THE EFFECTS OF THREE INSECTICIDES ON OOTHECAL-BEARING GERMAN COCKROACH, Blatt.~.ll.a. ~.e..r.m.a..lJ..lla L. • (DICTYOPTERA: BLATTELLIDAE), FEMALES.

by

James Dale Harmon

Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

in

Entomology

APPROVED:

l<&<>"' l y c - w: R.D. Fell W. H Robinson

June, 1987 Blacksburg, Virginia THE EFFECTS OF THREE INSECTICIDES ON OOTHECAL­ BEARING GERMAN COCKROACH. Blattella ~manica L. (DICTYOPTERA:BLATTELLIDAE), FEMALES

by

James Dale Harmon

Committee Chairperson: Mary H. Ross

Entomology

(ABSTRACT)

German cockroach. :6.lattella i,ermanica L •• females .. of resistant and non-resistant strains carrying oothecae were exposed to filter paper impregnated with propoxur. malathion. and diazinon. Premature oothecal drop was monitored during the exposure period and for 24 hours thereafter. Dete.rminations of female mortality were also made 72 h post-exposure.

Oothecae from exposed fema.les were observed for percentage egg hatch. time from exposure to hatch. percentage nymphal emergence. nymphal survival. and the percentage of nymphs able to move about freely 24 hours post-emergence. The comparisons of these factors were made not only on prematurely dropped oothecae but also on oothecae retained by females. and . oothecae that were manually detached from females. Premature oothecae dropped and those manually detached were hatched on an insecticide treated surface.

Premature oot beca 1 drop occurred in a 11 experiments • but was delayed 24 b in expe~iments with organophosphates.

The mortality of treated females which prematurely dropped their oothecae was higher than females retaining them (73% vs. 53%).

Percentage nymphal emergence and survival were reduced when oothecae were placed on an insecticide treated surface.

The significantly higher survival of resistant strain nymphs

(Carver. 60%. and Lynn Haven. 99%) in a comparison to nymphs of a susceptible strain (VPI. 45%) on insecticide treated surfaces provides evidence for resistance in first instar nymphs.

A procedure for the quantitative comparison of the effects of different insecticides on ootbec a 1-bear ing females bas ·been developed. Insecticides which cause a higher percentage of premature oothecal drop may reduce regrowth of a cockroach population when compared to materials which do not. even if the insecticides cause similar mortality. The extent to which population regrowth occurs depends on whether oothecae batch on a treated surface. oothecal age. and environmental conditions. ACBOWLEDGEMEBTS

The culmination of a graduate program is not to be solely attributed to the grad]!ate student. Many thanks go to my committee members for their help and guidance over the last two years, Drs. W"• H Robinson and R.D. Fell. Of course, to my major advisor, Dr. Mary H. Ross, I owe a great debt of thanks for her support and guidance throughout my program.

While the committee members are usually thought of as a major part of a graduate student's career, there are many others within the department to which I will be forever grateful for their help and assistance. Drs. R.L.

Pienkowski and D.E. Mullins for teaching me the skills of being an effective communicator and instructor; Dr. J.A.

Weidhaas for an education in ornamentals and organization;

for all the help in the cockroach lab and the darkroom; and all of the faculty, staff, and students of this department for their support and friendship.

Many thanks also go to Deans R.A. Teekell, M.J.

Johnson, B. LaBerge, and the Graduate Student Assembly for all of their help, support, guidance, headaches given and shared, and for the experiences within the GSA which have greatly helped me in my development as an administrator.

Another debt of thanks is owed to my family and friends outside the Entomology Department for putting up with

l.V someone who studies "bugs and roaches ... Their continued

support in all times was a distinct asset to my graduate experience. Lastly. I would like to thank the members of

the Newman community. especially·

and for their support. guidance and

conversation during my own conversion and development.

v LIST OF TABLES

I able.

4.1. Effect of propoxur on mortality and oothecal drop on female .lL. ,i_ermanica carrying Stage XII oothecae (oot) ••••••••• 34

4.2. Effects of propoxur on hatch of a~d nymphs from Stage XII .lL. ~ermanica oothecae ( oot) •••••••••••••••••••••••••••• 35

5 .1. Effect of ma~athion on mortality and oothecal drop on female .lL. ,i_ermanica carrying Stage XII oothecae (oot) ••••••••• 50

5.2. Effects of malathion on hatch of and nymphs from Stage XII .lL. ~exmanica oothecae (oot) ••••••• ~··•••••••••••••••••• 51

5.3. Effect of diazinon on mortality and oothecal drop on female JL.. iermanica carrying Stage XII oothecae (oot) ••••••••• 52

5.4. Effects of diazinon on hatch of and nymphs from Stage XII .lL. ~anica oothecae (oot)•••••••••••••••••••••••••••• 53

v~ LIST OF FIGURES

Pyrex glass chimney (19 em X 14.5 em · inside diam.) lightly coated with vaseline around'the base and enclosing a treated filter paper •••••••••••••••••••• 23

3.2. Sealed 0.9 1 jar used for placement of prematurely dropped oothecae on treated filter paper until hatch •••••••••• 23

4.1. Nymphs. pigmented and unpigmented. still entangled in'embryonic cuticle 24 h afte~ emergence on a propoxur treated surface •••••••••••••••• 36

4.2. Fully pigmented nymphs being unable to completely shed embryonic cuticle on antennae (65X} ••••••••••••••••••••••••• 36

4.3. Pigmented (~live) and unp~gmented (dead) nymphs still encased in embryonic c u t i c 1 e • • • • • • • • • • • • • • • • • _. • • • • • • • • • • • • • • • • • 3 7

4.4. Nymph still attached~to ootheca by embryonic cuticle encasing metatarsi (27.7X).~••••••••••••••••••••••• 37 TABLE OF COBTEBTS

ABSTRACT •••••••• •-• ••••••••••••••••••••••••••••••••••••••• ii ACKNOWLEDGEMENTS••••••••••••••••••••••••••••••••••••••••• iv LIST OF TABLES~~·•••••••••••••••••••••••••••••••••••••••• vi LIST OF FIGURES ••••••••••••••••••••••••••••••••••••••••• vii

