM). Apparently, once inside, all larvae survived almost
equally well within any bean type tested.

Pooling bean class effects, the means for seed coat perforation effects on
larval survival are presented in Table 6. Beans with seed coat perforations had
signiﬁcantly higher larval survival than those with no perforation (P=0.01,
Bonferroni t-test). Furthermore, when effects of seed coat perforation were

pooled, light red kidney bean had a signiﬁcantly higher proportion of larvae

Table 5.
on the 5

Bean

Table 5.

Effects of seed coat perforation and bean class

 

 

 

 

on the survival of A. obtectus in a three choice test.
No. of Percent larval
Bean class perforations survival
per cheek of
bean N (Mean 1 SE)
1
Kidney 5 20 35.6 i 2.6 a
Great northern 5 20 28.4 i 2.4 a
Black 5 20 26.2 i 2.5 a
Kidney 1 20 39.5 i 3.1 a
Great northern 1 20 30.2 i 2.6 ab
Black 1 20 19.6 i 2.2 b
Kidney 0 20 43.9 i 4.3 a
Great northern 0 20 12.8 i 4.0 b
Black 0 20 3.8 i 1.7 b

 

1

Means followed by the same letter in a column in a given
section of the table are not significantly different at P =
0.01, Bonferroni t-test. Data were arcsine transformed
prior to analysis of variance (GLM).

52

Table 6. Effects, of’ seed. coat. perforation on larval
survival of A. obtectus in a three choice test.

 

 

No. of % Larval1
perforations survival
per cheek of

seed N (Mean i SE2)
5 60 30.0 i 1.5 a
1 60 29.8 i 1.8 a
0 60 20.1 i 3.0 b

 

1
Pooled data for light red kidney bean, great northern, and
black bean classes.

2

Means followed by the same letter in a column are not
significantly different at P = 0.01, Bonferroni t-test.
Data were arcsine transformed for a 'valid analysis of
variance (GLM).

53

surviving than did the other bean classes (Table 7).

W

Seeds presented singly with varying seed coat perforations yielded signiﬁcant
differences in percentage larval survival among the three different bean classes
(P< 0.001, ANOVA). Larval survival in intact seeds was higher for light red kidney
bean than the other two beans (Table 8). On the other hand, no statistical
differences in percentage larval survival were observed among the three bean
classes at both one and ﬁve perforations of the seed coat (P=0.01, SNK).
Differences. in larval survival among bean classes were reduced with perforation of
seed coats. The overall survival was signiﬁcantly lower (Table 9) for seeds with no
seed coat perforation than seeds with perforated seed coats (P=0.01, Bonferroni t-
test). Seed coat perforation remarkably improved larval survival of the bean

weevil.

EXPERIMENT 4

Suitability of six bean classes as hosts of the bean weevil.
M ri l l h

To determine the suitability of six bean classes in larval growth and
development of the bean weevil, an experiment was conducted using all six
commercial bean classes (light red kidney bean, black bean, navy bean, pinto bean,
great northern bean and cowpea). In a 60 x 15 mm glass petri dish, one neonate
bean weevil larva was presented with only one seed of one bean class. This was

repeated for all bean classes. A block in this experiment consisted of 12 dishes

Table
obtect

Gre

Poole

Mean;
Si9n:
Data
(Gm

54

Table 7. Effects of bean class on larval survival of A.
obtectus in a three choice test.

 

 

 

Bean % Larval
class survival1
N (Mean 1 SE2)
Kidney 60 39.6 i 2.0 a
Greatnorthern 60 23.8 i 2.0 b
Black 60 16.5 i 1.7 h
1
Pooled data for beans having 0, 1, and 5 perforations.
2
Means followed by the same letter in a column are not
significantly different at P = 0.01, Bonferroni t-test.

Data were arcsine transformed prior to analysis of variance
(GLM) .

Tab.
on

55

Table 8. Effects of bean class and seed coat perforation
on larval survival of A. obtectus in a no-choice test.

 

 

 

No. of % Larval
Bean class perforations survival
per cheek of 1
bean N (Mean 1 SE )
Kidney 5 20 90.5 i 2.8 a
Great northern 5 20 87.5 i 3.3 a
Black 5 20 79.3 i 3.1 a
Black 1 20 80.9 i 3.4 a
Great northern 1 20 77.1 i 4.2 a
Kidney 1 20 76.0 i 5.5 a
Kidney 0 20 48.9 i 8.2 b
Great northern 0 20 8.9 i 5.0 c
Black 0 20 4.0 i 2.9 c
1
Means followed by the same letter in a column are not
significantly different at P = 0.01, SNK test. Data were

arcsine transformed prior to analysis of variance.

56

Table 9. Effects of seed coat perforation on larval
survival of A. obtectus in a no-choice test.

 

 

No. of % Larval
perforations survival1
per cheek of

seed N (Mean : SE2)

5 60 85.9 i 1.9 a
1 60 77.0 i 2.6 a
0 60 20.6 i 4.2 b

 

1

Bean classes were pooled.

2

Means followed by the same letter in a column are not
significantly different at P = 0.01, Bonferroni t-test.

Data were arcsine transformed prior to analysis of
variance.

where:
set of si
Perforat
assessme
on lanai
in prexic
Be,
and the '
Adults u
at 65 0(
develOpi
(SAS In
analyzer

(SAS Im

57

where: 1) one set of six dishes contained intact bean seeds only, and 2) the other
set of six dishes had one seed coat perforation in both cheek areas of the seed.
Perforation of seed coat was included as an additional factor to allow separate
assessment of effects of seed coat and those of nutritional quality of bean cotyledons
on larval host-suitability. Seed coats were perforated using the same technique as
in previous experiments. The 6 x 2 factorial experiment was replicated 10 times.
Beginning with the third week of the experiment, dishes were observed daily
and the number of emerging adults and the emergence date for each was recorded.
Adults were placed individually in vials at -15 0C for 2 days. They were then dried
at 65 0C for 24 h prior to obtaining individual dry weights. Dry weights and
developmental periods were analyzed by a general linear models procedure (GLM)
(SAS Institute 1985). The numbers of larvae surviving to the adult stage were
analyzed using a two-way cross-tabulation or contingency table for the 2 variables

(SAS Institute 1985).

Igrvﬂ survival

Effects of bean class were not independent of effects of seed coat perforation
(Log-Likelihood Ratio Chi-square: G=12.148; df =5; P=0.033 (0.01>P>0.05)) in
this experiment as well. This suggests an interaction between the two factors
affecting larval survival. The distribution of adult bean weevil emergence varied
among the six beans with the two seed perforation treatments (Figure 3). larval
survival was low in seeds without perforation. N 0 adults were recovered from
black, navy, and great northern beans with intact seed coats, however, in beans with
one seed coat perforation in each cheek, adults emerged from all six classes.

Suitability of different bean classes for larval survival, particularly during seed entry,

Fl;

58

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g 12. Intact Seed Coat

g . Cl Perforated Seed Coat

E 104 —

2 . _ __ __

Lu

5 8- — -—

D

9 5.

LL

0 1

>- .

o 4 §

5 ‘ V § §

E o1 \I S I ﬂ I \1
K N G B C

 

BEAN CU LTIVARS

Figure 3. Distribution of Acanthoscelides obtectus adults emerging from seeds of six
bean classes with and without seed coat erforation treatment. Bean class
codes: K = light red kidney; P = pinto; = navy; G = great northern; B =
black; C = cowpea. Log-likelihood ratio for contingency tables signiﬁed
lack of independence between bean class and seed coat perforation
treatments (P < 0.033; df = 5).

59

may have been hindered by the seed coat condition (hardness, thickness and

chemistry), or seed size, or shape of a given seed or bean class.

Whirlwind

Developmental periods of A. obtectus for each bean class were identical
(Table 10). The same is true for effects of bean class with the two seed coat
perforation treatments (Figure 4). When bean classes were pooled, seed coat
perforation did not signiﬁcantly affect developmental time of the bean weevil larvae
(P=0.89, GLM). Mean number of days taken for development (:SE) by ﬁrst
instar larvae from introduction to adult emergence was similar for both seed coat
treatments, i.e., 29.9 :t 0.6 days for beans with intact seed coats, and 29.9 t 0.3 days

for perforated beans.

A l w i h

Adult dry weights of the emerging bean weevils were not signiﬁcantly affected
by bean class, seed coat perforation, or by the interaction of the two factors (Table
11). Dry weights among the six bean classes were unaffected by the two
perforation treatments (Figure 5).

Host suitability of the six bean classes did not differ once the larvae entered
the seed. The main problem in host colonization by the ﬁrst instar larvae appears
to be associated with the entry process through the seed coat. In addition, stability
of a single seed in a ﬂat dish was not achieved with all beans tested in this
experiment. Some beans (e.g. navy bean) were occasionally observed to rock as

the larva attempted boring from the side of seed facing the bottom of the petri dish.

60

Table 10. Effects of bean class on developmental period of
A. obtectus larvae.

 

Elapsed time until1

 

Bean class N adult emergence (days)
(Mean 1 SE)
Black 9 29.6 i 0.3 a2
CowPea 13 28.6 i 0.4 a
Kidney 11 30.5 i 0.7 a
Navy 8 30.5 i 0.7 a
pinto 13 30.2 i 0.4 a
Great northern 9 30.3 i 0.5 a

 

1
Number of days from larval introduction to been seeds until
adult emergence.

2

Means followed by the same letter were not significantly
different at P = 0.01, SNK test. Seed coat perforation
effects were pooled.

61

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

’79: so

9‘: Intact Seed Coat

o 40,, E] Perforated Seed Coat
93

E O a a o a a 0 a a
2’ 30- -I- § F‘ -I- *- .. § F1!
g a a
o \ \
.1 \ \
.. a s
B 101 § §
° s s
g 0 s s
2 ' 13 N E; it c

BEAN CULTIVARS

Figure 4. Inﬂuence of seed coat perforation and bean class on the developmental
period (mean t SE) of Acanthoscelides obtectus larvae. Bean class codes
are given in Figure 3. Developmental period = number of days from larval
introduction onto beans to adult emer ence. Means were not signiﬁcantly
different at P=0.01, Student-Newman- euls’ test (SNK).

Table 11.

62

General linear' models procedure
analysis of variance of adult dry weights of

(SAS) for
obtectus

when larvae were exposed to six bean classes with two seed

coat perforation treatments.

 

 

Source df MS F P
Bean class 5 0.21 1.02 0.417
Seed coat perforation 1 0.23 1.08 0.304
Interaction 5 0.08 0.39 0.676
Error 54 0.21

 

 

(I‘ll-‘1

Fl

63

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5.0
’g“ . Intact Seed Coot
v 40- El Perforated Seed Coat
e . .
a
g 0 a a a a n; g-
; 3.0- Ti“ er {7 {a .1. $
‘ \
o 2.0- S
a t
D \
g 1.0- §
2 a
000 I I I T I U
K P N G B C

BEAN C ULTIVARS

Figure 5. Inﬂuence of seed coat perforation and bean class on dry weights (mean 2
SE) of the emerging Acanthoscelides obtectus adults. Bean class codes are
given in Figure 3. Means were not statistically different at P=0.01, SNK.

at
01’

sta

eac
and
am

64

EXPERIMENT 5

Effects of bean seed stability on larval performance of the
bean weevil in six bean classes.

Materials and Metheds

In the previous experiment wherein a single seed was presented individually in
a ﬂat dish, some larvae were observed to rock small bean seeds while attempting
boring, and this was more obvious with rounder seeds. Under natural storage
conditions, however, beans are stored in bulk and thus are tightly packed. These
seeds are relatively stationary when the bean weevil larvae attempt boring. Bean
stability during larval boring particularly on seeds with intact seed coats might be
necessary for successful penetration of ﬁrst instar larvae into bean seeds in stores.

To conﬁrm this, an experiment was set up where bean seeds were set in a dish
so as to provide additional purchase to larvae during boring. One seed of a given
bean class was placed in a 60 x 15 mm glass petri dish; one end of the seed was
attached to the side of the dish by means of melted wax (Figure 6) so that one cheek
or a part of it lay tightly against the ﬂoor of the petri dish. The bean was then
stationary and in contact with another surface. Light red kidney, black, navy, pinto,
and great northern beans as well as cowpea were used in this experiment. For
each bean class, a bean seed in a dish was either: 1) intact and unattached, 2) intact
and attached, 3) perforated once per check (as in previous experiments) and
attached, or 4) perforated once per cheek and unattached. The 6 x 2 x 2 factorial
experiment was replicated 10 times.

To each dish with one seed, a day-old larva was released close to the bean.

Larvae used in the experiment were from a stock culture of bean weevils maintained

65

Bean seed

 

 

 

U
Wax =E§;:::> ~r Petri dish

 

 

Figure 6. Bean seed attached to glass petri dish by melted wax. Part of one cheek
of the bean is in contact with the ﬂoor of the dish.

66

for 20 generations in the laboratory. Observations for adult emergence began by
the third week after experimental setup, and the numbers of adults emerging and
dates of emergence were recorded daily. Adults were placed individually in vials
for storage at -15 0C for at least 2 days, and were dried thereafter at 65 0C for 24
hours, and weighed. A three-way general linear models procedure (GLM) (SAS
Institute 1985) was used for analysis of the bean weevil developmental periods and
dry weights of adult bean weevils. Contingency tables (SAS Institute 1985) were

employed in the analysis of larval survival.

WW1

larval survival

The presence of an interaction or association between seed attachment and
seed coat perforation effects on larval survival of the bean weevil was strongly
evident in this study (Likelihood Ratio x2 = 14.99; df = 1; P<0.001). Seed
stabilization by attachment to a dish alone increased larval survival 19 times when
compared to survival on unattached beans (Table 12). On the other hand, even
though seed stabilization contributed substantially to better survival, seed coat
perforation alone had a higher impact on larval survival. The combination of seed
stabilization and seed coat perforation gave the highest survival of the bean weevil
larvae (Table 12).

An association between effects of seed attachment and effects of bean class on
larval survival was not obvious in this treatment combination (Pearson’s x2 = 3.06;
df = 5; P = 0.69). In other words, differences in the pattern of larval survival with
the two seed attachments were not highly evident among the bean types (Table 13).
Likewise, the pattern of larval survival was similar among the six bean classes with

two different seed coat perforation treatments (Likelihood Ratio x2 = 9.04; df = 5;

Table 12.

attachment on larval survival

67

Effects of seed. coat jperforation. and seed
(number' and. percentage of

adults emerged) of the bean weevil: a contigency table.

 

Seed coat

ADULT EMERGENCE1

 

Seed attachment

 

 

 

 

perforation Unattached Attached Total
0 1 (0.8%) 19 (16%) 20 (16.8%)
1 46 (38.7%) 53 (44.5%) 99 (83.2%)
Total 47 (39.5%) 72 (60.5%) 119 (100%)
1

Bean class data were pooled.
test for the contigency table: G = 14.99;

0.001.

Likelihood ratio chi-square

df ==Il; P <

68

Table 13. Effects of bean class and seed attachment on
larval survival (number and percentage of adults emerged) in
the bean weevil: a contigency table.

 

ADULT EMERGENCEI

 

Seed attachment

 

 

 

 

Bean class Unattached Attached Total
Black 7 (5.9%) 16 (13.5%) 23 (19.3%)
Cowpea 7 (5.9%) 13 (10.9%) 20 (16.8%)
Kidney 6 (5.0%) 13 (10.9%) 19 (16.0%)
Navy 9 (7.6%) 10 (8.4%) 19 (16.0%)
Pinto 8 (6.7%) 10 (8.4%) 18 (15.1%)
Great northern 10 (8.4%) 10 (8.4%) 20 (16.8%)

Total 47 (39.5%) 72 (60.5%) 119 (100%)

1

Seed coat perforation data were pooled. Pearson's chi-

square test for the contingency table: Chi-square value =
3.06; df = 5; P = 0.69.

69

P = 0.107) (Table 14). Larval survival was over two times higher in beans with
perforated seed coats than those without perforations. Again, seed coat
perforation improved survival of the bean weevil in beans irrespective of class
differences.

The stabilized seed simulated beans found in containers under normal storage
conditions. Bean stabilization improved larval survival perhaps by providing some
leverage during initial boring on the bean surface. Furthermore, perforation of the
seed coat exposed the cotyledon and subsequently improved survival of A. obtectus

through the removal of a barrier, the seed coat.

W

Although signiﬁcant effects of seed attachment with seed coat perforation
were not detected in this test (Table 15), arithmetically slight but statistically
signiﬁcant variation in the developmental period of the bean weevil among the six
classes of beans was observed (F=3.62; df =5; P=0.005; GLM) (Figure 7). The
quickest larval development occurred in cowpeas. There is a possibility that seed
coats and cotyledons of the cowpea and some other beans are easier to bore through
than others, and this among other factors may have contributed to the observed

differences in the developmental rates of the bean weevil.

W

Adult dry weights of the emerging bean weevils did not differ signiﬁcantly with
different bean classes, seed coat perforation or seed attachment treatments (Table
16). The weights of the F1 adults of A. obtectus were similar for insects reared on
the six bean classes regardless the presence or absence of seed coat perforation or

stabilization of the seed (Figure 8).

Table 14.

70

Effects of bean class and seed coat perforation

on larval survival (number and percentage of adults emerged)
in the bean weevil: a contingency table.

 

ADULT EMERGENCEI

 

Seed coat perforation
per cheek of bean seed

 

 

 

 

Bean class 0 1 Total
Black 6 (5.0%) 17 (14.3%) 23 (19.3%)
Cowpea 6 (5.0%) 14 (11.8%) 20 (16.8%)
Kidney 4 (3.4%) 15 (12.6%) 19 (16.0%)
Navy 1 (0.8%) 18 (15.1%) 19 (16.0%)
Pinto 2 (1.7%) 16 (13.4%) 18 (15.1%)
Great northern 1 (0.8%) 19 (16.0%) 20 (16.8%)
Total 20 (16.8%) 99 (83.2%) 119 (100%)
1

Seed attachment data were pooled.
square test for the contigency table: G = 9.04; df = 5; P =

0.107.

Likelihood ratio chi-

71

Table 15. General linear ‘models ‘procedure (SAS) for
analysis of effects of seed attachment, bean class and seed
coat perforation on developmental time of A. obtectus
larvae.

 

D:
H)

Source MS F P

 

Bean class (B) 5 0.01 3.62 0.005
Seed coat perforation (P) 1 <0.01 0.21 0.647
Seed attachment (A) 1 <0.01 0.00 0.976
BXP 5 <0.01 0.88 0.499
BXA 5 <0.01 0.20 0.960
PxA 1 <0.01 0.13 0.717
BXPXA 0

Error 100 <0.01

 

Data were log (x+1) transformed.

Figm

72

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

9.

o

S

D 40' O b

O + 0 0b Ob Ob b

E F—'_ .—.——

i 301

'—

Z

“2‘ 20-

O.

