BOLL WEEVIL: EFFECT OFDIFLUBENZURON ON REPRODUCTION

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University Microfilms

International 300 N. Zeeb Road Ann Arbor, Ml 48106

1325275

Miller, Gina Teresa

BOLL WEEVIL: EFFECT OF DIFLUBENZURON ON REPRODUCTION

The University of Arizona M.S. 1985

University Microfilms

International 300 N. Zeeb Road, Ann Arbor, Ml 48106

BOLL WEEVIL: EFFECT OF DIFLUBENZURON

ON REPRODUCTION

by

Gina Teresa Miller

A Thesis Submitted to the Faculty of the

DEPARTMENT OF ENTOMOLOGY

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 8 5

STATEMENT BY AUTHOR

This thesis has been fillment of requirements for University of Arizona and is Library to be made available the Library.

submitted in partial ful-an advanced degree at The deposited in the University to borrowers under rules of

Brief quotations from this thesis are allowable without special permission, provided that accurate acknow­ledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED:

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

Theo F. Watson Professor of Entomology

L // s's-(/ Date

ACKNOWLEDGEMENTS

I would like to express my most sincere

appreciation to my major professor, Dr. Theo Watson, for

his guidance and encouragement throughout the duration of

this research. I wish to thank Dr. Leon Moore and Dr.

Larry Crowder for serving with Dr. Watson on my committee

and for their valuable suggestions while reviewing the

manuscript.

My colleague, Doug Bergman offered significant

insight and support throughout the project for which I am

very grateful. I wish to thank my sister, Myrna Paul, for

her patience and understanding while typing the manuscript.

Special thanks go to Michael Trosset, in the Department of

Statistics, for his expertise in developing the unique

mathematical function for analysis of pertinent data.

My brother, Keith Miller, and my mother, Reenie

Breece Miller, through their wit and wisdom provided the

much appreciated positive atmosphere conducive to the

completion of the project.

iii

TABLE OF CONTENTS

Page

LIST OF TABLES v

LIST OF ILLUSTRATIONS vi

ABSTRACT vii

INTRODUCTION 1

Status of the Boll Weevil in Arizona 1 Diflubenzuron 4 Objectives 19

MATERIALS AND METHODS 20

Weevil Dip 20 Boll Dip 21

Constant Exposure 21 Limited Exposure 22

Tarsal Contact 22 Statistical Analysis of Data 23

Weevil Dip 23 Tarsal Contact 24

RESULTS 25

Weevil Dip 25 Adjuvant Effects 25 Diet and Age Effects 27 Concentration Effects 27

Boll Dip 33 Tarsal Contact 37

DISCUSSION 39

REFERENCES CITED 46

iv

LIST OF TABLES

Page

1. Acute mammalian toxicity of technical and formulated diflubenzuron 15

2. Percent (%) egg-hatch inhibition and days to maximum inhibition for boll weevils dipped in diflubenzuron with (W/A) and without (W/0) adjuvant 26

3. Linear regression coefficients of recovery for boll weevils dipped in diflubenzuron with adjuvant 28

Linear regression coefficients of recovery for boll weevils dipped in diflubenzuron without adjuvant 29

5. Percent (%) concentrations required for 60. 80 and 100% egg-hatch inhibition (1-20 days) for boll weevils dipped in diflubenzuron either collected in the field or reared on artificial diet 30

6. Percent (%) egg-hatch inhibition for young and old boll weevils dipped in diflubenzuron ... 31

7. Summary statistics of concentration effects on boll weevils dipped in diflubenzuron using the TDE equation 34

8. Egg-hatch inhibition for boll weevils feeding on bolls dipped in a 0.05% concentration of diflubenzuron for 2-10 days 35

9. Egg-hatch inhibition for boll weevils feeding on bolls dipped in a 0.025% concentration of diflubenzuron for 1 or 2 days 36

v

LIST OF ILLUSTRATIONS

Page

1. Comparative chemical structures of dichlobenil, DU 19111. and diflubenzuron 5

2. Biosynthetic pathway of chitin synthesis 18

3. Egg-hatch inhibition percentages for four concentrations of diflubenzuron over 1-22 days posttreatment using the TDE equation 32

4. Total (1-10 days) egg-hatch inhibition percentages for tarsal contact experiment with 95% confidence intervals 38

vi

ABSTRACT

In the laboratory, diflubenzuron (1-(2»6-difluoro-

benzoyl)-3-(4-chlorophenyl) urea. Dimilin^, TH-6040) was

effective in preventing hatch of eggs laid by female boll

weevils. Anthonomus grandis Boheman, after three different

methods of exposure: dipping, ingestion, and tarsal

contact. Dipping female boll weevils in concentrations of

diflubenzuron ranging from 0.01 to 0.10% resulted in an

average (1-20 days) egg-hatch inhibition from 23 to 98%.

respectively. Limited exposure (1-20 days) of female boll

weevils to bolls dipped in concentrations of 0.025 and

0.05% diflubenzuron resulted in average (1-20 days) egg-

hatch inhibitions of 66 to 100%. Exposure of female boll

weevils to a dry film of 0.10 to 0.65% concentrations of

diflubenzuron resulted in 12 to 66% average (1-10 days)

egg-hatch inhibitions. The experimental adjuvant, UA 101,

used in conjunction with diflubenzuron increased egg-hatch

inhibition at concentrations at or below 0.025% difluben­

zuron and decreased inhibition at concentrations above

0.025% diflubenzuron.

vii

INTRODUCTION

Status of the Boll Weevil in Arizona

The boll weevil. Anthonomus grandis Boheman, has

been an economic concern in the United States since the

1890's (Cross 1976). State and Federal regulations banning

stub cotton (ratoon) and setting mandatory plow-down dates

prevented the establishment of economic infestations in

Arizona cultivated cotton, Gossypium spp. (Fye 1968; Fye

and Parencia 1972). In 1978, these regulations were

relaxed and by 1981, widespread economic infestations of

boll weevils (Mexican form, after Burke 1968) occurred in

southwestern Arizona cultivated cotton (Bergman,

Henneberry, and Bariola 1982).