I • INTRODUCTION ••••••• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1 II. LITERATURE REVIEW BIOLOGY OF L i e rman i.s:..a. • ••••••••••••••••••• 3 EFFECTS OF INSECTICIDES ON THE LIFECYCLE OF L ~ermanica. • • • • • • • • • • • • 4 EFFECTS OF INSECTICIDES ON REPRODUCTION AND OVIPOSITION OF INSECTS OTHER THAN COCKROACHES •••••••••• • • • • • • • • • • • • 5 RESISTANCE••••••••••••••••••••••• ...... 6 Physiological Resistance •••• • • • • • • • • • • 6 Behavioral Resistance ••••••• ...... 7 OOTHECAE ••••••••••••••••••••••••• ...... 8 EFFECT OF ENVIRONMEN~ ON OOTHECAL DROP AND HATCH IN L ~ermanica ••••••• 10 EFFECTS OF INSECTICIDES ON OOTHECAL DROP AND HATCH IN L ~ermanica •••••••••••• 12

III. MATERIALS AND METHODS CHEMICALS •••••••• ...... 15 COCKROACH STRAINS ••••••••• • • • • • • • • • • • • • • • • • • • • • 15 TECHNIQUES••••••••••••••••••••••••••••••••••••• 16 Cu l.ture •••••••••••••••••••••••••••• ••••••• 16 Selection of Oothecal Bearing Females ••••• 17 Dosage Determination •••••••••••••••••••••• 18 Tarsal Contact Method••••••••••••••••••••• 18 EXPERIMENTAL PROCEDURES•••••••••••••••••••••••• 19 CALCULATIONS ••••••••••••••••••••••••••••••••••• 20 DATA ANALYSIS•••••••••••••••••••••••••••••••••• 21

IV. EXPERIMENTS USING PROPOXUR INTRODUCTION•••••••••••••• • • • • • • • • • • • • • • • • • • ••• 24 MATERIALS AND METHODS ••••• • • • • • • • • • • • • • • .... • • • 26 RESULTS •••••••••• . ... • • • • • • • • • • • • • • • • • • • • • ••• • • • 26 DISCUSSION•••••••••i••••••• • • • • • • • • • • • •• • • . . • • • 29 v. EXPERIMENTS USING ORGANOPHOSPHATES INTRODUCTION •••••••••• • • • • • • • • • • • • • • •• • • • • . . . .. 38 MATERIALS AND METHODS ••••••••• ...... • • • • •• • • • •• 39 RESULTS ••••••••• •• • • • • • • • • • • • • • • • • • • • • • • • • •• • • • 40 DISCUSSION.•••••••••••• • • • • • • • • ...... • • •• •• • • • 45

VI. DISCUSSION AND CONCLUSIONS...... ~ ...... 54

VII. LITERATURE CITED••••••••~••••••••••••••••••••••••••• 62

l.X l. IBTR.ODUCTIOB.

The German cockroach. Blattellll ~ermanli11 ( L •)

(Dictyoptera: Blattellidae). is one of the most common cockroach pests in human dwellings (Cornwell. 1968). The

German cock roach. t bought to have or ig ina ted in east Asia

(Roth. 1985). has become worldwide in distribution.

The reproductive potential of the German cockroach is such that a population. if uncontrolled. can increase 24 to

28 times in 3 months (Ross. et. al •• 1984). The obvious need for control of this pest has prompted control efforts using insecticides. Selection pressure from such efforts has resulted in the development of resistance to many insecticides. Resistance in the German cockroach has been well known since the early 1950's (Cornwell. 1976).

A considerable amount of information is available on the effects of lethal dosages of insecticides on cockroaches • however. comparably little is known about the effects of sublethal dosages on reproduction and oviposition. The inducement of premature oothecal drop in gravid female German cockroaches has been recognized as an effect of sublethal insecticide dosages. but very little is known about it. The ootheca is the most environmentally resistant stage in the life cycle of the German cockroach

(Van den Heuvel and Shenker. 1965). Information on

1 2

insecticide induced premature oothecal~ drop is very

important to the planning and implementation of pest control strategies. Variations of irisecticide induced premature

oothecal drop both within ,and between resistant and susceptible populations. and how it affects the survival of

eggs and nymphs represent areas in need of study.

The objectives of this investigation were to evaluate

the effects of propoxur. malathion. and diazinon on

premature oothecal drop in susceptible and insecticide resistant strains. Variables studied included female mortality. hatch of oothecae. and survivorship of the nymphs

from oothecae prematurely dropped or retained. 2. LITERATURE REVIEW

In the current literature, the batch of eggs of an ootheca is termed oot beca batch. This terminology will be ' used throughout this manuscript.

2.1 BIOLQGX or B. aeraauica

The German cockroach is a small species of cockroach, that bas been described taxonomically by various authors including Cornwell (1968) and Roth (1985). It prefers a warm environment, 25 to 30 °C, with a relative humidity of

50 to 75% (Cornwell, 1968).

The nymphal development period ranges from 40 to 50 days (Willis, et al., 1958). Adult females mate 5 to 6 days after ecdysis and produce an ootheca 1 to 2 days afterwards.

They live for up to 200 days and produce 4 to 8 oothecae during this period (Gould and Deay, 1940; Cornwell, 1968).

Formation of the ootheca takes 15 hours after oocyte maturation (Tanaka, 1976). Lack of either food or water during the time of oocyte maturation can lead to delayed and decreased fecundity (Kunkel, 1966; Mueller, 1978; Durbin and

Cochran, 1985). Biology of the ootheca is discussed in section 2.5. Low temperatures have been reported to prolong the nymphal period, 1.3 days for each decrease in degrees

Fahrenheit below 68°F, and decrease oothecae formation and hatch (Tsuji and Mizuno, 1972).

3 4

2.2. Effects Of Insecticides On The Lifecycle Of B. aermanica.

Morphological differences 'due to insecticide selection have been reported by various authors. Grayson (1951. 1953.

1954) reported on a DDT and benzene hexachloride resistant and a nonresistant strain of German cockroach. Body size and weight. reproductive success. and size of the egg cases of the resistant strain were significantly lower than those of the susceptible strain (Grayson 1951. 1953. 1954).

Wright (1968) reported similar results using chlordane resistant and nonresistant strains of German cockroaches.