O

.1

g 10- (19) (20) (1a) (19) (23) (20)

E o I I I I j 1
N G P K B C

BEAN CULTIVARS
Figure 7. Inﬂuence of bean class on developmental period (mean t SE) of

Acanthoscelides obtectus. Bean class codes are given in Figure 3. Data were
square-root transformed prior to ANOVA. Seed attachment and seed coat

data were pooled before separation of means. Means accompanied by the
Numbers m

same letter are not significantly different (P=0.01, SNK).

parentheses = numbers of beetles weighed.

73

Table 16w General linear' models procedure (SAS) for
analysis of effects of seed attachment, bean class and seed
coat perforation on the resulting dry weight of the adult
A. obtectus.

 

 

Source df MS F P
Bean class (B) 5 <0.01 0.40 0.849
Seed coat perforation (P) 1 0.02 1.73 0.191
Seed attachment (A) 1 <0.01 0.16 0.689
Exp 5 0.01 0.62 0.683
BxA 5 0.02 1.71 0.140
PxA 1 <0.01 0.09 0.759
BxPxA 0

Error 100 0.01

 

Data were log(x+1) transformed.

Fig

74

 

E O O O O O a

g 3' + + + + + +
Q

‘51

E 2‘

D

5

8

g 1‘ (19) (20) (18) (19) (23) (20)
a

2

o I ' T ' I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m4
0

N G P K
BEAN CULTIVARS

Figure 8. Adult dry weights (mean t SE) of emerging Acanthoscelides obtectus as
affected by different bean classes. Dry weight data were log(x+ 1)
transformed prior to ANOVA. Seed attachment and seed coat perforation
effects were ooled prior to mean separation test. Means were not
significantly different among the six bean classes (P=0.01, SNK). Numbers
in parentheses = numbers of beetles weighed.

fac

5Z0<

COUe
were

[USllll

[.

SWIM]

75

EXPERIMENT 6

Effects of larval density on growth and development of the
bean weevil in six bean classes.

Mri Mh

To determine the impact of larval density on larval growth and development in
the presence of a limited resource (bean seed), an experiment with the following
treatments was conducted: 1) six bean classes (light red kidney, black bean, navy
bean, pinto bean, great northern bean and a cowpea), 2) four larval densities (1, 3,
10, or 30 larvae per seed), and 3) two seed coat perforation treatments (intact or
one seed coat perforation in each cheek of the seed).

One bean seed was attached to each 60 x 15 mm glass petri dish (see
Experiment 5) to ensure seed stability during larval boring attempts into the seed.
One larval density was provided per dish. There were four blocks in the 6 x 4 x 2
factorial experiment. All day-old larvae were obtained from the same bean weevil
stock reared in the laboratory for 22 generations. The experiment was terminated
4 weeks after initial adult emergence. Data on adult emergence and weights were
collected as described above in Experiment 5. Survival and adult dry weight data
were analyzed using three-way general linear models procedure (GLM) (SAS
Institute 1985).

RlnDi in
magnum

Varying larval densities with different seed coat perforation levels affected
survival of the bean weevil larvae significantly (P<0.01, ANOVA) (Table 17).

76

Table 17. Effects of seed coat perforation and larval
density on larval survival of A. obtectus.

 

 

 

1
No. perforations No. of % survival
per cheek of larvae/bean
seed seed N (Mean i SE)
2
0 1 24 58.3 i 10.3 bcd
3 24 77.8 i 6.6 ab
10 24 53.8 i 5.6 cd
30 24 32.5 i 1.7 d
1 1 24 95.8 i 4.2 a
3 24 93.1 i 3.5 a
10 24 75.4 i 3.9 bc
30 24 35.1 i 1.1 d
1

Percentage larval survival data were arcsine transformed
for ANOVA.

2

Means followed by the same letter are not significantly
different at P = 0.01, SNK test. Bean class effects were
pooled.

SU
w},
gre
Per (

P617?

77

With no perforation in bean seed coats, percentage survival was highest at a larval
density of 3 larvae per seed. However, in beans with perforated seed coats, no
significant difference was observed in percentage larval survival at larval densities of
1 and 3, however, both differed signiﬁcantly from larval densities of 10 and 30
(P=0.01, SNK). When bean class and seed coat perforation were pooled,
percentage larval survival was best at lower densities (3 or 1 larva per seed) but
diminished signiﬁcantly at and above 10 (P_<_0.01, AN OVA) (Table 18).

Signiﬁcant variation in larval survival also occurred among the six bean classes
with different seed coat perforation treatments (P=0.04, ANOVA). As shown in
Figure 9, the light red kidney bean with perforated seed coat had the highest
survival and the great northern bean with intact seed coat had the lowest survival
when compared with other treatment combinations. Again, larval survival was
greater in bean seeds with perforated coats than in seeds with intact coats.
Percentage larval survival (mean t SE) was 74.9 t 3.0 for seeds with one

perforation per cheek, and 55.6 1 3.7 for seeds with intact seed coats.

A l w i

Adult dry weights did not vary signiﬁcantly among the six bean classes within
larval densities of 1, 3, and 10 larvae for a given seed (Table 19), but signiﬁcantly
declined in beans within larval density of 30 larvae to a seed (P = 0.01, SNK).
Within the larval density of 30, adults emerging from light red kidney beans were
the heaviest and those from black beans were the lightest. Pooling seed coat
perforation and bean class effects, adult dry weights at larval densities of 1, 3, and 10
were not signiﬁcantly different; but they varied signiﬁcantly from those of 30 larvae
per seed (Table 20). Seeds with larval density of 30 produced adults with relatively

lower dry weights than those produced at other density levels.

78

Table 18. Effects of larval density on larval survival of
A. obtectus.

 

 

 

1
No. of % survival
larvae/bean
seed N (Mean i SE)
1 48 77.1 + 6.1 a2
3 48 85.4 i 3.8 a
10 48 64.6 i 3.7 b
30 48 33.8 i 1.0 c
1
Percentage larval survival data were arcs ine transformed
for a valid ANOVA. Bean class and seed coat perforation

effects were pooled.

2
Means followed by the same letter are not significantly
different at P = 0.01, SNK test.

gun

‘11

F'

- . LC<FZhUkMQ 252

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

_l
<
E 120' Intact Seed Coot
3 [:1 Perforated Seed Coot
m 100- o
g 80- ab _} {- l “b 1
-1- ob _}
0.: ab
3 60- ab
b
E
o 40"
tr
LIJ
0- 20-
E
I 0 I "A _ I I
C K N P G

BEAN CULTIVARS

Figure 9. Percent survival (mean 1- SE) of Acanthoscelides obtectus when larvae
were exposed to six bean classes with two seed coat perforation treatments.
Bean class codes are 'ven in Figure 3. Data were arcsine transformed for
ANOVA. Density eficts were pooled. Means followed by the same letter

are not statistically different at P=0.01, SNK.

80

Table 19. Effects of larval density and bean class on
adult dry weight of A. obtectus.

 

No. of Adult
larvae/bean Bean class N dry weights
seed (mg)

(Mean i SE)

 

 

 

 

 

1 Black 7 2.71 i 0.14 a
Cowpea 6 2.15 i 0.21 a
Kidney 8 2.76 i 0.21 a
Navy 6 2.92 i 0.27 a
Pinto 5 2.42 i 0.46 a
Great northern 5 3.04 i 0.15 a
3 Black 22 2.52 i 0.10 a
Cowpea 20 2.39 i 0.11 a
Kidney 24 2.48 i 0.10 a
Navy 20 2.66 i 0.10 a
Pinto 20 2.48 i 0.09 a
Great northern 19 2.66 i 0.10 a
10 Black 52 2.48 i 0.09 a
Cowpea 52 2.43 i 0.07 a
Kidney 63 2.49 i 0.07 a
Navy 45 2.56 i 0.07 a
Pinto 57 2.64 i 0.06 a
Great northern 41 2.56 i 0.06 a
30 Black 84 1.91 i 0.07
Cowpea 87 2.32 i 0.06
Kidney 88 2.63 i 0.06
Navy 65 2.36 i 0.08
Pinto 85 2.49 i 0.05
Great northern 80 2.54 i 0.06 ab
1
Means followed by same letter in a column within a section
are not significantly different at P = 0.01, SNK test.

Density x bean class interaction was significant at P5,
0.001, GLM. Effects of seed coat perforation were
pooled. N = number of emerging adults weighed.

81

Table 20. Effects of larval density on adult weights of A.
obtectus.

 

 

 

No. of Adult
larvae/seed N dry weights
(109)
(Mean i SE)
1 37 2.67 i 0.11 a1
3 125 2.53 i 0.04 a
10 310 2.52 i 0.03 a
30 489 2.37 i 0.03 b
1
Means followed by the same letter are not significantly
different at P = 0.05, Tukey's Studentized Range (HSD)
test. Effects of seed coat perforation and bean class
were pooled. Density effects were significant at

P 5 0.001.

82

Signiﬁcant differences in dry weights of the emerging beetles were also
observed among each combination of bean class and seed coat perforation
treatments (P_<_0.001, GLM) (Table 21). Figure 10 further illustrates the pattern of
adult dry weights among the six bean classes in relation to the two seed coat
perforation treatments. Adult weights were 2.52 i 0.03 mg for intact seed coats,
and 2.40 z 0.02 mg for perforated seed coats (P<0.001, GLM). Perhaps more
larvae entered perforated beans than intact beans, and competition for a limited
resource may have lowered adult weights in some bean classes. Pooling larval
density and seed coat perforation effects, adult dry weights were signiﬁcantly higher
in light red kidney bean and great northern bean than the other beans (Table 22).
Black bean produced adults with the lowest weights in this test. In addition to
larval density effects on larval growth, bean size and perhaps the relative ease of
penetration into certain seeds of some bean types by neonate larvae may have also
contributed to the variations in the resulting dry weights.

It is known that more than one larva may enter the same bean seed (Labeyrie,
1960; Pankanin-Franczyk, 1980; Stamopoulos and Desroches, 1981) and since bean
weevil larvae are unable to migrate to other beans once they enter a seed,
competition between or among larvae is likely. A similar situation has also been
observed in other bruchids and in other stored-product insects.

At higher densities, competition among the larvae may drastically decrease
larval growth and survival in insects. For example, in the broad horned ﬂour
beetle, Gnathocems cornutus (F.), mortality rates increased highly with increased
larval densities (Tsuda and Yoshida, 1985). Because of contest competition for
pupation sites by this stored product insect, survival of G. comutus larvae decreased
from 97.5% at density of one larva per vial to 6.2% at the density of 64 larvae per

vial containing the same amount of wheat flour as that of lower larval densities.

83

Table 21. Effects of bean class and seed coat perforation
on adult weight of A. obtectus.

 

 

No. of Adult
perforations dry weights
Bean class per cheek of N (mg)
bean seed (Mean i SE)
1
Black 0 82 2.18 i 0.07 e
1 83 2.23 i 0.08 de
Cowpea 0 78 2.47 1 0.05 abcde
1 87 2.26 i 0.06 cde
Kidney 0 91 2.69 i 0.06 a
1 92 2.44 i 0.05 abcde
Navy 0 52 2.75 i 0.07 a
1 84 2.34 i 0.06 bcde
Pinto 0 80 2.56 i 0.05 abc
1 87 2.52 i 0.05 abcd
Great northern 0 54 2.56 i 0.07 abc
1 91 2.58 i 0.05 abc

 

1

Means followed by the same letter are not significantly
different at P = 0.01, SNK test. Effects of larval
density were pooled.

. t It: IF 2 _C< 262

Figure

84

 

4.0
Intact Seed Coat
3.5- I: Perforated Seed Coat

3.0 -
2.5 . FI'
2.0 4
1.5 -
1.0 -
0.5 -

0.0 . . . .
B C K N P G

BEAN CU LTIVARS

MEAN ADULT DRY WEIGHTS (mg)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d
d

Figure 10. Inﬂuence of bean classes and seed coat perforation on dry wei ts
(mean t SE) of emerging adults of Acanthoscelides obtectus. Bean 0 ass
codes are given in Figure 3. Density effects were pooled for mean
se aration. Means followed by the same letter were not signiﬁcantly

' erent at P=0.01, SNK (also see Table 21).

85

Table 22. Effects of bean classes on adult weights of
A. obtectus.

 

 

 

Adult
dry weights
Bean class N (mg)
(Mean 1 SE)
Kidney 183 2.57 i 0.04 a1
Great northern 145 2.57 i 0.04 a
Pinto 167 2.54 i 0.04 ab
Navy 136 2.49 i 0.05 ab
Cowpea 165 2.36 i 0.04 bc
Black 165 2.20 i 0.05 c
1
Means followed by the same letter are not significantly
different at P = 0.01, SNK test. Effects of larval

density and seed coat perforation were pooled. '

86

Weight of emerging beetles is of particular signiﬁcance among internally
feeding granivorous insects e.g., Callosobruchus spp. (Smith and Lessells, 1985); the
heavier the beetles at emergence, the higher the fecundity (Giga, 1982) and
longevity (Smith and Lessells, 1985). In the bean weevils, adults do not appear to
feed under storage conditions and therefore the energy reserves accumulated during
larval feeding may determine their reproductive capacity. Among some of the
bean classes used in this study, survival and weight of the bean weevil were
signiﬁcantly affected by higher larval density, particularly above 10 larvae per seed,
apart from bean seed factors. Higher larval densities and consequently
competition for space and food within a limited resource affected larval survival,
emerging adult weight, and perhaps the fecundity levels of the emerging adults (not

examined in this study).

GENERAL DISCUSSION

Experimental considerations

The major purpose of conducting these experiments was to establish baseline
information necessary for future behavioral studies on the bean weevil in the
laboratory. Uniformity of light intensity and dish type in studying ovipositional
behavior of the bean weevils is essential. In addition, when testing for varietal
differences in susceptibility or suitability to bean weevil larvae, the use of single
unstabilized seed instead of stabilized seed (or a compact layer of seeds) in a flat
dish may distort results from the behavioral studies, particularly in smaller and
rounder seeds. Under normal storage conditions, beans are stacked compactly
within a container and are generally more stable. Extreme care to prevent sliding

of beans within petri dishes is also essential when conducting tests with neonate

cht
a It

bee

behalf
glass (1

87

larvae. First instar larvae within dishes have been observed to be easily injured by
sliding beans when the a dish was accidently tilted. Further, when evaluating host
resistance due to seed coat factors among bean classes or cultivars, integrity of the

seed coat could be crucial to accuracy.

Practical considerations

Some of the factors affecting adult and larval bean weevil behaviors may be of
signiﬁcant practical value to agriculture. Based on results of this study, it appears
that bean weevil populations in dry beans can be reduced if: 1) prior to storage
beans are handled carefully to prevent or reduce seed coat damage and
consequently reduce the ease of seed entry by the A. obtectus larvae; 2) stored
beans are "disturbed" periodically to create the unstable conditions for boring and in
turn interrupt larval boring activity; 3) beans are exposed to high light intensities
that may affect ovipositional rates in the adult beetle. Exposure of beans (spread
out in a single layer) to direct sunlight may reduce both adult and larval activities via
both light intensity and heat.

The knowledge of host choice in the bean weevils and of variation in host
choice within a strain of bean weevil is of great importance as well. It may provide
a logical approach for ﬁnding and developing improved control techniques that may

be efﬁcient and safe.

CONCLUSIONS

Environmental factors evaluated for their inﬂuence on adult and larval
behaviors of A. obtectus included petri dish type and lighting conditions. Beans in

glass dishes received more eggs and subsequently better adult emergence than those

88

in plastic dishes. On the other hand, beans under light (16L:8D) received fewer
eggs than those under total darkness. larval survival or adult emergence was not
affected by lighting conditions.

The six bean classes varied in their acceptability as ovipositional hosts and as
hosts for larval survival when presented in two-choice tests against the black bean.
Black beans were most highly favored for oviposition while cowpeas had the highest
adult emergence. In general, the pattern of larval survival did not correspond well
with that of ovipositional host acceptance in the Tanzania strain of bean weevil.

In two-choice tests, some of the neonate larvae of the bean weevil emerging
from eggs deposited within one dish section containing one bean type tended to
move away from the point of deposition to distant sites within a petri dish. When
cowpeas were tested against the black beans, higher infestation levels and hence
more adults emerged from the cowpeas than from the black bean, despite the egg-
placement site. On the other hand, when seeds were perforated and eggs were
placed between cowpeas and black beans in another two-choice test, seed
infestation and adult emergence were greater in black beans than in cowpeas.

Differences in larval survival among light red kidney, great northern and black
beans were considerably reduced with increasing number of perforations in the seed
coat. Once larvae entered beans successfully, development and growth of larvae
were generally similar among the bean classes tested. The same was true for the
ﬁve bean classes and the cowpea tested for their suitability for larval development,
growth and survival. Seed coat perforation greatly improved larval survival in all
bean classes.

Stabilizing beans within the dish also improved larval survival of A. obtectus
in intact seeds. However, with seed coat perforation, larval survival was even

greater in all beans irrespective of the seed stabilization treatment.

89

Larval density affected both larval survival and emerging adult weights. At
the higher density of 30 larvae per seed, larval survival and adult dry weights were
signiﬁcantly lower than those of one, three, and ten larvae per bean seed. This
effect was more pronounced for smaller seeds such as the black bean than for larger
light red kidney bean seeds. However, intact beans produced slightly heavier

beetles than perforated seeds at higher larval densities.

CHAPTER 2

Ovipositional preference and larval performance of a
U.S.A. vs. Tanzania strain of the bean weevil,

Acanthoscelides obtectus (Say) as inﬂuenced by bean class.

90

pYEfe
But,
COITe

(1003
1975;

of bru

INTRODUCTION

Preference for and acceptance of ovipositional hosts have been demonstrated
in bruchids either when hosts were presented simultaneously or individually.
Schoonhoven et a1. (1983) observed differences in preference and acceptance of
ovipositional hosts in both A. obtectus (the common bean weevil) and Zabrotes
subfasciatus (Boheman) (the Mexican bean weevil), when beetles were presented
with cultivated and noncultivated P. vulgar-is lines in a choice or no-choice situation.
Less resistant cultivated beans were more preferred over than wild and more
resistant accessions. Ovipositional preference behavior has also been demonstrated
in another bruchid, the cowpea weevil, Callosobmchus maculatus (F.). Nwanze
and Horber (1976) found that cowpea cultivars with smoother seed coats stimulate
greater oviposition then do those with rough seed coats.

Generally, females of insects with relatively less mobile larvae are assumed to
select suitable hosts for their offspring since these larvae will often feed on hosts
close to where the eggs were deposited (Rausher, 1979). In the cowpea weevil,
eggs are glued to seeds selected by the parent. Females of C. maculatus appear to
prefer seeds that enhance larval survival, possibly maximizing ﬁtness (Wilson, 1988).
But, this may not hold true at all times for many other insects. Existence of poor
correspondence between host preference and host suitability in insects has been
documented (e.g., Waldbauer, 1962; Hsiao and Fraenkel, 1968; Wiklund, 1974,
1975; Wasserman and Futuyma, 1981).

Inter-strain differences in oviposition and larval development in some species

of bruchids have been demonstrated. Dick and Credland (1984) reported

91

b.

