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This is to certify that the
thesis entitled

IMPROVING MATING DISRUPTION PROGRAMS FOR
THE ORIENTAL FRUIT MOTH, GRAPHOLITA MOLESTA (BUSCK):
EFFICACY OF NEW WAX-BASED FORMULATIONS AND
EFFECTS OF DISPENSER APPLICATION HEIGHT AND DENSITY

presented by
Frédérique M. de Lame

has been accepted towards fulfillment
of the requirements for the

degree' m Entomolm

MajHProfessors’ Sfiflatures
x ’1‘1’03 X—ij

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IMPROVING MATING DISRUPTION PROGRAMS FOR THE ORIENTAL
FRUIT MOTH, GRAPHOLITA MOLESTA (BUSCK): EFFICACY OF NEW WAX-
BASED FORMULATIONS AND EFFECTS OF DISPENSER APPLICATION
HEIGHT AND DENSITY
By

Frédérique M. de Lame

A THESIS
Submitted to
Michigan State University
in partial fulfillment Of the requirements
for the degree of
MASTER OF SCIENCE
Department of Entomology

2003

ABSTRACT
IMPROVING MATING DISRUPTION PROGRAMS FOR THE ORIENTAL
FRUIT MOTH, GRAPHOLITA MOLESTA (BUSCK): EFFICACY OF NEW WAX-
BASED FORMULATIONS AND EFFECTS OF DISPENSER APPLICATION
HEIGHT AND DENSITY
By

Frederique M. de Lame

Emulsified wax dispensers (EWDS) of insect pheromones are economical and
biodegradable. An improved EWD formulation, EWD II-OFM, was developed for
control of Grapolita molesta (Busck) (Lepidoptera: Tortricidae) via mating disruption.
This formulation provided season-long control Of G. molesta with only one application,
versus two applications of Confuse-OFM, a commercial EWD; it was as effective as one
application of Isomate-M 100, the industry standard for control of this pest. Using a
simple Spatula applicator, EWD II—OFM could be applied in half the time required for
Isomate-M 100 or Confuse-OFM.

Confuse-OFM and paraffin disks were used as model dispensers tO determine
whether application time could be reduced by altering dispenser distribution, while
maintaining a high level of control. Orientational disruption of G. molesta was achieved
when Confuse-OFM was applied at a convenient height Of 1.2-1.8 m in peach and apple
trees as tall as 5.5 m. Additional studies revealed that the number of male moths
successfully orientating to pheromone traps in plots treated with a set number of
dispensers at a single height increased asymptotically towards 100% as the number Of
point sources per area increased. These data indicate that dispenser point source density

should be maximized for control Of G. molesta.

Copyright by
FREDERIQUE M. DE LAME
2003

ACKNOWLEDGEMENTS

Thanks to my advisors: Larry Gut, Jim Miller, and Cindy Atterholt, for their
availability and excellent guidance throughout my degree. Additional thanks to Larry Gut
for all of the freedom and resources that I was given to follow investigations of high
interest to me. Innumerable thanks to all of the research assistants, without whose good
work this labor-intensive research could not have been accomplished: Katie Bosch,
Clarrissa Chavez, Chad Hipshier, Mathew Jolman, Emese Karacsonyi, Creela Overton,
Travis Reed, Danielle Schield, and Vicki Walter. Especially warm thanks to Travis Reed,
for his initiative, friendship, and excellent assistance, and for continuing the “wax wor ”
at MSU. Thanks to Mike Haas, especially for all his help during my first field season, and
to Piera Giroux, for teaching me how to use the GC, a tool valuable to this research.
Thanks to Peter McGhee and the rest of the technical staff at CIPS for their assistance
and to all of the good people in the MSU Department of Entomology who contributed to
making these past years a pleasant experience. Thanks to Gary Van Ee, Richard
Ledebuhr and their students: Erik Arbut, Lindsey Brown, and Larry Morden, from the
MSU Department of Agricultural Engineering for their good work and good humor,
while helping us design, build, and maintain our applicators. Thanks to John Wise and
the MSU Trevor Nichols Research Complex staff for their warmth and maintenance Of an
excellent fruit research facility. Thanks to Bill Chase at the MSU Horticultural Teaching
and Research, Viticulture and Enology Farm and to our cooperators: Randy Bjorge, Jim

Calderwood, Chris Whitlow, Randy Willmeng, and Rodney Winkle, for making their

iv

orchards available to us. Thanks to my family and to Abbas Tamijani for their love,

support, and companionship.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ............................................................................................................ x
Chapter 1: Literature Review ............................................................................................... 1
Biology of Grapholita molesta ................................................................................ 1
Management Of Grapholita molesta ........................................................................ 2
Mating disruption of Grapholita molesta ................................................................ 4
Effective controlled-release devices for pheromones in mating disruption
programs ............................................................................................................ 6
Pheromone controlled-release technologies ............................................................ 8
Paraffin wax and release of active agents from wax- -based dispensers ................. 12
Paraffin wax-based dispensers designed for releasing insect pheromones ............ 15
Objectives of this thesis ......................................................................................... 18

Chapter 2: Development and evaluation of an emulsified paraffin wax dispenser for
season-long mating disruption of Grapholita molesta in commercial peach orchards in

Michigan, USA .................................................................................................................. 19
Introduction ............................................................................................................ 19
Materials and Methods ........................................................................................... 20

2001 field tests ........................................................................................... 20
Efficacy of Confuse-OFM ............................................................. 20

Field release rate of Confuse-OFM ................................................ 22
Developing EWD II-OFM ......................................................................... 24
2002 field tests ........................................................................................... 26
Efficacy of EWD II-OFM .............................................................. 26

Field release rate of EWD H-OFM with and without adhesive ..... 29
Efficiency of EWD applicators .................................................................. 30
Statistical analyses ..................................................................................... 31
Results and Discussion .......................................................................................... 32
Confuse-OFM trials ................................................................................... 32
Efficacy .......................................................................................... 32
Longevity ....................................................................................... 37
Drawbacks ...................................................................................... 40
Developing EWD II-OFM ......................................................................... 40
Evaluation of EWD H-OFM ...................................................................... 42
Efficacy .......................................................................................... 42
Longevity ....................................................................................... 48
Application ..................................................................................... 52

Cost ................................................................................................ 54
Potential for commercializing EWD technology ....................................... 55

Chapter 3: Ability of Grapholita molesta to locate pheromone traps, as influenced by
vertical positioning of traps and hand-applied pheromone dispensers .............................. 58

vi

Introduction ............................................................................................................ 58

Materials and Methods ........................................................................................... 61
Experimental design ................................................................................... 61
Pheromone formulation ............................................................................. 62
Shoot growth measurements ...................................................................... 63
Statistical analyses ..................................................................................... 63

Results .................................................................................................................... 64
Peach blocks ............................................................................................... 64

Pheromone traps ............................................................................. 64
Virgin female traps ........................................................................ 67
Description of indigenous G. molesta populations ........................ 67
Apple blocks .............................................................................................. 67
Wide apple block pheromone traps ................................................ 67
Narrow apple block pheromone traps ............................................ 69
Virgin female traps ........................................................................ 72
Description of indigenous G. molesta populations ........................ 72
Shoot growth in the 2002 peach and apple blocks ..................................... 74

Discussion .............................................................................................................. 74
Vertical placement of pheromone traps ..................................................... 74
Vertical placement of pheromone dispensers ............................................ 78

Chapter 4: Impact of varying density of pheromone point sources on the success of
mating disruption of Grapholita molesta using two paraffin wax-based pheromone

dispensers ........................................................................................................................... 84
Introduction ............................................................................................................ 84
Materials and Methods ........................................................................................... 85

Experimental design ................................................................................... 85
Confuse-OFM point sources .......................................................... 85
Paraffin disk point sources ............................................................. 86
Measuring treatment effects ........................................................... 88

Pheromone release rates from paraffin disks ............................................. 89

Statistical analyses ..................................................................................... 90

Results and Discussion .......................................................................................... 91

Point source experiments ........................................................................... 91
Confuse-OFM ................................................................................ 91
Paraffin disks ................................................................................. 92

Dispenser release profiles .......................................................................... 93
Confuse-OFM ................................................................................ 93
Paraffin disks ................................................................................. 93

Density of point sources, release rate, and success of mating disruption ..96
Varying point source density ......................................................... 96
Generalizability of Figure 4.2 ........................................................ 99
Varying release rate ..................................................................... 102

Suggested study: varying point source density and release rate .. 105

vii

Chapter 5: Suggestions for further research ..................................................................... 107

Building upon EWD II-OFM ............................................................................... 107
Dispenser distribution .......................................................................................... 109
Concluding remarks ............................................................................................. l 10
Literature Cited ................................................................................................................ 112
Appendix A: Additional Tables ....................................................................................... 122
Appendix B: S.P.L.A.T. Pheromone Applicator ............................................................. 127
Appendix C: Record of Deposition of Voucher Specimens ............................................ 159

viii

LIST OF TABLES

Table 2.1: Sum of Grapholita molesta caught per plot in bucket traps during the 2001 and
2002 field trials comparing the efficacies of Confuse-OFM and EWD II-OFM to two
commercial G. molesta pheromone formulations .............................................................. 36

Table 2.2: Grapholita molesta caught per three days in virgin female traps during the
2002 field trial comparing the efficacy of EWD H-OFM to Confuse-OFM and Isomate-M

100 ...................................................................................................................................... 44

Table 3.1: Description of blocks used in tests of the effects of vertical placement of
pheromone traps on Grapholita molesta captures and of dispensers on orientational
disruption of G. molesta to pheromone and virgin female traps ........................................ 60

Table 4.1: Numbers of Grapholita molesta caught in pheromone and virgin female traps
in apple plots treated with no pheromone, Confuse-OFM (74 g AI/ha) or paraffin disks
(106 g AI/ha) evenly distributed on all trees or a fraction of the trees .............................. 87

Table I: Descriptions of peach plots used to determine the efficacy.of Confuse-OFM
versus two commercial G. molesta pheromone dispensers in 2001 ................................ 123

Table H: Pesticides applied in peach plots used to determine the efficacy.of Confuse-
OFM versus two commercial G. molesta pheromone dispensers in 2001 ....................... 124

Table HI: Descriptions Of peach plots used to determine the efficacy.of EWD II-OFM
versus Confuse-OFM and Isomate-M 100 in 2002 .......................................................... 125

Table IV: Pesticides applied in peach plots used to determine the efficacy.of EWD H-
OFM versus Confuse-OFM and Isomate-M 100 in 2002 ................................................ 126

LIST OF FIGURES

Figure 2.1: Grapholita molesta caught in pheromone traps during the 2001 field trial
testing the efficacy of Confuse-OFM versus other commercial G. molesta pheromone
dispensers ........................................................................................................................... 34

Figure 2.2: Percent of peach shoots and fruits sampled with internal, larval-caused injury
during the 2001 field trial testing the efficacy of Confuse-OFM versus other commercial
Grapholita molesta pheromone dispensers ........................................................................ 35

Figure 2.3: Z8—122Ac remaining in Confuse-OFM dispensers aged in the field ............... 38

Figure 2.4: 28-12:Ac remaining in Confuse-OFM and three experimental EWDs aged in
a laboratory fume hood ...................................................................................................... 41

Figure 2.5: Grapholita molesta caught in pheromone traps during the 2002 field trial
testing the efficacy of EWD II-OFM versus Confuse—OFM and Isomate-M 100 ............. 43

Figure 2.6: Percent of peach shoots and fruits sampled with internal, larval-caused injury
during the 2002 field trial testing the efficacy of EWD H-OFM versus Confuse-OFM and
Isomate-M 100 ................................................................................................................... 46

Figure 2.7: 28-12:Ac remaining in EWD H-OFM dispensers with and without adhesive
aged in the field .................................................................................................................. 49

Figure 2.8: Average number of Grapholita molesta caught per pheromone trap per half
week in all treatments at each sampling date during the 2002 field trial comparing the
efficacy of EWD II-OFM versus Confuse-OFM and Isomate-M 100 ............................... 50

Figure 2.9: Comparison of time necessary to apply one hectare of: 1) EWD II-OFM using
the S.P.L.A.T., Swipe, or Spatula applicators, 2) Confuse-OFM using a paint-marking
gun, and 3) Isomate-M 100 ropes ...................................................................................... 53

Figure 3.1: 2001 peach block. Numbers of Grapholita molesta caught in pheromone traps
placed in the top third of the canopy (high traps) and in the bottom third of the canopy
(low traps) in plots treated with no pheromone, pheromone dispensers placed in the
bottom third of the canopy (low dispensers) and pheromone dispensers placed in the top
third Of the canopy (high dispensers) ................................................................................. 65

Figure 3.2: 2002 peach block. Numbers of Grapholita molesta caught in pheromone traps
placed in the top third of the canopy (high traps) and in the bottom third of the canopy
(low traps) in plots treated with no pheromone, pheromone dispensers placed in the
bottom third of the canopy (low dispensers) and pheromone dispensers placed in the top
third of the canopy (high dispensers) ................................................................................. 66

Figure 3.3: 2002 peach block. Numbers of Grapholita molesta caught in virgin female
traps placed at 1.2-1.8 m in tree canopies in plots treated with no pheromone, pheromone
dispensers placed in the bottom third of the canopy (low dispensers) and pheromone
dispensers placed in the top third Of the canopy (high dispensers) for three days ............. 68

Figure 3.4: Wide apple block. Numbers of Grapholita molesta caught in pheromone traps
placed in the top third of the canopy (high traps) and in the bottom third of the canopy
(low traps) in plots treated with no pheromone, pheromone dispensers placed in the
bottom third of the canopy (low dispensers) and pheromone dispensers placed in the top
third of the canopy (high dispensers) ................................................................................. 70

Figure 3.5: Narrow apple block. Numbers of Grapholita molesta caught in pheromone
traps placed in the top third of the canopy (high traps) and in the bottom third of the
canopy (low traps) in plots treated with no pheromone, pheromone dispensers placed in
the bottom third of the canopy (low dispensers) and pheromone dispensers placed in the
top third of the canopy (high dispensers) ........................................................................... 71

Figure 3.6: Numbers of Grapholita molesta caught in virgin female traps placed at 1.2-
1.8 m in tree canopies in the wide and narrow apple blocks in plots treated with no
pheromone, pheromone dispensers placed in the bottom third of the canopy (low
dispensers) and pheromone dispensers placed in the top third of the canopy (high
dispensers) for three days ................................................................................................... 73

Figure 3.7: Seasonal shoot growth measured in the top half of the canopies and the
bottom half of the canopies Of trees in the 2002 peach, wide apple, and narrow apple
blocks ................................................................................................................................. 75

Figure 4.1: 28-122Ac remaining in paraffin disk dispensers aged in the field .................. 94

Figure 4.2: Percent orientational disruption of Grapholita molesta as a function of point
sources of pheromone per hectare ...................................................................................... 97

xi

Chapter 1
Literature Review
Biology of Grapholita molesta

Grapholita molesta (Busck), the oriental fruit moth, is a gray microlepidopteran
belonging to the family Tortricidae (Chapman and Lienk 1971). G. molesta originated
from Northwest China. The spread Of this pest from Asia began early in the 20th century
and it is now found in most of the stone fruit-growing regions of the world (Rothschild
and Vickers 1991).

G. molesta larvae feed on trees belonging to the family Rosaceae and favor
members of the genus Prunus (stone fruit trees). The larvae attack young Shoots of their
hosts early in the season and feed on fruits once the shoots harden. G. molesta larvae will
feed on the shoots and fruits of both stone fruit trees and apple trees (Chapman and Lienk
1971). They will also feed on pear and quince fruits. G. molesta is a major pest of stone
fruits worldwide (Rothschild and Vickers 1991) and the primary internal feeder of
peaches and nectarines in Michigan. This pest is also problematic in young trees not yet
bearing fruit, where feeding by G. molesta can result in abnormally bushy growth of the
trees.

Depending on temperature, photoperiod, and the availability of appropriate hosts,
three to five generations of G. molesta per season may occur in North America (Hogmire
1995). In Michigan, there are three generations of G. molesta per season. The spring
flight of adults generally begins in mid April, about three weeks before apples are in
bloom. The second and third flights generally begin in late June and mid August,

respectively. Adults of each generation are active for a period of six to eight weeks.

Management of Grapholita molesta

Biological control of G. molesta was initially attempted in many Of the countries
where it became introduced. Although introductions of foreign parasites were not
successful (Roehrich 1961, Rothschild and Vickers 1991) in many cases, local parasitic
species colonized various stages of the insect, resulting in varying amounts of parasitism
in those regions (Allen 1943, Dustan 1961, Roehrich 1961, Phillips and Proctor 1970,
Bailey 1979, Rothschild and Vickers 1991). Nevertheless, biological control was not
sufficient as a Stand-alone control for G. molesta (Allen 1943, Dustan 1961, Phillips and
Proctor 1970).

Management of G. molesta has traditionally been achieved using insecticides.
Successful control was achieved for the first time with DDT in the 19403.
Organophosphate compounds and carbaryl (a carbamate) replaced DDT soon after their
appearance on the market. Azinphosmethyl has been by far the most widely used
insecticide for G. molesta control (Rothschild and Vickers 1991). Although broad-
spectrum insecticides provided an easy way to control this insect, both greater incidences
of resistance of G. molesta to these materials and increased regulation of pesticides have
gradually reduced their use.

Free et al. (1998) reported widespread economic losses in peach orchards in the
early 19905 in Ontario, Canada, as a result of fruit damage by G. molesta that had
developed resistance to organophosphates and carbamates. Usmani and Shearer (2001)

also found increased resistance to azinphosmethyl in G. molesta in New Jersey orchards

as the season progressed.

The 1996 Food Quality Protection Act (FQPA) gave the Environmental
Protection Agency (EPA) the tasks to: 1) review all pesticides which were on the market
in 1996 by 2006 and 2) to set strict tolerance levels for these pesticides in foods
(http://www.epa.gov/oppfeadl/fqpal). The result has been a phasing out of broad—
spectrum insecticides in agriculture. New, safer pesticides generally target a narrow range
of insect species and are slower-acting than Older chemistries. Furthermore, since
agrochemical companies have little interest in registering their products for use on crops
with relatively small markets, registration of pesticides for minor crops, such as fruit
crops, is fully dependent on the IR-4 program. The outcomes are fewer effective
pesticides and increased time necessary for new pesticides to be registered for G. molesta
control.

AS will be discussed below, research to develop a mating disruption program for
G. molesta began in the 19705 and the results of research efforts since then have been
quite promising. Mating disruption of insects is a reduced-risk alternative to the use Of
pesticides for control. Advantages of this technique include a low impact on the
environment and other animals and a potentially faster registration by the EPA. Limits to
this technique have included the need for a greater understanding of insect behavior,
reduced efficacy when pest populations are high, problems with releasing pheromones
into the environment for an extended period of time, and the high cost of pheromones.
Over time, however, some of these problems have been resolved (Justum and Gordon

1989), allowing for the commercial adoption of mating disruption.

Mating disruption of Grapholita molesta

The major component of the pheromone of G. molesta, (Z)-8—dodecenyl acetate
(28-12zAc), was first identified by Roelofs et al. in 1969. The full pheromone blend was
later determined by Carde’ et a1. (1979) and included three other components: (E)-8-
dodecenyl acetate (E8-12:Ac), (Z)-8-dodecen-1-ol (ZS—12:0H), and dodecanol (12:0H).
The pheromone blend typically released from G. molesta dispensers is a 93:6:1 blend of
28-12:Ac : E8-12:Ac : 28-12:OH.

G. molesta was one of the first insects for which the mating disruption technique
was developed. Early mating disruption trials took place primarily in Australia
(Rothschild 1975, 1979). Small scale mating disruption trials were also conducted early
on in the United States (Gentry et a1. 1974, 1975, Cardé et a1. 1977). Rothschild (1975)
conducted a first series of key experiments identifying conditions necessary for
successful mating disruption of G. molesta. He determined that mating disruption of this
pest was successful when its pheromone was released at 5 mg/ha/h and that pheromone
dispensers placed in a mid-canopy position or at the tree crowns were equally effective at
reducing the ability of G. molesta to orient to traps. Furthermore, there was no significant
difference in the percent orientational disruption to pheromone traps achieved in small
plot trials when dispensers collectively releasing pheromone at or below 5 mg/ha/h were
distributed as 25 to 200 point sources per hectare.

Based on Rothschild’s studies, a rope dispenser, Isomate-M (essentially, a
commercial formulation of the polyethylene microcentrifuge tubes originally used to
dispense G. molesta pheromone in the Australian mating disruption trials) was developed

by Shin-Etsu Chemical Co., Ltd., Tokyo, Japan (Vickers 1990). During 1985 to 1987,

this rope dispenser was extensively tested in commercial peach orchards in California,
USA. (Rice and Kirsh 1990). Field trials with Isomate—M were conducted for two years
in Virginia, USA, as well (Pfeiffer and Killian 1988).
. Various G. molesta pheromone formulations were also tested during this time in
stone fruit production regions throughout the world. In France, Audemar et a1. (1989)
reported seven years of mating disruption trials with three hand-applied dispensers:
Isomate—M, plastic laminate dispensers (Hercon Laboratory Corp.), and polyethylene
bulb dispensers (BASF). In Georgia, USA, Gentry et a1. (1980) tested the efficacies of
plastic laminate dispensers and microcapsules for mating disruption of G. molesta.
Isomate-M was also tested in peach orchards in Ontario, Canada (Pree et al. 1994). All
trials reported control of G. molesta with mating disruption at least as effective as control
Of this pest with insecticides, provided certain conditions were met: the orchards were
adequate with respect to size and other physical parameters, the formulations released the
necessary amount of pheromone for the period of the trial, and populations Of G. molesta
were not too high. Based on the outcomes Of these trials, Cardé and Minks (1995) cited
G. molesta mating disruption as an example of the successful development of this control
technique for a moth pest. In this review, they noted the increasing use of mating
disruption for controlling G. molesta following the initial, promising trials worldwide.
They also reported the successful use of mating disruption to control this pest on 1200 ha
of peaches and nectarines in South Africa in the early 19903, soon after G. molesta was
first found in this country (Blomefield and Geertsema 1990).
The consistent success of mating disruption for control of G. molesta has made

this management system an ideal model for large-scale development of the mating

disruption technique. In 1979, Vickers and Rothschild reported the success Of a district-
level mating disruption program. A limitation to the mating disruption technique is the
ability of mated females to move from untreated to pheromone-treated areas, where they
then lay fertile eggs (Sanders 1997). Audemard et a1. 1989 noted reductions in the
efficacy of mating disruption for G. molesta when plots were in close proximity to
untreated areas harboring large G. molesta populations. The application Of pheromone to
larger, contiguous orchards was thought to be a means to reduce the incidence of mated
female migration into treated areas and increase the success of mating disruption.
Following Vickers and Rothschild’s trial (1979), an area-wide mating disruption program
established in Australia in 1996 has continued to show successful results up to the present
time (Il’ichev et a1. 2002). District-level mating disruption of G. molesta has also been
successful in Italy (Cravedi and Molinari 1996).

More recent studies have also documented the effectiveness of incorporating
mating disruption of G. molesta into integrated pest management programs seeking to
minimize the use Of pesticides to control pests in peach orchards in Ontario, Canada
(Trimble et al. 2001) and in New Jersey, USA. (Atanassov et a1. 2002). As discussed
below, adoption of mating disruption as a viable pest control technique on its own or as a
part of an integrated pest management program has been a goal of proponents of this
technique from its inception.

Effective controlled-release devices for pheromones in mating disruption programs

AS the mating disruption technique has been developed, a set of conditions has
been identified that greatly influences the control of a pest insect by this technique. These

conditions have included both economic and scientific considerations.

Factors that impact the successful commercialization Of the mating disruption
technique have been primary concerns of scientists studying semiochemicals from the
start (Wood et al. 1970, Shorey et a1. 1977, Ritter 1979, Mitchell 1981, Kydonieus and
Beroza 1982, Ridgeway et a1. 1990, Cardé and Minks 1997). The cost of commercial
development, regulatory obstacles to registering pheromone products, the Skepticism of
growers and high costs of pheromone products to growers, and the need for efficacious
controlled-release pheromone formulations have most often been cited as hindrances to
commercialization. Improvements have occurred in all of these areas. Although various
pheromone formulations have been developed to date, nevertheless, the latest review of
pheromone research still mentions the development of adequate controlled-release
pheromone formulations as an area where mating disruption could be improved (Wyatt
1997). In one of many reviews of the status of and obstacles to pheromone
commercialization, Plimmer (1981) lists a set of criteria that a controlled-release
pheromone formulation should seek to meet: a pheromone formulation should provide
constant, reproducible release rates, dispense pheromone economically, be cheap to
produce, cheap and easy to apply, and non toxic. The development of single formulation
that meets all of these criteria optimally is a daunting challenge. Nonetheless, increasing
the variety of formulations available for dispensing semiochemicals will expand the
Options available for developing effective formulations for each target species in a
particular crop or crop type (Plimmer 1981).