Female boll weevils oviposit in squares and bolls

by puncturing the petal or carpel and inserting an egg into

pollen sacs or developing lint. The puncture may be sealed

with a moisture barrier of frass (Mitchell and Cross 1969).

Larvae developing in squares and bolls are protected from

conventional non-systemic insecticides as well as predators

and parasites. Therefore, control measures must be

directed toward the adult stage.

Overwintering adults migrate into seedling cotton

early in the season and can successfully lay eggs in

1

2

ten-day-old squares. Southeastern investigators have had

success controlling boll weevil populations by applying

insecticides just prior to the appearance of cotton squares

(Fye, Hopkins, and Walker 1961). In 1983, the University

of Arizona Cooperative Extension Service recommended

applying insecticides just prior to the appearance of the

first cotton squares susceptible to boll weevil oviposition

(Moore 1983). Low weevil populations and the tendency to

cluster make field sampling in seedling cotton ineffective.

Therefore, the recommendation for determining the need for

early season insecticidal control rests with 2 criteria.

Treatment is suggested in any field which was infested the

previous fall or if one boll weevil is caught in

pheromone-baited traps in the two-week period prior to the

first ten-day-old squares (Moore et al. 1984). Recommended

insecticides for early-season control include

D azinphosmethyl (Guthion ), methyl parathion, malathion,

phosmet (Imidan^), and oxamyl (Vydate^) (Borth 1984). The

number of applications necessary (2-3) depends on the

density of the overwintering population.

There are several drawbacks to early-season control

strategies using organophosphate and carbamate insecti­

cides. Early-season applications coincide with peak

numbers of beneficial insects in Arizona cotton fields

(Bergman et al. 1980). Many species of insect predators

3

feed on the eggs and larvae of the tobacco budworm,

Heliothis virescens Fabricius (McDaniel and Sterling 1979).

Natural control such as this serves to delay or even

prevent economic infestations of the tobacco budworm, as

well as other potentially damaging secondary pests. Rummel

et al. (1979) reported that early season applications of

azinphosmethyl in west Texas resulted in high mortality of

insect predator populations. Not only are organophosphates

detrimental to beneficial species, Ware (1983) stated that

this class of insecticides is the most hazardous to humans

in terms of dermal toxicity.

Another major concern with prolonged use of these

chemicals is the threat of resistance. Roussel and Clower

(1955) reported boll weevil resistance to certain

chlorinated hydrocarbons in Louisiana after 10 years

wide-scale use of these chemicals on cotton.

Organophosphates became the replacement insecticides in

boll weevil control in 1956. Resistance to the organo­

phosphates has not been documented in the boll weevil;

however, Graves et al. (1967) present data which show the

potential for resistance and cross-resistance to this class

of insecticide. Preliminary data in south Texas indicate

that boll weevils have already developed some levels of

resistance to some of the standard organophosphate

materials (Kepple 1984).

4

Pi flubenzuron

Diflubenzuron (1-(2,6-difluorobenzoyl)-3-(4-

chloropheny1) urea. Dimilin » TH-6040) is an insect growth

regulator (IGR) or developmental inhibitor which interferes

with the synthesis of chitin (Mulder and Gijswijt 1973)

which is an essential component of the insect exoskeleton.

Diflubenzuron is an analog of DU 19111 which is a

derivative of the herbicide dichlobenil (Figure 1).

The insecticidal properties of DU 19111 were first

described by van Daalen et al. (1972) at the research

laboratories of N. V. Philips-Duphar in the Netherlands.

These scientists concluded that DU 19111 acted as a

larvacide with stomach-poison activity only. Further in

vivo studies at these laboratories by Post and Vincent

(1973) revealed DU 19111 to inhibit chitin synthesis

without interfering with the synthesis of cuticular

protein. The use of radiolabeled glucose in treated and

untreated larvae resulted in practically no labelled

glucose incorporated in the cuticle of DU 19111-treated

larvae compared with significant amounts in the control

group.

Mulder and Gijswijt (1973) present histological

proof of major disturbances in the endocuticular matrix of

Pieris brassicae (L.) larvae after ingestion of difluben­

zuron. Normal endocuticle of P. brassicae more than

5

CN

D ich loben i l (he rb ic ide )

H I

N C ii

o

D U 1 9 1 1 1

D i f l u b e n z u r o n

Figure 1. Comparative chemical structures of dichlobenil. DU 19111 and diflubenzuron.

6

doubles in thickness within 48 hours of ecdysis whereas it

remains constant in larvae which ingested diflubenzuron.

If treatment starts at least 24 hours before ecdysis, the

newly-formed cuticle will consist only of epicuticular and

exocuticular tissue, which is not properly attached to the

epidermis. This condition results in an unsuccessful molt

because the new cuticle is unable to resist the muscular

traction and increased turgor which occurs during the

molting process. Marks and Sowa (1974) obtained similar

results with diflubenzuron on cockroach leg regenerates as

Post and Vincent (1973) did with DU 19111 on in vivo

studies with P.. brassicae. Diflubenzuron inhibited the

synthesis of chitin but did not appear to upset the other

processes involved in cuticle deposition.

The ovicidal effects of diflubenzuron were first

elucidated by Ascher and Nemny (1974) when they observed

that insect eggs failed to hatch after adults ingested

TH-6040. Subsequently, Moore and Taft (1975) reported the

same effect in the boll weevil, Anthonomus grandis Boheman

after ingestion or contact with diflubenzuron. It was

determined that eggs produced from untreated females mated

with treated males had reduced hatch but was not as

dramatic as the effect on the treated female. McLaughlin

(1976, 1977, 1978) commented extensively on the transitory

nature of these ovicidal effects suggesting that the

7

chemical is either used up in the process of egg

production, is eliminated from the tissues, or is

detoxified in situ. Moore et al. (1978) further confirmed

that maximum inhibition did not occur immediately. Bull &

Ivie (1980) found the same inhibition pattern and

correlated it with the level of diflubenzuron present in

the eggs. It is their conclusion that secretion of

unmetabolized diflubenzuron into eggs caused the observed

inhibition of egg hatchability.