Izutso. et al. (1970) reported insecticide susceptibility was increased when individuals were reared in high population densities.

Third generation insecticides have also been reported to affect the reproductive success of German cockroaches.

Avermectin B1 when fed to German cockroach females at sublethal dosages inhibited mating and reproduction

(Cochran. 1985). Das and Gupta (1974. 1977) reported on the effects of three juvenile hormone analogs on German cockroach metamorphosis and reproduction. They reported treated females ·did not mate and that the ovarioles of these females were either atrophied or hypertrophied. Under these conditions. oocyte maturation is prevented as is the formation of an ootheca. 5

2.3. Effects Of Insecticides On Reproduction And Ovip2Ii~ Of Insects Otlter Than Cogroaches.

Sublethal insecticide treatment of Musca domestics (L.) adults induced decreased reproduction and longevity

(Pimentel. et al •• 1951; Ouye. et al •• 1957; Hunter. et al ••

1958. 1959). Afifi and Knutson (1956) studied the effects of a single treatment of dieldrin on the parent and subsequent three filial generations of the house fly. They reported a 5 to 6 day postponement of mating and a consequent tim• lag in th~ reproduction of the treated flies. The F 1 generation had a significantly higher number of young as compared with untreated controls. but the F 2 and

F3 generations did not show marked dif£erences in fecundity. Knutson (1955) reviewed the effects of insecticides on reproduction in variotis insect species. He tested JL. me lanoiaS ter (L.) on dieldrin and reported that treated adults had a longer lifespan and produced more larvae per fly than the untreated controls. although the numbers of larvae produced per unit time were similar fo~ both groups.

Laboratory selection for insecticide resistance has been reported to suppress fecundity and egg hatch in several insects (Georghiou. 1972). Reduced fecundity and egg hatch occur · in ~.§. aeiypt i (L.) selected with DDT (Abedi. and

Brown. 196 0) • parathion and malathion (Inwang. 1968).

Tribolium castaneum: (Duval) with DDT (Bhatia and Pradham. 6

1968), Musca .d.2mestica (L.) with DDT (Derbeneva-Uhova. et

al •• 1966) and malathion (Harris. et al •• 1961). and

Tetranychus ~~ (L.) with demeton induced resistance

(Dittrich. 1960) •

.2..1LB.eaistance.

Resistance to insecticides is due to the detoxification

of the insecticide material (Georghiou, 197 2). This fact

has led to the characterization of resistance into 2

categories; biochemical artd physiological (Georghiou, 1972).

A third category, behavioral resistance. is not as well known or understood. Reduced penetration and transport of

the insecticide to the site of action. increased storage

capacity at inert sites and faster excretion all contribute

to physiological and/or biochemical· resistance (Georghiou,

1972). Behavioral resistance on the other hand results from

avoidance behaviors. Lockwood. et al. (1984) reported that

the three catego~ies of resistance cannot be thought of as

individual mechanisms. but that each may interact with and

affect the other. Th~y have proposed a model which

recognizes the fundamental continuity of both behavioral and

physiological resistance.

2.4.1 PHYSIOLOGICAL B.ESISTAB.c.L.

Physiological resistance is generally considered to

include the biochemical and physiological categories of 7

resistance. Taken together they may enhance the ability of

the organism to survive an insecticide treatment (Georghiou.

1972).

Resistance has been confirmed in the German cockroach

to DDT (Grayson. 1953). chlordane (Grayson. 1966). and

aldrin and dieldrin (Clarke and Cochran. 1959; Cochran.

1961). As early as 1960. resistance to carbamates (Grayson.

1961) and to organophosphates (Grayson. 1963; Bennet and

Spinks. 1968; Cochran. 1973a. 1975) was apparent. Pyrethrin

resistance has also been documented for the German cockroach

(Cochran. 1913b; Keller. et al •• 1956).

2.4.2 BEHAVIORAL RESISTANCE.

Behavioral resistance can be divided into two basic

groups; (a) stimulus-dependent avoidance behaviors (e.g.

irritability. repellency) which require the sensory

stimulation at the instant the behavior is expressed. and

(b) stimulus-independent avoidance behaviors which refers to

natura 1 avoidance of a certain env ironment or host by a

fraction of the population (Georghiou. 1972). Insecticide

induced selection can enhance both types of resistance.

although increased repellency or irritability relies on an

insecticide stimulus. Other types of behavior patterns which enhance the selection of resistant individuals. but which are not related to the above two groups. may occur in

a population. 8

Repellency and dispersal can be considered as one type of behavioral response to insecticides. Repellency in

German cockroaches has received considerable attention

(Ebeling. et al •• 1966. 1967. 1~68; Cornwell. 1976; Ebeling.

1978t Pluthero and Singh. 1984). Bret and Ross (1986). for example • reported that the d ispers a 1 of a sus cept ib le laboratory strain increased more than that of a resistant field strain in response to exposure to a 1% propoxur formulation in oil.

The effects of sublethal dosages are often not considered when looking at the insecticide resistance of an insect. In crop pests. the exposure to a sublethal dosage of an insecticide which enhance the evolution of behavioral resistance has been reported by Gould (1984). Similar reports of behavioral resistance in the German cockroach were not found.

2.5 OOTBECAia

The ootheca of the German cockroach is 7-9 mm long. tan in color with distinct indentations outlining the individual eggs. Eggs lie in a double row and are covered with a cuticle (Roth and Willis. 1955a). Ootheca formation requires an average of 15 h (Tanaka. 1976). First and second oothecae contain 40 to 50 eggs which hatch 24 to 26 days after formation (Tanaka. 1976). 9

The German cockroach was classified by Roth and Willis

(1954) as having a type 3 oviposition behavior in that the ootheca is. - ••• extruded and carried externally by the female until. or shortly before ' the eggs hatch (<24 hours).-

The ootheca is formed initially with the keel pointing upward. but after completion it is rotated 90° to the right. a position in which it is carried by the female during embryonic development (Roth and Willis. 1954: Willis. et al •• 1958: McKittrick. 1964). unless the ootheca is premature 1 y dropped. Natural premature oothecal drop is rare (Muller and Coch. 1975).