Rothschild (1981) identified various physical factors that influence the success of
mating disruption for any species. Sanders (1997) provided a more recent review of the

subject. The most important factor Rothschild (1981) identified for successful mating

disruption of a lepidopteran species was the behavior of that species; non-migratory,
oligophagous insects would be the best candidates for control by mating disruption. G.
molesta was identified as a Species with behavioral traits that made it amenable to
disruption. The second most important factor he identified was the appropriate release of
the semiochemical in the environment; inadequate release or uneven distribution of the
compound in the environment could lead to a failure to achieve mating disruption. The
spacial arrangement of dispensers in the environment greatly influences the distribution
of pheromone. The effectiveness of mating disruption for a species can be influenced
both by the vertical and horizontal distribution of dispensers. The optimal height for
dispenser placement varies for different moth species and may vary when mating
disruption is undertaken in different crops (Chapter 3). Whether mating disruption is
equivalent when pheromone dispensers releasing low amounts of pheromone are placed
in close proximity to one-another versus when large amounts of pheromone are dispensed
from widely-spaced dispensers should be a subject of debate (Chapter 4).
Pheromone controlled-release technolong

A variety of systems and materials has been developed to dispense pheromones
for mating disruption for research purposes. Weatherston (1989) gives a thorough review
of sOme of these devices. Some interesting examples include the use of rice seed to
dispense Dendroctonus frontalis (Zimm) pheromone and rubber tubing to release the
pheromones of Adoxophyes orana (F.v.R.) and Cydia pomonella (L.). Several types of
commercial pheromone formulations are currently available for mating disruption, each
with advantages and disadvantages. Weatherston (1990) has compiled a comprehensive

list of commercial pheromone formulations and their manufacturers.

The first pheromone formulation registered in the United States was a hollow-
fiber formulation for control of Pectinophora gossypiella manufactured by Albany
International in 1978. Hollow-fiber dispensers consisted Of one or more hollow fibers
arranged as a tape and hand-applied or chopped fibers that were mixed with an adhesive
and broadcast. The biggest disadvantage to these dispensers was the need for Specialized
equipment for application of chopped fibers to crops. However, the versatility Of hollow
fiber dispensers made them modifiable for both hand and broadcast application, thus
increasing the variety of crops to which they could be applied (Weatherston 1990). It was
initially thought that these dispensers released pheromones like capillary tubes: following
an initial burst of pheromone, they maintain a pseudo-zero order release rate by a
mechanism of evaporation from the liquid surface in the hollow fiber, followed by
diffusion to the end of the fiber, and subsequent release by convection from the fiber’s
opening. In reality, this theory did not hold for the polymer fibers; the dispensers released
pheromone at much higher rates than predicted and release rates from individual fibers
varied widely (Swenson and Weatherston 1989). Albany International eventually stopped
manufacturing this dispenser.

Trilaminate or plastic laminate dispensers were also among the first commercial
dispensers for pheromones. They consist of a pheromone-impregnated layer sandwiched
between two barrier polymeric layers. In this case, the boundary layers are permeable to
the pheromone and release occurs by absorption into the boundary layers, then diffusion
through those layers, and desorption from the boundary layers, finally making the
pheromone available for evaporation to the environment. These devices have first order

release kinetics: pheromone release rate depends primarily on the concentration of

pheromone in the devices and the thickness Of the plastic barriers. A disadvantage of
laminate dispensers is the retention of pheromone beyond the effective period of the
formulation (Quisumbing and Kydonieus 1989, Weatherston 1990). However, the
formulation is versatile, being available in the form of tape or squares for hand-
application, or chips for broadcast application (Quisumbing and Kydonieus 1989). AS for
hollow fibers, broadcast-application requires the use of specialized equipment and
mixture with an adhesive (W eatherston 1990).

Microcapsules consist of a droplet of pheromone encased in a polymeric coat. The
diameter of a microcapsule typically ranges from one to one hundred micrometers.
Microcapsules release pheromone both by permeation through the capsule wall as well as
capsule rupture. Rupture can result from the thin microcapsule wall breaking down in
sunlight. In theory, microcapsules can have zero-order release profiles. Although
microencapsulated pheromone formulations have generally had first order release
kinetics, new formulation are achieving closer to zero order release. The ease of
application of microcapsules makes these pheromone dispensers especially attractive.
Microcapsules can be applied using conventional spray equipment; thus, they can be
applied using existing machinery and in combination with other agrochemicals, such as
pesticides and fungicides. They can be sprayed on any crop, from field, to orchard, to
forest (Hall and Marrs 1989). Disadvantages of these formulations are that they have a
short lifetime (a few weeks) in the field and can retain a large amount of pheromone
beyond their period of efficacy (Weatherston 1990).

The most widely used commercial formulation is the rope, or twist-tie dispenser

(Shin—Etsu Chemical Co., Ltd., Tokyo, Japan). It consists Of a polymer tube(s) containing

10

the pheromone. A metal wire is incorporated into most dispensers to facilitate
application. More recent rope dispensers, however, consist of a wireless, twin-tube
design. For this version of the rope dispenser, two tubes are attached at the ends. The
twin-tube dispenser can be easily slipped over tree branches or plant leaves. Weatherston
(1990) reports that the initial rOpe dispensers had a first order release profiles. Recent
formulations, however, have improved, close to zero-order release kinetics for much of
their lifetime (Sexton and Il’ichev 2001). Rope dispensers can only be applied by hand,
which limits the range of crops for which this formulation can be used. For example, use
of this dispenser in forests may not be practical. A notable advantage of rope dispensers
is that they typically remain effective for an extended period'of time (W eatherston 1990),
reducing the negative impact of their labor-intensive application. A single application of
Isomate-M Rosso, the latest G. molesta rope formulation, is effective for 120 days, as
reported by the manufacturer.

Another hand-applied commercial dispenser is the Checkmate dispenser (Suterra
LLC, Bend, OR, USA). This device consists of a plastic pheromone pouch, from which
the compound is released through a membrane. Zero order release from these membrane
dispensers should be theoretically possible. A specially-designed clip makes application
of these dispensers relatively quick at both high and low heights. This dispenser is suited
to application in the same crops as are the rope dispensers.

Weatherston (1990) also describes liquid-flowable pheromone formulations.
These consist of the pheromone, bound to a particulate, which is then suspended in a
liquid for spray application. Early tests show that after a latency period, these

formulations have zero order release rates and can release pheromones for an extended

ll

period of time. However, a high percentage of the active ingredient is retained on the
particulate.

A last group of dispensers that has garnered considerable attention are the aerosol-
type high release, low density devices. Puffers (e.g. Shorey and Gerber 1996b) are a
commercially-available product of this type. Puffers and other aerosol systems release
large amounts of pheromone into the environment from a minimal number of locations.
Advantages of these devices include that they protect the pheromone from degradation,
drastically reduce dispenser application time, and could be re-loaded and re-used from
year to year, thus decreasing the cost of the device (Isaacs et a1. 1999). Release rates of
pheromone from these devices can be close to zero order. The efficacy of these devices,
however, has been inconsistent (e.g. Shorey and Gerber 1996, Giroux et al., submitted,
Chapter 4).

The most popular pheromone formulations used for mating disruption of G.
molesta by Michigan fruit growers are the Shin-Etsu rope dispensers, because of their
high level of efficacy, Often maintained for the entire season, and the sprayable
formulations, because of their ease of application.

Paraffin wax and release of active agents from wax-based dispensers

According to Bennett (1975), “...a wax is...an unctuous solid with varying
degrees of gloss, slipperiness, and plasticity, which melts readily.” Numerous natural and
synthetic waxes exist. Paraffin waxes are byproducts of petroleum refining. They are
composed of hydrocarbons containing 26-30 carbon atoms, most of which are straight-

chain molecules and contain large, well—formed crystals. Paraffin waxes are colorless to

12

white, have neither odor nor taste, feel slightly greasy, and are relatively non-sticky. The
melting points of paraffin waxes range from 43.3 to 65.5 °C (Bennett 1975).

Waxes have been used as components of controlled-release devices for a variety
of applications including for the release of medical drugs (e.g. Somerville et a1. 1976,
Vilivalam and Adeyeye 1994, Aronov et al. 1996, Walia et a1. 1998), pesticides (Quaglia
et al. 2001), and fertilizers (Kakoulides and Valkanas 1994). The pheromone of
Anthonomus grandis (Boheman) has also been released from paraffin wax in trapping
studies (Hedin et al. 1976).

The paraffin wax dispensers developed and tested in the research described in this
thesis research belong to the “matrix-type” or “monolithic” category of controlled-release
devices. They are defined as devices where the active ingredient is dispersed or dissolved
in a polymer matrix (Fan and Singh 1989). Release of the active ingredient from a
monolithic device occurs by diffusion and can be described, macroscopically, by Fick’s
Law. Fick’s law states that the movement of a molecule by diffusion is directly
proportional to the concentration of that molecule in a system. Microscopically, if we
follow the movement of a molecule of an active agent through a matrix, this molecule can
begin its journey in one of two ways. If it is dispersed in the matrix, it begins its journey
by dissociating from other molecules in its crystal cell and solubilizing into the polymer
phase. If it is dissolved in the matrix, then this step is bypassed. The molecule then
diffuses through amorphous regions in the matrix that comprise the free volume Of the
system. The molecule can move through the matrix in one of two ways as well. If it is
very small compared to the size of the amorphous spaces in the matrix, then it will

diffuse through the matrix by moving from one such space to another. If it is very large

13

compared to the size Of those spaces, then segments Of the polymer comprising the matrix
will have to be rearranged for diffusion of the active agent molecule to occur. Crystalline
regions in the matrix are virtually impermeable to molecules of the active agent. Upon
reaching the surface of the matrix, it will be released into the environment (Fan and Singh
1989).

A series of factors influences the rate of release of an active agent from a
monolithic device and includes properties of the matrix material as well as properties of
the active agent. The temperature of the matrix influences release of the active agent; at
higher temperatures, the free volume is increased, and diffusion occurs faster. At lower
temperatures, the free volume is decreased, and diffusion is slower. The thermal history
of a polymer can also increase or decrease the free volume of the system and lead to
changes in the diffusional rate of an active agent (Fan and Singh 1989). The surface area
of the device also influences its release rate. Paraffin dispensers with larger surface areas
release active agent at faster rates (Atterholt 1996).

The property of the active agent having the greatest influence on its release rate is
its molecular weight. Generally, larger molecules take more time to make their way
through the free space of a matrix. Branching in a molecule can also decrease its rate of
diffusion through a matrix. The partition coefficient of the active agent between the
matrix and the environment can also influence the release rate of that agent. If the agent
readily partitions to the environment, then its rate of release will be diffusion-controlled
and first order. If, however, partitioning of the active agent to the environment is
relatively slow, then its partition coefficient will determine its release rate from the

matrix and the device will exhibit zero order release kinetics (Chien and Lambert 1974).

14

The partitioning of the active agent to the environment is a function Of the solubility of
the active agent in the matrix; compounds more soluble in the matrix are released to the
environment more slowly (Fan and Singh 1989). Paraffin disks and emulsions in a field
environment exhibit diffusion-controlled release.
Paraffin wax-based dispensers designed for releasing insect pheromones

In the early 19905, Atterholt and colleagues set out to develop a biodegradable
pheromone dispensing system. They found that under controlled conditions, a solid
paraffin wax dispenser (paraffin wax, vitamin E, and pheromone) released G. molesta
pheromone at a fairly constant rate for over one year (Atterholt 1996, Atterholt et al.
1998). In small-scale field tests in an almond grove, this dispenser completely inhibited
moth captures in pheromone traps until harvest, a period close to 200 days. A liquid wax
formulation was next designed, with the goal that it would be easier to apply to fruit trees.
This formulation consisted of 30% paraffin wax emulsified in water (Vitamin E and soy
oil were added in small amounts as an antioxidant and extender, respectively) and was of
the consistency of toothpaste (Atterholt 1996). It released above-threshold levels Of
pheromone for an extended period of time. The efficacy of the emulsion, applied with a
grease gun, was directly compared to that of two applications of hand-applied Hercon
trilaminate dispensers, two applications Of Checkmate dispensers, pesticides, and no
treatment, each applied to 2 ha peach plots. Trap shutdown was maintained in the paraffin
emulsion and shoot injuries from G. molesta in that plot were equivalent to those tallied
in the plot treated with Hercon dispensers and less than those tallied in the plot treated
with Consep dispensers. The trial lasted 100 days. A subsequent study focused on

developing a spray applicator for the emulsion (Delwiche et al. 1998). A large grease

15

pump was modified tO spray the emulsion onto trees. Tests showed that using this system,
75% Of the emulsion sprayed was deposited on the tree. In small scale trials in an almond
grove, the emulsion, applied in this manner, was as effective as Shin-Etsu rope dispensers
for 75 days, as measured by trap shutdown (Delwiche et a1. 1998). The emulsion
developed was patented by the University of California at Davis (US Patent 6,001,346,
Delwiche et a1. 1999). Subsequently, several companies successively attempted to
commercialize the emulsion. Most recently, Gowan Co. (Yuma, AZ) acquired the rights
to the emulsion and marketed the product as Confuse-OFM. Knapp manufacturing
prepared the wax emulsion base for the Confuse-OFM dispensers using a different
protocol than originally developed by Atterholt (1996). Confuse-OFM had the
consistency of white, liquid glue and was applied using a plastic squirt bottle. Gowan Co.
discontinued the product in 2002.

Although to date, paraffin disks and emulsions have been mostly tested as
dispensers of G. molesta pheromone, there are several reports Of other, usually limited
trials using paraffin formulations similar to the ones developed by Atterholt (1996) to
dispense other insect pheromones. Rice et a1. (1997) used paraffin emulsions to release
Quadraspidiosus pemiciosus (San Jose scale) and Anarsia lineatella (peach twig borer)
pheromones in stone fruit orchards. Control of A. lineatella with the formulation was
ineffective. Application of Q. perniciosus pheromone with the emulsified wax
formulation reduced crawler populations for two generations. Sanders (1999) used
paraffin disks and emulsions to dispense Cydr'a pomonella (codling moth) pheromone. In
an orchard with a relatively low population of the moth, the disks maintained trap

shutdown for ten weeks, while the emulsion maintained trap shutdown for only three

16

weeks. Only the major component Of the pheromone of this moth, E8,E10-1220H, was
released from the dispensers and the short duration Of efficacy of the dispensers was
attributed to this, as well as the low viscosity and resultant high surface area of the
emulsion dispensers. Only the release rate of pheromone from the paraffin disks was
quantified in the field. Sanders reported that negligible amounts of pheromone were
detected in the dispensers after week eight of the study. Release rates of the emulsion in
the field were not reported. Meissner et al. (2000) used paraffin disks and a paraffin
emulsion to dispense Platynota ideausalis (tufted apple budmoth) pheromone in mating
disruption trials. The disks and the emulsion were as effective as polyvinyl chloride
spirals, as measured by disruption of male orientation to pheromone traps. The emulsion
was effective for a longer period of time than the PVC spirals. Analyses of pheromone
release from the dispensers showed that the ratio of the components of the pheromone of
P. ideausalis remained more constant when the pheromone was released from paraffin
disks, versus the polyvinyl chloride spirals. The disappointing results obtained when
using emulsions to dispense the pheromones of A. lineatella and C. pomonella may have

been due to the low molecular weights of those pheromones (Chapter 2).

17

Objectives of this thesis

Wax-based formulations were used to investigate three ways to reduce product or
application costs of pheromone dispensers for mating disruption Of G. molesta. The
Specific objectives were to:

1. Develop an effective EWD dispenser that provided season-long control of G.
molesta with one application, comparable to what could be achieved with the
best commercial hand-applied dispensers.

2. Determine whether dispensers could be placed at a convenient height, in the
bottom third Of tree canopies, and provide control of G. molesta.

3. Identify the relationship between decreasing numbers of point sources of
dispensers and efficacy of mating disruption. Could control of G. molesta be

achieved with few, widely-Spaced, high-release pheromone point sources?

18

Chapter 2
Development and evaluation of an emulsified paraffin wax dispenser for season-long

mating disruption of Grapholita molesta in commercial peach orchards in Michigan,
USA

Introduction

The availability of a controlled-release device to dispense pheromone for an
extended period can be a limiting factor to the development of a practical mating
disruption program (Plimmer and Inscoe 1978, Sidall 1978, Rothschild 1979, Jackson
1989, Weatherston 1990). Although it is unlikely than any one dispenser would meet all
of the following criteria, an ideal dispenser should: release pheromone economically and
effectively for the duration of the target insect’s seasonal activity, be cheap and easy to
apply, cheap to manufacture, and environmentally-friendly (Plimmer 1981). Isomate-M
100 and M Rosso (Shin-Etsu Chemical Co., Ltd., Tokyo, Japan) are currently the most
effective pheromone dispensers for mating disruption of Grapholita molesta (Busck)
(Lepidoptera: Tortricidae), the oriental fruit moth, in Michigan.

Atterholt (1996) and colleagues (Atterholt et a1. 1998; Delwiche et al. 1998)
developed a paraffin-based emulsified wax dispenser (EWD) that released pheromone at
a nearly steady rate for over 130 days under controlled laboratory conditions. In a four
month-long field trial in peaches, this emulsion was as effective as Hercon and Consep
hand-applied dispensers, as measured via trap shutdown and shoot flagging counts
(Atterholt 1996, Chapter 1). The emulsion was patented by the University of California at
Davis (US Patent 6,001,346, Delwiche et al. 1999). Subsequently, several companies
attempted to commercialize this emulsion. Gowan Co. (Yuma, AZ) was the last to

acquire the license to this patent and marketed the product as Confuse-OFM. Knapp

l9

Manufacturing prepared the wax emulsion base for the Confuse—OFM formulation using
a different protocol than originally developed by Atterholt (1996). Confuse-OFM was
less viscous than Atterholt’s (1996) formulation; it had the consistency Of white, liquid
glue and was applied using squirting devices, such as forestry paint-marking guns and
plastic squirt bottles.

This chapter reports on: 1) the efficacy of Confuse-OFM, as supplied by Gowan
Co., versus the efficacies Of two other commercial G. molesta disruption formulations, 2)
the development of an improved paraffin wax-based EWD formulation (EWD II-OFM)
with enhanced longevity as compared to Confuse-OFM and adapted to the cold Michigan
climate, 3) the efficacy of EWD II-OFM as compared to the efficacies of Confuse-OFM
and Isomate-M 100, 4) the release profiles of Confuse-OFM and EWD II-OFM under
field and laboratory conditions, and 5) the development of a cheap and effective
applicator for EWD II-OFM.
Materials and Methods

2001 field tests. Efi‘icacy of Confuse-0PM. The efficacy of Confuse-OFM
(Gowan Co., Yuma, AZ) was directly compared to those Of Isomate—M 100 (Shin-Etsu
Chemical Co., Ltd., Tokyo, Japan), Checkmate OFM-F (Suterra LLC, Bend, OR), and a
comparison pesticide treatment. Confuse-OFM was applied at the recommended rate of
74g Al or 580 deposits per hectare per G. molesta generation using a one liter plastic
squirt bottle. Each deposit consisted of approximately 2.7 ml (2.6 g) of Confuse-OFM
and was applied to the upper surfaces of tree branches at a height of 1.5 to 1.8 m.
Isomate-M 100 was hand—applied at the recommended rate Of 57g A1 (250 dispensers)

per hectare at the same height as Confuse-OFM once at the beginning of the season.

20

Checkmate OFM-F, a sprayable formulation, was applied at 74g AI/ha per G. molesta
generation with an airblast sprayer. The active ingredient in all formulations was a 93:6:1
blend of 28-122Ac : E8-12:Ac : Z8—12:OH.

The trials were carried out in commercial peach orchards in Berrien Springs and
Coloma, MI. Treatments were replicated on four farms and blocked by farm. Treated
plots ranged from 0.8 to 3.6 ha and most were planted with peach varieties harvested in
early to mid August (Table 1, Appendix A), coinciding with the beginning of the third
flight Of G. molesta in Michigan. The peach trees in all plots were approximately 3 m tall.
Insecticide sprays were not eliminated from pheromone-treated plots (Table H, Appendix
A), but the numbers of sprays, were reduced in most plots.

Each orchard was monitored for activity of male G. molesta via 3-5 pheromone
traps (Scenturion, Inc., Clinton, WA) and five attractant-baited bucket traps. Each trap
was placed in the plot so as to provide a similar coverage area to other traps Of its kind,
while minimizing interference with all other traps. Pheromone traps were monitored
biweekly. At each sampling date, males moths caught in the traps were counted and
removed. Lures were replaced once per G. molesta generation (ca. 6-8 wk). Sticky trap
liners were replaced when soiled or at least once per G. molesta generation. Bucket traps
were monitored once weekly. These green, yellow, and white traps (Great Lakes IPM,
Vestaburg, M1) were filled with ca. 500 ml of a liquid bait composed of: 10 ml terpinyl
acetate (Aldrich Chemical Co., Inc., Milwaukee, WI), 1 ml Tween 20 emulsifier (Aldrich
Chemical Co., Inc., Milwaukee, WI), 1.8 kg brown sugar, and 18.9 L water (Atanassov et

a1. 2002). Each bucket trap was emptied, rinsed, and refilled with bait at each sampling

21

date. All traps were hung at approximately 1.5-1.8 m heights in the outer third of tree
canopies.

G. molesta injury to shoots was evaluated at the ends of the first and second
generations. Three hundred and sixty to 400 Shoots were examined in the plot interiors
(defined as trees not located in the outer two rows nor the outer three trees within rows)
for evidence of larval feeding in the tips. Ten randomly-chosen shoots in the top half and
ten randomly-chosen shoots in the bottom half of randomly-chosen non-adjacent trees in
non-adjacent rows were examined for shoot flagging. Flagged shoots were subsequently
dissected to reveal G. molesta larvae. Larval voucher Specimens are deposited at the
Michigan State University Entomology Museum (Appendix C).

Levels of G. molesta fruit infestation were evaluated just prior to harvest by
examining 540 to 600 fruits in the plot interiors. Although it is desirable to cut open all
fruits evaluated for internal worm injury (Trimble et a1. 2001), a small peach crop in
Michigan in 2001 and 2002 and a high value for the available fruits limited the number of
fruits that could be removed from these commercial orchards. Thus, the current fruit
injury evaluations entailed carefully examining the fruits without removing them from
trees. Fruits with any external evidence of insect feeding were picked and cut to confirm
tunneling or the presence of a G. molesta larva.

Field release rate of Confuse-OFM. The release rate of Confuse-OFM was
determined in an apple orchard at the Trevor Nichols Research Complex, Fennville, MI.
Approximately 2.7 ml of Confuse-OFM was evenly applied as an ca. 12.5 cm x 1 cm x 2
mm (length x width x depth) deposit to hardwood tongue depressors (15.2 cm x 1.9 cm)

using one liter plastic squirt bottles. Initial weights Of depressors plus deposits ranged

22

from 5.00 to 5.50 g. The treated tongue depressors were left to dry on a horizontal surface
for approximately 12 h. Binder clips (1.9 cm) were permanently affixed at 30.5 cm
intervals to 3 m long plastic ropes. Two ropes were hung on the north sides of five 3.8 m
tall apple trees at approximately 1.8 In from the ground and within the outer third of tree
canopies. A single tongue depressor was hung vertically on each binder clip. All wax
deposits faced east. Experiments were initiated in early June. Five depressors treated with
Confuse-OFM were randomly selected for collection at time zero. Subsequently, five
treated depressors, one randomly selected from each tree, were collected at weekly
intervals over 11 wk. Immediately following collection, the treated depressors were
individually placed in 500 ml clear, wide-mouthed French Square Bottles with tinfoil-
lined black phenolic caps (Qorpak, Bridgeville, PA) and kept frozen at —10 to -20 °C until
analysis.