Several field studies were conducted between 1975

and 1978 to determine the effectiveness of diflubenzuron on

the boll weevil in cotton. Taft and Hopkins (1975) com­

bined TH-6040 at 4 and 8 ounces AI/A (active ingredient/

acre) with an invert sugar bait to obtain a 98% reduction

in adult emergence from infested squares in two-acre exper­

imental plots. Although the number of egg and feeding

punctures was much higher in the fields treated with TH-

6040 than the control fields which were treated with con­

ventional insecticides, the number of shed squares was much

lower. Fye, McMillian, and Hopkins (1959) contends that

squares containing larvae fall more readily from the plants

than those containing only feeding punctures. The results

obtained with implantation of larvae and injection of water

homogenates of second- and third-instar larvae demonstrated

that these two larval instars caused the reaction in the

8

plant that resulted in abscission of the squares (Coakley,

Maxwell, and Jenkins 1969). Injection of eggs or homogen-

ates of first-instar larvae (less than 6 hours after hatch­

ing) gave no difference in abscission from the control.

So, treatments with TH-6040 may permit a much higher pro­

portion of egg and feeding punctures but, because the eggs

do not hatch, much less damage is incurred by the plant.

Laboratory experiments conducted by Lloyd, Wood,

and Mitchell (1977) revealed cottonseed oil as the most

effective carrier for TH-6040 when compared with water, oil

spray (Bayol 72), raw cottonseed oil bait, Cabosil bait,

and invert sugar-molasses bait. Also, total inhibition

of egg hatch was observed for 1 to 7 days after females

were exposed for 3 days to cotton plants treated with a

cottonseed oil formulation at a volume of 3 gallons per

acre. In the field, TH-6040 (4 oz. AI/A) in cottonseed oil

applied every 4 or 5 days from initiation of squaring in

mid-June until late August to a 35 acre field either caused

all eggs to fail to hatch or caused emerging larvae to die

in the first instar. As a result, no first field genera­

tion boll weevils could be detected by in-field trapping

until August 20.

A North Carolina field experiment in 1975 resulted

in 99% reduction in reproduction in diflubenzuron-(2, 4,

and 8 oz. AI/A) treated plots through the F£ generation

9

compared with no-treatment check plots. Twelve foliar

applications were made on a 5 day schedule (Ganyard et al.

1977).

Nine applications at 1 or 5 day intervals of 4

different formulations of diflubenzuron (2 oz. AI/A) were

sprayed on 2 hectare replicated plots of cotton in Mexico.

All treatments were significantly different from the check

and were highly effective in inhibiting boll weevil egg

hatch although there were no significant differences in

effectiveness among treatments (Johnson et al. 1978). This

study revealed peak inhibition to occur after the fourth

application of each treatment and all treatments remained

relatively active for as long as 2 weeks after treatments

ceased.

House et al. (1978) discovered a pronounced dosage

rate response to diflubenzuron. A lower percent adult

emergence was observed in fields treated with the highest

dose and a higher adult emergence in fields treated with a

lower dose. The reduction in weevil emergence was most

pronounced in the generation. This was the first field

study conducted in nonisolated cotton which probably

accounts for the rebound in pest populations during the

latter part of the season. The increased emergence from

field-collected squares during this time was attributed to

movement of untreated weevils into the treated fields.

10

A wide-area field test of diflubenzuron for control

of an indigenous, low to moderate, boll weevil population

resulted in greater than 90% control of reproduction for

the and F2 generations (Ganyard, Bradley Jr., and Brazzel

1978). The test area included 76 fields for a total of 262

hectares. Ten treatments of diflubenzuron (2 oz. AI/A) at

7 day intervals began at the pinhead square stage. Based

on estimated adult boll weevil populations of over-wintered

versus the F generation, there was a slight decline in the

diflubenzuron test area, whereas there were twelve times

the increase in numbers of adult boll weevils in the con­

ventionally-treated area. An assay of incubated square

samples collected during the first 3 weeks of diflubenzuron

application revealed 80% of the mortality occuring during

the newly-eclosed larval stage with the remaining 20% in

the egg stage. During weeks 4 through 9 of the treatment

period, egg-stage mortality increased to 90%, with 10%

occurring during the newly-eclosed larval period. Appar­

ently, there is a cumulative effect which results from

repeated weekly applications. Mortality remained high 2

weeks after the last treatment which confirms the results

of Johnson et al. (1978). A significant aspect of this

test was the early maturity (1-2 weeks earlier) of the

cotton crop treated with diflubenzuron compared with the

cotton crop treated with organophosphate insecticides.

This phenomenon can be attributed to the fact that methyl

parathion and certain other organophosphates have been

shown to delay maturity (Bradley and Corbin 1974) and

extensive honeybee pollination of a cotton crop can result

in early maturity (McGregor and Todd 1956). Honeybee

abundance in the test fields was 6 times that of the

conventionally-treated control area. The restricted or

neglected use of organophosphate insecticides and the

resultant early maturing crop is advantageous for

subsequent cultural control of the pink bollworm,

Pectinophora gossypiella (Saunders), as well as potentially

reducing the number of insecticide applications required to

adequately protect the crop (Watson et al. 1974).

An important attribute of diflubenzuron is its

relatively non-toxic effect on beneficial arthropods in

cotton fields compared with conventional insecticides.