The anterior end of the ootheca is attached to the female within the genital chamber. This end is permeable and some materials pass through it to the embryos (Roth and

Willis. 1955a. 1955b). Roth and Willis (1955a. 1955b) reported the percentage of water within the ootheca remained constant at 62% for 4 days and then increased steadily to

76% by the time emergence occurred. During this period. dry matter within the ootheca decreased only slightly. On the fifth of sixth day of development. the wet weight of the ootheca drops slightly but increases steadily afterwards.

This increase occurs because the embryos are obtaining water from the female (Roth and Willis. 1955a. 1955b).

Cochran (1983) reported that females drink and feed more during oocyte maturation than once the ootheca is formed during the first four oviposition cycles. Females 10

carrying completed oothecae drank once every 3 days as compared to once every 1.1 to 1.3 days for females before formation of oothecae. He hypothesized that the presence of the ootheca in the genital ,chamber of the female serves as an inhibitory stimulus to feeding and drinking as well as to corpora a1lata function~

Neural inhibition over the secretion of the corpus allatum hormone. oocyte development. and subsequent secretion of c~lleterial proteins is related to the presence of an ootheca in the genital opening o~ the female (Roth and

Stay. 1962; Adiyodi and Adiyodi. 1974). It has been sugg~sted that inhibito~y nervous impulses prevent the female from prematurely dropping her ootheca (Roth and Stay.

1962; Adiyodi and Adiyodi. 1974). Premature dropping of the ootheca causes the female to produce another ootheca more

quickly than if the ootheca had b~en dropped normally.

Females not treated with insecticides and carrying unfertilized oothecae may either prematurely drop or carry the oothecae longer than the normal period (Roth and Stay.

1962).

2. 6 EFFI~ OF IHYIRQNMJSNT ON OQTBJSCAL · JtB.Q.l_ ·AID HATC1L.I.IL.L.

sermanica

While humidity has little effect on the number of oothecae dropped • it does play a role in the subsequent hatch of the eggs. Barson and Renn (1983) reported a direct 11

correlation between the age of the ootheca when dropped and

the hatch of the eggs. Older oothecae have a greater

percentage hatch than younger ones. At humidities of 45%

and 70% RH. 21-day old egg cases had a greater proportion

hatch than in 16-day old egg cases. Sixteen day old

oothecae incubated at 27°c. and 70% RH had a higher

percentage hatch than 21 day-old oothecae incubated at 27°C.

45% RH.

Manually detached oothecae only 1-day old or more than

seven days old may complete development and hatch when

incubated at 27°C. 90% RH (Barson and Renn. 1983). If

detached in an environment of 0% relative humidity. 60% of

the water in an ootheca can be lost within 20 days (Roth and

Willis. 1955b).

The amount of water lost from an ootheca varies

considerably with age. Little is lost from eggs of oothecae

detached when one day old. increasing to a maximum value by

six days old. and then falling to a constant level after

seven days (Roth and Willis. 1955a). The fate and hatching

of oothecae prematurely dropped by the German cockroach

depends on three factors: 1) the amount of water present in

the ootheca. 2) the age of the ootheca. and 3) the

saturation deficit of the ambient air (Cornwell. 1968). 12

2A1 EllECT 0! INSECTICIDES OB OOlBECA DRQl ABD BATCH IB B. au:rmanica.

Little is known of the effects of insecticides on the oviposition behavior of the German cockroach. specifically

the effects such as premature drop of the ootheca. and the

subsequent hatch of the eggs. Woodbury (1938) reported that

pyrethrum sprays and n-butyl carbitol thiocyanate caused

adult females to prematurely drop their egg cases. while

rotenone had no apparent effect. Parker and Campbell (1940)

reported similar results. with pyrethrum sprays causing 82 to 95% oothecal drop and n-butyl carbitoi thiocyanate

causing only 10 to 23% drop. At high dosages. both chemicals appeared to have a direct insecticidal effect on

the hatch of the dropped ootheca. Parker and Campbell

(1940) postulated that the difference in oothecae drop

resulted from induced muscular contractions by pyrethrum with the subsequent ejection of the ootheca. while n-butyl carbitol thiocyanate did not cause such contractions.

Parker and Campbell (1940) also reported that· eggs within

. . prematurely dropped ootheca could possibly hatch if given

proper environmental conditions. The age of the oothecae was unknown and the envir6nmental conditions were not

controlled in either of the above studies.

Smittle and Burden (1965) reported that treatment with dichlorvos had no effect on egg case hatch. Whether these

egg cases were prematurely dropped or retained by treated 13

females was not reported. Russell and Frishman (1965)

reported a 50% drop of oothecae in a susceptible and a resistant strain within 24 h after treatment with different dosages of dichlorvos resin st~ips (higher for the resistant

strain). Percentage drop in the susceptible strain was

significantly higher than in the resistant strain

immediately after and 24 h after exposure when both strains were exposed to the same dosage. Percentage hatch of

prematurely dropped oothecae was consistently lower than in

the controls. Muller and Coch (1975) reported that

dichlorvos. trichlorphon. propoxur. and a combination of

pyrethrins. piperonyl butoxide and lindane caused premature

oothecal drop. Dichlorvos significantly decreased hatch in

6 dayold oothecae while none of the insecticides tested had

any effect on 12 or 20 day old oothecae.

Van den Heuvel and Shenker (1965) studied the effects

of diazinon and pyreth~ins plus piperonyl butoxide on

oothecae of dieldrin-resistant females. They reported that

the death of the females (100%) did not seriously affect

hatch. and that all females tested pr~maturely dropped their

oothecae. The fate of these oothecae depended on whether

they remained on a treated surface and on environmental

conditions.

Chadwick and Evans ( 197 3) reported that oothecae five

days prior to hatch were dropped within 24 h after treatfuent with bioresmethrin. In less than 24 h after emergence. 68 14

to 71% of the nymphs that emerged from eggs of the treated oothecae died. In untreated gravid females. 95 to 100% of the oothecae hatched. Thirteen out of 20 'oothecae were dropped 1 h after treatment wl}.en females were treated and allowed to drop their oothecae naturally. Oothecae retained by the females were manually detached and then treated.