Pheromone was extracted from the dispensers using a method modified from
Meissner et a1. (2000). A shaker water bath (Orbit Shaker Bath Model 3540, Lab Line
Instruments, Inc., Melrose Park, IL) was heated to between 70 and 75 °C and left to
equilibrate at that temperature for at least 1 h. The bottles containing the dispensers were
removed from the freezer and left to thaw at room temperature. Four hundred ml of
acetonitrile (HPLC grade, EM Science, Merck KGaA, Darmstadt, Germany) were added
to each bottle, followed by 62.6 mg of tridecanoic acid methyl ester (98%, Sigma-Aldrich
Co., St. Louis, MO) (serving as the internal standard), in 1 ml acetonitrile. The bottles
were placed in the water bath for 10 min, then shaken in the water bath at 150 rpm for 30
S. The bottles were then left in the water bath for another 3 min followed by shaking for

another 30 s. The bottles were removed from the water bath and placed directly into a

23

freezer (-20 °C) for at least 24 h, during which time the wax solidified and settled to the
bottom. After 24 h, the bottles were removed from the freezer and left to thaw.
Approximately 1 ml of the solvent was removed from each jar via disposable glass
Pasteur pipette. This sample was filtered through another disposable glass Pasteur pipette
fitted with an ca. 5.5 cm x 5.5 cm piece of Kimwipe (Kimberly-Clark Corp., Roswell,
GA) folded to form a plug at the tapered end of the pipette, into a 1.5 ml GC vial
(Supelco, Bellefonte, PA). Pheromone in the sample was quantified by gas
chromatography using a 30 m HP DBWAXETR polar column. Helium, the carrier gas,
was passed through the column at a constant pressure of 20 psi. The instrument was set at
an initial temperature of 130 °C. The temperature was then ramped to 160 °C at a rate of
2.5 °C/min, then ramped to 230 °C at a rate of 40 °C/min. The pheromone content of the
analyzed samples was calculated using the internal standard method (McNair and Miller
1998).

Developing EWD II-OFM. The release rates of four experimental EWD
formulations and Confuse-OFM were determined in the laboratory environment. The
formulations consisted of the following ingredients (ratios given below): paraffin wax
(Gulf wax, Royal Oak Sales, Inc., Roswell, GA), soy Oil (Spectrum Naturals, Inc.,
Petaluma, CA), Span 60 (Sorbitan monostearate, Sigma-Aldrich Co., St. Louis, MO),
vitamin E ([:]—a-tocopherol, Sigma Chemical Co., St. Louis, MO), G. molesta
pheromone (93:6:1 blend of Z8-12:Ac : E8-122Ac : Z8-12:OH, Shin-Etsu Chemical Co.,
Ltd., Tokyo, Japan), and de-ionized water. The formulations were prepared as per
Atterholt (1996). The procedure consisted of heating the paraffin wax to 60-65 °C, while

separately heating the water to 65-70 °C. When both had reached the designated

24

temperatures, the remaining ingredients were incorporated into the wax, followed by the
addition of the hot water. The whole was mixed for ca. 5 min in an industrial laboratory
blender (Waring Commercial, Torrington, CN). The emulsion was gradually cooled to
room temperature by placing the mixing bowl in a bucket of cold water. Intermittent
mixing during the cooling process was necessary to ensure that the final emulsion was
smooth and flowable.

The release rates of Confuse-OFM, 30% wax EWD (30% wax, 5% G. molesta
pheromone, 4% soy oil, 1% vitamin E, 2% Span 60, 58% de-ionized water), 35% wax
EWD (35% wax, 5% G. molesta pheromone, 4.7% soy Oil, 1% vitamin E, 2.3% Span 60,
52% de-ionized water), and 40% wax EWD (40% wax, 5% G. molesta pheromone, 5.3%
soy oil, 1% vitamin E, 2.7% Span 60, 46% de-ionized water) were determined by aging
in chemical fume hoods. Three ml of each formulation were applied to 3.8 cm x 35 cm
strips Of heavy-duty aluminum foil. The bottom of each strip was fitted with two pennies
(5-6 g total weight) to stabilize the foil in the air flow of the hoods. The experimental
EWD dispensers were applied as ca. 2.5 cm diam., 0.5 cm thick dollops in the middles of
the aluminum foil strips using a 20 ml syringe with a cutoff tip (2 cm diam. opening).
This method of application gave the dispensers a shape very similar to that realized in the
field when applied with the S.P.L.A.T. applicator (Appendix B). Confuse-OFM was
applied 3 cm from the top Of the foil strip using an unaltered syringe. Foil strips on which
Confuse-OFM was applied were immediately held vertically to allow the deposit to run
down the foil strip, as it was observed to do when applied to tree limbs in the field. In no

case did material drip from the foils before hardening. The dispensers were left to dry on

25

a horizontal surface for approximately 12 h before being hung vertically in the fume
hoods.

Five strings (1.6 m long) were stretched tightly across the lengths Of each of two
fume hoods, ca. 98 cm above the work surfaces inside the hoods, so that the bases Of the
foil strips, when these were hung vertically, were several centimeters above the fully
Open windows of the fume hoods. Four sets of foil Strips containing each treatment were
randomly chosen for collection at time zero. The remaining treated aluminum strips were
hung side by side along the lengths of the strings (40 foil strips per string) and secured to
these using small paper clips. Fourteen collections were made over 24 wk. On each
collection date, two randomly selected strips of each treatment were collected from each
hood. Temperatures in the hoods were recorded weekly and remained between 22 and 24
°C for the duration of the experiment. The collection and analysis protocols were
identical to those described for the determination of the release rate of Confuse-OFM in
the field (page 23).

2002 field tests. Efi‘icacy of EWD II-OFM. The 40% wax EWD tested in the
laboratory (page 25) was modified to create EWD II-OFM. EWD II-OFM was made
using the same protocol as described previously (page 24) and consisted of, by weight:
40% paraffin wax, 5% soybean oil, 2.7% Span 60, 2.7% vitamin E, 5% G. molesta
pheromone, and 46% de-ionized water. Multiple batches of EWD II-OFM were stored in
19 L plastic buckets and held at room temperature until use. During the development of
EWD H-OFM, it was observed that as a result of freezing and thawing in the winter and
Spring, many of the dispensers dislodged from the trees soon after application. Paraffin

waxes are relatively non-sticky (Bennett 1975). The relatively liquid Confuse-OFM, due

26

to its high surface area Of contact, adequately adhered to tree bark. The more viscous
EWD H-OFM formulation had reduced contact with the bark. At temperatures below 0
°C, EWD II-OFM dispensers were easily dispensers dislodged from the trees. Adding
Premium Indoor/Outdoor Carpet Adhesive 6700 (Roberts Consolidated Industries, Inc., a
Q.E.P. Company, Boca Raton, FL) to the formulation resolved this problem. Just prior to
application in the field, a quantity of this adhesive equal to 2% or 5% of the weight of the
emulsion was thoroughly mixed into the emulsion using a power drill-driven paint mixer.

Except for the differences noted below, these trials were carried out in the same
manner as the 2001 efficacy trials. The experiment was replicated on four commercial
peach farms in Coloma, MI. The peach plots were Similar to those used in the 2001 trials
(Table III, Appendix A). AS in 2001, insecticide treatments were not eliminated from the
pheromone-treated plots, but were reduced in most of these plots (Table IV, Appendix
A).

The formulations tested were: Confuse-OFM, Isomate-M 100, and EWD II-OFM.
The Confuse-OFM treatment differed from that in the 2001 test; it was applied as 3 ml
(2.8 g) deposits with forestry paint-marking guns (essentially, heavy duty squirt bottles)
(Idico Products Co., New York, NY) for a total of 520 deposits per hectare. EWD II-
OFM was applied at 74 g A1 (590 dollops) per hectare as 3 ml (2.5 g) dollops using a
Swipe applicator developed by agricultural engineers at Michigan State University (page
30). The EWD II-OFM formulation initially applied contained 2% adhesive. Due to
adhesion problems at low temperatures early in the season, some EWD H-OFM dollops
were replaced with new material containing 5% adhesive as late as 6 wk following the

initial application. Specifically, on farms one and two, at the end of May, ca. 12% and

27

15% of dollops, respectively, were replaced. Between early and mid May, all dollops on
farm four, and ca. 50% of dollops on farm five were replaced. EWD H-OFM was applied
on these farms until ca. 21:30 on 19 April, when the sun set. Those treatments had little
chance to dry before experiencing freezing nighttime temperatures; ca. 33% of dollops
applied in those plots on 19 April had fallen when the plots were surveyed on 3 May.
Four pheromone traps and four bucket traps were placed in each plot.
Additionally, male G. molesta activity was monitored in all plots via virgin female traps.
Virgin females originated from a colony collected as larvae in an infested apple orchard
in Fennville, MI, in July 2001. Voucher specimens are deposited in the Michigan State
University Entomology Museum (Appendix C). This colony was reared on a pinto bean-
based diet (Shorey and Hale 1965) at 24 °C and 16:8 L:D. Pupae were sexed (George
1965) and females were reared individually, under natural light conditions, in 118 ml
plastic cups containing a 2 cm cotton wick soaked in 5% sucrose solution. Two females,
at most three days Old, were placed in a wire screen cage and provided with a source of
sugar water. The screen cage was placed in a delta trap (Scenturion, Inc., Clinton, WA).
Three to four times during the peak flight period of the first and second G. molesta
generations, five virgin female traps were placed in random locations in the interior of
each plot and at least 8 m from any other trap. The traps were monitored after 3 d for live
females and counts Of captured males. Data gathered from traps in which one or more
females were absent or dead were not used in the analyses (ca. 20% Of traps in each
generation); data was successfully collected from 6-13 and 10-14 traps in each plot in

generations one and two, respectively.

28

To estimate levels Of G. molesta injury to shoots, 400 shoots were examined in
the interior of all plots. Levels of internal fruit injuries in each plot were evaluated by
examining 600 fruits in plot interiors.

Field release rate of EWD II-OFM with and without adhesive. Field release rates
were determined for EWD II—OFM mixed with no adhesive, 2% adhesive, and 5%
adhesive. The experiment was initiated in early June. Three ml dollops of each
formulation were applied onto 10.2 cm x 2.9 cm x 0.3 cm pieces of wood using the same
procedure employed to apply the experimental EWD formulations to the aluminum strips
for the laboratory release rate experiment (page 25). The dollops were left to dry for 24 h.
A 1.8 m x 3.7 cm x 1.8 cm piece of wood was screwed into the north side of each of five
3.8 m tall apple trees at a height of ca. 1.5-1.8 m in the outer third of the canopy.
Subsequently, the smaller, dollop—treated pieces of wood were screwed horizontally, 1.2
cm apart, onto the larger pieces of wood, with the wax material facing up. Five dollops
from each treatment were randomly selected for collection to determine the amount of
pheromone they initially contained. One randomly selected dollop of each treatment was
collected from each tree (five dollops per treatment total) weekly for 10 wk, then every
other week for another 12 wk.

The collection and analysis protocols were identical to those described for the
field release rates of Confuse-OFM (page 23), except that the extraction procedure was
modified to reduce the volume of solvent used. Specifically, only 200 ml acetonitrile
were added to the sample bottles prior to pheromone extraction. After filtering the

extraction solvent, 0.5 m1 of that solvent was diluted by half with acetonitrile before

29

pheromone quantification. In preliminary GC analyses, results using this modified
procedure were comparable to those obtained using the original procedure.

Efficiency of EWD applicators. Three applicators were developed to dispense
EWD II-OFM. The S.P.L.A.T. applicator (Appendix B) consisted of a metal barrel
containing a piston powered by a modified cordless finishing nail gun. EWD II-OFM was
loaded into an electric grease gun attached to a backpack. When the trigger was
depressed, the piston rapidly Shot a dollop of EWD II-OFM out of the barrel. Following
the shot, EWD II-OFM was automatically loaded into the barrel again. The S.P.L.A.T.
applicator was fitted with a laser sight and could accurately fire at distances as great as 2
m.

The Swipe applicator consisted of an electric grease gun (PowerLuber, Lincoln,
St. Louis, MO) fitted with a 1 m long rigid stainless steel pipe (6 mm internal diam.). A
metal disk (3 cm diam.) at the end of the nozzle, created a “foot”. The foot was placed
against a tree branch. The trigger was depressed to dispense the desired amount of EWD
II-OFM. Using a swiping motion, a dollop of EWD II-OFM was deposited on top Of that
branch.

The Spatula applicator was a laboratory spatula with a 10 x 2 cm stainless steel
blade. When the spatula was dipped in a container of emulsion to a depth designated by
an engraved line, ca. 2.5 g (3.0 ml) of EWD II-OFM was consistently loaded on the
spatula. The loaded spatula was placed against a tree branch and then pulled downwards,
leaving a dollop of EWD II-OFM on the branch.

Times for application of EWD II-OFM using these three applicators were

compared with those to apply Confuse-OFM with a paint-marking gun and Isomate-M

30

100 rope dispensers by hand. The application timing experiment was conducted in
October 2002 in a 0.2 ha portion of a mature mixed peach and plum orchard at the
Michigan State University Horticultural Teaching and Research, Viticulture and Enology
Farm, East Lansing, MI. The experiment was replicated four times and blocked by the
person making the application. All formulations were applied at a rate of 537 dispensers
per hectare.

Statistical analyses. Moth captures in pheromone traps and bucket traps were
log-transformed (log [x+l]) prior to analysis. Shoot and fruit injury data and percent
mated females caught in bucket traps were arcsine-transformed (arcsine [x]) prior to
analysis. Moths captured in pheromone traps, moths caught in bucket traps in 2001,
percent mated females caught in bucket traps in 2002, and shoot and fruit injury data
were analyzed by analysis of variance (ANOVA) for a randomized complete block
design. Means were separated using Tukey’s test (SAS version 8.2, SAS Institute 1999).

A two-factor ANOVA for a randomized complete block design was used to
analyze the numbers of male and female moths caught in bucket traps in 2002; treatment
and generation were the two factors analyzed. Too few females were caught in generation
one to analyze the percent mated female data in this manner. All'means were separated
using Tukey’s test (SAS version 8.2, SAS Institute 1999).

Data on times required for dispenser applications were analyzed by AN OVA for a
randomized complete block design. Means were separated using Tukey’s test (SAS
version 8.2, SAS Institute 1999).

For release profile determinations, the amount of pheromone remaining in the

dispensers was plotted over time and exponential curves were fitted to the data. In some

31

cases, the best fit was achieved with two curves to data from one formulation. Fan and
Singh (1989) state that monolithic dispensers (devices in which the active agent is
distributed in a polymer matrix), such as EWD dispensers, have biphasic release rates.
Initially, the release rate is proportional to thi—I—nE; thus, the total amount Of material
released at any point in time is proportional to the square root of time. After most of the
active ingredient has been released from the device, the release profile then becomes
exponential. An attempt was made to fit curves to the data indicative of the two phases of
this relationship (Fan and Singh 1989). Generally, exponential curves best fitted these
data. The primary objective in fitting curves to the data was to use these to accurately
calculate the release rate of pheromone from the dispensers at any point in time.
Differential equations were used to calculate how long dispensers continued to release
pheromone at a rate equal to or greater than the threshold release rate of 5 mg/ha/h
established by Rothschild (1975) for disruption of male G. molesta orientation to
pheromone traps. Although other authors have Offered estimations of the release rate Of
pheromone necessary per area for mating disruption of G. molesta to be effective (6.25
mg/ha/h Cardé et a1. 1977, 0.01 mg/ha/h Charlton and Cardé 1981, 20 mg/ha/h Audemard
et al. 1989), the threshold release rate determined by Rothschild (1975) is most consistent
with the data gathered in the present studies. To convert release rates per dispenser to
release rates per hectare, the amount of active ingredient applied per hectare was taken as
74 g.
Results and Discussion

Confuse-OFM trials. Efi‘icacy. Two applications of Confuse-OFM (148 g AI/ha)

were at least as effective as one application of Isomate-M 100 (57g AI/ha) and two

32

applications of Checkmate OFM-F (148 g AI/ha) in controlling G. molesta (Figure 2.1).
Although a small number of moths was caught in all pheromone—treated plots in both
generations, significantly fewer moths were caught in pheromone-treated plots vs. the
comparison plot. Equivalent numbers of moths were caught in the Confuse-OFM and
Isomate-M 100 plot and significantly fewer moths were caught in the Confuse-OFM plots
than in the Checkmate OFM-F plots in generation one. In generation two, equivalent
numbers of moths were caught in all pheromone treatments, indicative of a similarly-
reduced ability of male G. molesta to orient to pheromone sources in those plots
(Generation 1: F = 18.92, df = 3, P = 0.0003; Generation 2: F = 41.05, df = 3, P <
0.0001).

Percentages of shoot injury quantified at the ends of generations one and two and
fruit injury at harvest did not significantly differ across the pheromone or comparison
pesticide treatments (Generation 1 Shoot: F = 0.90, df = 3, P = 0.48; Generation 2 Shoot:
F = 0.45, df = 3, P = 0.72; Harvest Fruit: F = 0.52, df = 3, P = 0.68) (Figure 2.2).
Although there was considerable variability in the percentage shoot and fruit injuries
quantified in each treatment, the levels of injury recorded were quite low. Most
importantly, the highest percentage of fruit injury reported at harvest for any plot was
only 0.4 percent, a level acceptable to Michigan growers.

In generation two, the numbers of males and female G. molesta caught in bucket traps in
the Confuse-OFM and comparison treatments were not significantly different (Male: F =
1.90, df = 1, P = 0.26; Female: F = 0.43, df = 1, P = 0.56) (Table 2.1). Apparently, there
were equivalent moth populations in these plots. Atanassov et al. (2002) also found no

Significant differences in the numbers of male and female G molesta caught in bucket

33

 

I Comparison

I Confuse-OF M

CI lsomate-M 1 00

E Checkmate OF M-F

Males Per Trap
N 03
O O

—L
O

 

 

 

Os

Generation 1 Generation 2

Figure 2.1: Grapholita molesta caught in pheromone traps during the 2001 field trial
testing the efficacy of Confuse-OFM versus other commercial G. molesta pheromone
dispensers. Treatments labeled with the same letter, within a generation, are not
significantly different (Tukey, P < 0.05).

34

9°
0

 

     

 

 

 

 

 

 

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(”g I Confuse-OF M
+1 .. Ulsomate-M 100 a
g 2,0 - ECheckmate OFM-F
é
a
.3
E“
g: 1.0 ~
C
(D
8
(D
0. fl
0.0 _ .9... 4:: I
Generation 1 Generation 2 Harvest Fruit
Shoot Shoot

Figure 2.2: Percent of peach shoots and fruits sampled with internal, larval-caused injury
during the 2001 field trial testing the efficacy of Confuse-OFM versus other commercial
Grapholita molesta pheromone dispensers. For each of the evaluations, there were no
significant differences between the treatments (Tukey, P < 0.05). aInjuries for Checkmate
OFM-F in generation two and at harvest are for three plots only. '

35

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36

traps placed in pesticide-treated plots and mating disruption plots treated with lsomate-M
or Isomate-M 100 dispensers.

The moth populations on the farms were moderate. The most moths caught per
pheromone trap per half week trapping interval in the comparison plots during
generations one and two was four on farm one, seven on farm two, 14 on farm three, and
five on farm four. Moths were caught in these pheromone traps throughout the season,
except during the later part of generation two. Following diapause, the life cycles of first
generation larvae are fairly synchronized. A period of low adult moth activity, following
the bulk of mating and egg-laying and during which time second generation larvae are
developing, is expected. Moth catches in the pheromone-treated plots usually coincided
with peak catches in the comparison plots.

There were both advantages and disadvantages to testing the efficacies of these
formulations on commercial peach farms. On-farm trials permitted use of relatively large
plots (Tables I and 111, Appendix A), thereby minimizing the likelihood of immigrating
moths masking treatment effects. However, growers were unable to risk their high—value
fresh-market peaches; overall, insecticide sprays were only slightly reduced in the
pheromone plots versus the comparison (insecticide only) plots (Tables II and IV,
Appendix A). Notably, azinphosmethyl, a very effective insecticide control for G.
molesta (Wise et a1. 2003) was not applied in the pheromone-treated plots.

Longevity. The release kinetics of pheromone from Confuse-OFM in the field
were first order (Figure 2.3). The initial release rate was high; approximately two thirds
of the pheromone was dispensed in the first 21 d. The high release rate of this

formulation was likely a result of its high surface area and concomitant low thickness.

37

 

 

 

 

a: 140
(I)
H
E _ -0.049x
:100 , y— 1212.1e
02> r = 0.99
a) 80 ~
0.
.‘L’
e 60 —
a)
D.
2 40 s I y = 59.56-0'018)‘
§\_i r2 = 0.97
ab 20 ~ TM
N
CD
E O l I l T l l l
0 10 20 30 4O 50 60 70 80

Elapsed Time (d)

Figure 2.3: Z8-12:Ac remaining in Confuse-OFM dispensers aged in the field.

38

The release rate of compounds from monolithic devices follows Fick’s law Of diffusion
and is influenced by the surface area and the thickness Of the dispensing matrix (Fan and
Singh 1989, Atterholt 1996). Increasing the surface area and decreasing the thickness of
the matrix increases the release rates of active agents from these devices.

According to these data, the release rate of pheromone from Confuse-OFM
remained at or above the threshold release rate Of 5 mg/ha/h (Rothschild 1975) for 93 (1,
approximately the longevity of Isomate-M 100, as stated on the product label. This is an
overestimation Of the longevity of this formulation when it is applied to trees. If Confuse-
OFM truly remained effective this long, one application of this formulation would
provide season-long control of G. molesta in peaches in Michigan. However, Gowan Co.
recommended applying Confuse-OFM once per G. molesta generation (ca. 42-56 d).
Furthermore, when maintenance of trap shutdown was used as a measure of dispenser
longevity in 2002, the first application of Confuse-OFM lasted only 77 d and a second
application of Confuse-OFM was necessary to maintain trap shutdown until harvest (page
48).

For this release rate-determination, Confuse-OFM was applied to a predetermined
area of ca. 12.5 cmz, approximately one third of the area covered by Confuse-OFM when
this formulation was applied to a tree branch. The thickness of the Confuse-OFM
deposits applied to tongue depressors was correspondingly greater. According to Fick’s
law, since the Confuse-OFM deposits in this experiment had smaller surface areas and
larger thicknesses than those applied in the field efficacy trials, their release rates were

likely reduced, compared to the release rates of Confuse-OFM deposits applied to trees.

39

As a result, the longevity Of the Confuse-OFM formulation, as applied in this experiment,
was inflated.

Drawbacks. Serious deficiencies of the Confuse-OFM formulation were identified
during the 2001 studies. Although Confuse-OFM was an effective mating disruption
product, the need for two applications Of this formulation for season-long control Of G.
molesta was a serious drawback. Pheromone is costly; almost three times as much
pheromone was necessary for season-long control Of G. molesta with Confuse-OFM
versus Isomate-M 100 (148 g AI/ha versus 57 g AI/ha). Hand—applying dispensers is
time—consuming and expensive as well; performing this process twice per season would
also increase the cost of a mating disruption program.

Confuse-OFM application was problematic. Confuse-OFM was too liquid,
resulting in a substantial time requirement to apply it to a horizontal surface on the tree
bark and minimize the amount that dripped onto the ground. The applicators provided to
apply the emulsion were inadequate. Plastic squirt bottles and paint-marking guns were
not designed to apply a wax material; they broke or clogged after only a few hours of use.

Developing EWD II-OF M. EWD H—OFM showed considerable promise for
commercialization. In 2001, Confuse-OFM deposits were found to waste pheromone by
releasing it very quickly for the first three weeks following application (Figure 2.3). It
was postulated that the large surface areas of Confuse-OFM deposits were responsible for
this phenomenon. The release profiles of pheromone from the experimental EWD
formulations and Confuse-OFM measured under laboratory conditions were of the first
order (Figure 2.4). Thicker experimental EWDS, created by increasing the wax content of

these formulations, had less concave release profiles than Confuse-OFM. Furthermore,

40

 

I Confuse-OFMab
+1 140.0 A 30% wax EWDc
\ 0 35% wax EWDd
\ ° 40% wax EWD

—L
N
.o
0

100.0 - \ \
80.0 —— X x
60.0 ~ \
40.0 -

20.0 -

 

 

 

mg 28-12:Ac Per Dispenser (mean

 

.0
O

 

Elapsed Time (d)

Figure 2.4: Z8-122Ac remaining in Confuse-OFM and three experimental EWDS aged in

a laboratory fume hood. “Days 021: y = 120.9e’0'068", r2 = 0.98; days 21+, y = 64.030045",
r2 = 0.97. by = 121.2e'°-°3'*, r2 = 0.95. Cy = 126.3e'0-024", r2 = 0.98. dy = 137.6e0-024", r2 =

0.97.

41

the 35% and 40% wax EWD formulations had Similar release profiles that were less
concave than the profile for the 30% wax EWD. The 35% and 40% wax EWD
formulations released pheromone above the threshold release rate of 5 mg/ha/h
(Rothschild 1975) for 113 and 116 d, respectively, twice as long as Confuse-OFM (56
d).The longevity Of Confuse-GEM, as determined under laboratory conditions matched
the time this formulation lasted in field in 2002, based on shutdown of moth captures in
pheromone traps (page 48). In these laboratory experiments, Confuse-OFM was applied
in a way that more precisely mimicked Confuse-OFM applied to trees.