Keever. Bradley Jr., and Ganyard (1977) report no

significant differences in predator populations between

diflubenzuron treated and untreated fields with the

exception of Geocoris punctipes (Say). This was in

contrast to the highly adverse effects of conventional

insecticides upon predator populations. For 6 days after

collection, egg hatch in laboratory-held Hippodamia

convereens (Guerin-Meneville) was significantly lower in

females collected from diflubenzuron-treated cotton fields

than those from untreated fields. Another field study

conducted by Abies, Jones, and Bee (1977) showed no adverse

effect on populations of beneficial arthropods in

diflubenzuron-treated cotton fields. Laboratory studies

did show reduced egg viability, larval survival, pupation

and adult emergence in Chrvsopa carnea (Stephens) after

feeding and topical treatments at 5 ppm diflubenzuron. H.

convergens had similar effects after topical sprays of 7.7

ppm diflubenzuron, but both predatory species gradually

recovered after treatments were terminated. The parasitic

wasp Trichogramma pretiosum (Riley), was not affected in

the laboratory. Several non-target species were observed

in the field after practical applications in fruit

orchards, forests, and marshes. Hives encaged together

with apple trees in blossom which were subsequently

sprayed, revealed that diflubenzuron had no adverse effect

on adult bees, larval development or honey production.

Parasites, predators, and birds in treated orchards were

not adversely affected. Springtail populations decreased

after an excessively high rate (2.24 kg AI/A) of difluben­

zuron was applied to the soil. Earthworms, mites, millipeds

and centipeds were unaffected. In a severe test in a

coastal marsh, during an 18 month period, diflubenzuron was

applied 6 times at mosquito control rates. Of 75 genera

sampled only 5 taxa showed a statistically significant

13

reduction when compared with an untreated population.

Another desirable quality of diflubenzuron includes

its rapid degradation in soil (T 1/2 = .5 - 1 week). This

rate of degradation is highly dependent on the particle size

in the pesticide suspension. An aqueous dispersion of

particles averaging 2 microns is necessary to maintain the

short half life. If the suspension contains a mean particle

size of 10 microns, the half life jumps to 8-16 weeks

(Verloop and Ferrell 1977). The primary degradation

products are 4-chlorophenylurea and 2.6-di-fluorobenzoic

acid. In natural water, the degradation pathways of

diflubenzuron are similar to those in soils» but the half

life is approximately 4 weeks. There is very little

absorption of diflubenzuron in any formulation by cotton

leaves. Diflubenzuron appears to be highly resistant,to

decompostion, either by photodegradation on foliar surfaces

or by metabolism within the leaves. This compound would

most likely be lost from treated foliage through physical

effects such as wind abrasion or rain-washing or because of

the fall of senescent leaves. Metcalf, Lu, and Bowlus

(1975) examined the persistence of diflubenzuron in model

ecosystem organisms which included algae, snail, mosquito

and fish. The lowest concentration of the chemical was

found in the fish at the top of the food chain. The

concentration of the parent compound in mosquito larvae

(Culex) was relatively high which demonstrates the

remarkable affinity of diflubenzuron for the insect cuticle.

Unlike DDT or DDE. diflubenzuron does not show a high degree

of ecological magnification, partly due to its lower lipid

solubility and partition coefficient.

The acute mammalian toxicity of technical and

formulated diflubenzuron is summarized in Table 1. These

products have no appreciable sub-acute inhalation toxicity

as well as very low sub-acute dermal toxicity. A two-year

study on rats revealed higher methaemoglobin levels in both

sexes at 160 ppm with the next lower dose (40 ppm) having no

effect.

Still and Leopold (1978) found no evidence of

metabolism in weevils after surface or injection

applications. They concluded that diflubenzuron was

transferred through the cuticle into the hemolymph and then

excreted unchanged. In contrast. Chang and Stokes (1979)

found that as much as 18.7% of an injected dose of re­

labeled diflubenzuron was excreted in the form of water

soluble conjugates with only minor amounts of the parent

material recovered in the excreta. Bull and Ivie (1980)

report 3% metabolism within 48 hours after topical

application of diflubenzuron. Even though there are

discrepancies among these reports, which could be attributed

to inconsistant techniques, very little diflubenzuron seems

15

Table 1. Acute mammalian toxicity of technical and formulated diflubenzuron. 1/

LD50in mg/kg body weight after 14 days observation

route species sex

technical diflubenz­uron

Dimilin dispersible powder 25% expressed as a.i.

oral mouse male and female

>4,640 >10,000

oral rat male and female

>4,640 >10,000

intraper­itoneal

mouse male and female

>2,150

percuta­neous

rabbit male and female

>2,000 >4,640

1/ Source: Dimilin Technical Bulletin Philips-Duphar B.V. Amsterdam, Holland

to be metabolized. Evidence is presented by Bull and Ivie

(1980) that certain aryl hydroxylations of diflubenzuron,

which facilitate excretion of the intact molecule, also

destroy its biological activity. This information makes it

seem likely that secretion of unmetabolized diflubenzuron

into the eggs caused the observed inhibition of egg hatch.

Diflubenzuron exibits its toxic effect on the boll

weevil as an ovicide on contact with eggs or on contam­

ination of females through ingestion or contact. As the

boll weevil egg is inaccessible in the field, the practical

approach for boll weevil control requires contamination of

the adult female. The larva in the egg deposited by a

contaminated female develops fully but is unable to leave

the egg, though it sometimes ruptures the egg wall

(Grosscurt 1978). At marginal doses the egg hatches

sometimes and mortality occurs in the first larval instar.

The presence of diflubenzuron in the embryo reduces the

rate of production of chitin during cuticle deposition.