Only 4 out of 20 of these oothecae hatched; 90% of the emerging nymphs died soon after hatch. Of oothecae manually detached but untreated. 65% hatched and the mean time to hatching was considerably reduced. Untreated oothecae manually detached had a significantly higher number of nymphs left in the egg cases than in egg cases allowed to complete development normally.

Barson and Renn (1983) reported that 60% of 21 day old oothecae were dropped during a 24 hour exposure period to bendiocarb. Percentage hatch of prematurely dropped oothecae incubated at 25°C. 55-60% RH was significantly higher (P

90% RH) for oothecae manually removed from cockroaches and left in contact with a glass plate treated with bendiocarb.

Within oothecae detached prematurely. the proportion of embryos that failed to hatch at the anterior end of the oothecae was larger than in the controls. 3. MATERIALS ABD METHODS

3.1 CHE)IICALS.e

Chemicals used in this series of experiments included two carbamates. two organophosphates. trichloroethylene. ind

Risella oil. All insecticides were technical grade. The carbamates were propoxur. (0-isopropoxyphenyl met hylcarbama te). One organophosphate was malathion ( 0 • 0- dimethyl thiophosphate of diethyl mercaptosuccinate).. The other organophosphate was diazinon (O.O-diethyl-0-(2-

isopropyl-4-methyl-6-pyrimidyl) phosphorodithioate).

Trichloroethylene (CHCL:CCL 2 ) was used as a solvent for all prepa~ations. It leaves no residues after evaporation and volatilizes rapidly upon exposure to air. All active

ingredients were highly soluble in trichloroethylene.

Risella oil. a viscous oil produced by the Shell Oil

Company. was used as the carrier oil.

~2 COCKROACH STRAIBSe

German cockroach. Blattella .iJU,m~.a.. females were

obtained from the VPI strain. a laboratory susceptible

strain that has been in culture for approximately 180

generations. Field resistant strains included the Carver

strain. collected in Gainesville. Florida. in 1983 (LT50 determinations. l7.4X level resistance to propoxur. >60X to malathion. >90X to bendiocarb. and >SOX to pyrethrins

15 16

(Cochran~ persbnal co~municatiori)) and the Lynn Haven

strain. collected in Lynn Haven. Virginia in 1983 (LT 50

determinations. 7.6X level resistance to diazinon (Cochran~

personal communication)). A' susceptible field strain.

(Fairbanks). collected in Fairbanks. Alaska in 1985 was also

tested on propoxur for comparative prirposes.

l....L.U.C.lUIIQUBS •

3 • 3 8 1·· CULTURB •

Cockroach rearing conditions were as follows: 25-27°C.;

14D:l0L photocycle; and ambient humidity (approximately 55%

R.H.). Stocks were maintained in glass battery jars (3.8

1). Smaller jars (0.9 1) were used for single or small

groups of females. Sections of wire screen provided

barb or age • and water and dog food pellets· were available ad

libitum. Jars were cleaned • and water and food changed

weekly to prevent any accumulation of frass and exuvia which

can possibly lead to fungal infections or mite infestations.

Any colonies found to be infected or infested were

immediat~ly destroyed.

3 8 3 8 2 SBLBCTION QE OOTBBCAL-BBARIBG EBMALBS~

First· stage nymphs were separated each week from the

stock colonies or jars containing oothecae-bearing females.

This was accomplished by ligh'tly anesthetizing the 17

cockroaches within the jar~ with co 2 • placing them on a no.

7 wire acreen sieve. ~nd shaking gently. The nymphs falling

through the screen ~ere caught in a metal pan and wete then

placed in a new jar ~ith penic!llin treated wire screen for

harborage. The date of sieving was noted on the front of

the jar. These new colonies were maintained as described

above.

The week of adult emergence was noted for each colony.

Two weeks after emergence. the colonies were lightly

anesthetized with co 2 and all females with oothecae were removed to separate jar•. the date of removal recorded for each jar. Two to three weeks later. gravid females were

lightly anesthetized with ·co 2 and females with oothecae in middle to late Stage XI I of deve 1 opmen t were removed to

separate 0.9 1 jars. Stage XII is characterized by yolk

concentrated in the posterior half of the embryo. comprising

about 1/5 of its total length; eyes pigmented; ·and no visible segmentation (Tanaka. 1976). The selected females were held for 24 h prior to testing to allow for any

premature oothecal drop which might have occurred due to anesthesia effects. Oothecae were also discarded if they contained more than 4 undeveloped eggs/ootheca.

3.3.3 DOSAGE DBTBJHIJATIQB.

Propoxur was initially used to establish a dosage that would produce partial mo~tality and partial premature 18

oothecal drop of VPI strain females within a standard time of exposure (100 min). Three replicates of 10 oothecal­ bearing females were conducted using a range of dosages.

Females were exposed to the 'insecticide using a tarsal contact method described below. Mortality data collected at

72 h post-exposure were used to establish.a lpg-probit curve of the dose mortality relationship. The dosage causing approximately 65% mortality was selected for the remainder of the t~sts becauae not all of the treated females dropped their oothe~ae premat~rely. All mortalities of VPI strain females were not significantly different (P>0.05). A

.similar procedure was followed for each insecticide tested in order t~ determine a dosage that would give 65% mortality from a 100 min exposure period.

3.3.4 rARSAL COBrAcr HErBO~

Oothecal-bearing females selected for testing were exposed to the insecticide using the tarsal contact method of Barson and McCheyne (1978). The present experiments used modifications to this techinque that were reported by

Co c h ran ( 19 7 3 b ) •

Sheets of Whatman no. l. filter paper (15 em X 15 em) were impregnated with a 3 ml mixture consisting of Rise lla oil - trichloroethylene (1:2 v/v) with sufficient technical grade ins~cticide to provide a predetermined dosage for that insecticide. The treated filter paper was. allowed to dry 19

for 1 h. After drying, 10 - 12 females were confined on the paper for 100 min by a glass chimney (19 em X 14.5 em inside diam.) lightly coated with petroleum jelly around the base

(Fig 3.1). Treated f i 1 te r papers were used once and then discarded. Each experiment was replicated 4 to 10 times for a total of approximately 50 to 100 fema1e~/insecticide.

3.4 IX~RlMENIAL-EROCEDURE~

Oothecal-bearing females were observed for knockdown, drop of the oothecae. and whether each female was knocked down or not at the time of oothecal drop.