92

differences in oviposition and larval development among three geographically
isolated strains of C. maculatus. A Nigerian strain laid more eggs than the
Brazilian or Yemen strains of cowpea weevil. Additionally, the Yemen strain
produced fewer adults, yet the weights of the emerging adult beetles were about
twice that of the Nigerian and Brazilian conspeciﬁcs. Pirnbert (1985) on the other
hand, compared ovipositional preference in two populations of Z. subfasciatw, one
reared on P. vulgaris and the other on P. lunatus. When presented with a choice
between the two Phaseolus species, females from both stocks oviposited highly on P.
vulgar-is; however, the number of eggs deposited by each of the two populations
differed.

In this chapter, I report on choice and no-choice tests conducted to determine
if: 1) female bean weevils discriminate among hosts within the same species (and
those of other species), 2) variability exists in some components of larval
performance among the different hosts, 3) adult host choices are positively
correlated with larval host suitability, and 4) if ovipositional host choice and larval

host suitability vary between two strains of the A. obtectus.

MATERIALS AND METHODS

Insects and Host materials

The two strains of A. obtectus used in this study were established from ﬁeld-
infested red kidney beans from Morogoro, Tanzania (TZ strain) and from infested
navy beans from the Michigan bean areas (MI strain) (See General Materials and
Methods, Chapter 1). Bean weevils used in this study were reared under the same
environmental conditions for 27+ generations and were raised continuously on light

red kidney bean for 14 generations prior to these tests. Stock cultures of the two

93

strains providing beetles for tests were also age-synchronized. Mixtures of one to
six day-old adults were used in tests.

Five locally available commercial common beans and cowpeas used in the
experiments were: light red kidney bean (K), pinto bean (P), navy bean (N), great
northern bean (G), black bean (B), and the cowpea (C), black eye pea. The term
"bean class" was used in this study to represent both bean and cowpea classes.
Only intact seeds were used. Insect rearing and all tests were conducted under

conditions speciﬁed in General Materials and Methods in Chapter 1.

Oviposition: Six-choice test

Choice tests were used to examine ovipositional preferences in the bean
weevil. Glass petri dishes (Corning, Pyrex Brand) measuring 150 mm diam. x 15
mm high were partitioned into six equal sections by 2 mm high parafﬁn wax ridges
radiating from a wax ridge circle of about 2.2 cm diameter made at the center of the
dish (Figure 11). Equal weights of each bean type were spread in an even compact
layer, one bean type in each section of the dish. Twelve pairs of adult bean weevils
of one strain from the 27th generation culture were released at the center of the
petri dish. Bean class arrangement within dishes was randomized to permit
detection of possible nearest neighbor effects. The whole procedure was repeated
simultaneously for the other strain of bean weevil; the design was randomized
complete block. Each block consisted of four dishes for each bean weevil strain
and there were six blocks set up over time.

The tests were terminated after 72 h of adult weevil exposure to bean seeds.
This was. done to avoid possible early egg hatch after the third day. To facilitate
removal of beetles from dishes prior to counting eggs, dishes were set at -15 0C for 2

to 4 minutes to temporarily immobilize the insects. Egg numbers for each section

94

 

 

Wax ridge

Figure 11. Petri dish is divided into six sections by wax ridges. B = bean
placement area; I = insect introduction area.

95

of the dish were recorded separately. No evidence of nearest neighbour effects
was observed. Data from the two-factor RCBD experiment were log-transformed

for homogeneity of variance, and were analyzed by a two-way RCBD AN OVA.

Oviposition: No-choice test

Ovipositional acceptance in the bean weevil was examined in a no-choice
situation, using the same six bean types. Equal weights of six bean classes were
individually presented to five pairs of adult bean weevils of a given strain in 60 x 15
mm glass petri dishes (Pyrex Brand, USA). Bean seeds were spread evenly yet
compactly within each dish to provide conditions close to those under natural
storage situations for the bean weevil. Adult beetles of each of the two strains
were randomly assigned each to the six bean classes comprising each of 12 replicates
set up over time. Beetles used in the test were from the 28th generation. Seventy
two hours after introduction, all beetles were removed from the dishes, and the eggs
were counted. AN OVA was performed on the data, and means were separated by

Tukey’s studentized range (HSD) test (SAS Institute 1985).

Oviposition: 'I\vo-choice tests

Additional paired-host ovipositional tests were conducted to further evaluate
the effects of certain bean classes on oviposition of the two bean weevil strains in a
limited choice situation. The two-choice experiments tested the ovipositional
stimulation of: 1) light red kidney bean against black bean, and 2) light red kidney
bean against navy bean.

Comparison between black bean and light red kidney bean was done to

examine the response of bean weevil females to either bean under limited choice

96

situation. Comparison between light red kidney and navy bean was conducted to
conﬁrm observations made under the six-choice test in this chapter that navy bean
appeared to be more favored for oviposition than light red kidney bean by the M1
strain, and that light red kidney bean was favored over navy bean for oviposition by
T2 strain. In other words, both strains of A. obtectus were tested simultaneously in
a two-choice situation (navy bean vs. light red kidney bean) to determine if: 1) the
"incident" observed under the six-choice situation was pure chance, and that 2) 16
generations of rearing the beetles continuously on light red kidney bean did not
change the original preference of the two strains of bean weevil for their locally
available and / or abundant hosts.

For each of these two tests, a 60 x 15 mm glass petri dish was allocated to one
bean weevil strain. The dishes were divided into two equal sections by two 2 mm
high parafﬁn wax ridges (Walnut Hill Company) dividing the dish in half. In each
test, equal weights of beans of a given class were spread compactly on one side of
the dish, and beans of the other class on the other side. Six pairs of bean weevils
from the 30th generation culture were released at the center of a dish between the
wax ridges. The beetles were introduced into each dish in a randomized fashion.
The tests were replicated 12 times.

Seventy two hours later, the beetles were removed from the dishes and eggs
from each section of the dish were counted separately. Ovipositional data from the
factorial experiment (2 bean weevil strains x 2 bean classes) were analyzed using a
two-way AN OVA. T-tests (SAS Institute 1985) were employed to analyze the

ovipositional rates of the bean weevils.

97

Host suitability: Six-choice test

The bean arrangement of the six-choice ovipositional preference test was used
for host-suitability tests, with the exception that 54 neonate larvae of a single strain
of bean weevil were introduced to the center of the dish. This procedure was
repeated simultaneously for the other bean weevil strain and the experiment was
replicated 12 times. Larvae were from eggs of the 27th adult generation.

Two weeks after larvae were set in dishes, each group of beans from each
section was transfered to individual petri dishes for monitoring adult emergence.
All emergence dishes were held within the same glass cage in the environmental
chamber. Emerging adults were removed, counted, placed in vials, and stored at
-15°C for 24 h. The beetles were later dried in an oven for 24 h and dry weights
were obtained by using a Mettler AElOO balance. Data for larval survival were
normalized by log transformation before being subjected to ANOVA. Adult dry
weights were analyzed by General linear models procedure (SAS Institute 1985).

Host-suitability: No-choice test

The experimental set up was similar to that for no-choice ovipositional
preference; however, 20 neonate bean weevil larvae from eggs laid by the 28th
generation culture were introduced into the dish instead of adults. The larvae
were placed into different dishes of the six bean classes in a randomized fashion.
The six dishes comprised a replicate; and there were 12 replicates set up over time
for each strain of bean weevil.

Daily observation for adult emergence was initiated about four weeks after
larval introduction to beans. Emerging adults of bean weevils were collected daily.

counted, placed into vials, and stored at -15 °C for 24 h. The beetles were later

98

dried at 65 0C and weighed. Adult emergence was followed for three weeks.
Numbers of adults emerged were subjected to log transformation prior to ANOVA.
Dry weights of the beetles were analyzed by General linear models procedure
(GLM) (SAS Institute 1985).

RESULTS AND DISCUSSION

Oviposition: Six-choice test

When offered a choice, A. obtectus discriminated among P. vulgaris hosts and
V. unguiculata (F = 23.9; df = 5; P < 0.001) (Table 23). On average, more eggs
were laid on P. vulgaris classes (e.g. light red kidney bean) and least on the V.
unguiculata, the cowpea. Pankanin-Franczyk (1980) also observed preferential
oviposition in A. obtectus when the bean weevil was offered different bean species.

A highly signiﬁcant strain x host bean interaction was also observed in this test
(F = 9.19; df = 5; P < 0.001); this means the pattern of ovipositional preference for
the same host bean classes varied between the two bean weevil strains. The T2
strain of bean weevil deposited most eggs among light red kidney bean seeds (Table
24) while the MI strain laid the most eggs among navy bean seeds. Moreover, with
pooling of bean classes, arithmetically more eggs were laid by the T2 strain
(51813.8, mean:SE) than the MI strain (45.5 t 3.1 eggs), however, the difference
was not statistically different (F = 0.07; df = 1; P = 0.78). Also, the number of
eggs laid per female per day for each strain of A. obtectus did not vary signiﬁcantly
(T=-1.609; df = 23; P > |T| = 0.12, Cochran t-test). The ovipositional rates were
8.6 t 0.6 eggs per day for the T2 strain and 7.6 t 0.3 eggs per day for the MI strain.

99

Table 23. Effects of bean class on oviposition of A.
obtectus in a six—choice test.

 

No. eggs laid
Bean class
(Mean 1 SE)

 

 

1

Kidney 87.8 i 7.1 a

Pinto 64.5 i 6.2 ab

Navy 58.0 i 6.3 b

Great Northern 29.6 i 3.1 c

Black 28.4 i 2.3 c

Cowpea 23.6 i 2.8 c
1
Means followed by the same letter are not significantly
different at P = 0.05, Tukey's studentized range (HSD)
test. Data were log(x+1) transformed prior to ANOVA and
mean separation. (N = 48) . Data for T2 and MI bean

weevil strains were pooled.

100

Table 24. Numbers of eggs laid by two A. obtectus strains
in a six-choice test.

 

 

 

Bean weevil Bean class No. eggs laid
strain
(Mean i SE)
1
Michigan Navy 77.6 i 8.5 a
Kidney 66.8 i 9.0 ab
Pinto 46.4 i 6.5 abc
Great Northern 36.8 i 4.4 bc
Black 24.7 1 2.4 cd
Cowpea 20.8 i 4.4 d
Tanzania Kidney 108.7 1 9.2 a
Pinto 82.6 i 9.4 a
Navy 38.3 i 7.5 b
Black 32.2 i 3.7 b
Cowpea 26.4 i 3.5 b
Great Northern 22.3 i 4.0 b

 

1

Means followed by the same letter within a section are not
significantly different at P = 0.05, Tukey's studentized
range (HSD) test. Data were log(x+1) transformed prior
to ANOVA and mean separation. (N = 24).

101

Oviposition: No-choice test

Signiﬁcant differences in number of eggs laid among the six bean classes by
female bean weevils was also detected in the no-choice test (P = 3.87; df = 5;
P=0.003). The cowpea received the fewest eggs (not statistically different from
pinto and light red kidney beans, Table 25). On the other hand, when host
acceptability was measured for each strain of bean weevil, the number of eggs laid
did not vary signiﬁcantly among the six bean types, however, the beetles tended to
deposit fewer eggs among the cowpeas (Table 26). The tendency to deposit more
eggs among the P. vulgar-is classes than the cowpea suggests that the cowpea may not
be among the highly acceptable or preferred hosts for oviposition. In other words,
the bean weevils appear to be more adapted to ovipositing among P. vulgar-is.

In the earlier choice test, the differences in ovipositional preference among
the ﬁve conspeciﬁc bean classes and between the beans and cowpeas may imply that
ovipositional stimulation in the bean weevil varied with different host-bean class
characteristics. However, these differences vanished in the no-choice test,
probably because deprivation of the optimal host increased acceptance of hosts only
slightly lower in the rank order of acceptability.

Overall oviposition of A. obtectus strains differed signiﬁcantly between the
strains (F = 22.49; df = 1; P < 0.001) in the no-choice test. On average, females
of the T2 strain laid 136.3 s 5.4 eggs and the M1 strain laid 104.8 2 4.4 eggs
(mean: SE). Accordingly, ovipositional rate was 9.1 t 0.4 eggs per female per day
in the TZ strain, and 7.0 t 0.3 eggs per female per day in the M1 strain (T = - 4.53;
df=142; P > |T| <0.0001, t-test).

102

Table 25. Effects of bean class on oviposition of A.
obtectus in a no-choice test.

 

No. eggs laid

Bean class
(Mean i SE)

 

 

1

Great Northern 134.1 i 7.9 a

Navy 130.6 1 8.7 a

Black 129.4 1 8.0 a

Pinto 121.6 : 8.4 ab

Kidney 117.0 i 8.4 ab

Cowpea 90.5 i 10.6 b
1
Means followed by the same letter are not significantly
different at P = 0.05, Tukey's studentized range (HSD)
test. Data for T2 and MI strains of bean weevil were

pooled: (N = 24).

103

Table 26. Number of eggs laid among six bean classes by
each strain of A. obtectus in a no-choice test.

 

 

 

Bean weevil Bean class No. eggs laid
strain
(Mean _ SE)

Michigan Great Northern 117.7 1 10.7 a
Navy 114.0 1 7.4 a
Kidney 111.9 1 9.7 a
Black 103.8 1 9.7 a
Pinto 101.2 1 10.3 a
Cowpea 80.5 1 14.3 a

Tanzania Black 155.1 1 7.3 a
Great Northern 150.6 1 9.7 a
Navy 147.2 1 14.5 a
Pinto 142.1 1 10.7 a
Kidney 122.1 1 14.1 a
Cowpea 100.7 1 15.7 a

 

1

Means followed by the same letter within a section are not
significantly different at P = 0.05, Tukey's studentized
range (HSD) test. N = 12.

104

Oviposition: Two-choice tests

Groups of females of both A. obtectus strains exposed to light red kidney
beans and black beans in a two-choice test deposited signiﬁcantly more eggs
(91.4166) among light red kidney than among black beans (41.5 i 5.2) (F = 33.79;
df = 1; P < 0.001). Oviposition between the two bean classes was not signiﬁcantly
affected by strain differences (F = 0.05; df = 1; P = 0.82). Consequently, the
ovipositional rates did not vary between strains. Numbers of eggs laid by each
female per day were 7.5 t 0.5 for the T2 strain and 7.3 i 0.4 for the M1 strain of
bean weevil. Eggs laid by the two A. obtectus strains among beans of the two
classes are presented in Table 27. No signiﬁcant interaction between strain and
bean class effects occurred (F = 0.05; df = 1; P = 0.94). Both MI and T2 strains
of A. obtectus readily accepted light red kidney bean, their current host bean for
more than 14 generations, over black bean, their earlier host in the laboratory.

On the other hand, when light red kidney and navy beans were offered to the
two strains of bean weevils in two-choice tests, differences in ovipositional responses
between the strains were signiﬁcant (F = 3.87; df = 1; P = 0.55). The T2 strain,
on average, laid more eggs (65.8 t 6.2) than the M1 strain (50.3 t 6.2 eggs).
Similarly, the ovipositional rates of the strains were statistically different (T=-2.189;
df = 22; P = |T| =0.039; T-test). Numbers of eggs laid per female per day were
7.3 t 0.5 for the TZ strain and 5.6 t 0.6 for the MI strain. Further, when the bean
weevil strain data were pooled, number of eggs laid did not vary signiﬁcantly with
bean classes. However, egg distribution between the bean classes was inconsistent
with the two strains of bean weevil, hence there was a statistically signiﬁcant strain x
bean class interaction (F = 13.61; df = 1; P = 0.001) in the two-choice test. T‘Z
females deposited most eggs among light red kidney beans than navy beans (Table

28), whereas MI females laid more eggs among navy beans than among light red

105

Table 27. Numbers of eggs laid by two strains of A.
obtectus in a two-choice test.

 

 

 

 

Bean weevil Bean class No. eggs laid
strain
(Mean 1 SE)
1
Michigan Kidney 90.8 1 10.6 a
Black 40.2 1 7.2 b
Tanzania Kidney 92.1 1 8.5 a
Black 42.8 1 7.7 b
1
Means followed by the same letter within a section are not
significantly different at P = 0.05, Bonferroni t-test.

(N = 12).

Tat

106

Table 28. Numbers of eggs laid by two strains of A.
obtectus in a two-choice test.

 

Bean weevil Bean class No. eggs laid
strain
(Mean 1 SE)

 

 

 

1

Michigan Navy 65.2 1 9.5 a

Kidney 35.5 1 5.5 b

Tanzania Kidney 79.8 1 9.4 a

Navy 51.7 1 6.0 b

1

Means followed by the same letter within a section are not
significantly different at P = 0.05, Bonferroni t-test;

(N = 12).

107

kidney beans. Although a similarity was observed between the beetle strains in
their ovipositional behavior in the presence on light red kidney and black beans,
interstrain differences were noted when the two strains were offered light red kidney
and navy beans in a choice situation.

Other bruchids are known to exhibit such behaviors. Three Callosobruchus
species, C. maculatus, C. analis, and C. chinensis exhibited preference for different
chickpea varieties (Cicer arietinum L.) when presented in choice or no-choice tests,
respectively (Raina, 1971). Furthermore, other insects, such as the cabbage
butterﬂy (Pier-is rapae L.), are also known to discriminate not only between species
but also among cultivars of a species. The Australian cabbage butterﬂies preferred
certain crucifer species as well as certain varieties within a crucifer species (Ives,
1978). The Brassica oleracea (Brussels sprouts, cabbage, kale) were preferred
over Raphanus sativus (radish) by P. rapae. These reports suggest that preferential

behaviors in presence of choice of ovipositional hosts exist in a number of insects.

Host Suitability: Larval survival

Six-choice Lest

Bean classes signiﬁcantly affected survivorship of bean weevil larvae to the
adult stage when presented simultaneously in the six-choice test (F = 23.53; df = 5;
P < 0.001). More adults emerged from light red kidney beans than from navy
beans (Table 29). Conversely, no signiﬁcant effects of strain on larval survival
were detected (F =0.47; df = 1; P = 0.49). The number of adults emerging from
beans infested by M] A. obtectus was 3.7105 and that from beans infested by the
T2 strain was 3.8105. Furthermore, interaction between strain and bean class

was not detected (F = 1.03; df = 5; P = 0.4) in this experiment. Patterns of adult

108

Table 29. Effects of bean class on larval survival of
A. obtectus in a six-choice test.

 

 

 

Bean class No. adults emerged
(Mean 1 SE)
1

Kidney 7.5 1 0.9 a

Pinto 5.3 1 0.9 a

Cowpea 4.7 1 0.7 ab

Black 2.6 1 0.6 bc

Great Northern 2.3 1 0.5 c

Navy 0.3 1 0.1 d
1
Means followed by the same letter are not significantly
different at P = 0.05, Tukey's studentized range (HSD)
test. Data were log(x+1) transformed prior to ANOVA and
mean separation. Data for MI and T2 strains were pooled;

(N= 24).

th

nit

7711‘

109

emergence for the two strains were very similar across the six bean classes (Table
30).
NQ-ghoice 1951

When conﬁned to one host, numbers of neonate larvae surviving to the adult
stage varied signiﬁcantly across the six bean classes (F = 18.30; df = 5; P<0.001)
(Table 31). Signiﬁcantly fewer adults emerged from great northern and navy beans
than other bean classes. On the other hand, strain of A. obtectus did not
signiﬁcantly affect larval survival (F <0.01; df = 1; P = 0.97). Pooling bean class
effects, mean number of adults emerged was 4.6 1 0.4 for the MI strain and
4210.4 for the T2 strain. In addition, the rank order of larval survival or adult
emergence among the six bean classes for the MI and T2 strains matched (Table
32).