There are many hypotheses concerning the molecular

basis of this disruption of insect cuticle. Ishaaya and

Casida (1974) suggested that the reduced amounts of chitin

in diflubenzuron-treated house fly larvae was the result of

enhanced chitinase acivity. However, Deul, DeJong, and

Kortenbach (1978) repeated the experiments using £.

brassicae larvae and found no effect on chitinase activity,

but chitin deposition was affected. Hajjar (1979) presents

strong support for a mode of action involving a direct

metabolic block of chitin synthesis. His in vitro model

system allows direct examination of diflubenzuron action on

chitin biosynthesis. Using isolated adult abdomens of

Oncopeltus fasciatus (Dallas), Hajjar observed a

significant accumulation of {HC} uridine-diphospho-N-

acetylglucosamine (UDPAG) as well as a significant decrease

in {WC} chitin when diflubenzuron was added to {|4C}

N-acetylglucosamine. This indicates that diflubenzuron

blocks a step in chitin biosysthesis beyond UDPAG, possibly

chitin synthase (Figure 2). Mayer, Chen, and DeLoach

(1980) isolated chitin synthase in cell-free preparations

and demonstrated that diflubenzuron had no effect on the

enzyme. The biosynthesis of deoxyribonucleic acid (DNA)

was inhibited in female A.. erandis treated with

diflubenzuron. Ribonucleic acid and protein synthesis were

unaffected. Some of the treated males revealed inhibition

of testicular growth which suggests diminishment of sexual

function may result in part from inhibition of the

biosynthesis of DNA by diflubenzuron. At this time there

is no conclusive evidence for any suggested mode of action

for this developmental inhibitor.

18

GLUCOSE

I GLUCOSE 6-PHOSPHATE

I D-GLUCOSAMINE 6-PHOSPHATE

I N-ACETYLGLUCOSAMINE 6-PHOSPHATE

UDP-N-ACETYLGLUCOSAMINE

CHITIN SYNTHASE

•Jc

POLY-N-ACETYLGLUCOSAMINE (CHITIN)

PROTEIN

LAMINATED CUTICLE

Figure 2. Biosynthetic pathway of chitin synthesis (Marks and Sowa 1974).

19

Objectives

The use of insect pest management techniques are

becoming increasingly important in pest control systems.

The development of resistance* excessive residues in the

environment, high mammalian toxicities, and disturbance of

the beneficial arthropod:pest ratios have all contributed

to the need for the implementation of an insect pest

management program which combines all available techniques

to maintain pest populations below economic levels.

Diflubenzuron is a chemical which has the potential of

being incorporated into an insect pest management program

for control of the boll weevil in Arizona cotton. The

objectives of this laboratory study were to: 1) compare the

effects of diflubenzuron on the boll weevil using 3 methods

of,exposure: dipping, ingestion, and tarsal contact; 2)

determine the relationship between concentration and the

rate of recovery; and, 3) determine the effect of an

experimental adjuvant used in conjuction with

diflubenzuron.

MATERIALS AND METHODS

Weevil Dip

The original intention of this experiment was to

hold constant all variables except concentrations of

diflubenzuron and use of an experimental adjuvant (.025%

UA101) (Carasso and Briggs 1982). However, complications

in the artificial rearing of boll weevils and the limited

availability of field-collected weevils necessitated

c l a s s i f y i n g w e e v i l s a s y o u n g ( 1 - 2 we e k s ) o r o l d ( 2 - 7

weeks), and also as field-reared or artificially-reared.

The artificially-reared weevils were originally collected

in pheromone traps placed in cotton fields near Yuma,

Arizona. Larval development was completed on a cottonseed

meal diet (Hilliard and Keely 1984). Field reared weevils

were dissected from bolls as pupae. Newly-emerged adults

were placed with squares and bolls in .5 liter containers

in a 50:50 sex ratio and held in a growth chamber main­

tained at 30°C and 50% RH with a 14L:10D photoperiod.

Technical diflubenzuron (95% pure) was dissolved in

a 50% acetone:water solution. Using forceps, female

weevils were dipped in the opaque suspension for 5 seconds.

Immediately following the dip, the weevils were placed on

tissue wipes and dried with a fan. Twenty females were

20

21

dipped per treatment. Twenty control weevils were dipped

in a 50% acetone:water solution. All weevils for each

treatment were placed together in plastic cups with a

minimum of one boll per 4 females. Every second day for 20

days, bolls were dissected and eggs transferred to

moistened filter paper in 9 cm diameter plastic petri

plates. Eggs were held at 25°C, 50% RH, and a 10L:14D

photoperiod. Filter paper was moistened daily to minimize

dehydration and collapse of eggs. Eggs were monitored for

5 days after dissection from bolls to determine if hatch

had been inhibited.

Boll Dip

The boll dip technique was similar to the weevil

dip technique except that 12 - 20 day old bolls were dipped

in the diflubenzuron suspension.

Constant Exposure

Twenty females (2-7 weeks old. F^) per treatment

were placed with the treated bolls and subjected to the

same conditions as in the weevil dip experiment. Fifty

bolls were treated for each concentration tested at the

onset of the experiment for replacement every other day for

20 days. Bolls were refrigerated until needed.

Limited Exposure

Twenty females (1-2 weeks old, field-reared) were

exposed for 24 and 48 hours to bolls dipped in a .025%

concentration of diflubenzuron. Eggs were monitored for 20

days posttreatment. Twenty females were exposed to bolls

dipped in the solution without diflubenzuron to serve as

the control. Another experiment involved 2- 4- 6- 8- and

10-day exposures to bolls dipped in a .05? concentration of

d i f l u b e n z u r o n . E a c h t r e a t m e n t i n v o l v e d 9 f e m a l e s ( 1 - 6

weeks old, field-reared and F-| ).

Tarsal Contact

Filter paper circles (9 cm diameter) were placed

in glass petri plates (10 cm diameter) to serve as a tarsal

exposure medium for female boll weevils. Using a pipette,

1 ml of a specified concentration of a diflubenzuron

suspension (50% acetone:water) was metered onto the filter

paper and allowed to dry. Five replicates of 5 fertile

females (pheromone trap collected) were exposed for 24

hours to the treated surface. Twenty-five control weevils

were exposed to the acetone:water solution alone. Females

were immediately transferred to bolls, and eggs were

recovered every other day for 16 days or until egg-hatch

proportions had recovered to control levels.