Evaluation of EWD II-OFM. Efi‘icacy. As evidenced by the results of
assessments using a variety of techniques, EWD H-OFM was as effective as Confuse-
OFM wax emulsion and Isomate-M 100 ropes for G. molesta control. Although large
numbers of moths were caught in the comparison treatments in both generations during
this trial, only a small number of moths were caught in the pheromone-treated plots in
both generations (Figure 2.5). In all generations, significantly fewer moths were caught in
pheromone traps in all pheromone-treated vs. the comparison plots and there were no
significant differences in the numbers of moths caught in the different pheromone
treatments (Generation 1: F = 17.72, df = 3, P = 0.0004; Generation 2: F = 41.09, df = 3,
P<0mmn.

Data from virgin female traps agreed with data from the pheromone traps. While
male moths were caught in virgin female traps placed in comparison plots during both the
first and second generations of G. molesta, no males were caught in virgin female traps
placed in any of the pheromone-treated plots during those times (Table 2.2). Rothschild

(1981) stated that in general, more male moths were caught in pheromone traps than in

42

 

 

 

 

 

 

80

I Comparison
I Confuse-OFM

Q 60 - a D lsomate-M 100

g I: EWD ll-OFM

'—

6'.’ 40 4

(I)

.512

c0

2 20 -

b b b b b b
0 ‘ .
Generation 1 Generation 2

Figure 2.5: Grapholita molesta caught in pheromone traps during the 2002 field trial
testing the efficacy of EWD H-OFM versus Confuse-OFM and Isomate-M 100.
Treatments labeled with the same letter, within a generation, are not significantly
different (Tukey, P < 0.05).

43

Table 2.2: Grapholita molesta caught per three days in virgin female traps during the
2002 field trial comparing the efficacy of EWD II-OFM to Confuse-OFM and Isomate-M
100.
Generation Males per trap in comparison plots Dates traps placed in orchards
(mean t SE)21
1 2.0 i 0.7 23-May, 27-May, 5-Jun
2 5.9 i 4.3 9-Jul, 20-Jul, 26-Ju1
aNO G. molesta were caught in pheromone-treated plots.

 

 

 

44

virgin female traps. This was certainly true in this trial. Moreover, pheromone traps
provided a more sensitive measure of the ability of male G. molesta to orient to
pheromone sources in plots protected by mating disruption. Virgin female traps were
more labor-intensive to maintain and provided no more information than was obtained
with pheromone traps. Virgin female traps seem superfluous in assessing orientational
disruption of G. molesta.

Although levels of injury to shoots and fruits were generally higher in 2002 than
in 2001 (Figure 2.6), nonetheless, data trends remained the same. As in 2001, there was
considerable variation in the injury quantified in all treatments and no significant
differences between the treatments for any of the injury evaluations conducted
(Generation 1 Shoot: F = 0.93, df = 3, P = 0.46; Generation 2 Shoot: F = 1.43, df = 3, P =
0.30; Harvest Fruit: F = 0.67, df = 3, P = 0.59). (Figure 2.6). Two plots at harvest had
percent fruit injuries in excess of one percent (one percent fruit injury would be
considered excessive by Michigan growers and processors): the comparison plot on farm
five had 1.8 percent fruit injury and the Confuse-OFM plot on farm one had 1.2 percent
injury. High levels of injury in some locations on the borders of those plots were
evidence of mated females immigrating from nearby abandoned apple orchards;
immigration of mated females may have led to higher injury levels in these plots.

Whereas in 2001, bucket traps were only placed in the Confuse-OFM and
comparison plots beginning in the second generation of G. molesta, in 2002, bucket traps
were placed in all plots beginning in generation one. Nevertheless, the data from these
traps replicated what was Observed in 2001 (Table 2.1). The numbers of male and female

moths caught in bucket traps in the different treatments were not significantly different

45

 

 

   

 

 

10.0
a IComparison
05 T I Confuse-OFM
8 0 ~
H ' D lsomate-M 100
§ ca EWD ll-OFM
g 6.0 -
2‘
:3
g 4.0 I
*5
<1)
9 2.0 ~
m ,.
0.
0.0 w . '
Generation 1 Generation Harvest

Shoot Shoot Fruit

Figure 2.6: Percent of peach shoots and fruits sampled with internal, larval-caused injury
during the 2002 field trial testing the efficacy of EWD H—OFM versus Confuse-OFM and

Isomate-M 100. For each of the evaluations, there were no significant differences
between the treatments (Tukey, P < 0.05).

46

(Male: F = 2.68, df = 3, P = 0.07; Female: F = 2.99, df = 3, P = 0.05). However, there was
an increase in the numbers of moths caught in bucket traps in generation two, versus
generation one (Male: F = 21.09, df = 1, P = 0.0002; Female: F = 64.22, df = 1, P <
0.0001). The interaction between treatment and generation was not significant (Male: F =
0.87, df = 3, P = 0.47; Female: F = 1.84, df = 3, P = 0.17). In addition, the percentages of
mated females in the comparison and pheromone-treated plots were not significantly
different (Generation 1: F = 15.49, df = 2, P = 0.06; Generation 2: F = 1.02, df = 3, P =
0.42). Rothschild (1981) stated that although bucket traps seemed to be more attractive to
mated females, than to virgin females, small, but Significant reductions in the percentages
of mated females were detected in pheromone-treated plots using this technique. Such
differences were not identified in the current experiment. Atanassov et a1. (2002) also
found no significant differences in the percentages of mated female G. molesta caught in
bucket traps placed in insecticide-treated versus pheromone-treated plots. Although these
data do not reveal treatment differences, they are evidence that moth populations in these
plots were equivalent.

As in the Confuse—OFM efficacy trial, pesticide treatments were only slightly
reduced in the pheromone-treated plots versus the comparison plots. Likewise,
azinphosmethyl sprays were eliminated from the pheromone-treated plots, (Tables 11 and
IV, Appendix A). Nevertheless, the high numbers of moths caught in the comparison
plots and equivalent numbers of moths caught in bucket traps placed in all treatments in
2002 indicated the presence of a relatively large G. molesta population in the

experimental plots. Even under relatively high pest pressure, trap Shutdown in all

47

pheromone-treated plots was successfully achieved. Thus, EWD II-OFM was as effective
as the other commercially-available dispensers for orientational disruption of G. molesta.

Longevity. A 40% wax EWD similar to EWD H-OFM released pheromone for
twice as long as Confuse-OFM in the laboratory (Figure 2.4). AS judged by the similar
slopes of the fitted curves, adding adhesive to EWD H-OFM did not alter the release rate
of pheromone from this dispenser (Figure 2.7). The rate of pheromone release from 3 ml
dollops of EWD H-OFM in the field remained at or above the threshold release rate of 5
mg/ha/hr (Rothschild 1975) for ca. 85 d, which is close to the 90 d lifetime of Isomate-M
100, as stated on the dispenser label.
Moth captures through the season can also provide an indirect measure of the longevity
of a disruption formulation. Figure 2.8 reveals the average numbers of G. molesta caught
per pheromone trap per week throughout the season in the 2002 plots. Following the first
applications of the pheromone formulations, on 19 April, moth catches were substantially
inhibited or completely shutdown in the EWD II-OFM plots until 9 August, 112 d
following application. Trap shutdown was maintained in the Isomate-M 100 plots until 6
August, 109 d following application, and until 5 July in the Confuse-OFM treatment, 77
(1 following application. Confuse-OFM was reapplied on 8 July and trap shutdown was
maintained until harvest. Thus, trap Shutdown in the pheromone-treated plots was
maintained until harvest with one application of EWD II-OFM or Isomate-M 100 or two
applications of Confuse-OFM.

Once the fruits were harvested in a plot, moth catches increased in both the
pheromone and bucket traps in that plot. Most plots were harvested in early to mid

August (Tables I and 111, Appendix A), 100-120 (1 post-application of pheromone. It is

48

 

I no adhesiveab
140 " A 2% adhesive
0 5% adhesivec

 

 

 

 

mg 28-122Ac Per Dispenser (mean :1: SE.)

 

0 10 20 30 40 50 60 70 80 90 100 110120
Elapsed Time (d)

Figure 2.7: Z8-12:Ac remaining in EWD II-OFM dispensers with and without adhesive
aged in the field. 3Days 042: = 119.7e'0'037", r2 = 0.93; days 42+, y = 49.660016", r2 =
0.82. bDays 0-42: = 117.5e'0' 6", r2 = 0.98; days 42+, y = 53.160024", r2 = 0.98. cDays 0-
42: y = 114.461” X, r2 = 0.98; days 42+, y = 57.16109", r2 = 0.95.

49

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50

hard to judge whether moth catch was first recorded in those plots around harvest time
because the dispensers had lost their efficacy or because the plot was being harvested. It
seemed that once the fruits were harvested, movement of moths increased. Females may
become more mobile following harvest, in search of suitable egg-laying sites. Male
moths, in search of females, may have become more mobile as a result. An exception to
the occurrence of higher catches in conjunction with harvest was in the EWD H—OFM
plot on farm one, which was harvested on 23 August. Only ca. 12% of the dispensers
were replaced with new dispensers in that plot on 30 May. Trap shutdown in that plot
was not maintained until harvest; catches increased on 15 August, 118 d following
application (Figure 2.8). In this case, it appears that moths were caught in this plot
because the release rate of pheromone from the EWD II-OFM dispensers fell below
threshold at this time.

The longevity of EWD II-OFM, based on maintenance of trap shutdown, was ca.
33 (1 longer than the estimate based on maintenance of release rates above the 5 mg/ha/h
threshold (Rothschild 1975). The longevity of Isomate-M 100, according to Shin-Etsu
Chemical Co., Ltd. (Tokyo, Japan), is 90 d. This is 19 d less than the longevity obtained
in southwest Michigan in 2002. From these data, it can be concluded that 3 ml dollops of
EWD II-OFM remained effective for a period equivalent to Isomate-M 100 and as much
as twice as long as Confuse-OFM. The first application of Confuse-OFM lasted an
unexpectedly long time, 77 d. The beyond-expected longevity of the first applications of
all pheromone dispensers in Michigan was very likely a result of the low temperatures in
April and May, when the dispensers certainly released less pheromone than during the

hotter summer temperatures (Fan and Singh 1989, Atterholt 1996).

51

A zero-order release rate has long been recognized as a desirable quality for a
pheromone dispenser. Monolithic dispensers, such as EWDs, have first order release
kinetics (Fan and Singh 1989). This property of EWDs might actually be desirable. Early
in the season, when temperatures are lower (Rothschild 1975) and foliage is not fully
flushed for pheromone absorption (Sauer and Karg 1998), the higher initial release rates
of EWD formulations may provide adequate pheromone in the air for mating disruption.
Further reducing the surface area and increasing the thickness of the dispensers could
also bring the dispenser release rates closer to zero order. Recent studies (de Lame,
unpublished data) have demonstrated that 5 to 10 g dollops, with reduced surface area to
volume ratios, have less concave release rates than 1 and 3 g dollops.

Application. The Spatula applicator enabled the application of EWD II-OFM in
almost half the time as Isomate-M 100 ropes or Confuse-OFM deposits (Figure 2.9) with
virtually no waste of the formulation. The Spatula was the simplest and cheapest
applicator developed for EWD H-OFM.

The time required to apply a disruption formulation to one hectare of a mixed
peach and plum orchard ranged from 41 min to 1 h 15 min (Figure 2.9). Applying EWD
II-OFM with the Spatula applicator was faster than with either the S.P.L.A.T. or Swipe
applicators. In contrast, application time was greatest for Confuse-OFM, Isomate-M 100,
and EWD II-OFM with the S.P.L.A.T. applicator (F = 32.86, df = 4, P < 0.0001).
Application time for hand—applied formulations is minimized when the person applying
the dispensers does not stop walking to prepare material for application to the next tree;

only the Spatula applicator could be quickly loaded while walking between two trees.

52

 

100
I EWDIl-OFM

E] Confuse-OFM
I lsomate-M 100

  

bc

 

60—

40*

20*

 

 

 

 

 

 

Application Time Per Hectare (min)

S.P.L.A.T. Swipe Spatula Paint- Ropes
Marking Gun

Figure 2.9: Comparison of time necessary to apply one hectare of: 1) EWD H-OFM using
the S.P.L.A.T., Swipe, or Spatula applicators, 2) Confuse-OFM using a paint-marking
gun, and 3) Isomate—M 100 ropes. Bars labeled with the same letter are not significantly
different (Tukey, P < 0.05). IrDue to mechanical problems, application with the
S.P.L.A.T. gun was only replicated three times.

53

A further advantage of the Spatula versus the other applicators tested was its
simplicity; it never broke down. During field trials, all other applicators employed
experienced some equipment failures (these were not included in the data of Figure 2.9).
Also, there was virtually no waste of EWD II-OFM when it was applied with the Spatula.
Waste of product was a serious issue with Confuse-OFM squirted onto trees. Deposits
had to be carefully applied; nevertheless, runoff of Confuse-OFM from the bark still
occurred. Although product waste was reduced, some EWD II-OFM dripped from the
Swipe guns in between applications and EWD H-OFM was wasted when dollops shot
from the S.P.L.A.T. applicator missed the tree. Peach tree limbs are relatively narrow, so
that a shooting applicator was not well-adapted to this crop. The Spatula applicator could
easily be modified to measure volume or to apply EWDs higher in tree canopies, which is
required for some other pests, most notably, Cydia pomonella, the codling moth
(Weissling and Knight 1995).

Cost. Paraffin wax emulsions are inexpensive to produce. The bulk of the
dispenser consists of wax and water. Paraffin wax is a byproduct of petroleum refining. It
is produced in large quantities annually, and is cheap to purchase (Bennett 1975). The
most expensive ingredient used to make the blank emulsion is vitamin E, which can be
replaced by BHT, a cheaper antioxidant (C.A. Atterholt, personal communication). The
wax emulsion-making process does not require the use of specialized equipment and
could be scaled up to make large batches of emulsion rapidly and with minimum labor.

Thus, the cost of commercially-producing a wax pheromone emulsion versus other hand

applied release systems may be considerably lower.

54

The Spatula applicator is cheap and virtually eliminates wastage of EWD II-OFM
during application, further reducing the cost of this system to a grower. EWD II-OFM
may be the cheapest mating disruption pheromone dispenser developed yet.

Potential for commercializing EWD technology. EWD technology offers some
practical advantages over other technologies for dispensing large quantities of pheromone
into cropping systems. EWDs are comprised entirely of biodegradable ingredients,
including the carpet adhesive (Roberts Consolidated Industries, personal
communication). This contrasts with commercially-available disruption formulations.
The most widely used dispensing system, polyethylene ropes, do not biodegrade and are
seldom removed from the crop at the end of the season. In this sense they can be
considered an environmental contaminant.

EWD formulations may provide good protection for fragile pheromones. Since
the active ingredient is present as only a small proportion of the overall dispenser and the
dispenser is opaque, pheromones that polymerize, such as the pheromone of Cydia
pomonella (Millar 1995) or degrade in other ways, when exposed to sunlight, may be
better protected in this dispenser than in other formulations.

Formulations with greater versatility can be aimed at a larger market. In addition
to extensive efforts with G. molesta pheromone, paraffin wax emulsions have been
tested, though not extensively, to release the pheromones of other pests, specifically:
Quadraspidiosus perniciosus (San Jose scale) and Anarsia lineatella (peach twig borer)
(Rice et al. 1997), C. pomonella (codling moth) (Sanders 1999), and Platynota ideausalis
(tufted apple budmoth) (Meissner et al. 2000). Control using the emulsion was deemed

successful only for Q. perniciosus and P. ideausalis. The variable results using wax

55

emulsions may be a result of the physico-chemical properties of the pheromones. Small
changes in the molecular weight of molecules have a large effect on the rate at which
these will diffuse through a matrix (Baker 1986, Fan and Singh 1989). The molecular
weights of Q. perniciosus, G. molesta, and P. ideausalis pheromones are 224, 226, and
254 g/mol. In comparison, the molecular weights of C. pomonella and A. lineatella
pheromones are 181 and 198 g/mol. The wax emulsions used to release these compounds
were not significantly altered to fit their specific chemical properties, thus their lack of
success is not surprising. Modifying the EWD formulation to fit particular pheromones
can favorably alter the release rates of those pheromones from the dispensers. In another
study, an EWD made with microcrystalline wax (also a byproduct of petroleum refining)
released G. molesta pheromone at half the rate as the same formulation made with
paraffin wax.

Perhaps formulations can be designed that are adapted to release pheromones of a
particular size. Large batches of blank emulsion suited to several chemically—similar
pheromones could be made to which the pheromone of interest could later be added. This
strategy was used by Gowan Co. and was successful in other emulsion studies conducted
in this laboratory. Manufacturing the formulation in this way could reduce costs by
decreasing the batches of emulsion that are made and perhaps reducing the equipment
necessary for manufacture or facilitating cleaning of this equipment prior to making a
different pheromone formulation. This process could also reduce the likelihood of
contamination between formulations.

Increasing the variety of crops on which a dispenser can be applied also increases

the market for that dispenser (Weatherston 1990). To date, EWD formulations have been

56

used mostly in tree fruit crops. This dispenser may be suitable for application in other
fruit mom and some field and vegetable crops, provided there is a surface to which to
apply the dispenser. This surface could be provided by a large plant, or another structure,
such as a stake in a tomato field. A viable applicator could be developed based on the
S.P.L.A.T. applicator (Appendix B) and used to apply these dispensers in forest systems.
However, a simple, low-cost EWD formulation may find sufficient interested buyers in
the world tree fruit market alone.

Industry representatives expressed concerns about the need to add an adhesive to
the emulsion, especially since it was added just prior to application in the field.
Modifying the EWD formulation might eliminate the need to add adhesive to the
emulsion; in another study, an EWD formulation made with microcrystalline wax and to
which no adhesive was added was as sticky as EWD H-OFM with adhesive. It appears
that the type and amount of wax used to make an EWD formulation has a large impact on
the properties of that formulation. The large variety of waxes available commercially
(Bennett 1975) could be used to make an innumerable number of EWD formulations.

EWD II—OFM, at this time, is fit for commercialization. Commercialization of this
formulation should lead to further exploration of the still largely undiscovered potential

of EWD formulations for dispensing pheromones for mating disruption of various pests.

57

Chapter 3

Ability of Grapholita molesta to locate pheromone traps, as influenced by vertical
positioning of traps and hand-applied pheromone dispensers

Introduction

Understanding the biology and behavior of a pest insect is essential to optimizing
a mating disruption-based control program (Suckling and Karg 2000). Several studies of
field-crop (e.g. Kaae and Shorey 1973), orchard (e.g. Sharma et al. 1971, Suckling and
Shaw 1992, Barrett 1995) and forest (Sower and Daterman 1977) pests have identified
the locations in crop canopies where mating or catches of males in traps is most likely to
occur in the presence or absence of a pheromone treatment. Obtaining this type of
information is an important step in the development of effective pheromone-based
monitoring and control programs.

Numerous investigations have addressed these aspects of male behavior for the
codling moth, Cydia pomonella, the most important pest of apples worldwide (Hamilton
and Steiner 1939, Riedl et al. 1979, Charrnillot 1980, Howell 1981, McNally and Barnes
1981, Thwaite and Madsen 1983, Ahmad and Al-Gharbawi 1986, Howell et al. 1990).
The greatest captures of C. pomonella males have usually been recorded in traps placed
high in tree canopies, suggesting that males may be more numerous or active there. Based
on this information, the optimal height for placing hand-applied pheromone for control of
C. pomonella was considered to be the upper tree canopy. However, it was well after
mating disruption had become an established control for this pest that researchers actually
demonstrated that activity of male and virgin female C. pomonella was greatest in the top
region of apple and pear orchards and that vertical positioning of dispensers influenced

the level of mating disruption. Specifically, Weissling and Knight (1995) recorded a trend

58

of increased mating of females tethered higher in the canopy when pheromone dispensers
were placed in the bottom of the tree canopies. This pattern was less evident when
dispensers were applied high in the canopy. These authors concluded that dispensers for
mating disruption of C. pomonella should be positioned in the upper portion of tree
canopies.

Investigations of whether Grapholita molesta (Busck) (Lepidoptera: Tortricidae),
oriental fruit moth, males were more likely to orient to pheromone sources placed at
particular heights in tree canopies yielded mixed outcomes (Beroza et al. 1973, Gentry et
al. 1974, Rothschild and Minks 1974, Rothschild and Minks 1977, Atterholt 1996). In
general, little evidence has been gathered that G. molesta are more numerous or active in
a particular part of the tree canopy. Rothschild (1975) conducted an unreplicated trial
investigating the effect of placing pheromone dispensers high or low in peach tree
canopies on the ability of G. molesta males to orient to traps. He found no differences in
moth captures when dispensers were hung in tree crowns or at mid-canopy. Despite his
findings, the industry-recommended application height for hand-applied dispensers for
control of G. molesta is in the upper part of tree canopies, which can increase the
difficulty and cost of dispenser application.

The current study reinvestigated the possible effects of trap and dispenser
positions in tree canopies on G. molesta trapping and mating disruption in two peach
orchards and two apple orchards (Table 3.1). Although traditionally classified as a pest of
stone fruits, this tortricid moth has recently become a major pest of apples (Chapman and

Lienk 1971). The effect of canopy position on the behavior of G. molesta in apples has

59

 

 

 

 

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60

not been examined. Experiments in tall apple trees permitted a stronger test of the effects
of trap and dispenser heights than was possible with peach trees in Michigan. These
studies were carried out for entire seasons to account for the possible effect of the time
during which the trial was conducted on the results obtained.

Materials and Methods

Experimental design. This study was conducted over two seasons utilizing a
total of four orchards. In 2001, experiments were set up in an abandoned peach orchard in
Fennville, MI. In 2002, a follow-up experiment was conducted in a commercial peach
orchard and two abandoned apple orchards in Coloma, MI (Table 3.1). The experimental
design was completely randomized and tested two factors: pheromone treatment and trap
height. The three levels of the pheromone treatment were: no pheromone, pheromone
dispensers applied in the bottom third of the canopy (low dispensers), and pheromone
dispensers applied in the top third of the canopy (high dispensers). The two levels of the
trap height were: traps placed in the bottom third of the canopy (low traps) and traps
placed in the top third of the canopy (high traps).

Four plots in the peach blocks and three plots in the apple blocks were treated
with each of the three pheromone treatments. A single delta trap baited with a pheromone
lure (Scenturion, Inc., Clinton, WA) was placed in each of two trees, located at least 11 m
apart, in the center of each plot. One pheromone trap was placed high and the other was
placed low in the canopy. The traps were checked every 3-4 d, at which time the high
trap was moved to the low position in the same tree and the low trap to the high position

in the same tree. Traps were placed at least 1 m from a pheromone dispenser. Pheromone

lures were replaced once per G. molesta generation (ca. 6-8 wk).

61

Also, in 2002, 4-5 virgin female traps were placed in all plots 3-4 times during the
peak flight of each G. molesta generation. These traps were situated at 1.2—1.8 m heights
and placed only on non-border trees that were not adjacent to a tree containing a
pheromone trap. Virgin females were obtained from a colony of G. molesta originally
collected as larvae in an infested apple orchard in Fennville, MI, in July 2001. They were
reared on a pinto bean-based diet (Shorey and Hale 1965) at 24 °C and 16:8 L:D. Pupae
were sexed (George 1965) and females were reared individually, under natural light
conditions, in 118 ml plastic cups and provided with a 2 cm cotton wick soaked in 5%
sucrose solution. Voucher specimens of adults and larvae are deposited at the Michigan
State University Entomology Museum (Appendix C).

Pheromone formulation. All experiments were conducted using a commercial
emulsified wax pheromone formulation, Confuse-OFM (Gowan Co.,Yuma, AZ).
Confuse-OFM is a matrix-type or monolithic dispenser (Fan and Singh 1989) with first
order release kinetics and efficacy equivalent to that of Isomate-M 100 (Shin-Etsu
Chemical Co., Ltd., Tokyo, Japan), the commercial standard (Chapter 2). Confuse-OFM
deposits were applied using a one liter plastic squirt bottle in 2001 and a forestry paint-
marking gun (Idico Products Co., New York, NY) in 2002. One deposit consisted of ca.
2.7 ml (2.6 g) Confuse-OFM in 2001 and ca. 3 ml (2.8 g) Confuse-OFM in 2002.
Applications were made once at the beginning of each G. molesta generation at 74 g
AI/ha or ca. 580 deposits per hectare per application in 2001 and 520 deposits per hectare
per application in 2002. Three applications were made for experiments conducted in the

2001 peach, wide apple, and narrow apple blocks. Only two applications were made in

the 2002 commercial peach block because the fruits were harvested very early in the
flight of the third generation of G. molesta.