Despite the differences in numbers of eggs laid by each strain of bean weevil,
the number of larvae surviving to the adult stage was generally similar between the
two strains. Also, patterns of adult emergence among the six hosts were similar
within both choice and no-choice treatments in the two strains of bean weevil.

This indicates that larvae of both strains responded similarly to the same host cues.

Host Suitability: Adult dry weights

Six-choice :95;

Dry weights of emerging adults varied among hosts and between the T2 and
MI strains of bean weevil. In the six-choice test, the pattern of adult dry weights
among the six beans varied with the two strains of bean weevil (Table 33); hence, a

signiﬁcant interaction was seen (F = 2.70; df = 5; P = 0.02). Likewise, when

Tab]
stre

Bea'

Mic

Tar

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110

Table 30. Effects of bean class on larval survival of two
strains of A. obtectus in a six-choice test.

 

 

 

 

Bean weevil Bean class No. adults emerged
strain
(Mean 1 SE)
1
Michigan Kidney 7.3 1 1.5 a
Cowpea 5.7 1 1.2 ab
Pinto 4.1 1 1.2 ab
Black 2.5 1 1.0 bc
Great Northern 2.1 1 0.5 bc
Navy 0.4 1 0.2 c
Tanzania Kidney 7.6 1 1.1 a
Pinto 6.4 1 1.2 ab
Cowpea 3.7 1 0.6 ab
Black 2.7 1 0.7 bc
Great Northern 2.6 1 1.0 c
Navy 0.2 1 0.2 d
1

Means followed by the same letter within a section are not
significantly different at P = 0.05, Tukey's studentized
range test; N = 12. Data were log(x+1) transformed prior
to ANOVA and mean separation.

111

Table 31. Influence of bean classes on larval survival of
A. obtectus in a no-choice test.

 

 

 

Bean class No. adults emerged
(Mean 1 SE)
1

Kidney 7.3 1 0.7 a

Cowpea 5.8 1 0.5 a

Pinto 5.0 1 0.7 a

Black 4.7 1 0.6 a

Navy 1.9 1 0.5 b

Great Northern 1.8 1 0.4 b
1
Means followed by the same letter are not significantly
different at P = 0.05, Tukey's studentized range (HSD)
test. Data were log(x+1) transformed prior to ANOVA and
mean separation. Data for MI and TZ strains were pooled:

(N = 24).

112

Table 32. Effects of bean class on larval survival of the
two strains of A. obtectus in a no-choice test.

 

 

 

Bean class Michigan strain Tanzania strain
(Mean 1 SE) (Mean 1 SE)
1

Kidney 7.3 1 1.1 a 7.4 1 1.0 a
Cowpea 5.7 1 0.7 a 5.9 1 0.7 a
Pinto 5.6 1 1.3 a 4.5 1 0.6 a
Black 5.5 1 0.9 a 3.9 1 0.7 ab
Navy 2.0 1 0.9 b 1.8 1 0.6 b
Great Northern 1.8 1 0.6 b 1.8 1 0.5 b

1

Means followed by the same letter are not significantly
different at P = 0.05, Tukey's studentized range test.

Data were log(x+1) transformed prior to ANOVA and mean
separation.

113

Table 33. Effects of A. obtectus strain and bean class on
dry weights of emerging adults in a six-choice test.

 

Bean weevil strain: MI

 

Adult

dry weights (mg)

 

 

 

 

Bean class N Mean 1 SE Range
1
Great northern 25 2.87 1 0.09 a 1.6-3.6
Navy 5 2.94 1 0.17 ab 2.4-3.4
Kidney 90 2.57 1 0.05 ab 1.3-4.0
Cowpea 68 2.45 1 0.06 b 1.3-3.8
Pinto 49 2.42 1 0.07 b 1.0-3.4
Black 30 2.23 1 0.12 b 1.1-3.6
Bean weevil strain: TZ
Adult

dry weights (mg)

 

 

 

Bean class N Mean 1 SE Range

0 i 1
K1dney 92 2.45 1 0.05 a 1.3-3.8
Pinto 77 2.29 1 0.09 ab 1.4-3.7
Great northern 31 2.29 1 0.09 ab 1.4-3.3
Cowpea 44 2.15 1 0.07 ab 1.2-3.3
Navy 2 2.10 1 0.50 ab 1.6-2.6
Black 32 2.10 1 0.09 b 1.4-2.9
1

Means followed by the same letter are not significantly

different at P

test.

Tukey's studentized range

(HSD)

th.
523
35 ).
UO-(
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114

strains of A. obtectus were pooled, dry weights of emerging adults differed
signiﬁcantly among bean classes (F = 6.83; df = 5; P <0.001) (Table 34). On
average, black beans produced lighter adults than the other beans. Differences in
the bean weevil strains were also highly signiﬁcant (F = 23.15; df = 1; P <0.001).
On average, adult dry weights were 2.51 1 0.03 mg. and 2.30 1 0.03 mg. for M1

and T2 strains respectively.

N-i

In no-choice tests, both bean class and beetle strain had major effects on the
resulting adult dry weights of A. obtectus. Dry weights of beetles emerging from
bean seeds differed signiﬁcantly among the six bean classes (F = 7.52; df = 5;
P<0.001), suggesting differences in host suitability among beans. When the strain
effects were combined, adults emerging from light red kidney bean were heavier
than those emerging from the other bean classes. However, they only differed
statistically from adults emerging from black beans, navy beans and cowpeas (Table
35). Differences in weights of adults emerging from the different bean classes in
no-choice tests might result from variation in case of penetration through the seed
coat by neonate larvae, number of larvae per seed, seed size and possibly nutritional
aspects of the bean types, apart from insect factors. The possibility of larval
overcrowding within a bean seed (see Chapter 1) thus producing smaller adults
cannot be totally dismissed in this case.

Highly signiﬁcant differences in adult dry weights between the two strains of
A. obtectus also occurred under the no-choice test (F = 25.89; df = 1; P < 0.001).
Adults of the M1 strain (2.29 1 0.03 mg.) were heavier than those of the T2 strain
(2.09 1 0.03 mg.). This trend was also noted in the six-choice situation. Presence

of a signiﬁcant interaction between the main effects was not detected in no-choice

115

Table 34. Dry weights of adult A. obtectus emerging from
various beans in a six-choice test.

 

Adult dry
weights (mg)

 

 

 

Bean class N Mean 1 SE Range

1
Great northern 56 2.55 1 0.07 a 1.4-3.6
Kidney 182 2.51 1 0.04 a 1.3-4.0
Pinto 126 2.35 1 0.04 ab 1.0-3.7
Cowpea 112 2.34 1 0.05 ab 1.2-3.8
Black 62 2.16 1 0.07 b 1.1-3.6
1
Means followed by the same letter are not significantly
different at P = 0. 01, Tukey's studentized range (HSD)

test. Bean weevil strain data were pooled.

 

Table 35’
beans in

 

Bean cla:

 

Kidney
Great n<
Pinto
Black
NavY

COWpea

\

l
“Gan.
diff
test

 

116

Table 35w Dry weights of adult A. obtectus emerging from
beans in a no-choice test.

 

Adult dry
weights (mg)

 

 

 

Bean class N Mean 1 SE Range
1

Kidney 176 2.35 1 0.04 a 1.4-3.5
Great northern 42 2.34 1 0.09 a 1.3-4.7
Pinto 121 2.18 1 0.05 ab 1.3-3.4
Navy 46 2.10 1 0.07 ab 1.2-3.1
Cowpea 139 2.03 1 0.04 b 1.0-3.6

1

Means followed by the same letter are not significantly
different at P = 0.01, Tukey's studentized range (HSD)

test. Bean weevil strain data were pooled.

 

 

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117

test, however, Table 36 presents effects of bean class on dry weights of emerging
adults within each bean weevil strain. In both cases, light red kidney ranked
among beans producing heaviest adults while the cowpea produced the lightest
individuals.

Variability in suitability of host cultivars has also been noted in another
bruchid, Callosobruchus chinensis L. When offered seven different broad bean
(Vicia faba L.) varieties in a test, survival of the beetle was not consistent among the
different hosts (Podoler and Applebaurn, 1968). Low host suitability in some of
these varieties was attributed to seed coat properties, since larval survival was equal
in all broad beans when decorticated. This situation was also observed in seeds
with perforated seed coats in Chapter 1 (this Thesis).

Some factors that may govern suitability of a seed as a larval host include
those of the seed coat (thickness, hardness, texture, volatiles, toxicity), cotyledons
(nutritional quality, toxicity, hardness), seed size, seed shape, and seed age. It can
thus be suggested that the biochemical and physical seed characteristics play an

important role in pulse suitability to seed beetles.

Correlation between larval survival and oviposition

In this study, females of A. obtectus oviposited on all six hosts provided
simultaneously, yet the egg distribution differed among the hosts. Similarly, larvae
of these bean weevils were able to survive on all six hosts; however, numbers of
surviving offspring differed. The patterns of larval survival among the six hosts did
not correspond well with those for oviposition in the bean weevils. In choice tests,
the ovipositional preference rank order among the six bean classes (light red kidney
(K) > pinto (P) > navy (N) > great northern (G) > black (B) > cowpea (C)) did
not match well with that of larval survival (K > P > C > B > G > N). Likewise,

I

C ] ‘IC 1 4

Table 36.

Effects of A.

118

obtectus strain and bean class on

dry weights of emerging adults in a no-choice test.

 

Bean weevil strain: MI

 

Adult dry

weights (mg)

 

 

 

 

Bean class N Mean 1 SE Range
1
Kidney 87 2.45 1 0.05 a 1.5-3.4
Great northern 21 2.31 1 0.09 ab 1.7-3.1
Black 66 2.30 1 0.06 ab 1.4-3.7
Navy 24 2.26 1 0.11 ab 1.3-3.1
Pinto 67 2.24 1 0.07 ab 1.3-3.4
Cowpea 68 2.13 1 0.07 b 1.3-3.6
Bean weevil strain: TZ
Adult dry

weights (mg)

 

 

 

Bean class N Mean 1 SE Range

1
Great northern 21 2.36 1 0.16 a 1.3—4.7
Kidney 89 2.25 1 0.05 a 1.4-3.5
Pinto 54 2.09 1 0.06 ab 1.3-3.3
Black 48 1.97 1 0.06 ab 1.3-3.0
Navy 22 1.94 1 0.09 ab 1.2-2.8
Cowpea 71 1.94 1 0.05 b 1.0-3.6
1

Means followed by the same letter are not significantly
d1fferent at P = 0.01, Tukey's studentized range (HSD)
test.

in m
not

aim

su.
hc
cf
SL‘

gt

119

in no-choice tests, ovipositional acceptance pattern (G > N > B > P > K > C) did
not match that of larval survival (K > C > P > B > N > G); the patterns were
almost reversed. The cowpea (C) which was less preferred and less acceptable as
an ovipositional host in the bean weevil was among the highly suitable hosts for
larval survival. In contrast, navy bean (N) which was among the highly preferred
and acceptable hosts for oviposition, was the least suitable for survival of A.
obtectus larvae. Apparently, cues used by adult bean weevils for oviposition did
not match host suitability for larval survival.

The low correspondence observed between oviposition preference and larval
survival may partially be attributed to lack of exposure of the bean weevils to all six
hosts over time, prior to these tests. Thus evolution may not have been given a
chance to operate so as to build congruence between adult preference and host
suitability. All bean weevils used were reared on light red kidney for more than 14
generations.

It is generally expected that ovipositional host preference and host suitability
in an insect should evolve to a coadapted state to maximize ﬁtness. In fact, the
correspondence between the two is often low (Wiklund, 1975). In A. obtectus,
Hereford (1935), found no correlation between ovipositional host choice of the
females and the nutritional quality of the seed types for larval development. This
author also noted that seeds of some species of legumes normally not infested by the
bean weevil had chemical compositions rendering them highly suitable for
development of the larvae.

These observations suggest that some suboptimal hosts used for oviposition
may probably be favored for indirect ecological reasons (Wasserman and Futuyma,
1981) such as host availability, host abundance (Futuyma, 1976; Benson, 1978,
Schneiderand Roush, 1986), phenology (Bernays and Chapman, 1976; Feeny, 1976),

or fewer natural enemies (Bernays and Graham, 1988), but not necessarily for their

direct suita

 

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120

direct suitability as larval hosts.

Inter-strain differences

Since the T2 and MI bean weevil populations were reared continuously on
light red kidney bean for 14 generations and more, it was expected that the bean
weevils would not only oviposit highly but survive best on this bean. This situation
was observed in the cowpea weevil, C. maculatus which developed an increased
preference for a host that it had been reared on for a number of generations
(Wasserman and Futuyma, 1981). When the A. obtectus in our study were
presented with choice of hosts, the M1 strain deposited most eggs on navy bean,
whereas the T2 strain oviposited most on light red kidney bean. It should be noted
however that the M1 strain was originally collected from ﬁeld-infested navy beans
in Michigan, and the T2 strain was from red kidney beans most commonly
cultivated by farmers in Mgeta, Morogoro, Tanzania. Perhaps an innate
ovipositional preference for their original hosts has been maintained in these two
bean weevil strains over time. It is particularly obvious in the MI strain, that

irrespective of being conﬁned on light red kidney bean for several generations,
beetles still preferred navy bean.

The differences between the T2 and MI strains in their ovipositional host
preferences may be related to the difference in availability and abundance of a
given host P. vulgan's in their geographical regions. In addition to variation in
ovipositional host preferences between the two bean weevil strains, differences in
overall egg numbers laid and in weights or size of the emerging adults between the
strains were also observed. More eggs were generally produced by the T2
A. obtectus strain than the M1 strain, yet, emerging adult bean weevils of the MI

strain attained greater weights than adults of the T2 strain. The differences in the

121

two strains must be genetic.

Inter-strain or intraspeciﬁc differences particularly in geographically different
populations of insects are not uncommon. They have been documented in: C.
maculatus strains (Dick and Credland, 1984), strains of Z. subfasciatus (Pimbert,
1985), and in Heliothis virescens (F.) (Schneider and Roush, 1986).

Recognition of inter-strain differences could be of great importance when data
are compared between different laboratories and different regions of the world. It
might also be necessary to consider strains separately when developing control
methods. For example, when breeding plants for resistance against a pest,
consideration of strain differences in relation to bean seed characteristics will be

useful for producing appropriate varieties for areas in question.

CONCLUSIONS

The six bean classes affected ovipositional behavior in the two strains of
A. obtectus differently, particularly in a choice situation. On average, the bean
weevils laid fewer eggs among the cowpea seeds (V. unguiculata) than among the
other bean classes (P. vulgaris) in both choice and no-choice situations. Where
choices were provided, the TZ strain generally laid the most eggs on light red kidney
beans while the M1 strain laid most on navy beans. The higher ovipositional
preference for light red kidney bean and navy bean in the T2 and MI strains,
respectively, were further conﬁrmed in a two-choice test. Given no choice, the six
bean classes were almost equally acceptable for oviposition within a given strain of
bean weevil, however, a decreasing trend was observed, with cowpea receiving the

fewest eggs.

122

Larval survivorship was generally lower in great northern and navy beans, and
highest in the light red kidney bean, under choice and no-choice conditions for both
strains of bean weevils. Pinto bean and the cowpea were also among the highly
suitable hosts for larval survival next to light red kidney bean. Black bean was
generally intermediate in its affect on larval survival. There was poor
correspondence between larval survival and ovipositional preference.

Larval survival was similar in the two strains of A. obtectus. Furthermore,
patterns of larval survival between the strains of bean weevil were almost the same.
However, among the six bean classes tested, the patterns of larval survival did not
correspond well with those of ovipositional host preference/ acceptance of adults.
Adult dry weights of the emerging F1 beetles varied between strains. The MI

strain produced heavier adults than the T2 strain in this study.

CHAPTER 3

Ovipositional behavior, larval growth, and survival of
the bean weevil, Acanthoscelides obtectus (Say):
Effects of bean seed damage.

123

INTRODUCTION

Susceptibility of stored grains or seeds to infestation by insect pests may be
inﬂuenced by seed variety, size, chemistry, and seed-coat characteristics. For
instance, cowpeas (Vigna unguiculata (L) Walp.,) with smooth seed coats received
more cowpea weevil (Callosobruchus maculatus (F.)) eggs than rough ones.
Likewise, smooth seed coats were more conducive to larval penetration (Nwanze
and Horber, 1976).

The seed coat of dry beans (Phaseolus vulgan's L.) has a strong inﬂuence on
seed penetration by the ﬁrst instar larvae of the bean weevil (Acanthoscelides
obtectus (Say)). The bean testa appears to act as a physical barrier (Pankanin-
Franczyk, 1980; Thiery, 1982) or as both physical and chemical barrier
(Stamopoulos and Desroches, 1981) against penetration of larval bean weevils.
When the seed coat is artiﬁcially punctured, penetration into seeds and survivorship
of bean weevil larvae was greatly improved (Pankanin-Franczyk, 1980; Stamopoulos
and Desroches, 1981; Chapter 1, this thesis).

This report reveals how different types of physical damage of the bean seed
affect ovipositional behavior, larval survival and size of emerging adults in the

Tanzania (T2) and Michigan (MI) strains of A. obtectus.

124

125

MATERIALS AND METHODS

Insects

Two strains of A. obtectus used in this study were from ﬁeld-infested bean
seeds from Morogoro, Tanzania and from East Lansing, Michigan. Both cultures
have been maintained in a walk-in environmental chamber for 30+ generations (for
rearing food history, see Chapter 1). Containers for all insect cultures and tests
were set inside separate glass cages under conditions speciﬁed in General Materials

and Methods, Chapter 1. Insects of similar ages were used in this study.

locally available commercial dry beans (Phaseolus vulgaris) of the light red
kidney bean class were used for all tests after storage within the environmental
chamber used for experiments. These beans were also the rearing host of both

bean weevil strains.

Oviposition: Choice-test

To set up a multiple-choice ovipositional test, a 150 mm diam. x 15 mm high
glass petri dish (Pyrex Brand, Corning) was divided into six equal sections by 2 mm
high wax ridges. About 14 - 15 variously treated seeds or cotyledons of light red
kidney beans were spread compactly within each dish section, according to a
randomized complete block design. The seed treatments were: 1) intact bean

seed (IB), 2) seed with cracked testa (CB): at either end of the seed and without

126

cotyledons showing, 3) bean seed with single exit hole placed in a dish with the hole
side up (HB): an artiﬁcial hole of about 2 mm diam. and 1.5 mm deep resembling
an adult bruchid exit hole was drilled into one side of the seed (to a depth less than
the width of a given cotyledon), 4) halved bean (cotyledon with testa still attached)
placed with inner side of cotyledon facing downwards (SD); 5) halved bean
(cotyledon with testa still attached) placed with inner side of the cotyledon facing
upwards (SU); and 6) "no bean" (NB) i.e. an empty section.