23

Statistical Analysis of Data

A moving average technique was incorporated to

smooth random fluctuations before a one-variable regression

was performed on various treatment groups to compare

recovery slopes. The moving average technique involved

averaging each datum point with the datum point on either

side in the time series. The first and last data points in

the series remained the same.

Weevil Dip

Confidence intervals (95%) were calculated for the

binomial distribution of egg-hatch inhibition at each time

interval (2 days) and for total egg-hatch inhibition over

time. A 3-parameter mathematical function, Truncated

Difference of Exponentials (TDE), was used to smooth the

observed data of random variation (Michael Trosset,

University of Arizona, personal communication). The

development of this equation was based on 4 biological

assumptions: 1) there is a natural rate of inhibition

which remains constant over time, 2) the inhibition due to

diflubenzuron is proportional to the diflubenzuron levels

in the eggs (Bull and Ivie 1980), 3) diflubenzuron

undergoes 2 processes to arrive at the site of action

(absorption through the cuticle and secretion into the

eggs) and, 4) the absorption and secretion times are

exponentially distributed. This equation was designed to

produce a weighted least squares criterion for choosing the

total amount of egg-hatch inhibition at time t (1-22 days).

The function evaluations were adjusted for a control

egg-hatch inhibition rate of 11.5%. Optimization of

parameters for the equation was accomplished on a VAX

11/750 computer.

Tarsal Contact

Confidence intervals (95%) were calculated for the

binomial distribution of total egg-hatch inhibition over

time.

RESULTS

Weevil Dip

The control weevils in this study yielded an

egg-hatch inhibition rate of 11.5% (±10.8) per two-day

interval.

Adjuvant Effects

Total egg-hatch inhibition (1-20 days post-

treatment) for concentrations at or below .0251 difluben-

zuron with and without experimental adjuvant resulted in

95% confidence intervals which did not overlap (Table 2).

The adjuvant increased total inhibition 16. 20 and 24% for

.025» .0175 and .01% concentrations of diflubenzuron,

respectively. Fewer days were required to reach maximum

inhibition with the use of the adjuvant at the .01 and

.025% concentrations. Maximum inhibition was also increas­

ed with the use of the adjuvant at the lowest concentration

(.01%). Higher concentrations of diflubenzuron (.05 and

.10%) showed the opposite effect. Total inhibition (1-20

days) was lower if adjuvant was added to both concentra­

tions. The adjuvant decreased total inhibition by 21 and

26% for .05 and .10% concentrations of diflubenzuron,

respectively.

25

26

Table 2. Percent (%) egg-hatch inhibition and days to maximum inhibition for boll weevils dipped in diflubenzuron with (W/A) and without (W/0) adjuvant. 1/

% Concen­ Days to Total % tration of Maximum % Maximum Inhibi tion

Diflubenzuron Inhibition Inhibition 1-20 Days

0.01 W/A 96 3 62 (443) 2/ W/0 64 5 38 (282)

0.0175 3/ W/A 100 3 91 (525) W/0 100 3 71 (457)

0.025 W/A 100 1 92 (380) W/0 97 9 76 (564)

1/ Experimental adjuvant UA 101.

2/ Number in parenthesis is the number of eggs checked for hatch.

3/ Older weevils were tested at this concentration.

Concentrations of diflubenzuron with or without

adjuvant affected the rate of recovery in both young and

old weevils (Tables 3 and 4). Adjuvant decreased the rate

of recovery with increased concentrations of diflubenzuron.

Without adjuvant, young boll weevils recover faster at

higher concentrations of diflubenzuron, but similar

comparisons of older boll weevils is less evident.

Diet and Age Effects

Consistantly higher concentrations of diflubenzuron

were required to produce equivalent egg-hatch inhibition in

artificially-reared weevils than for weevils reared in the

field (Table 5).

Diflubenzuron concentrations of .025% produced

higher miximum and overall inhibition for younger weevils

compared to older ones (Table 6). The 95% confidence

intervals for the total inhibition (1-20 days) do not

overlap.

Concentration Effects

Total egg-hatch inhibition (1-20 days) increased as

concentrations of diflubenzuron were increased in all

treatment groups. The Truncated Difference of Exponentials

(TDE) equation was used to fit the observed data in select

treatment groups to make comparisons between concentrations

(Figure 3). The 3 lowest concentrations of diflubenzuron

Table 3. Linear regression coefficients of recovery for boll weevils dipped in diflubenzuron with adjuvant.

Percent Concentration

of Diflubenzuron

Regression

Slope (b)

Coefficients

r

Young

0.01 -4.1 -.98 0.0175 -4.4 -.89 0.025 -1.1 -.89 0.075 -1.6 -.87

Old

0.05 -3.0 -.97 0.10 -2.0 -.92

Table 4. Linear regression coefficients of recovery for boll weevils dipped in diflubenzuron without adjuvant.

Percent Regression Coefficients Concentration

of Diflubenzuron Slope (b) r

Young

0.01 -1.3 -.40 0.025 -3.5 -.97

Old

0.01 -2.6 -.97 0.025 -4.3 -.99 0.05 -2.3 -.89

30

Table 5. Percent (*) concentrations required for 60. 80. and 100% egg-hatch inhibition (1-20 days) for boll weevils dipped in diflubenzuron either collected in the field or reared on artificial diet.

Total * Diflubenzuron Egg-Hatch Inhibition Field Reared 1/

60* 0.0175 0.0375 80% 0.0525 0.0675 100* 0.0875 0.0975

1/ One larval generation

31

Table 6. Percent (*) egg-hatch inhibition for old and young boll weevils dipped in diflubenzuron.

Total * % Concentration Maximum % Inhibition of Diflubenzuron Inhibition 1-20 days

0.01 Old 51 23 (428) 1/ Young 64 38 (282)

0.025 Old 93 54 (322) Young 97 77 (540)

1/ Number in parenthesis is the number of eggs checked for hatch.