Shoot growth measurements. These measurements were taken in early
September 2002. Samples were taken on transects from northeast to southwest through
each of the 2002 peach, wide apple, and narrow apple blocks. Ten shoots in the top half
of the canopy and 10 shoots in the bottom half of the canopy were cut in 10 randomly-
chosen trees along the transect. Shoot growth was measured as the distance from the first
growth scar for that year to the tip of the shoot. Current year’s growth was discerned
from previous years’ growth by the appearance and texture of the woody tissue.

Statistical analyses. Pheromone trap data from the 2001 peach block and the
narrow and wide apple blocks were transformed, log (x+1), then analyzed with a two-
factor (trap height and pheromone treatment) ANOVA. Means were separated using
Tukey’s test (SAS version 8.2, SAS Institute 1999). When the interaction between the
two factors was significant, comparisons were made of all cell means (LSMEANS
statement, adjust = Tukey, SAS version 8.2, SAS Institute 1999).

Pheromone trap data from the 2002 peach block and virgin female trap data from
all blocks could not be normalized and were analyzed using the Kruskal-Wallis test.
Means were separated using the Nemenyi test (Zar 1999). The Nemenyi test was more
conservative than the Kruskal-Wallis test; in some instances, although the Kruskal-Wallis
test identified significant differences between treatments at )8 < 0.05, the )8 value had to
be raised to 0.1 to find differences between means. The pheromone trap data from the

2002 peach block was examined graphically for the presence of a significant interaction

between the two factors (Zar 1999).

63

Shoot growth data were analyzed by ANOVA without transformation. Multiple
comparisons of all cell means for the shoot growth data were conducted as above (SAS
version 8.2, SAS Institute 1999).

Results

Peach blocks. Pheromone traps. In both years, there were no significant
differences in the numbers of moths caught in the low dispensers and high dispensers
treatments (Figures 3.1 and 3.2). However, significantly more moths were caught in the
traps placed in the no pheromone plots versus the pheromone-treated plots. The
interaction between trap height and treatment was not significant in 2001 (Generation 1:
F = 0.43, df = 2, P = 0.66; Generation 2: F = 1.18, df = 2, P = 0.33; Generation 3: F =
0.49, df = 2, P = 0.62) nor in 2002. In both 2001 and 2002, for all generations, there were
no significant differences in the numbers of moths caught in the high traps versus the low
traps (2001: Generation 1: F = 2.74, df = l, P = 0.11; Generation 2: F = 0.10, df = 1, P =
0.75; Generation 3: F = 0.56, df = 1, P = 0.46; 2002: Generation 1: x2 = 0.46, df = 1, P =
0.50; Generation 2: x2 = 0.95, df = 1, P = 0.33). However, there were significant
differences in the numbers of moths caught in the different treatments (2001: Generation
1: F = 11.20, df = 2, P = 0.0007; Generation 2: F = 10.52, df = 2, P = 0.0009; Generation
3: F = 11.56, df = 2, P = 0.0006; 2002: Generation 1: 12 = 19.01, df = 2, P < 0.0001;
Generation 2: x2 = 13.89, df = 2, P = 0.001). While the average percent pheromone trap
shutdown in the pheromone-treated plots in 2002 was high, 98.6 :t 0.4 percent (mean 1-

S.E.), the average percent pheromone trap shutdown in the pheromone—treated plots in

2001 was fairly low, 85.4 i 2.4 percent (mean t SE.)

64

 

150

 

 

 

 

 

 

 

 

 

 

 

I No pheromone High Traps
I Low dispensers
100 . E1 High dispensers
50 _
a . , ..
[.—
- 0 _-
a)
D.
8 150
g Low Traps
100 4
a a
50 - b
b b b b b
. O - L I I .
Generation 1 Generation 2 Generation 3

Figure 3.1: 2001 peach block. Numbers of Grapholita molesta caught in pheromone traps
placed in the top third of the canopy (high traps) and in the bottom third of the canopy
(low traps) in plots treated with no pheromone, pheromone dispensers placed in the
bottom third of the canopy (low dispensers) and pheromone dispensers placed in the top
third of the canopy (high dispensers). Bars labeled with the same letter within a
generation are not significantly different (Tukey, P < 0.05).

65

 

150
I No pheromone High Traps
Low dispensers
100 . [:1 High dispensers

 

 

 

 

 

 

 

 

 

 

— a
8 50
:- _-b b b .
a) T r
o. o
(I)
:3 150 L T
2 ow raps
100-
a
50‘ a
J . I - . .
O“ I
Generation 1 Generation 2

Figure 3.2: 2002 peach block. Numbers of Grapholita molesta caught in pheromone traps
placed in the tOp third of the canopy (high traps) and in the bottom third of the canopy
(low traps) in plots treated with no pheromone, pheromone dispensers placed in the
bottom third of the canopy (low dispensers) and pheromone dispensers placed in the top
third of the canopy (high dispensers). Bars labeled with the same letter within a
generation are not significantly different (Nemenyi, x2 < 0.05).

66

Virgin female traps. The results using virgin female traps in 2002 (Figure 3.3)
corroborated those obtained using pheromone traps (Figure 3.2). The numbers of moths
caught in the different treatments were significantly different (Generation 1: x2 = 7.16, df
= 2, P = 0.03; Generation 2: x2 = 5.8, (if = 2, P = 0.05). Significantly more moths (x2 <
0.1) were caught in the traps placed in the no pheromone treatment versus pheromone-
treated plots. In both generations, the numbers of moths caught in the low dispensers and
high dispensers treatments were not significantly different.

Description of indigenous G. molesta populations. The 2002 block was
commercially—farmed and received three permethrin sprays; nonetheless, a sizable moth
population was present. The moth population, as estimated by the number of moths
caught per trap in pheromone traps placed in no pheromone plots during the first and
second generations, was similar in both peach blocks (Figures 3.1 and 3.2). The 2001
block, although abandoned, bore small, but abundant fruits during generation three.
Approximately twice as many moths were caught in generation three than in the previous
two generations in that block (Figure 3.1). Moth captures in the 2001 block fell close to
zero for a few weeks in between each of the generations. By contrast, the number of
moths caught per trap at each sampling date in the no pheromone plots in the 2002 peach
block, although it dipped in between generations, was above zero on most sampling
dates.

Apple blocks. Wide apple block pheromone traps. The interaction between trap
height and treatment was significant for generation one (Generation 1: F = 15.41, df = 2,
P = 0.0005; Generation 2: F = 0.66, df = 2, P = 0.54; Generation 3: F = 1.62, df = 2, P =

0.24). During generations two and three, there were no significant differences in the

67

 

8.0
I No pheromone

B Low dispensers
C] High dispensers

9’
O
l

Males Per Trap
.5
O

 

 

 

 

2.0 ~ a
a
_— b b - b b
0.0 - 1
Generation 1 Generation 2

Figure 3.3: 2002 peach block. Numbers of Grapholita molesta caught in virgin female
traps placed at 1.2-1.8 m in tree canopies in plots treated with no pheromone, pheromone
dispensers placed in the bottom third of the canopy (low dispensers) and pheromone
dispensers placed in the top third of the canopy (high dispensers) for three days. Bars
labeled with the same letter within a generation are not significantly different (Nemenyi,

x2<o.1).

68

numbers of moths caught in low traps versus high traps (Generation 2: F = 0.59, df = 1, P
= 0.46; Generation 3: F = 1.70, df = 1, P = 0.22) (Figure 3.4); significantly more moths
were caught in the no pheromone treatment than in the pheromone-treated plots, and
there were no significant differences in the numbers of moths trapped in the high
dispensers versus the low dispensers treatments (Generation 2: F = 8.39, df = 2, P =
0.005; Generation 3: F = 45.67, df = 2, P < 0.0001). Multiple comparisons of all cell
means for generation one data revealed no significant effect of trap height and no
significant effect of dispenser height, but a significant effect of pheromone treatment,
except for the comparisons next mentioned. Significantly more moths were caught in the
high traps placed in the low dispensers treatment versus the high traps placed in the high
dispensers treatment (P = 0.004). Significantly more moths were also caught in the high
traps placed in the low dispensers treatment compared to the low traps placed in this
treatment (P = 0.04) and in the low traps placed in the high dispensers treatment than in
the high traps placed in this treatment (P = 0.01). The average percent pheromone trap
shutdown was 96.8 i 0.9 (mean t SE).

Narrow apple block pheromone traps. The interaction between trap height and
treatment was significant for generations one and three (Generation 1: F = 4.02, df = 2, P
= 0.05; Generation 2: F = 3.11, df = 2, P = 0.08; Generation 3: F = 4.49, df = 2, P = 0.03).
In generation two, significantly more moths were caught in low traps than in high traps (F
= 8.70, df = 1, P = 0.01) (Figure 3.5), significantly more moths were also caught in the no
pheromone treatment than in the pheromone-treated plots. However, there were no
significant differences in the numbers of moths trapped when dispensers were placed

high or low (F = 59.50, df = 2, P < 0.0001). Multiple comparisons between cell means for

69

 

500

 

 

 

 

 

 

 

 

 

 

 

 

 

High Traps I No pheromone
400 ‘ ILow dispensers
300 — DHigh dispensers
200 - a
o.
S100‘llvci abb abb
g 0- T -. , -
0.
3 500
a Low Traps
2 400 ‘
300 -
200 ~
100 - a a
be b
_ Cd , -b b p - b
Generation 1 Generation 2 Generation 3

Figure 3.4: Wide apple block. Numbers of Grapholita molesta caught in pheromone traps
placed in the top third of the canopy (high traps) and in the bottom third of the canopy
(low traps) in plots treated with no pheromone, pheromone dispensers placed in the
bottom third of the canopy (low dispensers) and pheromone dispensers placed in the top
third of the canopy (high dispensers). Bars labeled with the same letter within a
generation are not significantly different (Tukey, P < 0.05).

70

 

 

 

 

 

   

 

 

 

 

 

 

 

 

500
High Traps I No pheromone
400 _ I Low dispensers
300 _ I] High dispensers
200 ~ a
5% 100
g b b
a 0 ~ ' ‘
(l
8 500
a LOW Traps
2 400 ‘
300 -
200 — ab
1m q—. a
0 _ d Cd f - b b I
Generation 1 Generation 2a Generation 3

Figure 3.5: Narrow apple block. Numbers of Grapholita molesta caught in pheromone
traps placed in the top third of the canopy (high traps) and in the bottom third of the
canopy (low traps) in plots treated with no pheromone, pheromone dispensers placed in
the bottom third of the canopy (low dispensers) and pheromone dispensers placed in the
top third of the canopy (high dispensers). Bars labeled with the same letter within a
generation are not significantly different (Tukey, P < 0.05). 1'‘Significantly more moths
were caught in the high traps versus the low traps.

71

generations one and three revealed no significant effect of trap height and no significant
effect of dispenser height, but a significant effect of the pheromone treatment, except that
significantly more moths were caught in the high traps versus the low traps placed in the
low dispensers treatment in both generations one and three. The average percent trap
shutdown was 97.0 i 0.8 (mean t SE).

Virgin female traps. The patterns of moth captures in virgin female traps (Figure
3.6) corroborated those from the pheromone traps (Figures 3.4 and 3.5). In both blocks,
in all generations, there were significant differences in the numbers of moths caught in
the three treatments (Wide: Generation 1: x2 = 6.00, df = 2, P = 0.05; Generation 2: x2 =
6.82, df = 2, P = 0.03; Generation 3: x2 = 6.00, df = 2, P = 0.05; Narrow: Generation 1: 12
= 6.00, df = 2, P = 0.05; Generation 2: x2 = 6.82, df = 2, P = 0.03; Generation 3: x2 =
7.62, df = 2, P = 0.02). In all blocks and all generations, significantly more moths were
caught in the no pheromone treatment than in the pheromone-treated plots and there were
no significant differences in the numbers of moths caught in virgin female traps placed in
the low dispensers versus the high dispensers plots (Generation 2: x2 < 0.05, Generations
one and three, )8 < 0.1).

Description of indigenous G. molesta populations. The apple blocks initially
harbored large populations of G. molesta. The moth population, as estimated by the
number of moths caught per trap in pheromone traps placed in no pheromone plots during
each generation, was approximately one and a half times larger in the narrow block than
in the wide block (Figures 3.4 and 3.5). Moth captures in both blocks rarely fell to zero.

Many moths were caught during the first generation, but the numbers of moths caught

a. Wide apple block.

 

8.0
I No pheromone

I Low dispensers
5,0 - DHigh dispensers

4.0 ~

N
o

 

 

a
a
a
b
J” lbs!”

6. Narrow apple block.

 

 

.0
o

 

Males Per Trap
90
O

 

 

 

a
6.0 ~
I
I
4.0 n
a

2.0 - a

0.0 — , e _"—‘ w ,

Generation 1 Generation 2 Generation 3

Figure 3.6: Numbers of Grapholita molesta caught in virgin female traps placed at 1.2-
1.8 m in tree canopies in the wide and narrow apple blocks in plots treated with no
pheromone, pheromone dispensers placed in the bottom third of the canopy (low
dispensers) and pheromone dispensers placed in the top third of the canopy (high
dispensers) for three days. Bars labeled with the same letter within a generation are not
significantly different (Nemenyi: Generation 2: x2 < 0.05, Generations l and 3: x2 < 0.1).

 

was approximately halved in the subsequent generations. Both blocks bore very few
fruits, so that the moths present were reliant on fresh shoots for feeding.

Shoot growth in the 2002 peach and apple blocks. The period of shoot growth
seemed to be shortest in the wide apple block, followed by the narrow apple block. By
contrast, shoot growth in the 2002 peach block continued well into the season. The
interaction between block and shoot growth was not significant (F = 1.07, df = 2, P =
0.35). Shoot growth in the three blocks was significantly different; shoot growth was
greatest in the 2002 peach block, followed by the narrow apple block, then the wide apple
block (F = 286.73, df = 2, P < 0.0001). Shoot growth was significantly greater in the top
third of the canopy than in the bottom third of the canopy (F = 22.78, df =‘ 1, P < 0.0001)
(Figure 3.7). Multiple comparisons of cell means showed this difference to be most
significant for the narrow apple block (2002 peach: P = 0.44; wide apple: P = 0.13;
narrow apple: P = 0.004).

Discussion

Vertical placement of pheromone traps. In plots not treated with pheromone,
there were few differences in the numbers of male G. molesta moths caught in
pheromone traps placed in the top third or the bottom third of the tree canopies (Figures
3.1, 3.2, 3.4, 3.5). This was consistent for all orchards and through all G. molesta
generations, except for the second generation flight in the narrow apple block. This lack
of an effect of trap location on moth captures is incongruent with the results of some
earlier investigations (Beroza et al. 1973, Gentry et a1. 1974, Rothschild and Minks 1974,
Rothschild and Minks 1977, Atterholt 1996). Several factors may have been responsible

for the discrepancies between our results and those of earlier findings. The placement of

74

 

 

 

 

 

 

50.0
I Top half of canopy

E 400 ~ Cl Bottom half of canopy
3
5 30.0 -
E
9
<5 20.0 ~
*5
2
0) 10.0 — i

 

 

 

 

 

2002 Peach I Wide Apple Narrow Apple

Figure 3.7: Seasonal shoot growth measured in the top half of the canopies and the
bottom half of the canopies of trees in the 2002 peach, wide apple, and narrow apple
blocks. Shoot growth was significantly different in all three blocks and greatest in the top

half of the canopy in all three blocks (Tukey, P < 0.05).

75

traps within the canopy, rather than outside the canopy, can have a substantial influence
on the numbers of moths captured. Riedl et al. (1979) and Howell et al. (1990) found that
significantly fewer C. pomonella moths were captured when traps were placed outside,
rather than within the canopy. Although canopy effects on moth catches have not been
reported for G. molesta, traps in the current study were consistently placed within the
outer third of the canopy. In contrast, in Gentry et al.’s (1974) study of the effect of trap
location on G. molesta captures, pheromone traps were placed in between trees. Since
this study was a continuation of the efforts of Beroza et al. (1973), it is likely that in the
original experiments, traps also were placed in between peach trees, rather than within
tree canopies.

Placing traps above or below the tree canopy can greatly reduce moth captures
(Riedl et al. 1979, Howell et al. 1990). In all previous G. molesta trapping studies, the
authors did not report the heights of the bases of the canopies of the trees in which they
placed their traps. Furthermore, only Rothschild and Minks (1974, 1977) and Atterholt
(1996) indicated the heights of the tops of the tree canopies. Although the differences do
not appear to be very large, Rothschild and Minks (1974) observed significantly more
moths orienting to both pheromone and virgin female traps placed at 2 m compared to
those placed at 4 m (as reported in Rothschild 1975) and very few G. molesta were
observed orienting to traps placed at 1 m. They reported an average tree height of
approximately 3 m, so that traps placed at 4 m were apparently outside of the canopy. It
is also likely that traps placed at 1 m were below the canopy, which could explain the
reduced number of moths caught at this height. The results of Rothschild and Mink’s

1977 trials were similar, with the greatest numbers of moths caught in traps placed at 2

76

m. Tree heights were 3-4, 2, and 4 m in three trials, respectively. Their experimental
design was not described in sufficient detail to judge the robustness of their results; e. g. it
was not clear whether traps were placed within, or outside of tree canopies or whether
multiple traps were placed in a single tree. Atterholt (1996) placed her traps in 7.6 m tall
almond trees. She caught equivalent numbers of moths in traps placed at 3.7 m and 5.5 m
and comparatively, very few moths at 1.8 m. While the traps she placed at 1.8 m were
within the tree canopy (Atterholt, personal communication), two factors may have
influenced the numbers of moths caught in the trap placed at 1.8 m. Placement of traps in
potentially competing positions of 1.8, 3.7, and 5.5 m in the same tree may have reduced
the numbers of moths caught in the 1.8 m trap. This competitive interaction was noted by
Thwaite and Madsen (1983) for C. pomonella. Fewer moths could have also been caught
at the low height due to the sparseness of the canopy at 1.8 m. Almond tree canopies are
not as full as apple or peach tree canopies. Although the study in almonds provides some
evidence that trap height within the canopy may influence G. molesta moth catches,
differences in the numbers of moths caught in most regions of the almond tree canopies
were not significant (Atterholt 1996).

Reduced shoot growth in the bottom half of the canopy (Figure 3.7) is one
possible explanation for the lower number of male moths caught in pheromone traps
placed low versus high in the canopies of trees during generation two in the narrow apple
block (Figure 3.5). Males are likely to co-inhabit areas of tree canopies where females are
present; females may spend more time in areas with more ovipositional sites. Differences
in the numbers of moths caught in the traps placed low and high in the canopy in the

narrow apple block first became apparent half way through the first generation, then

77

disappeared before the third G. molesta generation. This period of increased captures
high in the canopy coincided with the period of greatest shoot growth in this block. Shoot
growth accelerated when the weather warmed up, in early June. By early August,
following increasingly hot and dry weather, shoot growth terminated.

In conclusion, based on all data published to date, in most orchard environments,
male G. molesta are equally likely to be found at all heights in tree canopies in orchards
not treated with pheromone. This was previously observed for another orchard pest;
mating of tethered female Epiphyas postvittana (light brown apple moth) was equivalent
at 1.5 m and 3 m in an orchard never treated with pheromones (Suckling and Shaw 1992).
These authors did not report the trap locations in reference to the tree canopies, nor the
tree heights.

Vertical placement of pheromone dispensers. In all of the current experiments,
at least a few G. molesta were captured in pheromone traps in most of the pheromone-
treated plots (Figures 3.1, 3.2, 3.4, 3.5). The moth population in the 2002 peach block
was higher than those typically encountered in commercial peach orchards in Michigan
(Figure 3.2). Moth populations in the narrow and wide apple blocks were even greater
than those recorded in the 2002 peach block (Figures 3.4 and 3.5). Despite high G.
molesta pressure, the average percent pheromone trap shutdown values in the
pheromone-treated plots in the 2002 peach, wide apple, and narrow apple blocks were all
close to one hundred percent.

Interestingly, although the moth population in the 2001 peach block was no larger
than the moth populations in the other three blocks, only 85% pheromone trap shutdown

was achieved in that block. At this level of inhibition of moth captures, this pheromone

78

treatment would not protect a fruit crop from G. molesta injury. The low level of trap
shutdown may have resulted from reduced foliage in the 2001 peach block. This block
had not been cultivated for several years. The trees were suffering from drought and
pathogen infections, so that tree canopies were sparse. Foliage can serve an important
function in mating disruption by absorbing pheromone from the air and then releasing it,
thus enhancing mating disruption by maintaining pheromone in the crop (Sauer and Karg
1998).

In the peach blocks and the wide apple block, mating disruption, as measured by
the ability of male G. molesta to orient to pheromone traps placed in the top third or the
bottom third of tree canopies and virgin female traps placed at 1.2—1.8 m was equivalent
whether dispensers were placed in the bottom third or in the top third of tree canopies.
The only exception to a lack of a height effect of dispenser treatment was during the first
generation in the wide apple block, when more moths were caught in the high traps
placed in the low dispensers treatment and in the low traps placed in the high dispensers
treatment. A low release rate of pheromone from the dispensers during the low spring-
time temperatures (Fan and Singh 1989, Atterholt 1996) may have decreased the active
space of the dispensers at this time, so that pheromone traps placed above or below the
dispensers could more easily be detected by male G. molesta.

Rothschild (1975) conducted a study similar to the present one, where he attached
pheromone dispensers for G. molesta either at tree crowns (3.5 m) or in the centers of the
canopies (1.5 m) of 3-4 m tall peach trees in 0.4 ha plots. He used pheromone traps
placed at a single height (probably mid-canopy) to measure orientational disruption. This

trial was unreplicated and it seems likely that it was conducted over arelatively short

79

period of time. Nonetheless, Rothschild captured equivalent numbers of moths in plots
with dispensers placed at the crown or center of peach tree canopies. His results are
consistent with the present findings for the 2001 peach, 2002 peach, and wide apple
blocks.

In the narrow apple block, in all generations, equivalent numbers of moths were
caught in the virgin female traps placed in both pheromone treatments. However, while
equivalent numbers of moths were caught in low and high pheromone traps placed in the
high dispensers treatment, more moths were caught in the high traps than in the low traps
placed in the low dispensers treatment in generations one and three. A low concentration
of pheromone in the upper portion of the tree canopies may have caused this outcome. A
low concentration of pheromone high in the tree canopies in this plot may have resulted
from a combination of the large separation between the low and high positions in this
plot, the close proximity and dense canopies of the trees, and a larger pheromone active
space above, versus below the dispensers.

The 2.4 m separation between the high and low positions was greater than in any
other block (Table 3.1). Atterholt (1996) placed dispensers at 1.8 m and monitored moths
with pheromone traps placed at 1.8 m, 3.7 m, and 5.5 m in the canopy of the same
almond tree. Traps at 1.8 m and 3.7 m (1.9 m above the dispensers) caught equivalent,
low numbers of moths. However, traps placed at 5.5 m, 3.7 m higher than the pheromone
dispensers (an even greater separation between the trap and dispenser heights than in the
narrow apple block), caught significantly more moths, though less than the numbers of
moths caught at that height in no pheromone plots. These results are similar to those

obtained in the narrow apple block in this study.

80

The canopies of the apple trees in the narrow block were denser than in any other
plot. In addition, the trees were planted at close intervals, so that the canopies of adjacent
trees within a row touched and the canopies of trees in two adjacent rows were usually no
more than 1-1.5 m apart, and in some cases, touched one-another. McLaughlin and
Shorey (1972) dispensed hexalure from various heights in cotton fields. They found that
dispensers placed at the base of the cotton plants were more effective at disrupting male
orientation to pheromone traps in cotton plantings with open foliage, versus plantings
with dense foliage. Increased wind flow in open foliage may serve to disperse the
pheromone further in open canopies.

Interestingly, when the dispenser was placed in the top third of the canopy in the
narrow apple block, numbers of moths caught in the pheromone traps placed in the low
and high positions were equivalent. Karg et al. (1994), quantifying EAG responses to
apple leaves collected from positions up to 50 cm in the x, y, and z axes from two central
Shin—Etsu dispensers (Shin-Etsu Chemical Co., Ltd., Tokyo, Japan) dispensing the
pheromone of Epiphyas postvittana (a mixture of unsaturated 14 carbon acetates) found
that the concentration of pheromone was greatest below and south of the dispensers. They
attributed the higher concentration of pheromone on leaves located below the dispensers
to downdrafts through the canopy. The data gathered from these studies also suggest the
existence of a larger active space below the pheromone dispensers than above the
pheromone dispensers.