Females that prematurely dropped their oothecae were placed together in a 0.9 1 jar. Females that retained their oothecae at the end of the exposure period were placed separately in 0. 9 1 jars. All were observed for mortality at 72 h post-exposure~ Females retaining their oothecae were also observed for oothecal drop, time from drop to hatch (as days from the exposure period) • and hatch of the . oothecae.

The number of eggs/ootheca and the number of nymphs that emerged/ootheca were also recorded.

Oothecae prematurely dropped during the exposure period were placed individually on filter paper (7 em diam.) in 0. 9 1 jars (Fig.

3.2). Each paper was impregnated with a like dosage of the insecticide used in tests on adult females. A water source was placed 5 to 7 em above the bottom of the jar. A solid cover sealed the jar from the ambient air. This increased the humidity within the jars and thus 20

enhanced the chance that the eggs would hatch. Preliminary testing with a pistol psychrometer showed that the relative humidity within the jars with the solid top was held between 85 to 98% RH (x = 93%) for 7 days. The solid top was replaced with cheesecloth' following hatch of the oothecae. Oothecae were observed for time to hatch, number of eggs/ootheca, number of nymphs/ootheca that were able to emerge successfully, and number of nymphs able to move about freely 24 hours post-exposure. Nymphs were judged dead in the calculation of percentage nymphal survival 24 h post-emergence if no pulsation of the dorsal blood vessel was observed.

Oothecae were also manually detached from females and exposed to the insecticide treated surface as described above.

3.5 CAJ,C]JJAUOBS.

Percentage oothecal hatch, percentage nymphal emergence per ootheca, percent nymphal survival per ootheca and percent nymphs able to move about freely per ootheca were calculated as follows:

no. oothecae hatched . % hatch = ------total no. oothecae tested

no. nymphs emerged/ootheca % emergence/ootheca = ------no. nymphs emerged/ootheca + no. nymphs left in ootheca

no. nymphs alive/ootheca % survival/ootheca = ------no. nymphs emerged/ootheca 21

no. nymp~s entangled in % able to move the embryonic cuticle .about freely/ootheca =.1 - no. nymphs emerged/ootheca

In the calculation of the perceJ1tage of nymphs: able to move about

fre~ly 24 h. post-exposure • nymp}ls .were considered: unable to. move about

freely. if their 4ppendages . were entangled in tpe embryonic exuvium.

Data for this calc~lation were not. available, . for . exper.im~nts with

propoxur. ··

3.6 DATA AJAI~SXS.

All data were analyzed using the. Statistical Analysis

Serv'ice (SAS) (SAS. 1~~85) and Bio-Medical Data .. Processing

(BMDP) statistical. !JOftware av.ailable on the VPI & SU IBM mainframe compu~er.

The percentage·.· premature o.othecal drop. ·during: the

exposure analyzed .. using; Least Significant

Differences (LSD) pairw.ise comparison p.rocedure ( SAS. 1985).

Two-way frequency~tables to measure the.as~ociati9n between

111ort~l ity and premature ootheca 1 drop were set up using

BMDP. The null hypothesis was that post treatment mortality

among females that retained their oothecae did not differ

significantly from that of females that dropped their

oothecae prematurely. Loglinear 'models (BMDP) were set up

to.assemble the best statistical model to analyze the data.

Percentage hatch of the oothecae per treatment group

(example: propoxur treated females with Stage XIII oothecae) 22

was analyzed using a one-way ANOVA (F = 19.79; df = 1075; and P<0.05) (for an analysis Qf all treatment groups t oge the r) followed by a. LSD pairwise c ompar is on 'test. Data on time from the exposure period to hatch were analyzed using a general linear models procedure (F = 134.55; df = 893; and. P<0.05) (for an analysis of all treatment groups together) followed by a LSD pairwise comparison ·test. Means were calculated for each treatment group and sorted in ascending order. Significance groupings were assigned from the LSD tes t.s.

Data calculated for nymphal emergence. survival. and nymphs en tang led in the embryonic cuticle (percent age of nymphs able to move about freely) were first transformed using arcsine to account for non~homogeneity of variance of the data sets. The ~ata were then analyzed with a general linear models procedure followed by a LSD pairwise comparison test. Significance groupings were assigned to

I the means for each treatment group using the results of the analyses. 23

Figure 3.1. Pyrex glass chimney (19 em X 14.5 em inside diam.) lightly coated with vaseline around the base and enclosing a treated filter paper.

Figure 3.2. Sealed 0.9 1 jar used for placement of prematurely dropped oothecae on treated filter paper until hatch. 4. EXPERIMENTS WITH PROPOXUR

~Al Introduction

Populations of the Germin cockroach. .B. • ~~.t:.m.a.ni.c..a. • contain oothecal-bearing females. Nymphs hatching from oothecae that survive an insecticide treatment can ,provide for the re-establishment of an infestation. The problem of reinfestation from viable oothecae is of particular concern because hatch of oothecae can occur when the parent females have been killed by an insecticide treatment (Cornwell.

1976).

Insecticides cause female .n.. drop their oothecae prematurely (Cornwell. 1976). This was first demonstrated with pyrethrum spray (Woodbury. 1938; Parker and Campbell. 1940). and subsequently with dichlorvos vapors

(Russell and Frishman. 1965). topical applications of bioresmethrin (Chad~ick and Evans. 1973) and surface treatment with pyrethrins and diazinon (Van den Heuvel and

Shenker. 1965). bendiocarb (Barson and Renn. 1983) and propoxur (Muller and Coch. 1975). Differences in methodology in these studies preclude meaningful comparisons between the effects of different insecticides. although pyrethrins clearly cause extensive oothecal drop.

The fate of prematurely dropped oothecae depends upon several factors. including oothecal age. ambien~ temperature and humidity. and whether the ootheca is dropped on an

24 25

insecticide treated or untreated surface. Van den Heuve 1 and Shenker (1965). Chadwick and Evans (1973) and Barson and

Renn (1983) reported a decrease in oothecal hatch if the oothecae were on an insect i,c ide treated surface. but otberwise treatment of the females had little or no effect

on oothecal hatch and nymphal survival. Parker and Campbell

(1940) reported the number of oothecae that hatched was reduced in insecticide treated females. The majority of the

available data suggest .that treatment of the female does not have a direct effect on oothecal hatch and nymphal survival. however it may have an indirect effect because of the drop of immature and 'mature oothecae in unfavorable environments.

including insecticide treated surfaces.