Prior to initiation of the experiment, over 150 pairs of adult bruchids of each
strain were collected and placed in separate jars. Collections were made from the
32nd generation of both the Michigan and Tanzania strains. Twelve pairs of adult
bruchids of a single strain were transferred from the jar and released at the center of
each dish containing the six seed treatments. This procedure was repeated for the
other bean weevil strain. The experiment had 12 replicates.

The adults were removed from dishes after 72 h, and eggs from each dish
section were counted. Data from the 6 (seed treatments) x 2 (bean weevil strains)
factorial experiment were square-root transformed prior to two-way ANOVA (SAS
Institute 1985).

Larval survival: Choice-test

The experimental design was identical to that of Experiment 1 except that the
"no-bean" treatment was deleted. F ifty-four neonate larvae obtained from eggs
collected from the 32nd generation of A. obtectus cultures were placed at the center
of each petri dish containing the damaged seeds. Twelve replicates were
established for each bean weevil strain using the 5 x 2 factorial design.

By the end of the third week, bean seeds from each section of the dishes were

transferred to separate dishes for monitoring adult bean weevil emergence. Adults

127

usually begin emerging four weeks after the initial larval exposure to seeds
(personal observation). Emerging adults were counted daily and removed from
the dishes. Adult emergence was monitored for 14 days. A two-way AN OVA was

performed on the log-transformed data.

Larval survival: No-choice test

For each bean weevil strain, ﬁve 60 x 15 mm glass petri dishes (with interiors
not sectioned) were each provided with a different seed treatment. Treatments
were the same as in Experiment 2. Each dish contained 15 or 16 seeds or
cotyledons spread evenly yet compactly within the dish. Twenty neonate larvae
from eggs obtained from the 34th generation A. obtectus cultures, were placed in
each dish. The experiment was replicated 10 times. Emerging adults were
counted and removed from the dishes daily for 14 days. Collected adults were
stored in vials at -15 0C for at least 24 h. They were later dried at 65 0C for 24 h
and then weighed. The numbers of larvae surviving to adult stage and numbers of
infested seeds were each analyzed by two-way ANOVA. Dry weights were
analyzed by general linear models procedure (GLM) (SAS Institute 1985).

RESULTS
Oviposition: Choice test
Differences in oviposition between the bean weevil strains, and among seed
damage treatments were highly signiﬁcant for strain (F =25.62; df= 1; P<0.001) and

for seed treatment (F=88.68; df =5; P<0.001). When data for seed damage

treatments were pooled, numbers of eggs laid (mean 1 SE) by the Tanzania strain

128

(49.9157) were signiﬁcantly higher than those laid by the Michigan strain
(32.0149). When comparing mean number of eggs laid for each seed damage
treatment (data of both strains pooled), intact beans received more eggs than any
other seed treatment (Figure 12). Numbers of eggs laid among the different seed
treatments can be ranked in descending order: intact beans (IB) 1 beans with cracks
in seed coat (CB) > beans with one exit hole (HB) > halved bean with inner side
facing down (SD) 1 halved bean with inner side facing up (SU) > no bean (NB).
No eggs were laid in dish sections containing no beans. The intact bean treatment
was signiﬁcantly different from all other treatments except beans with cracks in the
seed coat. Figure 13 gives detailed mean egg distributions among each seed
damage treatment for each bean weevil strain. Interaction between the bean

weevil strain and seed treatment was not signiﬁcant (F = 1.95; df =5 ; P = 0.09).

Larval survival: Choice test

Signiﬁcantly more (F=4.67; df= 1; P=0.03) Michigan bean weevils survived to
the adult stage than did the Tanzania strain in this experiment. When data for
seed damage treatments were pooled, mean (1 SE) values of adult emergence were
8.0 1 0.8 for the Michigan strain and 7.0 1 0.8 for the Tanzania strain. Highly
signiﬁcant differences in larval survival of A. obtectus were observed among the
different seed damage treatments (F=65.90; df =4; P<0.001). Of the ﬁve seed
treatments, the most adult weevils emerged from the two halved bean treatments
(SD and SU) and least emergence was among intact beans (IB) (Figure 14). Beans
with one exit hole (HB) were intermediate in mean numbers of adults emerging.

The pattern of adult emergence for the MI strain was similar to that of T2
strain (Figure 15). In both cases, the halved beans treatments (SD and SU)

produced the most adults, followed by beans with one exit hole (HB) having

129

1 20
1 05
90
75
60
45
30
1 5

MEAN NUMBER EGGS LAID

 

IB CB HB SD SU NB
BEAN SEED DAMAGE TREATMENT

Figure 12. Oviposition (mean 1 SE) by Acanthoscelides obtectus among six bean
seed damage treatments (chorce test). Seed damage treatments: IB =
intact seed; CB = seed with cracked seed coat at both ends; HB = seed
with one "exit" hole; SD = cotyledon with inner side facing down; SU =
cotyledon with inner side facing up; NB = no bean. Data were square-root
transformed prior to ANOVA. Bars accompanied by the same letter are
not signiﬁcantly different at P=0.05, Tukey’s studentized range (HSD) test.

130

 

12° " a 9 . . .
Michigan Strain
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IB CB HB so 50 NB

BEAN SEED DAMAGE TREATMENT

Figure 13. Mean number of eggs laid (18E) by two strains of Acanthoscelides
obtectus among:six bean dama e treatments (choice test). Treatment codes
are given in igure 12. ata were square-root transformed prior to
AN OVA. Bars within each strain accompanied by the same letter are not
signiﬁcantly different at P=0.05, Tukey’s studentized range (HSD) test.

131

 

 

 

 

 

 

 

 

 

20
D
llJ
8 a
LIJ
5 15- a
E
D
9 10-
[K
35 b
3
El d It
2

0‘

IB CB HB so so

BEAN SEED DAMAGE TREATMENT

Figure 14. Adult emergence (mean 1 SE) of Acanthoscelides obtectus in ﬁve bean
seed damage treatments (choice test). Treatments were exposed to 54
larvae per dish. Treatment codes are given in Figure 12. Data were
log(x+ 1) transformed for ANOVA. Bars accompamed by the same letter
are not signiﬁcantly different at P=0.05, Tukey’s studentized range (HSD)
test.

132

N
O

 

Michigan Strain
- Tanzania Strain

 

_a
01

01

Cbc
§

CB HB SD
BEAN SEED DAMAGE TREATMENT

    

MEAN NUMBER ADULTS EMERGED
E3

 

 

V////////////////////4;;;

 

7////////”’; v

 

O

 

Figure 15. Adult emergence (mean 1 SE) of two strains of Acanthoscelides
obtectus in ﬁve bean seed damage treatments (choice test). Treatments
were exposed to 54 larvae per dish. Treatment codes are given in Figure 12.
Data were log(x+ 1) transformed for ANOVA. Bars within a strain
accompanied by the same letter are not statistically different at P=0.05,
Tukey s studentlzed range (HSD) test.

133

intermediate survival, and beans with cracks in seed coat (CB) and intact beans (IB)
with lowest survival. This pattern of larval survival was not congruent with that of
oviposition of the adult bean weevils. Apart from the HB treatment being
intermediate in number of adults emerging and number of eggs received, larval
survival was high in halved beans where oviposition was low, and low in whole beans

(CB, IB) where oviposition by adults was high in the bean weevils.

Larval survival: No-choice test

When larvae were presented with only one of the ﬁve seed damage treatments
at a time, both weevil strain and seed damage treatment effects were highly
signiﬁcant for numbers of larvae surviving (F=41.09; df= 1; P<0.001 for strain, and
F=54.71; df =4; P<0.001 for seed damage type) and for numbers of bean seeds or
cotyledons (halved beans) infested (F=30.08; df= 1; P<0.001 for strain, and F=38;
df =4; P< 0.001 for seed damage type). However, interactions between bean weevil
strain and seed damage treatment were not signiﬁcant (F =2.06; df =4; P=0.092 for
larval survival; F: 1.51, df =4, P=0.205 for seed infestation level). Table 37 shows
means of adult emergence and infestation levels for the TZ and MI strains of bean
weevil, and effects of the five bean seed treatments on larval survival are presented
in Figure 16. Signiﬁcantly more adults emerged from halved beans with inner side
facing down (SD), followed in order by: halved beans with inner side facing up (SU)
> beans with cracks in seed coat (CB) > beans with one "exit hole" (HB) > intact
beans (IB). Relatively few adults emerged from intact beans, while the most
emerged from damaged beans. Infestation levels correspond well with adult
emergence (Figure 17). On average, about two weevils emerged from a given

infested seed or cotyledon in the different seed treatments.

134

Table 37. Adult emergence and bean infestation levels of
two strains of A. obtectus when data for seed damage
treatments were pooled.

 

 

 

Bean weevil No. adults emerged No. beans infested
strain (Mean 1 SE) (Mean 1 SE)
1
Tanzania 14.7 1 0.6 a 7.4 1 0.3 a
Michigan 11.3 1 0.8 b 5.7 1 0.4 b
1
Means followed by the same letter in a column are not
significantly different at P = 0.05, Bonferroni t-test.
(N = 50) .

Table 38. Dry weights of A. obtectus adults emerging from
different seed damage treatments in a no-choice test.

 

Adult dry weights (mg.)

 

 

Seed

treatment N Mean 1 SE Range
HB 255 3.03 1 0.03 a1 0.9-4.3
18 117 3.00 1 0.05 ab 1.7-4.2
CB 270 2.94 1 0.03 ab 1.0-4.5
SD 342 2.91 1 0.03 bc 0.9-4.4
80 329 2.81 1 0.03 c 1.4-4.3

 

1

Means followed by the same letter are not statistically
d1fferent at P = 0.05, Tukey's studentized range (HSD)
test.

135

 

           

 

 

   

205' 20 -' Michigan Strain a

E - TanzanIa StraIn a a
I?! I) Ch \‘\‘ Ob

& 15 i' bC \ V ‘

S 10 - N N N

Z q \ ‘ \ \

BEAN SEED DAMAGE TREATMENT

Figure 16. Adult emergence (mean 1 SE) of two strains of Acanthoscelides
obtectus in ﬁve bean seed damage treatments (no-choice test). Each
treatment was exposed to 20 larvae. Treatment codes are given in Figure 12.
Bars within a strain accompanied by the same letter are not signiﬁcantly
different at P=0.05, Tukey’s studentized range (HSD) test.

136

 

 

 

 

 

12
a Michigan Strain
E 10 . - Tanzania Strain
E
Z
In 8 b
c:
B
m 6
a: c
B
2 4
3
z
z 2
El
2

IB CB HB SD SU

BEAN SEED DAMAGE TREATMENT

Figure 17. Mean number seeds infested (1 SE) by each strain of Acanthoscelides
obtectus in ﬁve bean seed treatments (no-choice test). Each treatment was
exposed to 20 larvae. Treatment codes are given in Figure 12. Bars within
a strain accompanied by the same letter are not statistically different at
P=0.05, Tukey’s studentized range (HSD) test.

137

Emerging adult bean weevils of the TZ strain weighed 2.95 1 0.02 mg.
whereas those of the MI strain weighed 2.89 1 0.02 mg. However, this difference
between the strains of A. obtectus was not highly signiﬁcant in this study (F = 4.60;
df = 1; P = 0.03, GLM). Figure 18 illustrates the distribution of means of adult
weights of the two strains of A. obtectus among the ﬁve seed treatments.
Although no signiﬁcant interaction between the main effects was detected in adult
dry weight data (F = 0.79; df = 4; P = 0.535, GLM), effects of different seed
treatments on dry weights of emerging adult bean weevils were highly signiﬁcant
(F=6.38; df = 4; P < 0.001, GLM) in this test. Whole bean seed treatments (HB,
IB, CB) produced considerably heavier adults than halved seeds (Table 38; p. 134).
Adult dry weights were lowest in the SU treatment. Obviously, the pattern of adult

dry weights was not in accord with that of larval survival in the ﬁve seed treatments.

DISCUSSION

Whole beans (IB and CB) were more acceptable to adults for oviposition than
were damaged beans. Such an adaptation in A. obtectus adults may be related to
the quality of host resource provided by whole beans for larval feeding, growth and
development. Beans with intact seed coats may restrict numbers of larvae entering
a seed at a given time, thus an even distribution of larvae within seeds might be
achieved. On the other hand, the beetles might have oviposited best on whole
beans because they were reared on whole beans (beans with intact and slightly
cracked seed coat) for 32 generations.

In this study, under a no-choice test, higher adult weights were attained for
beetles emerging from whole beans (HB, IB, and CB) than from halved beans.
Whole bean treatments had lower adult emergence when compared to halved

beans. Larvae developing within whole beans probably had more food and space

138

 

 

 

       

 

 

 

3 Michigan Strain
5 4. - Tanzania Strain
g 3. \0 ob ab ab 0 ob ab 0 b
3 ‘ ‘ *N \‘
\ 1‘ N “s
. N N N N I
. 2 N N N N
3 N N N N 2
s N N N N
“ N N N N
i N N N N
5 o N N N
IB CB HB SD SU

BEAN SEED DAMAGE TREATMENT

Figure 18. Dry weights (mean 1 SE) of two strains of Acanthoscelides obtectus
adults emerging from ﬁve bean seed treatments (no-choice test). Treatment
codes are given in Figure 12. Bars within a stram accompanied by the same
iI-tItSeDfIC not signiﬁcantly different at P=0.05, Tukey’s studentized range

test.

139

and consequently achieved heavier adult weights than those in the other treatments.
In addition, an intact seed coat also provides a more sheltered environment for the
larvae, e.g., prevents immature bruchids from falling out of seeds.

On the other hand, survivorship of neonate larvae of A. obtectus to the adult
stage appeared to be highly inﬂuenced by the severity of bean damage, particularly
in the choice test. Larvae survived better with increasing severity of damage.
More larvae survived in split bean seeds (halved bean treatments) than in other
seed damage treatments. In the halved bean treatments, more area without the
seed coat barrier was available to the insect for penetration than in other damaged
seeds. This suggests that the ease of penetration into seeds is a major factor
affecting larval survival of the bean weevil. Based on results of both oviposition
and larval survival, it can be concluded that the pattern of adult host acceptance did
not agree with that of larval survival in these bean weevils.

Physically damaged dry beans, particularly those with exposed cotyledons,
appear to be more susceptible to bean weevil infestation than intact beans.
Comparable observations have been made in other stored product insects.
Schoonhoven et a1. ( 1972, 1974, 1976) reported that higher maize weevil (Sitophilus
zeamais Motschulsky) infestations occurred in maize kernels with damaged pericarp
than in intact kernels. Similarly, when undamaged and damaged sorghum kernels
(with abraded pericarp) were exposed to Sitophilus oryzae (L.) (rice weevil), higher
survival of the insect occurred in damaged kernels than in sound ones (Williams and
Mills, 1980). White (1982), testing the effects of the degree of wheat grain damage
on survival and development of a beetle, Tn'bolium castaneum (Herbst), found that
exposure of the wheat germ through broken seed coats or broken grains was
necessary for larval survival of this insect. These reports suggest that higher losses
from insect infestations in stored grain or seed can be reduced by minimizing or

preventing mechanical damage to these products. The same can be said for

140

A. obtectus infestations of beans.

Differences between the two A. obtectus strains were observed in the overall
oviposition and larval survival in all three tests, despite rearing the two strains under
same conditions for more than thirty generations. Ovipositional and larval
performance differences between the Tanzania and Michigan strains were also
detected in experiments of the previous chapter. Inter-strain differences, however,
have been observed in other bruchids. Dick and Credland (1984), studying the
oviposition and development of three strains of Callosobmchus maculatus
originating from Brazil, Yemen, and Nigeria and reared continuously under same
conditions, found that the Yemen and Brazilian strains laid about 40 eggs while the
Nigerian strain laid about 75 eggs, when each was provided with a single cowpea
seed. Adult emergence among the three strains also varied. The Nigerian and
Brazilian strains had higher adult emergence than the Yemen strain. This was
partially explained by the larger adult size of the Yemen strain. When only one
larva occupied a seed, about 5% of the dry weight of a cowpea was consumed by
larvae of the Brazilian and Nigerian strains, but twice this amount was consumed by
the Yemen strain (Credland and Dick, 1987). The knowledge of existence of these
differences between strains is helpful to researchers developing resistant bean
varieties for use in more than one locality, and to entomologists working on the

biology of these bruchids.

CONCLUSIONS

Adult bean weevils preferred to oviposit on intact or whole bean seeds over
damaged seeds with exposed cotyledons. However, damaged seeds were more
suitable for survival of larvae than intact seeds. Most adults emerged from

damaged beans having exposed cotyledons. The numbers of bean seeds (whole 4-

141

halved beans) infested in the no-choice test did correspond well with the numbers of
larvae that developed to adults. However, distribution of weights of emerging
adults was inconsistent with that of larval survival.

Bean weevil strains differed slightly within each test. In overall ovipositional
choice-tests in this study, the Tanzania strain laid more eggs than the Michigan
strain. In the choice test for larval survival, the Michigan strain had higher adult
emergence than the Tanzania strain. The contrast was true for larval survival
under no-choice test. More adult beetles emerged from the Tanzania strain than
the Michigan strain. Weights of emerging adults also varied slightly between the

bean weevil strains.

CHAPTER 4

Bean attack behavior of neonate larvae of the bean weevil,

Acanthoscelides obtectus (Say).

142

INTRODUCTION

The bean weevil, Acanthoscelides obtectus (Say), infests seeds of a number of
tropical and subtropical legumes, however, it is most often found in common beans,
Phaseolus vulgaris L. The physically destructive stage of this insect is the larva.
After hatching, neonate larvae engage their three pairs of thoracic legs to ﬁnd a
favorable spot for boring into an appropriate bean. Whitish cotyledon powder is
indicative of penetration sites (Larson and Fisher, 1938).

In previous studies (Chapter 1) using one or more bean seeds with intact seed
coat, larvae bored into the cheek of the seed. Furthermore, these entry holes were
always on the check of the bean contacting the petri dish ﬂoor. In another
experiment (Chapter 1), keeping beans from moving during larval boring improved
penetration success and consequently survival of larvae to adult stage by 19 times
when compared to non-stabilized seeds.

Speciﬁc objectives of this study were to determine if: 1) bean areas other than
the cheek were also entered, and 2) the zone of contact established between a
stabilized intact seed and dish lid was necessary for successful bean entry by the
bean weevils (Tanzania (T2) and Michigan (MI) strains). Additionally, video
cinematography was used to determine: 1) various events or steps involved in the
boring behavior of neonate bean weevil larvae prior to complete entry into the
intact stabilized seed, 2) time taken by larvae to bore into bean seed, and 3) time

spent by larvae during different phases of bean boring.

143

144

METHODS

Methods devised for exposure of bean parts and establishing
contact between bean parts and lid of culture dish.