Suckling et al. (1999) developed a model to predict pheromone concentrations in
orchards. According to this model, dispensers placed low in tree canopies yielded higher

peak concentrations of pheromone in the canopies versus dispensers placed higher in tree

81

canopies. This effect was attributed to higher wind velocities at increased heights in the
canopies. The model also predicted that as dispensers were placed lower in the tree
canopies, the maximum height at which pheromone released from these dispensers would
be detected would also decrease. Dispensers have fixed active spaces; if the upper part of
a tree canopy extends beyond the active space of the dispenser, pheromone will not be
detected in that area. Thus, in taller trees, dispensers may need to be placed higher in the
tree canopies and perhaps greater amounts of pheromone need to be released from those
dispensers for effective mating disruption throughout the tree canopy.

These predictions were realized in the narrow apple block in this study. In this
case, it seems that the release rate from the dispensers was sufficient to significantly
reduce male orientation to pheromone sources when dispensers were placed at the high
position. The ability of more male G. molesta to locate pheromone traps when these are
placed further away from the dispensers seems to be related to the active space of the
dispenser, rather than a unique behavior of G. molesta.

Based on the studies discussed, it is estimated that in trees up to 4.0 m tall, which
includes most commercial peach and apple trees in Michigan, a dispenser placed at 1.5-
2.0 m will adequately disrupt mating of G. molesta throughout the tree canopy. A more
conservative approach may be warranted in taller trees. In trees up to 5.5 m tall, the
dispenser should be moved to approximately 2.0 m from the top of the tree canopy. In
trees taller than 5.5 m, dispensers may need to be placed at two heights, in the top and
bottom thirds of the canopies, for adequate mating disruption of G. molesta. Dispensers
can easily be placed at a 1.5-2.0 m height without any special equipment, thus reducing

application costs for G. molesta dispensers in many commercial situations. Reducing

82

application height may also decrease the amount of pheromone carried out of the
orchards by winds, increase the peak concentration of pheromone in the canopy
(Suckling et al. 1999), and perhaps increase the longevity of the dispensers in the

orchard, thereby further reducing the cost of mating disruption for this pest.

83

Chapter 4
Impact of varying density of pheromone point sources on the success of mating
disruption of Grapholita molesta using two paraffin wax-based pheromone
dispensers
Introduction

The cost of mating disruption is often noted as one of the obstacles to greater
adoption of this technique for pest control (e. g. Plimmer 1981). The high cost of the
active ingredient and labor to apply the most widely used systems, hand-applied devices,
contribute to the overall high cost of this strategy compared to control by insecticides.
Labor costs for hand-applied formulations could be significantly reduced if fewer
pheromone dispensers were needed per area.

Grapholita molesta (Lepidoptera: Tortricidae), the oriental fruit moth, is a major
pest of stone and pome fruits worldwide (Rothschild and Vickers 1991) and mating
disruption of G. molesta using hand-applied pheromone dispensers has been relatively
successful (Cardé and Minks 1995). This strategy generally entails evenly-spacing
dispensers in an orchard at a density of 250 units per hectare. Although some growers in
most fruit production regions of the world have incorporated this tactic into their
management programs, greater adoption is likely limited, at least in part, by the need to
apply many dispensers per unit area. Only one study has been conducted to determine the
effect of varying the numbers of point sources per area on mating disruption of G.

molesta (Rothschild 1975). The current chapter reports on more extensive studies of the

effect of reducing point source densities on disruption of the ability of G. molesta to

84

orient to traps (orientational disruption) using two wax-based mating disruption
formulations.
Materials and Methods

Experimental design. The study was conducted in apple orchards at the Trevor
Nichols Research Complex, Fennville, MI and in Douglas, MI. The orchards ranged from
ca. 1.5-2 hectares with trees planted at densities of 300-414 per hectare. The study
entailed direct comparisons of the effect of varying numbers of point sources of
pheromone per area on G. molesta orientational disruption. The amount of pheromone
applied per area in all experiments was kept constant. The experimental design was
randomized complete block; each orchard was a blocks. Four plots (0.07 to 0.10 ha) were
set up in each block. A suite of treatments (described below) was randomly assigned to
the small plots within a block. There was a total of 4 blocks or replicates for all
experiments.

Confuse-OFM point sources. Confuse-OFM (Gowan Co.,Yuma, AZ), a
commercial emulsified wax formulation, was applied as 2.7 ml (2.6 g) deposits of
material using one liter plastic squirt bottles. A single deposit of Confuse-OFM generally
covered an area ca. 30-40 cm2, and was ca. 1 mm thick. Release rate profile of this
monolithic formulation is described in Chapter 2.

Treatments entailed distributing a total of 74 g A1 (580 deposits) per hectare
among a varying number of trees or point sources per hectare. Confuse-OFM was
reapplied at this rate at the start of each of three G. molesta generations (ca. every 6-8

wk). Treatments during the first G. molesta generation were: no pheromone, pheromone

85

applied to one ninth of the trees (1/9 trees treated), to one third of the trees (1/3 trees
treated), or to all of the trees (all trees treated). During the second and third generations of
G. molesta, treatments remained the same except that Confuse-OFM in the 1/9 trees
treated plots was applied to one half of the trees.

One of the objectives was to identify a threshold number of point sources above
which orientational disruption was maximized, a dispenser deployment strategy that
would likely control G. molesta in commercial orchards. During the first generation,
orientational disruption was much lower in the 1/3 trees treated treatment than when all
trees were treated. Half of the trees were treated to determine whether at this intermediate
number of point sources, orientational disruption was equivalent to the level of disruption
in the all trees treated treatment. Since the 1/9 trees treated treatment was least
efficacious (Table 4.1), this treatment was replaced with the 1/2 trees treated treatment.

Parafiin disk point sources. Paraffin disk dispensers were constructed as
described in Meissner et al. (2000). Each disk weighed ca. 10 g and consisted of 87%
paraffin wax (Gulf wax, Royal Oak Sales, Inc., Roswell, GA), 8.6% vitamin E ([i]-(1-
tocopherol, Sigma Chemical Co., St. Louis, MO), and 4.3% G. molesta pheromone
(93:6:1 blend of 28-12zAc : E8-122Ac : 28-12:OH, Shin-Etsu Chemical Co., Ltd., Tokyo,
Japan). The procedure for constructing disks consisted of heating the paraffin wax to 65
°C, adding the vitamin E and pheromone, then pouring the mixture into 60 mm x 15 mm
Petri dishes to a height of 5 mm. The filled Petri dishes were frozen until the wax

hardened. The disks were then easily tapped out of the Petri dishes. A hot nail was used

86

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87

to melt a hole (ca. 3-5 mm diam.) in the center of each disk. The disks were hung in the
trees using twist ties looped through this hole.

Disk point-source experiments were conducted during the second and third
generations of G. molesta. Treatments were: no pheromone, paraffin disks applied to one
ninth of the trees (1/9 trees treated), to one third of the trees (1/3 trees treated), or to four
fifths of the trees (4/5 trees treated). In this last treatment, one disk was placed at each
release location. In all pheromone treatments, 247 disks (106 g AI) per hectare per
application were evenly distributed among the treated trees. New disks were hung and old
disks were removed at the beginning of the third generation of G. molesta.

Measuring treatment effects. Male G. molesta captures in pheromone and virgin
female traps measured treatment effects. Two pheromone-baited delta traps (Scenturion,
Inc., Clinton, WA) were placed in opposite corners of each plot, each in the second tree
in the second row from the edge of the plot (5.9-9.1 m from the edge of the plot).
Captures of male G. molesta were recorded twice a week. Pheromone lures were replaced
once per G. molesta generation (ca. 6-8 wk). Trap liners were replaced when soiled or at
least once per G. molesta generation.

Virgin female traps were placed in each plot during the peak flight of the second
and third generations of G. molesta in the Confuse-OFM point source experiment and
during the third flight of G. molesta in the paraffin disk point source experiment. A total
of 9-12 virgin female traps was placed in each plot during each generation. To achieve
this, a set of 3-5 traps was placed in each plot 3-4 times during a generation. The number

of traps placed in the plots each time depended on the availability of female moths; but in

88

all cases, equal numbers were placed in all plots on a given date. Virgin female traps
were not placed on border trees, on trees adjacent to a tree with a pheromone trap or on
trees adjacent to another virgin female trap. Traps were checked every morning for three
days. The virgin female traps were constructed as described in Chapter 2. Each trap
consisted of a caged virgin female G. molesta (at most three days old) placed in a delta
trap. Virgin females came from a colony established from moths originally donated by
the Rutgers Agricultural Research and Extension Center, Bridgeton, New Jersey in the
spring of 2000. Once acquired, the colony was reared on a pinto bean-based diet (Shorey
and Hale 1965) at 24 0C and 16:8 L:D. Pupae were segregated by sex (George 1965) and
females were reared individually, under natural light conditions, in 118 ml plastic cups
and provided with a 2 cm cotton wick soaked in 5% sucrose solution.

Pheromone release rates from paraffin disks. The release profile of the paraffin
disks in the field was determined as described in Chapter 2 for quantifying the release
profile of Confuse-OFM in the field (page 23). Disks were hung at 30.5 cm intervals
along two 3 m long ropes attached on the north side of the canopies of five 3.8 m tall
apple trees at a height of ca. 1.8 m. Ropes were positioned so that dispensers resided
within the outer third of the tree canopies. Five dispensers were harvested at time zero.
Thereafter, one randomly selected disk was collected each week from each of the five
trees for a total of 11 wk. The retrieved disks were wrapped in aluminum foil and frozen
at ca. -10 to -20 °C until extraction.

Pheromone was extracted from the dispensers as described in Chapter 2, except

that due to the large size of the dispensers, only a known sample of each dispenser was

89

 

extracted. Each dispenser was quartered using a knife. A small rectangular slice,
weighing 230-320 mg, was cut along the radius of one of the quarters. To prevent
contamination between disks, the knife was heated in a Bunsen burner flame, then cooled
in clean water between the sampling of different disks. The disk slice was extracted in a
glass scintillation vial in 20 ml of acetonitrile with 5 mg of tridecanoic acid methyl ester
(98%, Sigma-Aldrich Co., St. Louis, MO) as the internal standard. The amount of
pheromone in each disk was calculated from the amount of pheromone quantified in the
disk sample.

Statistical analyses. Data from pheromone and virgin female traps for both
experiments were transformed to log (x+1), then analyzed by analysis of variance
(AN OVA). Means were separated using Tukey’s test.

Data from pheromone and virgin female traps for each experiment were plotted as
percent orientational disruption (percent reduction in moth catch in treated, versus
untreated plot, Rothschild 1975) versus numbers of point sources of pheromone applied

per hectare. The following equation was fit to the data:

X-k 100_y (Eqn. 1)

In Equation 1, x is the number of point sources per hectare, y is the percent orientational
disruption, k is the number of point sources per hectare at which 50% orientational
disruption is achieved, and a is an exponent that governs the slope of the equation. k and

a were obtained by plotting x vs. (10%;!) on a ln-ln scale. The linear equation

90

that could then be fit to the data was the following:

 

ln(105_))=alnx+lnk (Eqn. 2)

Using Equation 2, k and a could easily be determined from the data (A. A. Tamijani,

personal communication).

Results and Discussion

Point source experiments. Confuse-OFM. For all Confuse-OFM trap data,
including both pheromone and virgin female trap data, significantly more moths were
caught in the no pheromone plots than in the all trees treated plots (Table 4.1). There was
a consistent trend of declining average numbers of moths caught in traps as the number of
point sources of pheromone increased. Although the numbers of moths caught in the all
trees treated, 1/2 trees treated, and 1/3 trees treated plots were not significantly different,
between four and thirty one times more moths were caught in traps placed in the 1/3 trees
treated plots versus the all trees treated plots and up to four times more moths were
caught in the 1/2 trees treated treatment versus the all trees treated treatment.
Furthermore, the average percent orientational disruption (mean :1: S.E.), as calculated
from the combination of pheromone and virgin female trap data for thepheromone
treatments was: 35.0 (1/9 trees treated, single datum, so standard error cannot be
calculated), 64.3 i 10.1 (1/3 trees treated), 84.1 :t 1.9 (1/2 trees treated), and 94.9 :t 1.7
(all trees treated). Orientational disruption was greatest when Confuse-OFM was applied

to all of the trees. Thus, for effective control with Confuse-OFM at 74 g AI/ha, the

91

application should be distributed among at least 365 point sources per hectare, which in
peach orchards, would usually translate to a single pheromone source on every tree in the
plot. High numbers of G. molesta were caught in the no pheromone plots in this
experiment, indicating the presence of a large G. molesta population in the test orchards
(Table 4.1).

Paraffin disks. The number of moths caught in the no pheromone plots was
similar to the numbers of moths caught in plots not treated with pheromone on
commercial farms harboring relatively large G. molesta populations in Michigan (Figure
2.5, Chapter 2). For all trap data (with the exception of the pheromone trap data from
generation three) significantly more moths were caught in the no pheromone plots than in
the 4/5 trees treated plots (Table 4.1). However, the difference between the treatments for
the generation three data from pheromone traps was nearly significant (P = 0.06). Perhaps
due to the plot’s location, relatively large numbers of moths were caught in the 4/5 trees
treated plot in one block. The percent orientational disruption measured in generations
two and three with pheromone traps and in generation three with virgin female traps in
that plot varied between 80.4 and 89.1% (85.4 :1: 2.6, mean :t S.E.). By contrast, the
percent orientational disruption in the 4/5 trees treated plots in the other two blocks, as
measured by those traps, varied between 95.8 and 100.0% (98.6 1 1.4 and 99.4 :I: 0.6,
mean :l: SE, for each of the two blocks). It is likely that the variation in the results
caused by the data gathered in this plot led to the finding of insignificant differences
between the no pheromone and 4/5 two treatments. Values for the average percent

orientational disruption in the pheromone-treated plots (mean i S.E.), as measured by

92

both pheromone and virgin female traps and including all blocks, were: 67.7 i 5.3 (1/9
trees treated), 87.1 i 7.4 (1/3 trees treated), and 94.5 :t 0.6 (4/5 trees treated).
Accordingly, control of G. molesta in a commercial orchard would likely be achieved
with disks applied at a rate of 106 g AI (247 disks) per hectare and evenly distributed, on
at least 247 point sources per hectare.

Dispenser release profiles. Confuse-OFM. The average release rate of Confuse-
OFM during 7 wk (the average duration of one G. molesta generation) was approximately
50 mg/ha/h (range: 6 - 178 mg/ha/h), as determined from laboratory release rates for this
dispenser (Figure 2.4, Chapter 2). The field and laboratory release rates of a 40% wax
EWD during the same time period were comparable (field: 1.98 mg/d per dollop; lab:
1.94 mg/d per dollop) (Figures 2.4 and 2.7, Chapter 2); the release rate of Confuse-OFM
determined in the laboratory should be a good estimate of the release rate of this
dispenser in the field.

Paraflin disks. Paraffin disks are monolithic dispensers. By definition, their
release kinetics are first order (Fan and Singh 1989). Although a linear equation
described the paraffin disk release profile well (r2 = 0.93), an exponential equation was a
better fit to the data (Figure 4.1). The release rates from this dispenser were quantified
over only about one third of its predicted lifetime. In other release rate experiments, data
collected early in the experiments appeared linear. However, when the experiment was
conducted until no more pheromone could be detected in the dispensers, its first order

rate of release was unmistakable. Nonetheless, the release rate of the paraffin disks was

93

 

450

400 if

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94

much closer to linear than the release rates previously measured for other wax-based
dispensers (Chapter 2). The average release rate for the disk over 7 wk was 27 mg/ha/h
(range: 22 to 33 mg/ha/h) (Figure 4.1). Extrapolating from these data, this dispenser
released pheromone at a rate above 5 mg/ha/h (proposed by Rothschild 1975, as the
release rate of pheromone necessary to provide effective control of G. molesta) for 222 d.

For a given number of point sources per hectare, the percent orientational
disruption was consistently higher in the paraffin disk experiment than in the Confuse-
OFM experiment. This is counterintuitive, since the average release rate of pheromone
per hectare for the Confuse-OFM experiment was twice as high as the average release
rate of pheromone per hectare for the paraffin disk experiment. However, although the
average release rate per hectare for Confuse-OFM was higher than for the paraffin disks,
the range of release rates from this dispenser was much larger. Furthermore,
approximately three weeks following application (ca. the middle of each G. molesta
generation), the release rate per hectare of Z8-l2:Ac in the Confuse-OFM plots fell below
the release rate of Z8-122Ac in the paraffin disk plots. When percent orientational
disruption was plotted versus the release rate of pheromone for each trapping period for
both dispensers, the percent orientational disruption was consistently higher when the
release rate of pheromone per plot was greatest (de Lame, unpublished data). Thus, when
comparing the data from the Confuse—OFM and paraffin disk experiments, the average
release rate of pheromone per generation for each dispenser is not an accurate measure of

the overall release rate in each generation and the release rate of pheromone per hectare

95

 

for the paraffin disk experiment should be considered higher than the release rate of
pheromone per hectare for the Confuse-OFM experiment.

Density of point sources, release rate, and success of mating disruption.
Varying point source density. A common trend became evident with both Confuse-OFM
and paraffin disk data when percent orientational disruption was plotted versus numbers
of point sources per hectare for each experiment (Figure 4.2). The curves fit to the data
described a relationship where, as the number of point sources was increased, the
corresponding increase in orientational disruption became decrementally smaller. The
greatest percent orientational disruption (99.2%) was achieved with one point source per
tree. Although statistically significant differences were not always detected between the
numbers of moths caught in the treatments with varying numbers of point sources for the
dispensers tested, the relationship between percent orientational disruption and numbers
of point sources of Figure 4.2 reveals that point sources per area should be maximized for
mating disruption to be most effective. There was high variability in the percent
orientational disruption realized when few point sources were applied per hectare.
Reliability of a positive outcome increased with point source density, further stressing the
importance of applying pheromone dispensers at large numbers of point sources per area
in orchards.

Only one previous study described the effect of varying numbers of point sources
on orientational disruption of G. molesta. Rothschild (1975) tested the effect of

distributing dispensers that collectively released pheromone at or just below 5 mg/ha/h

96

 

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97

among 12.5 to 200 point sources per hectare in peach orchards. He found no significant
difference in the numbers of moths caught in pheromone traps when the dispensers were
applied at 25 to 200 point sources per hectare. Percent orientational disruption in the plots
he treated with pheromone varied between 73 and 88 percent. Interestingly, although he
calculated percent orientational disruption when pheromone was released from a wide
range of point sources, the trend in Figure 4.2 was not evident in his trial. Percent
orientational disruption did not consistently increase with increasing numbers of point
sources: 73% orientational disruption was achieved at 100 and 200 point sources per
hectare, 88% orientational disruption was recorded with 50 point sources per hectare,
81% orientational disruption was achieved with 25 point sources per hectare, and 40%
orientational disruption was achieved with 12.5 point sources per hectare.

Rothschild (1975) provides too few details of his experimental setup to allow
judgement of why his data do not seem to fit the patterns identified in this study (Figure
4.2). However, a probable cause is that his experiment was not replicated. Furthermore,
the data he presents are total catches of moths from three successive sampling periods,
which suggests that the trial was brief. Due to the high level of variability in these types
of data, high replication was essential in the current trials to develop the trends shown in
Figure 4.2. The release rate of pheromone per hectare in Rothschild’s experiment was
relatively low. When the release rate per dispenser was plotted versus the percent
orientational disruption for the present experiment, there was much more variability in
the percent orientational disruption when pheromone was released a low rate, versus a
high rate. This was because a high rate of pheromone emission led to the same level of

mating disruption relatively independently of the number of moths caught in the

98

corresponding no pheromone treatment. However, when a low rate of pheromone was
released, the percent orientational disruption was large when few moths were caught in
the no pheromone plots, and low when many moths were caught in the no pheromone
plots (de Lame, unpublished data). Rothschild’s (1975) experiment was carried out in a
peach orchard, whereas the current experiments were carried out in apple orchards. It is
probable that the results of point source experiments could vary depending on the type of
crop in which they were carried out; nonetheless, the trends presented in Figure 4.2
should hold.

Generalizability of Figure 4.2. I expect that the overall relationship between point
sources and percent mating disruption described for G. molesta (Figure 4.2) will hold for
other moth species in orchard and non-orchard environments. Differences in
experimental design, however, make comparing the current studies with others
investigating the effect of point source density on percent orientational disruption
difficult. Some important design differences have included the range of point source
densities tested, the release rate of pheromone from each dispensing device, and the
distribution of the devices in the crop. A recent experiment has revealed the same
relationship described in Figure 4.2 when the release rate per point source was kept
constant (de Lame et al., unpublished data). In light of this observation, experiments
conducted using the later two experimental designs are not differentiated.

Only a few studies to date have investigated the effect of varying numbers of
point sources on percent orientational disruption over a wide enough range of point
sources per hectare to note whether the data they present follows the relationship in

Figure 4.2. Using hand-applied dispensers with release rates comparable to Confuse-

99

OFM deposits and paraffin disks, Suckling and Angerilli (1996), working with Epiphyas
postvittana in 0.5 ha apple orchards, recorded increases in percent orientational
disruption as 100 rope dispensers (Shin-Etsu Chemical Co., Ltd., Tokyo, Japan) were
evenly distributed at one point source per plot, nine, and then 100 point sources per plot.
Interestingly, at only two point sources per hectare, the percent orientational disruption
inside the plots was relatively high (76%). Suckling and Angerilli (1996) found stronger
and more frequent EAG (electroantennogram) depolarizations for E. posrvittana in plots
with increased numbers of point sources of dispensers per hectare in the same plots where
they reported increased percent orientational disruption as numbers of point sources were
increased. They suggested that a more frequent detection of pheromone and detection of
larger concentrations of pheromone, as occurred with increasing numbers of point
sources per hectare, should increase orientational disruption. These researchers also
reported similar changes in the EAG response of E. postvittana with increased numbers
of dispensers applied per area in an earlier study (Suckling et al. 1994). In this case, the
treatments were 100, 200, and 400 dispensers per hectare; the rate of pheromone applied
per hectare was not held constant.

In a vineyard, Sauer and Karg (1998) measured higher concentrations of
pheromone by EAG with Lobesia botrana when the number of dispensers releasing the
pheromone of this species was increased. These authors applied BASF RAK2 dispensers
at rates of 200, 400, and 1600 dispensers per hectare. Interestingly, they noted that a log-
linear equation better described the relationship between increasing the number of
dispensers and the increase in the concentration of pheromone measured. This

relationship is similar to that described in Figure 4.2.

100

 

Most studies of the effect of point source density on orientational disruption have
investigated this relationship with numbers of point sources per area greater than those in
this study and concluded that when a constant amount of pheromone was released per
area, the number of point sources per area did not influence the percent orientational
disruption achieved. Figure 4.2 predicts that as the number of point sources at which
dispensers are applied is increased, it would become increasingly difficult to prove
statistically significant differences in orientational disruption; for dispensers releasing
equivalent amounts of pheromone per hectare as the paraffin disks, it is likely that no
statistically-significant difference in the percent orientational disruption would be found
when dispensers are applied at densities higher than ca. 200/ha. Meissner et al. (2000),
working with Platinota idaeusalis in apple orchards and using PVC spiral dispensers, did
not find statistically-significant differences in percent orientational disruption when point
sources were varied; the smallest number of point sources per area tested in this study
was 370.

Some point source density studies have used pheromone formulations that are
uniformly broadcast. Each dispenser releases much smaller quantities of pheromone than
those released by hand-applied dispensers such as the ones tested in the current study. In
the following two studies, the investigators released pheromone from hollow fibers and
Hercon flakes. Miller et al. (1990), in wind tunnel, small plot, and large plot trials,
determined that a larger number of point sources of an attracticide formulation were
visited by male Pectinophora gossypiella and there was more male mortality when the
number of point sources per hectare was increased. Their results suggest increased

inhibition of moth captures in traps would also result from an increase in the numbers of

101

 

point sources of pheromone placed in an orchard. The number of point sources per
hectare in the large plot field trials ranged from 1400 to 47,424. Alford and Silk (1983),
working with Choristoneura fitmiferana in a mixed balsam fir and white spruce woodlot,
reported increased percent mating disruption with decreased numbers of point sources
and concurrent increased release rates of pheromone per point source down to a rate of
500 point sources per hectare. It is possible that at the higher densities of point sources
tested, the release rate per point source was not sufficient to cause orientational
disruption. At the lowest number of point sources per hectare (182), there was a
significant decrease in orientational disruption for this species, which is in agreement
with Figure 4.2.