32

{• } M M t- M i M M M t M t

-I !T

X, K„

trv a. Tf'X.,

TT\ t X

1

" t

25^-

0

N.j

\ , , N »m

- ̂

J I 1 L

0 5 IS 1 ̂ 23 DAYS POSTTREATMENT

Figure 3. Egg-hatch inhibition percentages for four concentrations of diflubenzuron over 1-22 days posttreatment using the TDE equation.

33

resulted in delays of 4 to 9 days after treatment to reach

maximum effectiveness. The highest concentration reached

peak inhibition on day 2. Maximum percent inhibition and

extrapolated values of the days spent at greater than 50%

inhibition are presented in Table 7.

Boll Dip

The egg-hatch inhibition rate for control weevils

in this study was 6.5% (+5.3) per two-day interval. All 3

concentrations tested (.01» .025 and .05%) yielded total

inhibition rates (3-20 days) above 99% when weevils were

continuously exposed to treated bolls. When female weevils

were exposed to bolls treated with .05% diflubenzuron for

varying lengths of time (2- 6- 8- and 10-days)t overall

inhibition rates ranged from 66% at 2 days exposure to 100%

at 8 days exposure (Table 8). Boll weevils exposed from

4-10 days showed little recovery. Not only was recovery

delayed as length of exposure was increased but the rate of

recovery was decreased. The same experiment was conducted

with a lower concentration (.025%) and exposure times of

1-2 days. This experiment resulted in higher total

percentages of egg-hatch inhibition (1-20 days) than the

higher concentration previously discussed (.05%) (Table 9).

Weevils exposed for only one day to bolls dipped in a .025%

suspension of diflubenzuron began to recover on day 7

posttreatment although the inhibition percentage at day 19

Table 7. Summary statistics of concentration effects on boll weevils dipped in diflubenzuron using the TDE equation. 1/

Treatment Group 2/

Percent Concentration

of Diflubenzuron

Maximum Percent

Inhibition Days 50% Inhibition

Art-Yng-Adj 0.01 98 12.1 0.025 100 63.6

Art-Old-Adj 0.05 79 13.2 0.10 92 20.6

Fld-Yng 0.01 56 4.7 0.025 97 21.4

Fld-Yng-Adj 0.0175 100 17.5 0.075 100 31.1

Fid-Old 0.0175 91 19.3 0.075 99 65.4

1/ Truncated Difference of Exponentials.

2/ Artificial diet (Art) or Field collected (Fid), Young (Yng) or Old (Old), and with adjuvant (Adj).

Table 8. Egg-hatch inhibition for boll weevils feeding on bolls dipped in a 0.05% concentration of diflubenzuron for 2-10 days.

Linear Regression Estimates

Total % Coefficients Days Inhibition Days at Peak

Exposure 1-20 days Inhibition 1/ Slope r

Control 6 (459) 2/ 0 - -

2 66 (169) 10 -6.4 -.99

4 95 (269) 17 -2.5 -.99

6 99 (148) 18 - -

8 100 (62) 20+ - -

10 98 (92) 20+

1/ Inhibition greater than 80%.

2/ Number in parenthesis is the number of eggs checked for hatch.

Table 9. Egg-hatch inhibition for boll weevils feeding on bolls dipped in a 0.025* concentration of diflubenzuron for 1 or 2 days.

Days Exposure

Total % Inhibition 1-20 days

Linear Regression Estimates

Coefficients Days at Peak Inhibition 1/ Slope r

Control 6 (459) 2/ 0 - -

1 90 (247) 14 -1.9 -.90

2 95 (304) 18 -1.4 i •

OO

1/ Inhibition greater than 80%.

2/ Number in parenthesis is the number of eggs checked for hatch.

was 71%. A two-day exposure resulted in initiation of

recovery at 13 days posttreatment with a 19 day inhibition

rate of 89%. Recovery was again delayed as length of

exposure was increased but the rate of recovery was

essentially unchanged.

Tarsal Contact

Concentrations of .10 to .25% did not significantly

increase inhibition compared to the control. The .MO, .55

and .65% concentration showed a significant increase in

total egg-hatch inhibition (1-10 days) over the controls

(Figure 4). These concentrations returned to control

levels by day 12.

100

90

80

70

60

50

40

30

20

10

4.

38

I

• 1 1 i i

, 1 , ,

0.10 0.25 0.40 0.55 0.65

% CONCENTRATION OF DIFLUBENZURON

Total ( 1 -10 days) egg-hatch inhibition percentages for tarsal contact experiment with 95% confidence intervals.

DISCUSSION

The enhancement of ovicidal activity in the boll

weevil with the use of the experimental adjuvant was

encouraging in the laboratory. The findings of Bull and

Ivie (1980) confirm that the inhibition due to difluben­

zuron is proportional to the amount of diflubenzuron in the

eggs. Therefore, the increased inhibition due to the

adjuvant is a result of higher levels of diflubenzuron at

the site of action — the embryo within the developing egg.

McLaughlin (1977) suggests a 1-3 day delay in effectiveness

is due to slow cuticular absorption. The use of the

adjuvant with a .025% concentration of diflubenzuron

eliminated the delayed-time effect as well as increased the

egg-hatch inhibition throughout the posttreatment time

(1-20 days). Unfortunately* the adjuvant reduced the

inhibition due to diflubenzuron at concentrations above

.025%. concentrations at which diflubenzuron alone

exhibited a high egg-hatch inhibition. This antagonistic

effect could render the adjuvant inappropriate for use in

the field because it is unclear how much diflubenzuron boll

weevils assimilate in the field. It would be difficult to

arrive at an effective rate of diflubenzuron used in

39

conjunction with the adjuvant and be assured it would not

act as an antagonist.

The reason for the lack of effect on the time to

reach maximum inhibition at the .0175% level could be

attributed to age of the weevils; these were older than

those tested at the other 2 concentrations. Noble-

Nesbitt (1970) postulated that insects are more vulnerble

to toxicants when applied to thinner, less sclerotized

regions of the integument rather than to thicker more

sclerotized parts. Since newly-emerged weevils have not

completed the sclerotization process, it is conceivable

that the adjuvant might aid the entry of diflubenzuron

through the less sclerotized integument of younger weevils

more rapidly than through the more heavily sclerotized

integument of older weevils.