When insecticides cause premature oothecal drop, mature oothecae have the best chance of hatching under good conditions, which could occur soon after dropping, and quite

likely on a surface containing eoxic residues. More

information is needed on the effects of treated surfaces on

oothecal hatch and nymphal survival. Russell and Frishman

(1965), used females with oothecae of unknown age and found

less drop and a higher percentage hatch of oothecae from dichlorvos resistant than susceptible females.

The objective of the experiments reported here was to compare the effects of exposure to propoxur on females of

susceptible and resistant strains and on the oothecae. 26

4.2 Materials and Methods

Oothecal-bearing females were obtained from the VPI •

Carver. and Fairbanks strains. The culture and selection of oothecal-bearing females techniques were described in sections 3.3.1 and 3.3.2. Using Tanaka's (1976) developmental table. females bearing oothecae in late stage

IX and XII were selected.

Females were exposed to filter paper impregnated with \ 10 mg of technical grade propoxur (44.4 ug/cm2 ) using the tarsal contact method (sections 3.3.3 and 3.3.4). Data were recorded and analyzed during both the exposure period and 24 h post-exposure as described in sections 3.4. 3.5. and 3.6.

A susceptible field strain (Fairbanks) was tested and limited data were also obtained on the susceptibility and oothecal drop of females in this strain in order to compare laboratory and field-collected susceptible strains.

4.3 Results

Unexposed females did not drop their oothecae during the exposure period (Table 4.1). A single Carver strain female died. but the resulting mortality (3%) was not significantly different from that of the VPI strain (0%)

(Table 4.1). Among treated females. premature oothecal drop was significantly higher (P>O.OS) in the VPI than the Carver strain (Table 4.1). Over half of the VPI females dropped their oothecae. compared to only 6% in the Carver strain. 27

Mortality among VPI and Carver strain females that dropped their oothecae was significantly higher than in females that retained their oothecae during the exposure period (Table

4.1). Mortality and premature oothecal drop of VPI strain females with Stage IX oothecae did not differ significantly

(P>O.OS) from mortality of VPI strain females with Stage XII oothecae.

Mortality of Fairbanks strain females was 41% (n=32). significantly (P

The percentage of premature oothecal drop was also closer to that of the VPI strain than the Carver strain (42% vs. 57 and 6% respectively) (Table 4.1).

Data on untreated females indicated that manual removal of oothecae had no effect on either the number of oothecae that hatched or the time to hatch (Table 4.2). Hatch of oothecae retained by treated females was essentially identical to that of untreated females VPI strain oothecae that hatched on a treated surface showed a tendency towards a reduction in the percentage that hatched. Oothecae retained by treated females hatched after a significantly

(P

The mean number of nymphs per ootheca in the VPI strain did not differ significantly (P>O.OS) from that of the

Carver strain (40 + 3.8 and 41 ± 3.8. respectively). 28

Nymphal hatch and survival are summarized in Table 4.2.

Over 95% of the nymphs in oothecae of untreated females emerged successfully. except for the 90% emergence observed in detached oothecae of Carver' females. Inadvertent damage to the oothecae in the process of manual removal may have caused this difference. Emergence from oothecae retained by the treated females was not significantly (P>O.OS) different in the two strains. but showed a slight reduction when compared tb normal hatch (Table 4.2). Percentage emerg~nce

in both strains was reduced significantly (P<0.05) when oothecae were on a treated surface. regardless of whether the ootheca was dropped prematurely or manu a 11 y removed.

Percentage emergence from the Carver strain oothecae tended to be slightly higher than from VPI strain oothecae. while

the percentage hatch of Carver strain oothecae prematurely dropped was lower than VPI strain oothecae. Comparisons between dropped oothecae were limited by low numbers of

oothecae from the Carver females. In all cases. most of the embryonic death occurred in the anterior end of the

oothecae.

Nymphs that emerged onto an untreated surface. and

those emerging from oothecae retained by treated females • were alive and fully pigmented at 24 h after emergence.

Survival in both· strains was reduced ~ignificantly when newly-emerged nymphs hatched on a treated surface. Although

survivors were fully pigmented. they were either close to 29

the oothecae or they rem•ined attached to the oothecae. All survivors were knocked down but mortality was significantly less among nymphs from detached oothecae of the Carver strain females than the VPI fe~ales. A similar difference was not apparent among nymphs from prematurely dropped oothecae.

Nymphs that emerged onto an untreated surface moved away from the oothecae within 24 h after emergence. In contrast. many of the nymphs emerging on a treated surface were characterized by a failure to escape the oothecae (Fig

4.1). In many instances pigmentation was limited to the cerci and/or the last 2 abdominal segments. Nymphs still alive 24 h after emergence frequently had the antennae (Fig

4.2 and 4.3). and/or the metatarsal segments (Fig. 4.4) still encased in the embryonic cuticle.

L!_J)iscussion

Mortality from propoxur exposure to females carrying

Stage XII oothecae was higher in the susceptible strain

(VPI) than in the resistant strain (Carver). Decreased hatch and nymphal survival occurred .only when the oothecae hatched on a treated surface. Similar results have been reported for other in~ecticides (Barson and Renn, 1983;

Chadwick and Evans. 1974; and Van den Heuvel and Shenker.

1965). 30

While 9% of the resistant strain females dropped their oothecae prematurely. drop was common among susceptible laboratory (VPI) and field (Fairbanks) strain females. The field strain was more resistant than the VPI strain. and the frequency of oothecal drop was lower. This suggests that retention of oothecae by propoxur-exposed females is an ancillary effect of the resistance mechanism. If so. it is not surprising that mortality was higher among females that dropped their oothecae since they may represent the susceptible females of the population. Oothecal drop was not complete in the susceptible strain. but the dosage used only killed 65% of the females. Further study is needed to determine whether a discriminating dosage might be found that would distinguish genetically susceptible from resistant females on the bas is of oothecal drop.