Varying release rate. As an equal amount of pheromone per hectare was divided
among a decreasing number of point sources in the current trials, percent orientational
disruption decreased. However, as the release rate per point source increased, the level of
disruption with a low number of point sources could be increased as well. As a result,
with a higher release rate of pheromone per hectare, the number of release devices per
hectare could be reduced, while maintaining a high level of orientational disruption. The
dispensers used here had first order release kinetics, so that the release rate per dispenser
per generation could not be defined accurately. Furthermore, only two different rates of
pheromone release per area were tested, so that the current data cannot be used to
accurately predict the effect of changing the amount of pheromone released per area on
the percent orientational disruption achieved. This relationship should be similar to that
identified between the number of point sources per hectare and percent orientational

disruption (Figure 4.2); at a given, low number of point sources per hectare releasing

102

large amounts of pheromone, further increasing the amounts of pheromone released
would not greatly increase the percent orientation disruption achieved. When Rothschild
(1975) determined the threshold release rate of pheromone per hectare necessary for
mating disruption of G. molesta, he plotted a graph similar to Figure 4.2 that described
the relationship between increasing the release rate of pheromone and percent
orientational disruption. For these studies, polyethylene microcentrifuge tubes were
applied at rates of two per tree, one per tree, or one every other tree. Thus, when
relatively high and constant numbers of point sources were applied that released varying
amounts of pheromone, a relationship similar to the one depicted in Figure 4.2 was
identified. Although it seems likely that the relationship between release rate and percent
orientational disruption may be described by these curves, it is probable, however, that
when very few point sources are applied, the asymptote will fall below 100%
orientational disruption, so that greatly increasing the release rate of pheromone per
hectare will not provide high levels of orientational disruption necessary for pest control.
A series of studies by Shorey and colleagues describes the development and
testing of puffers, high release rate, low density pheromone emitters, for mating
disruption of moth pests (McLaughlin et a1. 1972, Shorey et al. 1972, Farkas et al. 1974,
Shorey et al. 1994, Shorey et al. 1995, Shorey et al. 1996, Shorey and Gerber 1996a,
1996b). Conclusions from early experiments with Trichoplusia ni and Pectinophora
gossypiella (McLaughlin et a1. 1972, Shorey et al. 1972) were that the release rate of
pheromone per hectare was the most important factor determining the success of mating
disruption and that this release rate per hectare could be divided among a limited number

of point sources per area. Shorey et al. (1972) achieved above 90% orientational

103

disruption with as few as 11 point sources per hectare. In trials using puffers (high
release, low density devices), Shorey et al. (1996) and Shorey and Gerber (1996a,
1996b), achieved high levels of orientational disruption with as few as 3 puffers per
hectare releasing a sum, low rate of 22.5 mg/ha/h of pheromone and placed along the
perimeter of large experimental plots (36 puffers evenly spaced around the perimeter of a
16 ha orchard) (Shorey and Gerber 1996b). These trials suggest that a high release, low
point source pheromone dispensing strategy could be effective. However, in trials with G.
molesta using microsprayers, dispensers similar to puffers, even after greatly increasing
the amount of pheromone released per microsprayer, when these were placed at rates of
two per hectare, percent orientational disruption remained around 80%, a level of
disruption too low for the treatment to control this pest (JR. Miller, personal
communication). In the case of the microsprayer experiments, it seems that the asymptote
did fall below 100%.

The Shorey experiments had several weaknesses. Early experiments were
conducted over only one night. Later ones were conducted only over one week. In early
experiments, treatments were re-randomized in the plots each night, so that pheromone
contamination on foliage from previous nights could have influenced subsequent results
(Sauer and Karg 1998). In later trials, the control plots were located at distances of 2-10
km from the treated plots. Although this was beneficial to avoid pheromones moving
from the puffer plots to the control plots, equivalence of moth population density in the
treated and control plots was unproven. Thus, it remains unclear how the aerosol

dispenser studies fit with studies of point source density effects using smaller hand-

applied dispensers.

104

Suckling and Angerilli (1996) showed that frequent contact with pheromone
plumes occurred in orchards where E. postvitanna orientation was most disrupted. When
point sources per hectare of pheromone are decreased, it is evident that there will be
increased areas of pheromone-free air within the plots, which would reduce the efficacy
of the treatment. It is possible that when dispensers are evenly deployed around the
perimeter of a plot, versus within the plot, areas of pheromone-free air are reduced, since
no matter which direction the wind blows, several dispensers will release their
pheromone within the orchard. However, more extensive studies should be conducted to
determine whether Shorey’s dispenser deployment strategy would be effective season-
long in a robust, replicated trial.

Suggested study: varying point source density and release rate. Determining the
relationships between the release rate of pheromone per point source, the release rate of
pheromone per hectare, the number of point sources of pheromone applied per hectare,
and the percent orientational disruption is invaluable to increasing our understanding of
mating disruption and how to minimize its cost and maximize its efficacy. To determine
this relationship, replicated trials should be conducted using pheromone dispensers with
close to constant release rates. Ideally, a two-factor experiment should be conducted, with
the factors of release rate per area and number of point sources per area, both of which
should be varied over a wide range. Because the population of moths increases to a peak,
then decreases during each generation, each experiment should be carried out over one
full generation to accurately determine the efficacy of the treatment. It is likely that many
of these relationships will be described by asymptotic equations. The two constants in

Equation 1 (page 90) can easily be determined from data (A. A. Tamijani, personal

105

communication). This equation should be useful in identifying and describing asymptotic

relationships between variables.

106

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Chapter 5
Suggestions for further research

Building upon EWD II-OF M. These studies generated valuable knowledge
about the characteristics and efficacy of paraffin-based emulsified wax dispensers
(EWDS). As designed in this research, EWD II-OFM is as effective as Isomate-M 100
(Shin-Etsu Chemical Co., Ltd., Tokyo, Japan), a leading commercial dispenser for mating
disruption of Grapholita molesta. The season-long efficacy of EWD II-OFM and
development of an effective applicator for this formulation was a big accomplishment for
this class of dispensers and a significant improvement from Confuse-OFM, the
commercial EWD formulation. However, the potential of EWD technology has not yet
been exhausted.

In on-going laboratory studies, the linearity of the release rate profile of EWD H-
OFM, and thus also the longevity of this formulation, was further increased by increasing
the size of individual dollops. This decreased the surface area and increased the thickness
of those dollops. Fick’s law of diffusion states that modifying the dispenser in this way
should decrease its release rates (Fan and Singh 1989, Atterholt 1996).

EWD formulations have been tested against a handful of pests other than G.
molesta. In most cases, mating disruption of these pests with the EWD formulations was
not successful. The low molecular weight of the pheromones released is the most likely
cause of their failure (Chapter 2). Adapting EWDs to release a variety of pheromones
will make these dispensers more marketable. From the experience gained working with
EWDs, it seems that modifying the wax type or wax content of EWDs has the most

pronounced effects on the properties of these dispensers. Paraffin wax is only one of

107

‘*“g‘" I t’A“.

innumerable types of waxes available commercially; all of them have different physico-
chemical properties (Bennett 1975). In an on-going study, a microcrystalline wax-based
EWD releases G. molesta pheromone at half the rate of EWD II-OFM. This formulation
is also stickier, so that it is able to adhere to bark under cold conditions without the
addition of adhesive. It is likely that by modifying EWD formulations, especially the type
of wax used to make these formulations, EWDs could successfully dispense virtually all
insect pheromones.

Studying the physico-chemical properties of different waxes will be invaluable in
designing EWDs to dispense a wide variety of pheromones. Several properties are
generally used to describe waxes, including: melting point and hardness. It seems logical
that a high melting-point, or a hard wax would dispense pheromone more slowly than a
low melting point, or a soft wax. It would be expected that in the first, less free volume
would be available for the molecule to diffuse to the surface of the dispenser, while in the
latter, more free volume would be available for the chemical to do so. By determining the
relationships between wax properties and their ability to release particular compounds,
one could eventually use the properties of waxes to identify potentially good waxes (or
mixtures of waxes) to release almost any compound. Bennett (1975) compiles
information on the properties and uses of an impressive number of industrial waxes, both
natural and synthetic.

The Spatula applicator designed in this research was cheap and could be used to
apply EWDs very quickly at heights that can be reached from the ground. Effective
applicators should be designed to dispense EWDs at greater heights so that these

dispensers could be used in a wider variety of crops and for mating disruption of pests,

108

Van-nmyHi-n

such as Cydia pomonella, for which pheromone must be applied high in tree canopies for
effective control (Weissling and Knight 1995). The market for EWDs could also be
increased by modifying these for use as attract and kill devices.

Many of the benefits of EWD II-OFM are a result of its thick, flowable design.
Although these dispensers have been made by emulsifying waxes, perhaps the same
benefits can be reached with non-wax formulations which mimic EWD H-OFM. Whether
this formulation would have added or reduced benefits to emulsified wax formulations is
unknown, but this may be a worthy line of investigation. Another interesting question is
whether an EWD or similar dispenser could be designed to form a crust following
application. EWDs are monolithic devices and have first order release kinetics (Fan and
Singh 1989, see Chapter two). If a thick, flowable dispenser could be designed to form a
crust, perhaps by polymerizing at the surface following exposure to air, or UV light, this
dispenser might effectively be transformed into a reservoir device, with the crust
controlling the rate of release of active agents from the device. Such a device would have
zero order release rates until the rate of diffusion of the compound to the crust became the
limiting step in the release process, at which time, the release rate of the device would
become first order (Fan and Singh 1989). This transition could be delayed by creating a
dispenser with a relatively loose internal matrix and/or incorporating larger amounts of
pheromone into the dispensers than were incorporated into EWD II-OFM.

Dispenser distribution. The current studies convincingly showed that disruption
of male G. molesta orientation to pheromone sources was successful when EWD

dispensers were applied at 1.2-1.8 m (heights at which application can be made from the

109

ground) in trees as tall as 5.5 m. This information can greatly reduce the time and cost of
application of dispensers for mating disruption of G. molesta (Chapter 3).

Studies of the effect of varying the horizontal distribution of dispensers applied at
a set rate per hectare in an orchard generated valuable information about the
mathematical relationship between increasing numbers of point sources and percent
orientatonal disruption achieved. Further, systematic studies should be conducted using
zero-order release dispensers, such as Isomate-M 100, to further elucidate this
relationship. A factorial experiment with two factors: point sources per hectare and
release rate per hectare, would be well-suited to this purpose. These tests should be
carried out over a wide range of release rates and point sources, mimicking dispensers
ranging from the high release-low density puffers and microsprayers to
microencapsulated formulations. The generation of a mathematical model or models
describing the relationships between release rate per hectare, and perhaps also release rate
per point source, point sources per hectare, and percent orientational disruption for G.
molesta and other pests would be useful in quickly identifying which pheromone release
strategies will and will not work for mating disruption of a particular moth pest. Perhaps
some strategies will not work for any pest. In those cases, the reasons for the failure of
that strategy would be identified and valuable research resources could be redirected to
develop new, effective dispensers. It would be interesting to document whether the
relationships between these variables are similar, or vary for different pests.

Concluding remarks. As is usually the case, this research has answered many
questions, but also generated many new and interesting questions. I am confident in the

EWD technology and hope to see it commercialized soon. I hope that further research on

110

the effects of dispenser distribution on mating disruption of moth pests will help increase
the success of this technique and generate knowledge needed to expedite the development

of effective pheromone dispensers.

111

 

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120

 

 

 

 

 

APPENDICES

121

APPENDIX A

Additional Tables

 

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APPENDIX B

S.P.L.A.T. Pheromone Applicator

127

 

 

S.P.L.A.T.

Pheromone Applicator

www.cgrmsu.edu/age/students.htm

   

May 22, 2002

Agricultural Engineering Department
Michigan State University

Design Engineers and Authors Faculty Advisors
Erik Arbut Dr. Gary R. VanEe, P.E.

Graduated, May 2002

 

Mr. Richard Ledebuhr
Lindsey Brown
Expected graduation, August 2002

 

Student Branch Advisor
Larry Morden
Expected graduation, December 2002 Dr. Daniel E. Guyer

 

 

128

 

S.P.L.A.T. Pheromone Applicator

EXECUTIVE SUMMARY

 

CONFUSE-OFM Pheromone is a biological control chemical used to combat the Oriental
Fruit Moth. The chemical must diffuse slowly into the atmosphere near the fruit trees.

At present the MSU Entomology Department researchers manually apply a pheromone-
based wax to tree trunks in orchards.

We have developed an accurate, easy-to-use catapulting device that can quickly apply the
wax substance to trees. This report has some prototype test data that attest to the
usefulness of our invention. At low volume manufacture, the device could sell for
$2,400, which is less than the $3,000 that orchard owners are willing to pay. The device
allows an ambulatory worker to administer pheromone to approximately 700 trees per
hour. The device can enter the market in less than two years. Conventional fabrication
tools were used in building the prototype; no sophisticated tooling is needed.

 

ACKNOWLEDGEMENTS

0 We would like to thank Freddy de Lame and Dr. James Miller from the
Entomology Department for helping us with this project.

0 We would also like to thank Nancy Aitcheson, Richard Ledebuhr, Steve Marquie,
Nick Tipper, Dr. Gary VanEe and Richard Wolthuis for their continued advice
and support.

0 A special thank you goes to Senior Design Professor, Dr. John Gerrish for sharing
all of his priceless knowledge and expertise.

129

TABLE OF CONTENTS

EXECUTIVE SUMMARY ............................................................................................. 129
1.0 INTRODUCTION
2.0 ESTABLISHMENT OF NEED

2.1 BACKGROUND INFORMATION .................................................................... 131

2.2 PROBLEM STATEMENT .................................................................................. 132
3.0 DESIGN OBJECTIVES & CONSTRAINTS

3.1 ALTERNATIVESCONSIDERED ...................................................... 133

3.2 EVALUATION OF ALTERNATIVES .............................................................. 133

3.3 SAFETY CONSIDERATIONS ........................................................................... 134

3.4 ECONOMIC CONSTRAINTS ............................................................................ 134
4.0 ENGINEERING DESIGN

4.1 GENERAL ........................................................................................................... 134

4.2 SYSTEM .............................................................................................................. 135

4.3 ERGONOMICS AND SAFETY ......................................................................... 136

4.4 ADAPT ABILIT Y ................................................................................................ 137
5.0 TESTING

5.1 LAB ...................................................................................................................... 137

5.2 FIELD .................................................................................................................. 138
6.0 ECONOMIC ANALYSIS

6.1 PROJECT COST ................................................................................................. 139

6.2 COST COMPARISON ........................................................................................ 139

7.0 CONCLUSIONS

8.0 RECOMMENDATIONS

9.0 REFERENCES

10.0 APPENDICES
Appendix 1: Specifications
Appendix 2: System Diagram
Appendix 3: Drawings
Appendix 4: Electrical Diagrams
Appendix 5: Calculations
Appendix 6: Economic Analysis

130

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1.0 INTRODUCTION

Oriental Fruit Moth (OFM) mating is the outcome of a sequence of behaviors of both
sexes that is initiated by the females’ release and the males’ perception of a specific
chemical, called a pheromone. Permeating the environment within which the pest lives
with sufficient “background” pheromone inhibits the male’s awareness of the small
amount produced by the female. This prevents mating and leads to a significant decrease
in reproduction and pest population. Consequently, damage done to fruit by these insects
is decreased.

A plastic, pheromone-laced “bread-tie” is currently used to saturate the OFM breeding
environment, but its application in trees is extremely labor-intensive and costly. Also, the
release rate of pheromone through the bread-tie is unfortunately high, necessitating too
many applications per season. In order to control the release of pheromone, MSU
researchers developed a wax formulation that offers a slow uniform release rate and
reduced breakdown due to ultra-violet in sunlight.

 

The Chemical Ecology Group from the Entomology Department at Michigan State
University supported our project to design an effective applicator for wax containing
Oriental Fruit Moth pheromone. Our client intends to use the applicator for seasonal
pest management. We anticipated a wider market and decided to make the applicator
lightweight and user-friendly for people of various sizes.

2.0 ESTABLISHMENT OF NEED
2.1 BA CK GROUND INFORMATION

Approximately 20-30% of all horticultural crops are damaged before harvest and never
reach market. The reasons for this include mishandling, disease, disorders, and damage
caused by insects. One such group of insects is the family of Lepidoptera, in particular,
the Oriental Fruit Moth. Even at relatively low population densities, OFM has the
capacity to cause economic damage to apple and peach crops.

Integrated pest management (IPM) is widely accepted as the environmentally safest
means of pest control. Appropriate measures are designed to maximize the effect on the
target species and minimize the disruption of beneficial species. With 1PM came the use
of synthetic sex pheromones in place of toxic chemical pesticides. Pesticides leave
residue in the soil and groundwater. They are also expensive to apply and may eliminate
natural predators and beneficial species.

The need is more general than the immediate need of our client, the MSU Chemical
Ecology Group. Therefore, as we proceeded, we had in mind the possibility of
addressing the industry’s need.

131

 

Researchers in the Entomology Department at MSU have been working on a new
pheromone emitter. They have tried a paraffin wax disk, which is hung on each tree
individually by hand, and releases pheromone at a slow rate. This product is tedious to
apply, although it needs only two applications per season.

The newest pheromone emitter is a fluid wax formulation. If the wax is thin enough to
spray through a typical household spray bottle, it tends to flow down the tree before it
hardens. MSU entomologists prepared a thicker wax formulation, which consists of
Gulfwax household paraffin wax, emulsifier (span 60), soybean oil, sorbitan
monostearate, (i)-alpha-Tocopherol, water, and pheromone.

With the thick wax formulation, a dollop forms after a wax projectile is launched.
Ideally, it lands as a hemisphere, about four centimeters (1.5 in) in diameter. This is
desirable because its low surface-to-volume ratio results in a slow release rate, and little
breakdown due to UV in sunlight. If the dollop is propelled through the air at a velocity
near 200 km/hr (120 mph), then it adheres to the tree throughout the growing season
resisting the “weathering” effect of temperature and moisture. Temperature seems not to
affect this substance significantly; therefore, it can be applied in the cool spring weather,
and hot mid summer weather. The wax mixture is harmless to people if it comes in
contact with their skin, but further tests are warranted.

2.2 PROBLEM STATEMENT

The entomology department had no way to apply the pheromone wax to trees except by
hand. They needed a more efficient method by April 30, 2002. The purpose of this design
was to provide an effective means for applying a pheromone product onto fruit tree bark
using an accurate and labor-efficient method. Application may occur no more than two
times per year. Eventually, the technique must be useful in many orchards, meaning that
a solution had to be manufacturable, economical for the user, and profitable for the
manufacturer.

3.0 DESIGN OBJECTIVES & CONSTRAINTS

We wanted a lightweight, easy-to-use loading and a carrying system, so that a worker
could carry the device on his/her back. We needed an accuracy of i 2 cm (0.8 in) at 3 m
(10 ft) distance. We wanted a “range” of approximately 300 trees.

The best design will satisfy all of the following: The device must provide accurate dollop
placement with 3mL of wax delivered to a target branch 8 cm (3 in) in diameter from a
distance of two to four meters (7-13 ft). The device must be labor efficient, user-friendly,
and made with long-lasting, easily maintainable and serviceable components. The device
must be ergonomically designed to fit 95 percent of male and female body shapes and

132

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have a mass less than 11 kg (weigh less than 25 lbs) when field ready. A low production
quantity of 24 units was desired at a costumer cost of no more than $2400.

3.] ALTERNATIVES CONSIDERED

We studied four conceptual designs: a spring-loaded catapult, a paintball gun, an electric

grease pump, and a cordless nail gun. There was a common theme among the alternatives
we considered. Each needed to catapult a dollop of long-lasting, thick, sticky pheromone

formulation onto a tree.

The positive features of the spring-loaded catapult included being hand-operated, having
a simple spring-driven plunger, and being easy to manufacture. We decided against this
application method because it did not have enough power to catapult the dollop more
than one meter (3ft), and it was heavy and difficult to maneuver.

The paintball gun was a practical possibility. It featured automatic loading at a high
velocity and power, which could prOpel the wax a long distance. It would be easy to
handle and transport, and it was accurate. We decided not to use the paintball gun
because of a few negative attributes. Loading the pheromone substance jarmned the
action of the gun. When the gun was shot, instead of forming a dollop, the pheromone
splattered on the target, causing an undesirable and excessive surface area.

The electric grease pump featured automatic, timed loading and was easy to use and
clean. It is a common tool that has inexpensive parts, and it is readily available. We chose
not to use this option because the pump was hand-operated, tired the operator because of
its weight, and was labor intensive. It also did not catapult the wax to form a truly
adhesive dollop.

The modified cordless nail gun met the power and velocity requirements to propel a
dollop at the desired range, and could be modified easily to automatically load and shoot
wax instead of nails. The nail gun was made with long lasting components for
continuous use. It was quick and easy to use, transport, maintain, and service. Safety
mechanisms were already built into the gun to prevent accidental injury. We had
concerns with wax entering the combustion chamber and the possibility of too much
power, but those concerns were overcome.

3.2 EVALUATION OF ALTERNATIVES

We performed pertinent calculations and made drawings for each candidate design. We
experimented by catapulting grease through plastic and metal syringes. These
experiments proved that we needed more power than the grease gun could deliver, but
not as much as the paintball gun. A modified cordless nail gun was our most—promising
method.

133

 

 

3.3 SAFETY CONSIDERATIONS

In the design, we considered how to protect the operator and bystanders during use and
mis-use of the applicator. For example, the operator should not be able to accidentally
discharge the gun.

The operator and assistants are to wear safety glasses. OSHA regulations govern design
decisions and operational procedures. A Materials Safety Data Sheet must be supplied
for the wax formulation, and the operator must be properly trained. The operator should
wear gloves, that are left in the orchard in order to prevent a bunch of eager male moths
following him/her home from work.

3.4 ECONOMIC CONSTRAINTS

The economics of this project are to be compared with the economics of hired immigrant
labor hand-placing the bread-tie pheromone dispensers. Since our application is
relatively new technology, the average grower would be willing to invest as much as
$3,000 for an initial prototype.

4.0 ENGINEERING DESIGN
4. I GENERAL

Firstly, we ran tests with “sawed-off” plastic syringes to evaluate the optimal length-to-
diameter ratio for a propelled dollop. We cut off the pointed tip of a syringe so that the
end was open and had the same diameter as the bore of the syringe. This interrupted the
cohesive force between the inside wall of the syringe and the outside layer of the wax,
and it kept the dollop in a cylindrical form while traveling through the air. We were able
to determine the relationship between dollop size and barrel diameter. If the diameter
were too large, then the three milliliters wax dollop would have too large a frontal area
with insufficient depth, making the dollop fracture while airborne. If the bore was too
small, then we tended to squirt a “noodle.” We found the best size to be a half-inch bore
with a length-to-diameter ratio of 1.87 for 3mL.

Secondly, we machined an aluminum plunger and steel barrel. To determine velocity, we
recorded projectiles with a digital camera and measured the velocity. We determined the
mean velocity to be 204 km/hr (127 mph) with a standard deviation of 13 km/hr (8.1
mph). This gave us a benchmark velocity for our modified nail gun.

Thirdly, we experimented with a pneumatic framing nail gun to confirm that it would
deliver enough power to propel a dollop at 204 km/hr (127 mph) velocity. Since the
pneumatic framing nail gun conveyed excessive power, we experimented with a lesser-
powered machine, a cordless finishing nail gun that had only two thirds the power of the

134

 

 

 

framing nail gun. We decided to work with the finishing nail gun, modifying it to match
the small power needed for our projectiles.

The graph in Appendix 32 shows a conceptual pressure-volume curve for a Paslode
Impulse® Cordless Finishing Nailer IMZSOII. The red curve represents the original nail
gun and the blue curve represents the nail gun after it had been modified. Prior to point
1, the chamber is closed, and the fuel and air is mixed by the fan located at the back of
the gun. When the trigger is squeezed, at point 1, a spark is created and the fuel air
mixture begins to combust. The initial volume of original nail gun is estimated to be 77
ml (4.7 in3), while the modified nail gun is estimated to have an initial volume of about
93 ml (5.7 in3). The increase in volume is due to the structure of the piston in the
modified nail gun, which was bored in such a way as to allow some gases to exhaust into
a secondary chamber.

After ignition, the gases rapidly combust and the pressure rises with little change in
volume. As the piston begins to move, and passes point 2, the “P-V” diagram follows an
adiabatic curve to point 3. This point is at a lower pressure for the modified nail gun
because of its greater initial volume. At point 3, the piston reaches the exhaust ports that
line the outside of the cylinder; the pressure drops immediately. At this point, the
original nail gun has a final volume of about 10 cubic inches, where as the modified nail
gun has a final volume of about 150 ml (9.3 in3). The reduction in chamber volume is
due to the much larger piston head of the modified nail gun.