As the concentration was increased, the rate of

recovery decreased in all treatment groups when the

adjuvant was used in conjuction with diflubenzuron. This

is also the case one would expect without the adjuvant.

The results show the opposite effect in young and old boll

weevils below the .025% concentration. The comparison of

the young weevils includes only 2 concentrations and the

.01% concentration reveals a poor correlation coefficient

(.40). The concentrations tested with the old weevils

however, reveal excellent correlation coefficients. The

rate of recovery, in fact, did decrease when the

concentration of diflubenzuron was increased from .025 to

.05%. The reason for the opposite effect from .01 to .025%

diflubenzuron seems to be attributable to the low overall

inhibition at .01% (23%).

The consistancy of increased inhibition in the

weevils reared in squares and bolls collected in the field,

over those reared for one larval generation on artificial

diet points to a curious possibility. Previous testing of

diflubenzuron in the laboratory has been on long-standing,

artificially-reared colonies of boll weevils. These

results could be understating the effect of diflubenzuron

in the field. For example, the laboratory study conducted

by Moore and Taft (1975) resulted in very high egg-hatch

inhibition levels after dipping in a .10% diflubenzuron

suspension with recovery commencing between 8 and 14 days

posttreatment. The present study shows a greater

effectiveness for the same concentration on weevils reared

for one larval generation on artificial diet. The

significant difference (.05% level) occurs for the time

period of 15-21 days. The present study reveals 100%

egg-hatch inhibition during this time period; whereas,

Moore and Taft (1975) report only a 72% inhibition rate.

It is possible that field-reared weevils, which have not

been selected for several generations on artificial diet,

are more susceptible to the effects of diflubenzuron.

As the concentration of diflubenzuron was in­

creased, average egg-hatch inhibition likewise increased

over the entire posttreatment period (1-20 days). This

measure can sometimes be misleading because of the

transitory nature of these ovicidal effects. For example,

Figure 3 illustrates egg-hatch inhibition percentages for 4

concentrations of diflubenzuron. Concentrations of .025

and .05% appear reversed for the first 6 days

posttreatment, with the .025% concentration exhibiting a

higher maximum inhibition than the .05% concentration. A

possible explanation for this reversal is that some larvae

are able to emerge from the egg and live up to 24 hours

after exposures to marginal concentrations of diflubenzuron

(Ganyard et al. 1978). The technique used in this study

did not incorporate that percentage which may have hatched

and soon died as a result of the diflubenzuron. As a

result, the inhibition percentages recorded for the

marginally effective doses could be understating the total

effect of diflubenzuron on the progeny of the adult female.

Once an effective dose is ingested, the length of

time of its effectiveness is not only dependent on the

amount of the original dose (McLaughlin 1976), but is also

dependent on the exposure time to the original dose. The

variable exposure/boll-dip experiment revealed that the

longer the exposure, the slower the recovery. Again, it is

unknown exactly how much diflubenzuron weevils in the field

are actually ingesting. Therefore, the best way of

ensuring maximum effectiveness would be to make the

chemical available for continuous uptake by the weevil. It

is interesting to note that a comparable concentration of

diflubenzuron produced a much more rapid recovery in the

study undertaken by McLaughlin (1976). McLaughlin used

artificial diet injected into cigarette filter tips for

exposure of diflubenzuron to female weevils rather than the

boll-dip method, but the differences in recovery rates and

commencement time are tremendous. These differences could

be partially due to weevils in the McLaughlin study being

selected for many generations on artificial diet, whereas

the weevils in this study were collected as pupae and

adults directly from squares and bolls in the field.

Another possiblity could be the Mexican

weevil present in Arizona (Burke 1968),

ciently different physiologically to be

diflubenzuron.

Significant increases in total egg-hatch inhibition

compared to the control occurred at .40, .55 and .65%

concentrations of diflubenzuron for ten days posttreatment

in the 2U hour tarsal contact experiment. The two lower

form of the boll

that is suffi-

more susceptible to

concentrations, .40 and .55% exhibited only 15 and 12%

increases, respectively, in egg-hatch inhibition whereas

the .65% concentration yielded a 56% increase over the

control. Even though the .40 and .55% concentrations

revealed significant increases in inhibition compared to

the controls for 1-10 days posttreatment, these differences

are not significant for 1-12 days posttreatment. The .65%

concentration remained significantly different for 1-12

days posttreatment. A .65% concentration is equivalent to

a field rate of 13 oz. AI/A at a volume of 14 gallons/A.

These studies indicate that for tarsal contact, it

would take at least 65X the effective dose of that for

ingestion to produce a comparable amount of egg-hatch

inhibition at 2 days posttreatment. This figure could

conceivably be much higher because the minimum dose

required for 100% inhibition after a 24 hour boll-dip

exposure was not determined. Equivalent average egg-hatch

inhibitions over 20 days posttreatment for the dip

technique would require at least 3X the dose needed after a

24 hour ingestion period.

Very small quantites of diflubenzuron (.01%) are

needed to have an impact on the progeny of the boll weevil

if the chemical is ingested rather than contacted (.65%).

The boll-dip experiment revealed cumulative effects of

continued exposure. Tarsal contact to a lower

concentration may also provide effective egg-hatch

inhibition if exposure to the treated surface is prolonged.

The experimental adjuvant has the potential for increasing

the effectiveness of a tarsally-contacted dose of

diflubenzuron.

The toxic effects of diflubenzuron on the boll

weevil are transitory regardless of the mode of entry.

Increased concentrations of diflubenzuron delay the onset

and rate of recovery. Translated to the fieldi continuous

exposure to diflubenzuon appears to be necessary for

effective long-term suppresssion of boll weevil

populations.

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