Special petri dish arenas were prepared as follows: General purpose parafﬁn
wax (Walnut Hill Company) was melted in a beaker and a dark blue concentrated
candle dye (Walnut Hill Company) was added at one square of dye (3.18 gm.) to
226.8 gm. of wax. The dye was used to provide a contrasting background for the
bean and particularly for viewing the tiny white larvae when off the bean. About
2 ml of the dyed wax was ﬁrst poured into the 35 mm diam. x 10 mm high plastic
tissue culture dish (Corning Brand) and allowed to begin setting. A single bean
seed was then appropriately positioned to yield a given treatment (see below)
before the wax had completely solidiﬁed. Once set, another layer of molten dyed
wax (ca. 1.5-3.5 ml) was added to the dish to afﬁx the seed and also smoothen the
surface adjacent to the seed. The bean areas (Figure 19) exposed were: cheek,
end, and keel of the seed. Beans were positioned so they either touched the dish
lid (Figure 20) or so there was a gap of about 3 mm. When beans were positioned
on end, a portion was excised from the bottom so the product would ﬁt the dish.

Prepared dishes were set aside at 75% RH for 24 h prior to larval introduction.

145

<— Hilum

 

 

 

Keel —~
Cheek

M
End ./

 

Figure 19. Side view of a bean seed showing various areas exposed for colonization
by Acanthoscelides obtectus larvae.

146

Zone of contact

 

 

Immersed bean ]

”In

L“ T 7‘13
Dyed wax \X\\\\\\\\\\\ \l

 

 

 

 

 

 

 

 

 

Figure 20. Bean positioned with the cheek touching the lid to create a zone of
contact (ZOC).

147

EXPERIMENT 1

Effects of different bean seed areas and zone of contact on
the boring behavior of the bean weevil larva.

Mri Mh

Three bean areas were exposed one at a time in conjunction with presence or
absence of the zone of contact (ZOC) to the ﬁrst instar larvae of either of the two
bean weevil strains. Intact light red kidney beans (Jack Rabbit Brand) used in the
experiment were from one batch purchased from a local grocery store.

A factorial design was used to test effects of: 1) different bean seed areas, 2)
contact, and 3) bean weevil strain on larval entry into beans. A complete block of
treatments was set up on ﬁve different dates. Thus, for each block, twelve
prepared dishes were each presented with three, one-day-old larvae of the
appropriate bean weevil strain. Larvae were transferred carefully onto the wax
surface very close to the bean via lightly moistened hairs of a ﬁne, paint bnish
(Liquitex). Dishes were immediately covered with the lids and sealed with
laboratory paraﬁlm (Paraﬁlm M) to discourage escape of larvae and reduce
moisture loss from seed. All dishes were grouped per block on a table with light
intensity 9;), 2300 lux in a growth chamber. Placing dishes on trays facilitated
simultaneous observations of larval behavior under a dissecting microscope. All
larvae in this experiment were from stocks of bean weevils of the 37th generation
and above. Larvae were obtained from eggs of females emerging during a three-
week period.

In addition to the main experiment, an attempt was made to determine if the

moisture content of beans at the beginning of the tests would improve larval

148

penetration into seeds. Larval boring into seeds hydrated within a dish for 24 h
was compared to that on dry seeds. The test was run simultaneously with the main
experiment and using only beans with check contact with the lid. Three larvae
were added to each dish for the two strains of bean weevil.

All tests were conducted within the environmental chamber at 25 0C and
photoperiod of 16L:8D. A ﬁber optics lamp was used only during ﬁxed times of
observations of larval behaviors particularly by and after the 12th h after larval
introductions. Various larval activities were recorded at half hour intervals for the
first 6 h, after every hour for the next 3 h, after another 3 h, and every ‘12 h
thereafter. (Some observations were also made at every hour from the 20th to
24th h). Tests were terminated after 48 h.

Components of the behavioral sequence of the ﬁrst instar larva used for
analysis included: 1) time at which the larvae were introduced, 2) time within
which they settled and initiated boring on seed, 3) time within which borings or
cotyledon powder ﬁrst appear near larva, and 4) time within which larvae
completely bored into seed (no abdomen visible). The 12th h after introduction of
larvae into the dish was used to determine number of larvae actively boring, and the

48th h was used to determine the number of holes consequently bored by these

larvae.

MW

Wm

In the absence of contact (Figure 20; p. 146) between seed and dish lid, no
larvae settled or positioned themselves for digging on any of the seed areas; and
consequently, no holes were dug. Boring occurred only when the seed touched the

dish lid. This phenomenon was also observed by Kannan (1919) and Thiefy

149

( 1982b). Thus the acceptability of the bean areas can only be evaluated in trials
with an available ZOC. When introduced larvae had a ZOC (N = 90 larvae),
52-2% bored continuously by 12 h, and 51.1% of the total eventually entered beans.
Tables 39 and 40 show the distribution of larvae digging by 12 h and holes bored by
48 h for each strain of bean weevil and bean seed part exposed. No relationship
was detected between insect strain and bean area contacted with respect to
sustenance of larval digging by 12 h (Pearson’s x2 = 0.428; df =2; P = 0.807) and
ﬁnal number of holes bored by larvae at 48 h (Pearson’s x2 = 0.465; df = 2; P =
0-793).

No boring occurred on bean areas without a ZOC even when seeds were
softened by pre-hydration for 24 h prior to testing. With a ZOC, larvae bored
holes in all three bean areas, indicating that all were susceptible to the bean weevil
larval attack, provided there was contact with another seed or other object. Under
natural situations, parts of seeds are either in contact with the pod walls or are in
contact with other seeds in storage. In both these situations, larvae are provided
with favorable boring sites.

In this experiment, the three bean seed areas did not differ signiﬁcantly in the
number larvae initially boring (,8 = 0.551; df=2; P<0.8) and the number of holes
eventually bored into seed (,3 = 1.351; df = 2; P>0.5). These no-choice results
may not exactly reﬂect those in the usual choice situation where large number of
beans are stored together. However, observations from a following experiment

Support this ﬁnding that any seed part can be attacked successfully as long as there
is a ZOC.

Strain of bean weevil had no signiﬁcant effect upon the boring behavior of
larvae nor in the number of holes dug. The x2 values were 0.532 (df = 1; P<0.5)
for lill'Vae digging by 12 h and 0.087 (df = 1; P>0.75) for ﬁnal number of holes
bored by 48 b. With bean seed areas pooled, frequencies of larvae boring by 12 h

150

Table 39. Influence of different bean seed areas on
sustenance of larval boring by the 12th hour in the two
strains of A. obtectus: a contingency table of bean seed
area vs. strain of bean weevil.

 

Numbers of larvae digging

 

 

 

 

 

Michigan Tanzania
strain strain
Bean seed
area exposed
Freq. (%) Freq. (%) Total (%)
Cheek 11 (23.4) 7 (14.9) 18 (38.3)
End 8 (17.0) 7 (14.9) 15 (31.9)
Keel 7 (14.9) 7 (14.9) 14 (29.8)
Total 26 (55.3) 21 (44.7) 47 (100)

~

Pearson's Chi-square test value = 0.428: df = 2; P = 0.807.

151

Table 40. Influence of different bean seed areas on the
ftilaal number of holes bored and entered by larvae of the two
strains of A. obtectus by the 48th hour: a contingency
1:211:1e of bean seed area vs. strain of bean weevil.

 

Numbers of holes bored

 

 

 

 

 

 

Michigan Tanzania
strain strain
Bean seed
area exposed
Freq. (%) Freq. (%) Total (%)
Cheek 11 (23.9) 8 (17.4) 19 (41.3)
End 7 (15.2) 7 (15.2) 14 (30.4)
Keel 6 (13.1) 7 (15.2) 13 (28.3)
Total 24 (52.2) 22 (47.8) 46 (100)
Pearson's Chi-square test value = 0.465; df = 2; P = 0.793.

152

were 26 and 21 for the MI and T2 strain, respectively; and frequency of holes bored
by 48 h was 24 for the M1 strain and 22 for the T2 strain. Numbers of larvae
observed digging at an earlier time may not always correspond exactly with the
number of holes ﬁnally bored into the seed. Some larvae may quit digging early
and subsequently enter a hole dug by another larva as did some larvae of the MI
strain; whereas other larvae not settling on a seed earlier, begin digging their own
holes subsequently (i.e. after 12 h) as was the case with some TZ larvae in this

experiment.

Effgggs of bgan moismre content

No relationship was detected between seed moisture and strain of bean weevil
with respect to larval boring behavior 03 = 0.019; df= 1; P=0.89 for sustenance of
larval boring by 12 h, and x2 = 0.422; df = 1; P = 0.516 for the ﬁnal number of
holes bored (Tables 41 and 42)). In addition, seed moisture did not greatly
inﬂuence initiation of larval boring behaviors in this test. Proportions of larvae
digging by 12 h were not statistically different between dry (51.4%) and hydrated
(48.6%) beans (X2 = 0.029; df = 1; P<0.9). Likewise, percentage of holes bored
by larvae by 48 h were also similar in each of the two seed treatments (Table 42).
Effects of seed hydration may have been obscured because observations were
generally made at ﬁxed time intervals; some larvae may have bored into seeds a
little faster than others, yet all were inside by the designated observational time.

The number of larvae initially boring was higher for M1 than TZ bean weevils;
however, the difference was not signiﬁcant (x2 = 1.4; (if = 1; P<0.25). The same

Was true for the eventual numbers of holes bored (x2 = 0.105; df = 1; P4175)-

153

Table 41. Effects of bean seed moisture content on
sustenance of larval boring in the two strains of
A. obtectus.

 

Larvae digging at 12 h

 

 

 

 

 

 

Michigan Tanzania
strain strain
Bean
condition Freq. (%) Freq. (%) Total (%)
Dry 10 (28.6) 7 (20.0) 17 (48.6)
Pre-hydrated 11 (31.4) 7 (20.0) 18 (51.4)
Total 21 (60.0) 14 (40.0) 35 (100.0)
Pearson's Chi-square test value = 0.019: df = 1: P = 0.89.
Table 42. Effects of bean seed moisture content on final

numbers of holes bored and entered by larvae of two strains
of A. obtectus.

 

Holes bored by 48 h

 

 

 

 

 

Michigan Tanzania
strain strain
Bean
condition Freq. (%) Freq. (%) Total (%)
Dry 9 (23.7) 10 (26.3) 19 (50.0)
Pre-hydrated 11 (28.9) 8 (21.1) 19 (50.0)
Total 20 (52.6) 18 (47.4) 38 (100.0)

 

Pearson's Chi-square test value = 0.422; df = 1: P = 0.516

154

i fl rv 1 e k ivi i

Estimates of timing for different events on the bean seed are presented in
Table 43. Excluding two "outliers" for settling time (6 h and 9.5 h) and for
complete entry (30.5 h and 40.5 h), elapsed time for settling was 1.8 1 0.1 h
(mean1 SE), and elapsed time the ﬁrst cotyledon borings appear near the larvae
was 3.8102 h. Time elapsed from settling to complete entrance of the larva into
seed was 21.8 1 0.1 b. On average, a neonate larva took a total of 24 h to locate a
favorable spot on the seed and enter. Furthermore, one or two other larvae from
the dish usually entered the same hole dug by the ﬁrst larva. All these larvae were
inside by 48 h. Distributions of larvae settling and consequently completely boring
into seed at different times are presented in Figures 21 and 22, respectively. The
two "outliers" are included for each variable. Even though the range of time taken
by the larvae to settle on seed and initiate boring was 0.5 to 9.5 b, about 87% of the
larvae settled within 3 h. Elapsed time for larvae to enter the seed completely

ranged from 19 to 40.5 h, however, about 82% of these entered within 20 to 23 h.

mm

In this experiment (includes data from moisture content trials) larvae were
categorized as: 1) pioneers (Umeya and Kato, 1970) - larvae initiating, digging and
entering their own hole into the bean; 2) followers (Umeya and Kato, 1970) - larvae
entering a hole dug by a pioneer larva; however, in this study, followers also include
larvae that initiate digging but quit early in the process to seek holes bored by other
larvae; and 3) escapees - larvae that left the arena and did not return by 48 h.
Escapees were usually found stuck between the paraﬁlm and dish. Followers were
often seen waiting close behind a digging pioneer. Of 120 larvae, 53.3% were

pioneers, 23.3% were followers, and 23.3% escaped. Of the 64 holes dug, 67%

155

Table 43. Timimg of stages of A. obtectus entry into
bean seeds as determined by periodic examination under a

d i s secting microscope .

 

Elapsed hours

 

 

Event N Mean 1 SE Range
Introduction
to settling 61 1.8 1 0.1 0.5-4.5
I nt roduct ion
to bean powder
appearance 19 3.8 1 0.2 2.5-5.0
Settling to
beam entry 61 21.8 1 0.1 19.0-24.0

156

 

 

 

 

 

B
E 100 o #0
.——-—--C——
(I) I
Bill /0
> 80‘ 0
C: I
S /o
‘5 60- o
'2 /
25'
m 40" O
LIJ
m 1
E 20- /
. o
‘1". /
g 0' ‘ ' ' ' ' ' ' 8 9 10
o 0 1 2 3 4 5 6 7
TIME(Hours)

Figure 21. Distribution of Acanthoscelides obtectus elapsed times for settling to dig
into beans (N = 63).

157

 

100-

O
O

S"

80-

60-

40-

o
o’
/
o
/
o
.l
20- /
o

CUMULATIVE PERCENT OF LARVAE
HAVING ENTERED COMPLETELY

1600'

18 2'0 2'2 2'4 2‘6 2'8 50 3'2 3'4 3'6 38 4'0 42

 

 

 

TIME (Hours)

Figure 22. Distribution of Acanthoscelides obtectus elapsed times for completing
bean entry (N = 63).

158

were entered by a single larva, 22% by two larvae, and 11% by three larvae. It
appears that some larvae are diggers and others are followers when presented with
same conditions. Whether these behaviors were physiologically, genetically or
environmentally based was not established in this study. In 7.5% of the 40 trials in
which there was a ZOC, none of the three larvae attempted to dig; all left the arena
despite the presence of ZOC and hydrated seed. Perhaps none of these larvae
were diggers or perhaps this result is just due to stochastic processes. Under
natural situations, they may have found more distant seeds or holes bored by other

larvae; or they may have died.

EXPERIMENT 2

Larval seed-boring behavior under ”natural” storage conditions.

Mriln

I wished to determine if the distribution of larval entry holes on various seed
areas under natural storage conditions (relatively large numbers of beans and
beetles) agreed with that observed in Experiment 1 (this chapter): that all areas of
bean in contact with other beans or container were susceptible to larval attack.
Bean seed areas examined were: cheek, keel-back, keel-front, end, and hilum zone
(see Figure 23 and Table 44 (pp. 159 and 161)). Available surface area for
each external bean seed part was also estimated by measuring the individual
demarcated zones of the seed as shown in Figure 23.

Locally purchased light red kidney beans (244 gm) with intact seed coats were
Placed in 800 ml glass jars. To each jar, 15 pairs of adult bean weevils from the

40+ generation culture were added. Only beetles of the Michigan strain were used

159

 

Hilum

 

---------------‘--

 

Keel
(Front)

 

End

Cheek

Keel
(Back)

1ﬁgure 23. Side view of a bean seed showing various areas used to estimate the

surface area for each bean part.

160

in this test. The jars were then placed on their sides within a glass cage in a walk-in
environmental chamber maintained at the conditions of Experiment 1. Beans
within jars were undisturbed for about 28 days after which dead adults were
removed and entry holes on various parts of beans were counted. Beans were
returned to jars for adult emergence. The test was replicated 4 times. Relative
frequencies were obtained for the numbers of entry holes on each bean part
examined, and also for the numbers of entry holes per whole bean. Further, the
distribution of entry holes per bean was also compared with a Poisson distribution.

The goodness of ﬁt was analyzed by a chi-square test.
Besultsaninscussion

iriinfnhl niffrnr fen

The distribution of entry holes among various locations on the bean seed
uIlder normal storage conditions is presented in Table 44. Only two replicates of
the experiment were used to determine the frequency of entry holes among the
different bean areas. In this experiment, neonate bean weevil larvae bored holes
in almost every part of the seed, however, the frequency appeared to vary greatly.
me cheek was by far the common entry site when compared with the keel, end and
hilum areas of the bean even when the entry hole distribution was adjusted for
slll’face area available for each bean part (Table 45). The cheek of the light red
ki(iney bean received approximately seven times more entry holes per unit surface
a~1‘ea than the end and hilum areas; and over two times more holes than the keel
areas. Further, in the hilum area, entry holes were found close to the edges of the
hilum rather than on the hilum itself.

The cheek of light red kidney bean has a larger surface area (Figure 24) and

Z0C; subsequently, it received more entry holes than the other bean areas. Also,

161

Table 44. Positions and relative fre uencies of Acartthoscelidar obtectus entry holes
on various parts of bean seeds (N = 01 entry holes).

 

 

Entry hole Bean part Relative
position or area frequency (%)

 

69 End 1.9
Hilum 2.2
@ Keeleront 6.8
0""

. Keel:Back 9.7
Q Cheek 79.4

162

Table 45. Number of A. obtectus entrance holes in seed
parts with normalization for differing surface areas of
respective parts.

 

 

 

 

Surface No. of % % No. of
Bean 1 area (cm ) entry Surface Entry entry holes
area available holes area (cm ) holes per unit
per bean per bean surface area
END 0.31 12 10.02 2.00 0.20
HILUM 0.33 13 10.61 2.16 0.20
KEEL-F 0.35 41 11.56 6.82 0.59
KEEL-B 0.41 58 13.56 9.65 0.71
CHEEK 1.66 477 54.25 79.37 1.46
TOTAL 3.06 601 100.00 100.00 3.16
1
Keel-F = front keel parts (2) : Keel-B = back keel of the
Seed . Hilum included areas immediately around hilum and
2hilum itself.

N = 20 beans for surface area estimates.

163

 

 

 

 

 

 

g 50- y
:2: %
§?é%%%%

BEAN SEED AREAS

Figure 24. Estimated surface area (mean 1 SE) for various bean seed areas or
zones of a 1i t red kidney bean (N = 20 beans). CHK = cheek; KLB =
ke31:back; = keel:front; HLM = hilum + areas close to hilum; End =
en area.

164

the angle between beans in cheek-to-cheek contact might be more favorable for
boring and the seed coat may be thinner or possibly less toxic in the cheek area than

in other seed areas (these factors were not examined in this study).

Diszribmion of gntg hgles among beans

When 2,076 beans distributed in four 800 ml glass jars were exposed to 30
beetles of mixed sex per jar, no entry holes were observed in about 52% of these
beans (Figure 25). About 32% of the beans had one hole per bean.
Furthermore, two entry holes per bean were found in about 12% of the beans.
More than two holes per bean were found in less than 10% of the beans.