The combusted gases exhaust out of the front of the gun and the pressure falls to near
zero at point 4. The bottom of the piston head hits the urethane bumper at the bottom of
the chamber and the piston begins to rebound towards its initial position again sealing the
combustion chamber. The remaining gases behind the piston head cool to form a
vacuum, which quickly draws the piston to the top of the chamber. When the cocking
lever is released, the combustion chamber is “opened” and fresh air increases the pressure
to point 1.

The area under the curve represents the energy of the system. There is a considerable
decrease in the power produced by the modified nail gun as compared with the original
nail gun.

4.2 SYSTEM

We modified the nail gun to load and shoot wax instead of nails. This required all the nail
system parts to be replaced with a new, machined piston and barrel. In order to prevent
friction damage in the mechanism, the barrel was manufactured from steel, a hard metal,
whereas the piston was machined from aluminum, a soft lightweight metal. The softer
metal can absorb the detached particles of the harder one. Aluminum was chosen to
reduce piston mass, which was desired for the primary moving component.

Vital—T .hm

 

The piston was designed to be larger than the nail driver’s piston. We wanted a shorter
stroke and a larger initial combustion volume than the nailer had. Holes and a channel
were bored into the aluminum piston head to allow combustion gases to flow through the
chamber exhaust ports during that part of the cycle. These changes created a significant
reduction in the power of the nail gun. A compression ring was placed at the bottom of
the piston head so that no excess gas is released during firing. An “O” ring was placed
near the front of the piston shaft to ensure all the wax was expelled.

The barrel length was determined (during preliminary testing) to produce the most
efficient velocity. The base was formed to fit the existing gun components. A hole was
drilled into the bottom of the barrel for loading. A pair of holes was drilled into the base
to allow the cocking mechanism to function.

A steel collar was placed on the outside of the barrel to hold the hose-barb without
interfering with the plunger. Two holes were tapped into the walls of this collar. One was
for hose-barb attachment, which connected to the loading hose; the other was for the
setscrew to keep the collar in place.

A “hand cocking” mechanism was created to replace the original safety seal for the
combustion chamber, and then load the fuel for the nail gun. This action also starts the
fan to mix the air and propane and electronically enables the trigger to ignite a spark.
When the trigger creates a spark, the fuel and air combusts, forcing the pressure to
increase rapidly with little change in volume. As the gases expand, the piston is driven to
the bottom of the chamber. Once the machined portion of the piston head reaches the
exhaust ports, the pressure decreases rapidly and the gases escape. The piston hits the
urethane bumper at the bottom of the chamber and rebounds slightly. Due to the cooling
gases behind the piston head, a slight vacuum is created, which draws the piston to the
top of the chamber. The trigger and cocking mechanism are released opening the
chamber and allowing fresh air to enter. The vacuum is destroyed, and the pressure and
volume return to their initial state. A system diagram is in Appendix B. 1.

We connected an adjustable time-delay relay to accurately produce a three-milliliter
dollop for each application. The timer is set for about two and a half seconds, which is
the time required for three milliliters of wax to be expelled from the grease pump. A plate
was machined to mount the timer onto the pump. Quick disconnections on the electrical
wires and load tube ensured easy maintenance, loading, and transport.

A pumping switch was mounted on the nail gun in order to activate the loading of the
wax. When the trigger of the nail gun is pressed, the pumping switch opens. Once the
trigger is released, the switch closes and the grease pump reloads the nail gun with wax.
4.3 ERGONOMICS AND SAFETY

We intended the applicator to be used by 95 percent of both men and women. The gun

can be easily operated with either the left or right hand. The backpack is fully adjustable
to correlate with various back and hip sizes. The system mass is 9 kg, field-ready, which

136

 

is less than our 11 kg limit (weights 20 and 25 pounds, respectively). We also designed
the cooking mechanism to be operated comfortably and with a four-finger force of 36 N
(8 ft-lbs), which is less than the standard maximum pull force of 10 N (2.2 ft-lbs) per
finger.

Our team built safety measures into the applicator. The separate cocking mechanism and
trigger requires a two-handed action, keeping the operator safe. Once the trigger is
squeezed, only one shot is fired, and the gun will not fire again until the trigger and
cocking mechanism have been released. The catapulted dollops do not travel with
enough velocity to cause bodily harm to anyone.‘ A dollop is shot only after the wax is _
loaded into the barrel from the grease pump. If"

Because the exhaust ports face towards the front of the gun, the operator does not inhale i
the exhaust fumes from combustion. A green light advises the operator when the battery 1
is in place and the gun is ready for use. The body of the gun is painted safety orange to .2
make people aware that this item might be dangerous. ' -

 

We added a 5 mW 635nm laser sight to help the operator acquire the target. This
addition means that the operator and others nearby must wear safety goggles that are
laser-proof.

4.4 ADAPTABILI TY

Our applicator is multi-functional; it can be used with different products. These could
include a pheromone application for the coddling moth, a deer repellent for rural areas to
impede the spread of bovine tuberculosis, and insecticidal bait.

5.0 TESTING

We ran tests in the laboratory and the field to determine several characteristics of our
final product. When considering which tests to perform, we determined what data our
client and customer would require, what performance information was needed, and how
our finished product would meet our design criteria.

5.1 LAB

In the lab, tests were conducted to find optimal velocity of the dollop as it was fired from
the nail gun and also the percent of the dollop that was lost due to a splatter effect once
the dollop hit its target. Repeated shots were fired from a machined syringe to produce
enough data to estimate how fast the dollop should travel. A graph is shown in Appendix
E. l , which represents the data by which we designed our nail gun.

137

The percent loss testing was done using the unmodified nail gun. We loaded three
milliliters of wax into a barrel clamped in a vise. The three milliliters was weighed
previously. Once the wax was loaded, a nylon plug was inserted after the wax. The nail
gun, loaded with finishing nails, was pressed to the back of the barrel, against the nylon
plug. When fired, the nail would propel the nylon plug to the end of the barrel, where it
would be stopped, and catapult the wax. A target was placed twelve feet away to catch
the propelled dollop. After the wax hit the target, we weighed it and calculated the
percentage of wax that was lost during impact. A graph in Appendix E.2 shows the
results.

A similar loss-test for the modified nail gun showed that less wax was wasted than with
the unmodified gun. Appendix E.3 shows the results. On days when we ran out of wax,
we tried grease. We discovered that grease behaved even better than the wax
formulation; the grease might carry other agents, for other applications, e. g. baits,
repellents, or lubricants. Appendix E.4 shows only about one percent loss for USDA H1
food-grade grease.

Since the chances of consistently hitting the center of the tree trunk are fairly low, we
implemented a target angle test to simulate off-center trunk shots. The wax dollops are
unaffected by targets rotated 30°; and the dollop was able to adhere to targets at an angle

of 60°.

Through other experimentation, we determined that a person could shoot twenty-four
three-milliliter dollops in one minute. The team tested this by rapidly firing the gun as
many times as possible in one minute. This proved to be 24 shots per minute (as
determined by timer calculations in Appendix E.5). We did this several times and we
compared it to the calculation in Appendix E.6. We also measured an insignificant
temperature increase in the barrel during rapid firing; therefore, no characteristics of the
wax will change because of rapid firing. Also, the dollop will stay on the target during
variable weather, temperature, and time.

5.2 FIELD

We were extremely pleased with our field results because we were able to accurately fire
dollops onto apple trees. It became apparent that, because of the accurate sighting and
high launch velocity, the operator was able to shoot through the outer branches of the tree
and hit the target. We also learned that the gun was capable of hitting smaller branches
of about a 5 cm (2 in) diameter, which were higher than 1.5 m (5 ft) from the ground.
This proved to be an important discovery. While the Oriental Fruit Moth will mate at any
height of the tree, Codling moth is known to mate toward the top of the fruit tree. This
has been one of the difficulties of using a behavior disruptor with Codling Moth.

Our client, the Chemical Ecology Group, has done some preliminary field-testing with

our system. They showed that our gun is able to produce a desirable dollop, and it is
significantly faster than competing techniques. They report that our gun was fun to use.

138

 

 

6.0 ECONOMIC ANALYSIS
6.] PROJECT COST

During the construction of this product, all costs were recorded. The total materials cost
was $915. Many of the materials used were stock items from the Research and
Development Shop; we replaced what we used. The nail gun itself was inherited from a
previous project, but its replacement cost is included. A complete accounting appears in
Appendix F. 1.

6.2 COST COMPARISON

We ascertained that our applicator was similar in cost to other applicators on the market.
An accurate price comparison was not possible because of the variability of the cost of
migrant labor. We can, however, justify high equipment costs because our product is a
much faster applicator than competitors’ applicators. This both reduces labor cost and
increases timeliness.

7.0 CONCLUSIONS

Our team satisfied the need of our client for a single operational prototype by 30 April.
We are confident that the device we have built and tested can be marketed. We
recommend the modified cordless nail gun and grease pump system because it can
accurately apply a precise amount of wax to fruit trees. This system is portable and easy
to load, maintain and service. It is cost-effective, and it is ergonomically correct for 95
percent of both men and women. During testing it proved to be the best applicator
because of its speed and accuracy, while providing safety for the operators.

8.0 RECOMMENDATIONS

The next step in this project would be to apply for a Small Business Research Grant.
With this grant, a low volume production run of the product would produce
approximately twenty-four units. Twelve of these units would be sold and distributed to
apple and peach growers and another twelve units would be distributed to twelve
researchers studying this issue. This distribution would allow feedback for future
improvements, and allow the manufacturer to introduce the product into the market. A
patent disclosure has been filed through MSU.

139

 

 

P‘S‘PP’N?‘

9.0 REFERENCES

Paslode Impulse Cordless Finishing Nailer operating manual, #IM250 F—16 II.

Macromatic Controls LLC, Time RangerTM Installation Instructions

Lincoln PowerLuber Model 1200 Series A operating manual

Dr. James Miller of Department of Entomology, Michigan State University
Freddy de Lame of Department of Entomology, Michigan State University
Dieter, George E. 2000. Engineering Design 3rd Edition, McGraw Hill Higher
Education.

, 1986. MIT Humanscale 5a, Ergonomic Design for People at Work.
Eastman Kodak.

140

 

 

10.0 APPENDICES

Appendix 1: Specifications

Paslode Impulse® Cordless Finishing Nailer IM2SOII

 

Physical Description

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lerggth 11 in
Width 3 3A in
Height 12 % in
Weight 6.75 lb
Functional Description
Battery
6 V Nickel-Cadmium
4,000 shots per charge
2 hours to recharge
Fuel Cell
Liquid Hydrocarbon
2,400 shots
Operating Range
20 °F to 120 °F
Hand Use
Ambidextrous
Lincoln PowerLuberTM 1242
Physical Description
Length 10 1A in
Width 3 in
Height l9 1/2 in
Weight 7.9 lb dry
Functional Description
Battery
12 V Nickel-Cadmium
400 shots per charge
1 hour to recharge
Loading
Cartridge (130 shots)
Filler pump
Suction
Hose*
3/8 in Reinforced PVC
sprayer hose
Fittings

 

 

 

Openjgort quick disconnect

 

141

 

 

 

Lincoln PowerLuberTM 1242 (cont.)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Timer
Macromatic Time RangerTM
Operating Range
0 °F to 120 °F
Backpack Frame
Physical Description
Length 15 in
Width 6 1/2 in
Height 31 1/2 in
Weight 3 lb
Functional Descgition
N on-gender specific

 

 

 

 

Universal Pistol Laser Sight

 

 

 

 

 

 

 

 

 

 

 

 

Physical Description
Weight 1.2 oz
Functional Description
Battery
Uses 3 A76 batteries
Lasts for 900 shots
Operation
GaA lAs diode at 635 nm
5 mW of continuous power

 

Total System

 

Physical Description
Weight 18 lb dry
20 lb field ready

 

 

 

Limiting Factors
Amount of wax

 

 

Lincoln PowerLuber can
carry 130 shots

 

Battery

 

Lincoln PowerLuber can
pump 400 shots

 

Operating Range

 

 

40 °F to 100 °F

 

 

 

* The hose used for this application had the largest possible diameter. This was a factor
because the wax that was used was a non-Newtonian shear sensitive fluid. If a smaller
diameter hose were used, the wax would loose its adhesion and become flaky.

142

 

 

Apgndix 2: System Diagram

Appendix 2.1 System Cycle

 

Don Safety
Equipment and
Carrying System

 

 

 

 

3

Load Fuel
(2,400 shots)

1

Load Fresh Paslode Battery
(4,000 shots)

H

Load Fresh Pump Battery
(400 shots)

1+ /""\

Load Wax Cartridge
(130 shots)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Last
Tree?

 

Cock

 

 

 

 

Aim

 

 

 

 

 

 

 

Auto. Fire

Load N N
R I
(3mL) e ease

 

 

 

 

 

 

 

N Trigger

? Shot 8-
Fired? Cocking
Mech.

  
   
 

  

Wax in
Barrel ?

 

 

 

Y

r\ —Fielease Trigger
8- Cooking Mech.

 

 

 

 

 

 

 

Y

 

 

Return Unit to Shop
for Troubleshooting
and Maintenance

 

 

143

 

 

 

Appendix 2.2 P-V Diagram

Conceptual Pressure vs. Volume Diagram
For a Paslode Nail Gun

 

T

Percent oF CyUndar Pressure

 

 

 

 

Percent of Volume

144

 

Appendix 3: Drawipgg lflmensions in Inches)

*Drawings are computer generated
All diameters and bores: Tolerance = i 0.0005 in
All lengths: Tolerance = :l: 0.005 in

Appendix 3.1 Machined Piston

 

 

[0.03130

0.1.000 3

"‘i

4.3?“50

 

 

 

“NH

 

 

 

 

 

  

0.0900 0.2500
003350 05350
11 II 11
9'2?
11 n H i ”0““ J
*15000'.‘
0.1350
fifl-lEEIO— E13500

 

 

 

145

 

Appendix 3.2 Machined Barrel

 

 

 

 

 

 

 

 

I
lllllllll

II

 

 

Loading Port

146

 

 

 

Appendix 3.3 Cocking Mechanism

 

.1

fl

7

 

 

 

 

 

 

 

 

 

 

 

 

147

Appendix 3.4 Battery Cover

 

.“7

a
._l

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

148

 

Appendix 3.5 Base Plate for Grease Pump

 

c3.
..___ 1"! ________________....
LI”)
-, r "‘y
W I77
d—u— CD
'3 U‘J
m ("7
-O-——-—- ‘If --—-l-
\D _
- L.)
"“ c—1
r-l r" +-
1"-
D
(D
0—4
v—J
V.
:3

 

 

 

 

 

 

6.5300

 

 

 

 

 

 

 

 

149

0 Id.

 

 

Appendix 3.6 Backpack Frame Attachment Plate

.0..
O

 

 

[U

01

 

 

HEEL
FEE-351 —Tr

‘\

\—0.8‘

 

 

KL

 

 

a
3.5
0 o [

 

 

 

150

 

Appendix 4: Electrical Diagrams

 

 

 

 

 

 

 

 

F‘— ‘T" “T" “‘ T“ “T “‘“i
l , _ l
I__ .... l'I'LL’liE .... ...:
E3 E3 8 Quick Dlsccnnect
i‘ ____________ ‘x ————————— "l
a/J— 7 11

 

 

 

 

 

 

 

 

 

 

I GREASE GUN TRIGGER

ice @—
TRIGGER

854 @—

 

 

 

 

 

 

 

 

151

r ' "2 . s.“
-I

 

 

Appendix 5: Calculations
Appendix 5.1 Calculation of Dollop Velocity (Modified Nail Gun)

*Measured velocity from digital camera in inches per hundredth of a second

22in *3600s* 1mile =125.0miley
0.013 lhour 63360in hour

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

v (in/0.01s) v (mph) 1 l l [ I
22 125.0 1 4 I ‘ 4 - __
23 130.7 Paslode Cordless Nailer (Modified)
22 125.0 :
21 119.3 160.0 _
21 119.3 140.0
22 125.0 120 0 . F047 __
24 136.4 E '
22 125 o 3 100° —
. ... L_
21 119.3 g 00.0 __
24 136.4 8 60.0 __
24 136.4 g 400 L.
23 130.7
2 . 2.0
21 119.3 0 0 __~
19 108.0 0'0 . . .
24 1364 0 5 10 I5 20 T—
23 130.7 Trial Number T’”
23 130.7 _ c“
24 136.4
Mean 127.2
Standard
Deviation 8.1

 

 

 

 

 

 

 

 

 

 

 

Stheviation

J17
8.1

Velocity: 127.2: 2 — = 1272:3132 (mph)
l0)

 

95% Confidence Interval = Mean 1- 2( j, where N is the number of trials

With a ninety five percent confidence level, the velocity of the dollop is calculated to be
between 123.4 mph and 131.0 mph.

152

 

 

Appendix 5.2 Calculation of Percent Loss (Pre-Modified Nail Gun)

OriginalMass — F inalMass

 

*100% = PercentLoss

 

 

 

OriginalMass
2.4 — 1.61
g——g *100% = 33%
2.4g
Original Final Percent
mass (9) mass (9) Loss Paslode Cordless Nailer (Pro-Modified)
2.4 63%
2.4 1.30 46%
2.4 1.30 46%
2.4 1.01 58%
2.4 1.61 33% 3
2.4 1.01 58% g
C
Mean 1 .18 51% :3
Standard °'
Deviation 0.27
Trlal Number

 

 

 

[MDe—j‘llmmn] , where N is the number of trials

95 % Confidence Interval = Mean i 2

Final Mass: 1.18 i 2[%) = 1 . 18 i 0.220 (grams)

With ninety—five percent confidence, the final mass of the wax dollop is between 0.96
grams and 1.40 grams. This converts to a percent loss between 41.7% and 60.0%. The
fifth trial showed the least amount of percent loss. During this trial, the nail gun was shot
immediately after cocking, and the fuel and air did not have the proper amount of time to
mix. This resulted in a lower power output. Overall, this testing showed that the power
of the nail gun would have to be reduced for application purposes.

153

Appendix 5.3 Calculation of Percent Loss (Modified Nail Gun)

OriginalMass — F inalMass
OriginalMass

 

*100% = PercentLoss

34.8g —29.5g “00% = 15%
34.8g

3mlwaxdollops (10)

Original Final Percent

"1355(9) "1355(9) '063
34.8

 

 

25.8 26 Paslode Cordless Nailer (Modified)
34.8 28.2 19
34.8 29.5 15 35
34.8 27.9 20
Mean 27.9 20
in
Standard 3 I3 ml
Devratlon 1.53 3
5 I 2 ml
§
1:.

2 ml wax dollops (10)
Original Final Percent
mass (9) mass (9) loss

 

 

 

 

 

 

26.1 21

26.1 19.1 27

26.1 21.4 18

26.1 18.3 30
Mean 20.0 24

95 % Confidence Interval: Mean i {mijvfi'flnflj , where N is the number of trials
. 1.53
Final Mass: 27.9 i 2 W = 27.9 :1: 1 .53 (grams)

With ninety-five percent confidence, the final mass of ten 3 ml wax dollops is between
26.37 grams and 29.43 grams. This converts to a percent loss between 15.4% and 24.2%.
With ninety-five percent confidence, the final mass of ten 2 ml wax dollops is between
18.51 grams and 21.49 grams. This converts to a percent loss between 17.7% and 29%.
These tests show decreased percent loss of wax with the modified nail gun. The tests
also showed that using 3 ml dollops resulted in less loss than using 2 ml dollops.

154

Appendix 5.4 Calculation of Percent Loss (Modified Nail Gun)

OriginalMass — F inalMass
OriginalMass

*100% = PercentLoss

 

 

23.5g - 23.2g *100% 21%

 

 

 

 

 

 

 

23.53
3 ml grease dollops (10)
Original Final Percent Paslode Cordless Nailer (Modified)
mass (9) mass (9) loss

23.5 23.2 1 2

23.5 23.5

23.5 23.7 -1 1 -

23.5 23.3 8

3 1

Mean 23.4 0 ‘5
Standard g 0 ~ 1 2
Deviation 0.22 °-

 

 

 

 

 

 

Trlel Number

 

 

 

R Food Grade Grease

 

 

95 % Confidence Interval = Mean i 2[ Sth§1;V_tatton] , where N is the number of trials

 

Final Mass = 23.4 3: 2(22) = 23.4 i- 0.220 (grams)

4;

With ninety-five percent confidence, the final mass of ten 3m] dollops of USDA H1
food-grade grease is between 23.18 grams and 23.62 grams. This converts to a percent
loss between 1.4% and —0.5%. These results show a significant reduction in percent loss
compared to the wax formulation. We recommend using a pheromone medium with fluid
properties similar to those of the food-grade grease. This will minimize the amount of
pheromone loss during application.

 

155

Appendix 5.5 Calculation of Timer Setting

Grease pump feeds at 76.4 milliliters per minute (Lincoln Operator’s Manual)

76.3.ml* 1min =1.27m%
1min 6OSec 53C

lsec
1 .27ml

* 3m] = 2.4 sec

 

Therefore, the timing relay must be set for approximately two and a half seconds.

Appendix 5.6 Rapid Shot Calculation

6086C * lshot _ 24shots
1min 2.58ec 1min

 

 

* 2.5 seconds is the time required to load 3 ml into the barrel from the grease pump

156

 

Appendix 6: Economic Analysis
Appendix 6.1 Prototype Production

 

 

 

 

 

 

 

 

 

 

...”.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Item Cost
Propulsion System
Paslode lmpulse® Cordless Finishing Nailer IMZSOII $379
Piston $22
Barrel $26
Feeding Collar $5
Battery Cover $9
Cocking Mechanism $14
| Total Cost $455
‘ Feeding System
Lincoln PowerLuberTM 1242 $200
Macromatic Time RangerTM $60
Switch and wiring $20
Base plate $3
Project box cover $8
Reinforced PVC sprayer hose (3/8 in) $9
Open-port quick disconnect $10
I Total Cost $310
Carrying System
Backpack frame $50
Attachment plate $5
Grease gun bracket $5
[ Total Cost $60
Sighting System
Universal Pistol Laser Sight $80
Attachment piece - $10
1 Total Cost $90

 

 

 

‘ Total System

 

Materials and modifications
- Labor

  

157

 

— $115

 

Appendix 6.2 Low Volume Production

   

      

Total System

Production Cost* $900

 

 

     

    

 

" if if i * —ce** —sz4oo

 

*Assumes volume discount on component purchases and production costs

 

**Calcu1ated using known formula that selling price is two and a half to three times more ;_
than the direct material and labor costs r

The next logical step to be taken with this project, from an economical standpoint,
would be to produce 24 units using a Small Business Research Grant. The products
would be sold and distributed to twelve researchers and twelve growers for feedback
purposes. Afterwards, any necessary changes could be made to the design and the
product would be further refined.

The selling price could be further reduced by expanding the market for this
product. Currently, other areas of use have been considered, including Codling moth
behavioral disruptor, deer repellent, and insecticidal bait.

158

 

APPENDIX C

Record of Deposition of Voucher Specimens

159

 

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.: 2003-08

Title of thesis or dissertation (or other research projects):

IMPROVING MATING DISRUPTION PROGRAMS FOR THE ORIENTAL FRUIT
MOTH, GRAPHOLITA MOLESTA (BUSCK): EFFICACY OF NEW WAX-BASED
FORMULATIONS AND EFFECTS OF DISPENSER APPLICATION HEIGHT AND
DENSITY

Museum(s) where deposited and abbreviations for table on following sheets:

Entomology Museum, Michigan State University (MSU)

lnvestigator’s Name(s) (typed)
Frédérigue M. de Lame

 

 

 

Date 14 August 2003

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in North America.
Bull. Entomol. Soc. Amer. 24: 141-42.

Deposit as follows:
Original: Include as Appendix C in ribbon copy of thesis or dissertation.

Copies: Include as Appendix C in copies of thesis or dissertation.
Museum(s) files.
Research project files.

This lonn is available from and the Voucher N0. is assigned by the Curator, Michigan State
University Entomology Museum.

160

 

Appendix C.1

Voucher Specimen Data

Page 1 of 1 Pages

 

 

 

meow 339.2. 3. 900

 

 

2.2025 990 598.2 05 c. .088

 

.o. 9.0:..0000 .020: 0250 05 u0>_0o0m

 

moéoow .oz

.0co:o>

080.. 0c .2 039.000.“.

6098 30:52 9.209.005.

9.000000: : 0.0050 .0550? 002

 

«com .8853

00.80 E0: 0060.60
.8200 E9. meow .0392 m “$.32
00.000 So: Sow .0392 ”0020..

..8 58.2. :2 0:320.

 

 

 

 

3w: 9 cm 08.9.80 5.0000: 0.9.0.2 .90.... 0.00.2: 03050.0
m e m s e
u r ..n. n a “0:02.00 9.0 000:
m m M m N .0 “3080.60 0:08.009». .0. Sun .30.. 29:3 .050 .0 00.003

 

 

 

 

161

   

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