The observed distribution of entry hole number per bean among total beans
was compared to the Poisson distribution. It was assumed that each entry hole was
distributed randomly and independently among beans. The calculated Poisson
(expected) values and observed data are presented in Table 46. A chi-square test
for goodness of ﬁt (Snedecor and Cochran, 1969) comparing the two distributions
indicated a significant deviation from a perfect ﬁt ()8 =75.23; df=4; P<0.005).
The signiﬁcant chi-square value can be ascribed to ﬁnding fewer beans than
expected with one hole per bean, and, more than the expected number of beans with
Zero or more than two holes per bean. Furthermore, the combined 5-7 categories
had a large contribution to the chi-square, however, their exclusion from the test of
ﬁt to the Poisson distribution did not reduce the chi-square value to a non-
Signiﬁcant level (XZ =12.67; df =3; P<0.01). These observations suggest that
deSpite the single hole per bean being most common, there may be a slight tendency
by neonate larvae to aggregate for bean entry. Perhaps the presence of digging
la“lac on a bean may be indicative of suitability of that bean for digging. On the
other hand, studies reported in Experiment 1 and 3 of this chapter, showed that

many A. obtectus larvae initiate their own holes upon ﬁnding suitable areas on beans

165

 

60

501

A

40‘)-

30-

I

N
N N N .....

0 r
0 1 2 3 4 5-7

NUMBER OF ENTRY HOLES PER BEAN

20¢

RELATIVE FREQUENCY
(Percent of Beans)

T

10-

 

 

 

 

 

Figure 25 . Frequency histogram for Acanthoscelides obtectus entry hole numbers
per bean under normal storage conditions (N = 2076 beans).

166

frable 46. Distribution of numbers of entry holes per bean
(N = 2,076 beans) with fitted Poisson distribution.

 

 

No. of Observed Expected Deviation
entry frequency frequency
holes (Poisson)

0 1085 1027.72 57.28

1 656 722.49 -66.49

2 239 254.32 -15.32

3 70 59.51 10.49

4 15 10.47 4.53
25 11 1.48 9.52

Total 2076 2075.99 0.01

167

for digging. These larvae have been categorized as pioneers.

EXPERIMENT 3

Videotaping bean weevil seed entry behavior.

M rial n Mth

Video recordings revealed the activities of larvae from introduction into the
dish to complete entry into the bean seed. Behaviors were recorded on T-160
VHS video cassettes using a JVC (Reg) BY-110 color camera ﬁrst set to capture all
larval activities on the whole bean and then set close-up. The camera was outﬁtted
with a Nikon lens (Micro NTKKOR 55mm) and 2 adapters (Nikon PK-13 (27.5) and
Nikon PK-12 (0.14)) and was mounted on a tripod. The video recorder was the
Panasonic (Reg) (Pro-Line) GX4 Multi-function model. A high resolution Sony
(Reg) Model No. PVM-2030 Trinitron color video monitor was used to observe
larval activities being ﬁlmed and later used to view play-backs of the larval
behaviors.

Day-old (0—24 h) ﬁrst instar larvae used were obtained from eggs of the T2
and MI bean weevils conﬁned in glass petri dishes until hatching. For behavioral
Observations, larvae were placed in 35 mm diam. x 10 mm high plastic tissue culture
diShes as in Experiment 1. Only the experimental setup in which the bean cheek
Was in contact with the dish lid was used, because the cheek had higher frequency of
laI'Val boring than other bean seed areas (Experiment 2, this chapter). Dishes were
Set at 75% RH for 24 h prior to larval introduction. During the ﬁrst 10 trials, only
One larva was placed in a given dish. However, due to low numbers of larvae

s“vttling, initiating boring, or actively boring, the next set of tests used three larvae of

168

the same strain in a given dish. The dishes were sealed with laboratory paraﬁhn to
discourage larvae from leaving.

At the beginning of a test, larvae were released on the wax surface close to the
bean within the dish. The dish was quickly sealed and positioned under the camera
to record ensuing larval activities. Thus the larval movements in the dish were
observed from above and through the transparent petri dish lid. A high intensity
fiber optics illuminator was placed about 10-15 cm above and to each side of the
dish to provide continuous lighting of approximately 38,700 lux for an entire trial.
On average, ﬁve 8-hour video cassettes were used for one trial. Trials in which all
larvae either did not settle, dug discontinuously, or left the arena for at least 8 h
were discontinued. The entire study was housed in a controlled-environment room
maintained at 25 oC and 50 1 5% RH. Insects used were of similar age as those
described for Experiment 1.

Behaviors analyzed were movements over a bean, settling at a point of boring,
and boring. Other aspects also noted were time taken by a "pioneer" to enter a
seed completely, time taken by a "follower" to enter the same hole, disturbance or
interruptions by other larvae during some of the activities, and interactions between
followers in multiple introductions. Not all behavioral components were detailed.

In addition to videotaping, some morphological features of ﬁrst instar A.
Obtectus that appeared to play some role in the boring process (e.g. prothoracic
Plates, mouthparts, tarsi, and sucker disk (tenth abdominal segment» were also

examined using a dissecting microscope at 65 and 70x.

Resglgs and Disgugsign

Major steps in the bean-entry process established from videotaping both

Pioneer and follower larvae of A. obtectus are illustrated in Figures 26 and 27

169

 

Walking/searching
on dish

1

Walking/searching
on Dean

1

Settling/initiating
boring

1

Actively boring

1

Inside bean

 

 

 

 

 

 

 

 

l ’ ll

 

 

 

 

 

 

 

 

 

 

 

 

Figure 26. Sequence of behaviors whereby pioneer Acanthoscelides obtectus larvae
colomze a bean contained in a petri dlSh arena.

170

 

Walking/searching
on dish

1

4 Walking/searching
a on bean

- 1

’ Finds freshly
bored hole

 

 

 

 

 

 

 

 

 

 

 

 

 

Encounter

with
'pioneer" ‘

and/or chasing b Attempting entry
secondary <

”follower" % J

 

 

 

 

 

 

 

 

 

 

 

 

 

Disturbed by
secondary
'follower'

 

Inside bean

 

 

 

Figure 27. Sequence of behaviors whereby primary follower Acanthoscelides
obtectus larvae colonize a bean contained in a petri dish arena.

171

respectively. Detailed description of the boring action is given for the initial

preparation of the hole on the bean seed by the pioneer larva.

ﬂg-bog'ng and settling of pignggr larvgg

Neonate larvae introduced to a dish with a bean seed spent 1.3 h on average,
walking about in the enclosed arena and/or over the bean before settling
completely to begin seed boring. Seemingly random search behavior was highly
altered when larvae encountered the crevice where bean and lid touched (ZOC).
Larvae frequently continued walking but restricted their paths so as to depart the
ZOC infrequently (Figure 28 a). All pioneering larvae became arrested in the
ZOC (Figure 28 b), presumably at a point conducive to digging. When multiple
larvae were introduced to one dish their sites revealed the boundaries of the ZOC
with great accuracy (Figure 28 b). A settled larva tightly wedged its head and
thorax within the bean/lid crevice (Figure 28 c) with mouthparts oriented towards

the bean; their long dorsal setae were bent ﬂat against the body.

b rin h vi r f i r1 r1 rv

The broadest part (9a, 0.2 mm) of the larval body includes the head, thorax,
and ﬁrst three abdominal segments. When preparing for boring, the head was
retracted under the thorax, and the posterior larval abdomen was advanced such
that the resulting compression moved body ﬂuids forward, thus swelling the broad
anterior. These adjustments may aid in anchoring the thorax so that scraping
Strokes with the mouthparts are translated into displacement of bean tissue rather
than advancement of the larval body along the bean surface. Moreover, these
hycirostatic forces might generate downward force for digging. Mouthpart strokes
during the initial digging were postero-anterior (Figure 28 (I). These were followed
by lateral or diagonal strokes (Figure 28 e) and by rotation of the head (Figure 28 f)

172

(a)

(b)

 

In“—

Figure 28. Movements and positioning of ﬁrst-instar Acanthoscelides obtectus
larvae entering a bean. ee text for explanation.

under

rnand.
dhhnn
reuar
Opera
onthe

abutn

 

the b
enny

(Kann

173

under the thorax.

The head of the ﬁrst instar larva is oval and elongate. Its heavily sclerotized
mandibles are situated at the anterior of the head, which face downwards during
digging. According to Pfaffenberger and Johnson (1976), the role of the
retractable head with the V-shaped neck fold is in extending and increasing the
operational angle of the larval mouthparts in the boring process. In addition, teeth
on the upper two arms of the prothoracic plates (Figure 29) are anchored against an
abutting seed or pod (Kannan, 1923), while the lower arms are positioned close to
the boring point. This plate is repositioned at equal distances around the point of
entry during the digging process, thus the characteristic circular shape of the hole
(Kannan, 1923).

After about 2.5 h of digging, borings of white cotyledon powder appeared on
one side of the larva. As they were generated, borings were moved under the body
posteriorly, and eventually were extruded to either side. Once appreciable borings
accumulated on both sides (Figure 28 g) and periodically thereafter, the larva began
a stepwise shift of its posterior (adhesive-bottomed 10th segment) along a semi-
circular track (Figure 28 h) as far as the given ZOC would allow. In this process of
repositioning its head, mouthparts, or prothoracic plate around the hole, the borings
were spread from the digging site by the dragging abdomen. During this action,
the larva did not remove its head from the hole. Body ﬂuids were periodically
moved from the posterior to anterior as the larva repositioned itself over the hole.
Forward body thrusts were also observed during the later half of the boring period
when the larva appeared to actively push forward and downwards. The adhesive
sucker disk of the 10th abdominal segment appeared to keep the larva attached to
the bean surface during the seed-boring activities.

Once the larva had most of its body inside the hole, its posterior yet outside

was no longer in contact with the lid (Figure 28 i). The larva occasionally rotated

FiEur

174

(a)

(Pfaffenberger & Johnson, 1976)

 

PA”

(Cl

 

F e 29. Prothoracic lates of the ﬁrst instar larva of Acanthoscelides obtectus:
lgur (a) Prothoracic plaIies with teeth onu uper arms and medians, (b dorsal vie;
of plates on larval prothorax, and( cposition of the plates on arva (later
view). Head of larva 1s retracted M in the thorax In Fb) and (c).

C0
ar.

til

Ira

175

in the hole as it bored further. A larva was considered to be completely inside
when its posterior was below the bean surface. Data from one of the trials of this
experiment, illustrate larval activities at selected times during the bean colonization
(Figure 30). In this experiment, elapsed time from introduction to entry into the
seed was 22.5 1 0.8 h (Table 47), which agrees well with Table 43 (p. 155).

Some larvae periodically paused during walking or boring on bean seed, and
this varied with the individual larva and time spent on the bean. For example, two
larvae walking on a bean paused three times prior to settling on the bean to dig.
One larva paused for 3.1 1 2.5 min (mean 1 SE) after every minute of walking and
ﬁnally walked continuously for 2 min and settled. Another larva paused for a
minute after an average of 1.3 1 0.3 min of walking and eventually walked
continuously for 8 min before settling. A few larvae did not appear to pause at all
and settled quickly or left the seed. Moreover, during digging, one larva paused 60
times within seven hours of digging; the pauses lasted about one minute after every
5 min of boring. After 10 h of seed boring, the larva bored for an average of 19
min (1 3.2 (SE)) prior to pausing for 2.0 1 0.4 min. Pausing was observed even

during the last stage in the entry process.

Pr - n in e h f l w

A searching follower larva walked about the seed and became arrested when it
found a hole already dug by a pioneer, or came upon a digging larva. When a
pioneer larva had not fully entered the seed, the follower usually waited close
behind or beside the borer, or left the site or seed temporarily or permanently.
Also, when a pioneer was inside the seed, yet its posterior was still close to the bean
surface, the follower attempted entry and left, or waited for the digger to bore in
farther. However, when a completed hole was found, the follower tried forcing its

body into the hole. In a few cases, the opening of the hole was slightly small. The

176

 

B 8
V) N)
M AC QA
(28 min) (35 min) (2h 40 min)
A settled B settled C climbs on bean

     

(2h 50 min) (4h) (4h 03 min)
Borings aroUnd A C settled

     

(16h 15min) (23h) (24h)
Active boring A inside All inside

Figure 30. Behaviors and their timing for three Acanthoscelides obtectus larvae
(A,B,C) during bean colonization.

Table 47. Timing of stages of

177

A.

obtectus

entry into

bean seeds as determined by continuous videotaping.

 

 

Boring behavior phases Mean 1 SE Range
Hours
Introduction to settling 0.9 1 0.5 0.01-4.00
Introduction to
bean powder appearance 2.9 1 0.1 2.50-3.08
Settling to bean entry 21.6 1 0.7 18.12-23.63
Introduction to entry 22.5 1 0.8 18.17-24.00

 

N = 7 larvae.

178

larva appeared to scrape the edges of the hole so as to enlarge it.

When several followers were present at the hole site, the ﬁrst follower
(primary follower) appeared not to abandon the hole for long. One larva was
observed to sit and cover part of the hole by its body, occasionally attempting entry,
until the hole was available for entry. On average, the follower took 9.9 1 1.5 h
(SE) from attempting entry into an already dug hole to complete entry into the seed
(range 5.8 to 15 h; N = 6 larvae).

Di rnefinr followr rin rin

A follower was normally observed to approach the digger (pioneer) from
behind but retreated upon contacting the digger’s setae or terminal segments. By
waiting behind the digger, the follower often got bumped in the face by the posterior
of the digger. When an aggressive follower initially got too close to the boring site,
the digger usually pushed (head against head) the follower away from the hole.
This reaction was observed mainly in disturbed diggers at earlier stages of the
boring where most of their bodies were still outside the seed. After such
disturbance, the digger usually returned to the boring site and resumed digging.
The intruder retreated or moved from the scene temporarily but then returned and
stayed close behind the digger or at some distance on the side of the digger. The
follower had to wait until the digger completed the hole and entered ﬁrst. Usually
the interruption or disturbance level observed in this situation was minimal. No

prolonged ﬁghts were observed.

'n l r h f 1] r
When more than one follower was present at a ﬁeshly dug hole often with the
pioneer’s setae still visible in the hole, larvae appeared to get excited and contests

for penetration into the hole ensued. In this study, ﬁve larvae were introduced

179

onto a bean with a hole just entered by the pioneer. A follower larva ﬁnding the
hole ﬁrst and attempting entry, appeared to protect it from the other follower
larvae. This resulted in constant ﬁghts and chases between the primary follower
and other larvae before seed entry was accomplished. A secondary follower in the
presence of others had to go through a process identical to the primary follower
prior to entry into the seed. Disturbances continued until only one larva was left
on the seed. Follower-follower aggression was more pronounced than that of
follower-pioneer.

Three categories of "disturbance" observed in the tests among followers were
fights, chases, and jabs.

ﬂglLt. A ﬁght involved a larva charging headﬁrst into an intruder, striking its
head or abdomen. Larvae attacked with their mandibles spread and then poked or
grabbed a soft part of the intruder’s body and sometime swung the intruder side to
side. Usually a fight began when a secondary follower approached and jabbed or
poked the larva attempting entry into the hole. More aggressive intruders fought
back and although they left the site temporarily, and they usually returned later for
another ﬁght, if the primary follower was not inside the hole.

Chm A chase involved a larva pushing away or chasing after intruders at
the hole site. Chases may occur soon after a ﬁght, after a physical contact, or just
to keep the boring area free of intruders (probably to give the larva more time to
bore into the seed). After a chase, the larva usually returned to the hole and
resumed entering, sometimes by squeezing into the hole, or if necessary by hole
enlargement.

M The secondary follower waiting behind jabbed at the primary follower
attempting seed entry. When the body of the seed-entering larva was more than a
third, or, half way into the seed, the other follower pushed and bit the exposed part

of the abdomen and/ or posterior of the seed-entering larva. If the entering larva

 

180

had most of its body outside, it fought back, but when a greater portion of its body
was inside, it generally did not stop the seed penetration process to ﬁght.
Secondary followers were observed to continue jabbing the exposed posterior of the
seed-entering larva (e.g. primary follower) until the larva was almost inside the seed.
This period of steady jabbing ranged from 15 to 26 min in this study. It appeared
that jabbing did no harm to the seed-entering larva but that this larva may have
been forced to enter faster than it would have if not jabbed. In one case, the
primary follower was inside the seed 30 min later following 26 min of being "pushed"
into the hole.

Despite the aggressive behavior in larvae presented with an already dug and
entered hole, individual variation exists among these larvae. Some were "hawks"
whereas a few were "doves". In one extreme case of a highly aggressive group of
four followers, no larva managed to enter an already dug hole due to constant ﬁghts
and chases among the larvae. All larvae eventually died without seed entry.
However, in most cases, the less aggressive larvae either waited until other followers
were inside the seed or entered the hole when other larvae left the site temporarily.

Frequency of disturbance involving ﬁghts and chases also varied with different
larvae in the case of multiple followers on a seed with a single freshly dug hole.
Table 48 presents the different types and total number of disturbances observed for
each larva and intervals between disturbances where the larva was able to continue
seed penetration. Overall interval (N = 36) with no disturbance during seed entry
averaged 18.9 1 4.3 (SE) min for each larva. The most common interval without
disturbance ranged from 1 to 10 min (Figure 31). This indicates that the seed-
entering larva in such a case was disturbed more often by other followers and thus
boring was delayed. In Table 48, larva 2 and 3 entered the holes within 6 h while
others took longer. These two larvae had relatively longer periods of hole entering

between disturbances.

181

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182

25

 

20-
15-

10-

FREQUENCY

 

 

 

N

1-10 11-20 21 -30 31-40 41 -50 51 -60 >60
SEED ENTRY TIME WITHOUT DISTURBANCE (min)

 

 

Figure 31. Frequency distribution of intervals without disturbance during seed
entry of the follower larvae.

183

GENERAL DISCUSSION

Factors that may affect boring success and the duration of
boring in intact bean seeds by the bean weevil larvae

The presence of the zone of contact between seeds, between seed and pod, or
between seed and seed container, and the stability of the seed itself while being
attacked by the larva are essential features for successful boring into an intact seed.
In seeds with perforated seed coats, some penetration is possible even without the
ZOC (Chapter 1).

Temperature and relative humidity in the storage environment may also play
an important role in duration and subsequent success of seed entry by larvae.
Thiery (1981) observed a mean seed coat-perforation time of 10 h when neonate
larvae of A. obtectus were exposed to beans at 26 0C and 70% RH. In this chapter,
the larvae took about 23 h on average to penetrate the bean testa at 25 OC and 50%
RH.

Bean testa moisture content and seed age have been shown to affect the bean
penetration process of ﬁrst instar bean weevils. Thiery (1982a) found that larval
penetration through the bean seed coat increased with increasing seed coat moisture
content; however, older beans (e.g. bean crop from the previous year) had lower
penetration frequencies than those of new beans, irrespective of moisture content
levels. Presence of a toxic chemical barrier was suspected in testa of older beans.

Age of the ﬁrst instar larvae may also determine the ability of these larvae in
successfully penetrating intact bean seeds. The amount of time spent walking
outside the bean is critical in the survival of the ﬁrst instar larvae. Prolonged
displacement prior to seed entry may exhaust most of the energy available for

digging through testa. Most larvae died in about a week when they were left in an

 

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Illa

184

empty glass dish without a bean. Further, it would be expected that the exertion of
boring would shorten life-span signiﬁcantly if entry were not attained. Therefore,
delays of any kind in bean penetration process may be costly. Longer time taken
for penetration by the pioneer larva will in turn delay entry of the follower.
Furthermore, the size of the hole dug by the previous larva may prevent or favor
faster entry of the follower or other larvae, depending on their sizes. Where the
follower is bigger than the pioneer, the follower may have to spend a little time to
enlarge the hole or squeeze in where possible. On the other hand, aggression by
the follower outside the hole on the follower about half way in the seed appeared to
hasten the borer’s entry into the seed. Perhaps this favors survival of the followers.

The ability of larvae to penetrate the seed coat can also be affected by various
stressful situations. Larvae exposed to stressful conditions such as high
temperatures and very low relative humidities, in addition to temporary host
deprivation, reduced bean penetration frequency by more than 50% for most larvae
(Thiery, 1982b).

Density (Chapter 1) and distribution of larvae on a given seed may contribute
to either success or duration of entry into a seed. When no hole is available, and a
pioneer larva is digging, a follower generally waited nearby quietly. However, high
density of follower larvae on a given freshly bored hole may prolong larval
penetration into the seed mainly by contesting the hole. The more followers, the
more fights. Aggressive followers generally disturbed the seed-entering follower
frequently, and entered seed earlier than less aggressive followers. Size of the
larva did not appear to be directly related to aggressiveness of the larvae. Some
small larvae were equally ﬁerce ﬁghters as long as they had ﬁrst claim to the hole.

Type of larvae present in a given group may also affect the penetration success
of larvae particularly on an intact seed. Presence of pioneer larvae in a group

increases the chance of survival for both types of larvae.

 

 

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185

Future research areas and applicability of study

One area that could be explored further is the relationship of bruchid larval
morphology to colonization behaviors. Perhaps there are structural differences
between pioneer and follower larval types that inﬂuence their seed entry behaviors.
likewise, another hypothesis could involve genetics; certain larvae may be
programmed for digging while others may be programmed as followers. This
hypothesis might be tested by selecting for one larval type and attempting to shift
the ratio of pioneers and followers in a population, if such pure categories of these
larvae truly exist. On the other hand, examination of physiological differences in
digger and non-digger larvae of A. obectus of the same age may possibly uncover
some causes for these larval behaviors observed.

Neonate bean weevil larvae need stabilized beans (Chapter 1) with a zone of
contact between seeds (this Chapter) in order to penetrate beans successfully.
Destabilizing the seed in a petri dish by agitation usually disrupted the boring
activity, injured or killed the tiny, delicate larva. Disruption of the boring process
in turn may reduce the population of this bruchid in stored beans.

It can be concluded that detailed knowledge of seed colonization behaviors in
the neonate larvae of the bean weevil is a prerequisite to development of techniques

for the control of the bean weevil and possibly other insects with similar behaviors.
CONCLUSIONS
First instar larvae of A. obtectus settled or positioned themselves for boring

and eventually bored into a seed with intact seed coat, only when a zone of contact

was present between the seed and culture dish lid. All three bean seed areas, the

 

 

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186

cheek, keel and end were susceptible to larval boring as long as there was a zone of
contact available for aiding the digging process of the larvae.

In this study, some larvae were diggers (pioneers) while others were followers.
In the ﬁrst experiment where only three larvae per dish were used, the ratio of
pioneers to followers was 2:1 respectively for beans artiﬁcially arranged to attain
zone of contact. Most holes dug in the seed were entered by one larva only. On
average, the first instar larva appeared to take Q. 23 h from the time it located the
bean until total penetration into the seed.

For dry beans under normal storage, neonate bean weevil larvae bored holes
in almost every part of the seed where there was a zone of contact. However,
cheek of the bean received the highest frequency (79%) of entry holes. About half
of the beans tested were infested. Among these, beans with only one entry hole
(9a, 66%) were the most common.

The frequency of entry holes among beans under normal storage conditions
did not fit the Poisson distribution. Apparently, there may be a slight tendency by
neonate larvae of Acanthoscelides obtectus to aggregate during the bean-entry
process. On the other hand, not all larvae entered beans in this fashion; some
settled on beans that had no other larvae or larval holes and initiated their own
holes.

The sequence of seed entering was generally the same in all neonate larvae
but differed slightly in time spent within the different phases of the boring process.
Upon introduction in a dish with seed, the larva climbed onto a seed, walked on the
seed in search of a favorable point, stopped and settled on the site for boring,
initiated boring and ﬁnally entered the seed. Some larvae settled, bored and
entered seed faster than others. In general, all larvae that successfully bored into
pre-hydrated and dry beans in this study, entered seed at least within 24 h.

Followers took generally less than half the seed entry time of the pioneer to enter

 

thes

(non
pres
atter
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187

the same hole.

Where only one follower was present, interruptions by the follower upon the
pioneer during digging were minimal. However, when more than one follower was
present at a hole that was freshly dug and entered by a pioneer, the follower
attempting entry into the hole was disturbed by other followers at various intervals
during the seed penetration process. The primary follower and other followers

engaged in ﬁghts and chases until the borer ﬁnally accomplished entry.

CHAPTER 5

Tumbling of beans as a control measure for the common

bean weevil, Acanthoscelides obtectus (Say).

188

INTRODUCTION

Common beans, Phaseolus vulgaris L., are a dietary protein staple in many
developing nations, particularly where animal protein is not very accessible. Most
of the dry beans consumed in these areas are grown by subsistence farmers who
store the crop in a diversity of containers. In many parts of Tanzania, for example,
most dry beans are stored in gunny sacks, clay pots and "debes" (15 liter tins)
(Quentin, 1984; Due et al., 1985).

The common bean weevil, Acanthoscelides obtectus (Say), is a cosmopolitan
pest of dry beans. Adults lay eggs inside maturing bean pods prior to harvesting
and among shelled dry beans in storage. Mobile larvae (0.2 mm diam. x 0.6 mm
long), developing from eggs laid loosely among beans, bore into and complete their
entire development within a seed. In tr0pics, the insect completes its life cycle in
about one month.

Explosive A. obtectus populations usually limit bean storage in the tropics to
about 1.5 months (Schoonhoven, 1976); thereafter, populations of this pest escalate
to the point of ruining the crop. "Weevily" beans are not only unattractive, but also
impart unpalatable ﬂavor when eaten. In addition, insect feeding also damages
embryos of seeds for planting.

Insecticides for common bean weevil control are rarely available or affordable
to the subsistence farmer. When an insecticide is available, there is appreciable
danger of abuse due to lack of knowledge about proper uses. Many peoples of the

world need readily accessible, safe, and reliable controls for A. obtectus.

189

190

Previous research on A. obtectus colonization of and development in bean
seeds (Chapters 1 and 4) indicated that the bean-entry phase might be sensitive to
disruption. The importance of interactions at the bean exterior (testa) was
supported by the ﬁnding (Chapter 1) that pin-perforated beans were signiﬁcantly
more colonizable than intact beans; moreover, testa perforation equalized normally
disparate suitabilities across a range of bean cultivars (Chapter 1). That this
insect/testa interaction had a critical physical component was established by the
ﬁnding (Chapter 4) that no larvae could penetrate beans with intact seed coat when
the exposed surface of seeds half-submerged in wax was untouched by an abutting
surface, e.g., another seed or the experimental container. Close-up video
cinematography (Chapter 4) through a transparent petri dish lid arranged to touch a
half-submerged bean revealed that larvae settle and bore successfully only where
they can wedge themselves between two resistive surfaces. Apparently, such a
"purchase" is required for a larva to: 1) develop adequate down-pressure towards a
bean for its scraping strokes, and/or 2) avoid being displaced forward by the
reactive force of a stroke while boring. It took 23 h or more of nearly continuous
scraping for these bruchid larvae to enter a dry red kidney bean (Chapter 4).

Consideration of the above facts led to the hypothesis to be tested in this
chapter: that A. obtectus might be controlled by a simme mechanical measure like
tumbling beans to dislodge attacking larvae and to reconﬁgure partially bored holes
so they fall out of register with abutting surfaces. Calculations based on
measurements of samples of beans undergoing bean weevil attack supported this
premise: By inspection, a typical red kidney bean was found to have 12 contact
points with adjacent beans; this result is supported by theory on spheroid close-
packing (Sloane, 1984). About 2 mm diam. A. obtectus entrance holes were found
between 0.72 and 0.92 mm from the point of contact of one bean with another.

Thus the suitable area for larval boring around such a contact can be estimated as a

191

1.04 mm2 ring. Since there are 12 contact points for an average red kidney bean
having a surface area of 285 m2, the probability that an initiated hole would fall
where boring could be continued after tumbling is estimated at (1.04 mm2 x 12) /285
mm2 = 0.043. The overall probability that a tumbled, initiated hole would actually
be continued would be 0.043 x the unknown probability (thought to be low) that a
properly reconﬁgured hole would be found by a capable larva. An underlying
assumption is that, for beans tumbled several times per day, larvae not injured by
tumbling beans would succumb due to exhaustion of energy reserves before gaining

access to suitable food.
MATERIALS AND METHODS
Experimental design

The effect of bean tumbling on A. obtectus populations was tested using a
design of paired main treatments (tumbling vs. stationary) progressively replicated
at three scales: 0.8 liter glass jars, 16 liter plastic food buckets, and 45 kg gunny
sacks; each container was 1a. half-ﬁlled with beans so that thorough tumbling of
contents could readily be achieved. To quantify possible effects of bean breakage,
beans for the glass jars were viewed under 3x magniﬁcation and divided into lots
virtually 100% intact and 50% damaged. Damage ranged from small cracks in the

testa -to- longitudinally or transversely halved seeds.
Insects and Rearing Conditions

A Michigan strain of A. obtectus laboratory reared 1a, 40 generations on light

red kidney beans was used in all replicates of this experiment. Rearing and

192

experimental conditions were 1a. 25 0C and 60% RH. The numbers of individuals
added to each container type are given in Table 49 (p. 194). The sex ratio for jars
was exactly 50:50, while that for buckets and sacks was undetermined, but thought to
be close to 50:50. Beetles used in jars had newly emerged from the laboratory
culture, while those used for the buckets had newly emerged from the stationary jar
trials. Beetles used for sacks were transferred from the bucket trials and had

already laid some eggs before transfer.

Beans

Light red kidney beans purchased from a local supermarket were used in jars.
Michigan-produced, dark red kidney beans (donated by the Northern States Bean
Co., Lansing, Michigan) were used in buckets and sacks; about one percent of these
bulk beans were broken. Quantities of beans used in each container type are given

in Table 49.

Containers and Tumbling Protocols

To prevent beans from sliding en masse inside the smooth glass jars when
containers were rolled, two plastic bafﬂes (0.5 cm diam. x 16 cm long) were installed
along the length of the inside walls of jars whose mouths were then closed with ﬁne
mesh screening. The high-density polyethylene buckets were thoroughly baked at
75 0C in an oven to remove odors. Each was then equipped with three 2.5 cm high
x 32 cm long smooth wooden bafﬂes, a 3 cm diam. hole (plugged with a rubber
stopper) for introducing beetles and making intermittent observations, and a tight-

ﬁtting plastic lid. Standard weave gunny sacks were tied at the mouth with twine.

193

Once replicates of this experiment were initiated, great care was taken that the
controls remained stationary. Every eight hours, cylindrical containers with beans
to be tumbled were manually rolled one circumference, and then back to their
original position. Sacks of beans were twice turned end-over-end two to three
times per day. Tumbling was faithfully conducted until about one week after
inspection revealed no live adults. Dead beetles were then removed to permit

correct assessment of the emerging F1 bean weevil adults.

RESULTS AND DISCUSSION

For all container types, regular bean tumbling dramatically lowered A.
obtectus populations by :11. 97% relative to stationary controls (Table 49). Rather
than the normal _c_a. 20-fold increase per generation, beetle populations in tumbled
intact beans fell to 1a. one third of the starting population. Presence of large
numbers of cracked and broken beans improved beetle survival only slightly (Table
49); control was excellent (95%) even for broken beans. Bean tumbling was done
for only one generation of the common bean weevil in this study.

It is intriguing that the number of beetles surviving rolling (3%) was close to
the calculated probability (4%) that a given initialized hole in a tumbled bean would
fall back into register. As each larva might start a new hole several times under
this regime of tumbling, perhaps the effect of increased density of initialized holes
was offset by inefﬁcient ﬁnding of initialized holes in tumbled beans.
Alternatively, some larvae in tumbled beans may have died from other causes.
Some smashed eggs and larvae were visible on the walls of the glass jars. Whether
longevity and fecundity of adults were affected by bean tumbling was not measured

in the present experiment. Preliminary measurements revealed no decrease in

194

Table 49. Influence of container rolling (tumbling) on
population increases of the common bean weevil, A. obtectus.

 

 

No. beetles % Reduction
introduced Mean relative to
per beetles stationary
Treatment N container emerged control
GLASS JAR
Beans Intact
(0.24 kg)
ROLL 4 30 12.3 1 2.8 c1 98
STATIONARY 4 30 623.0 1 54.0 a --
Beans
50% Damaged
(0.24 kg)
ROLL 6 30 30.2 1 5.4 b 95
STATIONARY 6 30 643.5 1 82.6 a --
PLASTIC BUCKET
Beans
> 98% Intact
(7.0 kg)
aorr 2 480 171 (160; 182)2 98
STATIONARY 2 480 7,622 (6,812; 8,431) --
GUNNY SACK
Beans
> 98% Intact
(22.7 kg)
TUMBLE 2 1,000 244 (228; 259) 97
STATIONARY 2 1,000 6,857 (5,101; 8,612) --

 

Grand Mean = 97%3

 

1

Means (Mean 1 SE) followed by a common letter are not
different at P=0.05 as determined by paired t-test.

2

Beetle counts for both trials are given in parentheses.

3

Reduction was significant at P < 0.001 according to paired
t-test on log-transformed data.

 

 

 

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195

germination due to tumbling.

Given these results, the recommendation can be advanced that, in addition to
local practices for managing A. obtectus populations, subsistence bean producers
and consumers: 1) store shelled dry beans in sturdy cylindrical containers
impermeable to adult A. obtectus, 2) ﬁll containers no more than 75% and
structure containers (e.g. add bafﬂes) so that beans tumble thoroughly when
containers are rolled, and 3) as a regular chore, roll or tumble containers one to
two circumferences every morning and evening; however, if adult bean weevils are
detected among beans, tumbling duration and frequency should be increased. It is
notable that knowledge is the only critical input enabling implementation of this
control method by susbsistence farmers.

Only imagination will limit variations of this control principle. The method
could be scaled up to tumbling or rolling sacks, sealed baskets, or drums of beans.
Where advanced equipment is available for bulk storage, beans could be tumbled
by, for example, angering them from bottom to tap of the bin until the whole mass
shifts. Moreover, with reliable sampling procedures, bean tumbling could be
limited only to periods bracketing A. obtectus oviposition and lifespan of ﬁrst instar
larvae.

This study represents the only known example of mechanical control
employing bean tumbling to restrain populations of the bean weevil in shelled dry
beans. Labeyrie (1957) and Labeyrie and Maison (1957) have recommended
picking and threshing of beans as soon as possible after maturation to reduce
oviposition by A. obtectus and to separate egg-bearing pods from seeds.

Mechanical control has been attempted for pests of stored grain. It has long
been recognized that periodic transfer of large stores of grain from one bin to
another noticeably reduced pest damage (Cotton and Gray, 1948; Joffe, 1963).
Although it was originally thought that grain transfer slowed growth of pest

196

populations by lowering temperatures and dissipating moisture, subsequent research
(Joffe and Clarke, 1963; Bailey, 1969; Loschiavo, 1978) established that the effect is
mainly due to damaging impacts to insects, particularly prepupae and pupae, inside
the grain seeds. For example, various Sitophilus spp. are killed by impact speeds of
11 4 m/s or greater (Bailey, 1969; Loschiavo, 1978). However, pest control via
mechanical impact has not been widely adopted for stored products, probably
because of concern over damage to seeds receiving adequate impact forces needed
to kill most pest with few iterations of the treatment. By contrast, the forces
applied to beans in our study were gentle and undamaging.

It remains to be seen whether this tactic of regular tumbling will prove widely
useful in control of storage pests of other pulses and grain. Tumbling is unlikely to
be as disruptive to host colonization by the cowpea weevil, Callosobruchus
maculatus (Fab.), whose larvae bore directly into beans from eggs glued to the testa.
Perhaps in this situation, protocols of mechanical disruption can be devised that will
maximize crushing of eggs during tumbling.

Tumbling should certainly be tried for storage pests meeting any one or more
of these criteria: 1) eggs, larvae, or any soft-bodied or otherwise fragile forms
appear outside the seed, 2) particular sites or seeds are selected for entry, 3) the
entrance hole or insect is small in relationship to the seed and entry requires special
purchase, 4) entrance into the seed takes more than 12 h, and, if entrance is not
gained within several days, death will result, 5) entrance is via scraping rather than
biting into the seed, and, 6) the seed exterior is slightly toxic (e.g. allelochemics or
residual insecticide) to the insect.

Even if such features of a pest’s biology were not completely known, regular
tumbling could be attempted, just to see if there were beneﬁt worth the modest
effort. However, the better approach is likely to be characterization of the pest’s

biology and behavior followed by informed attempts at control. Pests under close-

UP
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197

up surveillance may divulge completely unexpected vulnerabilities, as happened in

our studies of A. obtectus.

CONCLUSIONS

Tumbling beans every 8 h remarkably reduced the common bean weevil
populations in all bean containers tested by about 97% relative to stationary
controls. Larvae that did not succumb to the physical impact of tumbling beans
probably died from exhaustion resulting from forced repeated initiation of holes
after being displaced by tumbling beans. Some eggs were also smashed by
tumbling beans. The effect of tumbling on bean weevils among damaged beans
(50% of total beans in jars) was only slightly less (95% control) than that of intact

beans.

APPENDIX

APPENDI X 1

Record of Deposition of Voucher Specimens*

The specimens listed on the following sheet(s) have been deposited in
the named museum(s) as samples of those species or other taxa which were
used in this research. Voucher recognition labels bearing the Voucher
No - have been attached or included in fluid-preserved specimens.

Voucher No. : 1991-03

Title of thesis or dissertation (or other research projects):

Host-colonization behaviors of the bean weevil,
Acanthoscelides obtectus (Say), in stored beans.

Museum“) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

Other Museums:

Investigator's Name (5) (typed)

Hanna Erjca ﬂllgntin

 

 

 

Date 7-23-1991

4
tierence: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in
th America. Bull. Entomol. Soc. Amer. 24:141-42.

Dep031 t as follows:

original: Include as Appendix 1 1n ribbon copy of thesis or

Go dissertation.
Pies: Included as Appendix 1 in copies of thesis or dissertation.

Museum(s) files.
Research project files.

This
mehizzm is available from and the Voucher No. is assigned by the Curator,
:1 State University Entomology Museum.

198

 

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