Experiments by Muller and Coch (1975) suggest a relationship between resistance and oothecal drop and that premature oothecal drop occurs only when the dosage is sufficient to cause knockdown.

The primary effects of leaving oothecae in contact with the propoxur-treated surface were a reduction in the number of nymphs that emerged and also in those that survived for

24 h on tpe surface. The prevalence of dead embryos at the anterior end of the oothecae (the more permeable end (Roth and Willis. 195Sa. 1955b)) suggests propoxur penetrated into the oothecae and may have been partly responsible for a 31

reduction in nymphal emergence. The number of oothecae that hatched was not affected significantly. although a slight tendenc~ towards reduced hatch was apparent. Van den Heuvel and Shenker (1965) reported a ~eduction in oothecal hatch as well as nymphal emergence in experiments with diazinon and pyrethrins. but there was a question as to whether failure to hatch may have been due to mold on the oothecae.

The data regarding detached oothecae indicate that resistance c6nferred a survival adva~tage even in newly emerged nymphs and perhaps a slight advantage with respect to emergence. This seems to be contradictory to the nearly identical estimates of nymphal survival when oothecae were dropped on the treated surface. However. if premature drop is an indication of susceptibility. the very few resistant strain females that dropped their oothecae may have been susceptible members of the strain. It is unlikely that the field strain was completely homozygous for a major gene(s) conferring propoxur resistance.

In the VPI and Carver strains newly emerged nymphs were frequently attached to the ootheca due to their inability to free themselves from the embryonic cuticle. If still alive. this prevented escape from the treated surface.

Entanglement in the cuticle may have been due to the dehydrative effects of the insecticide on the nymphs.

Propoxur is metabolized by a ring hydroxyl~tion at po~ition

5 (Oonnithan and Casida. 1968). This reaction requires the 32

input of a hydroxl group. generally from water. To metabolize the insecticide. nymphs may have to utilize the water within their bodies. During the detachment of the epidermis from the old exoc,uticle water is needed to facilitate the movement of potassium ions to activate and buffer the molting fluid (Jungreis. 1978). Synthesis of compounds responsible for the sclerotization and darkening of the cuticle also requires water. Insufficient water for these processes due to detoxification could explain why nymphs were unable to shed the embryonic cuticle completely and also the failure to sclerotize the cuticle. However. non-pigmentation and presumably non-sclerotization were more likely due to death before these processes would normally occur.

The potential hatch of prematurely-dropped oothecae is higher in older than in younger oothecae (Barson and Renn

19 83). However. the advantage of ootheca 1 maturity may be nullified if oothecae drop on a treated surface. Propoxur causes rapid drop. thus increasing the likelihood of exposure to residues from a propoxur treatment. In the present experiments. survival of nymphs that emerged on the treated surface was decreased due to lethal effects of the insecticide coupled with entanglement in the embryonic cuticle. These deleterious consequences were mainly in the susceptible strain since very few resistant females dropped their oothecae. 33

The primary benefits of resistance in females was their higher survivorship and productivity. Higher productivity was due largely to the low frequency of premature oothecal drop. Retention of oothecae by females in a propoxur-

tr~ated field population could give the females time to

either seek a new harborage or possibly return to their

original location after dissipation of lethal residues and

before oothecal hatch. This was reported by Ross. et al.

(1984) following propoxur treatment of a shipboard

population of wild type German cockroaches. This advantage

could also be enhanced by the lengthened period of oothecal

retention that occurred following exposure of oothecal­

bearing females to propoxur. Table 4.1. Effect of propoxur on mortality and oothecal drop in feinale 1h gennanica carrying Stage XII oothecae (oat). a

% mortality--~~~--~~ ~- ~~~ - ~% oothecal drop Strain Treatment N . % mortalityb females w/oot females w/o oat . during exposure 24 h post . . . N % N %

VPI untreated 27 0 .A 0 Aa 0 0 a w .J:.. propoxur 79 65 B 5J Ac 7J Ba 45 57 b

Carver untreated J4 J A J A'b 0 0 a

propoxur 110 9 A 7 Ab 4J Bb 10 9 a

~eans in rows with the: same upper case letters do not differ significantly (P) 0 .05); means in columns with the same lower case letters do ~ot differ significantly (P ) 0.05). ~2 h after exposure to untreated or propoxur-treated filter paper. cDropped during exposure period. Table 4.2. Effects of propoxur on hatch of and nymphs from Stage XII. ~ germanica oothecae (oot).

b Experimental Time to Ootb Nymphs from hatched oot c conditions hatch (days)a N ~~ hatched % emergence % survival

Untreated females

oot attached VPI 2.0 b 27 100 a 96 a 100 a

Carver 2.7,b 34 94 a 96 a 100 a

oot detached VPI 2.1 b 37 97 a 95 a 100 a

Carver 2.3 b 29 100 a 90b 100 a w VI Treated females

oot attached VPI 3.5 a 34 94 a 94 a 1oo·a Carver 3.0 a 100 98 a 92 a 100 a oot dropped VPI 1.9b 45 91 a 77 c 75 1 Carver 2.5 .b 10 6ob 87 c 74 b

oot detached VPI 2.1 b 33 91 a 75 c 45 d Carver 1.3 b 59 95 a 80c 6oc

~rom end of exposure period (25 h and 4o min after oot stage identification) to hatch. bMeans with same letters within columns not significantly different (P) 0.05). cNymphs that emerged and were alive at 24 h after emergence. See text. 36

•

Figure 4.1. Nymphs. pigmented and unpigmented. still entangled in embryonic cuticle 24 h after emergence on a propoxur treated surface.

Figure 4.2. Fully pigmented nymph being unable to completely shed embryonic cuticle on antennae (65X). 37

Figure 4.3. Pigmented (alive) and unpigmented . (dead) nymphs still encased in embryonic cuticle.

Figure 4.4. Nymph still attached to oothecae by embryonic cuticle encasing metatarsi (27.7X). 5. EXPERIMENTS WITH ORGANOPHOSPHATES

5.1 Intr.QslllJ&.t.i.2ll

The prior chapter in ,this study established a definitive procedure for comparison of the effects of propoxur on oothecal-bearing females and their oothecae in two German cockroach strains. Exposure to propoxur caused females to drop their oothecae prematurely. Van den Heuvel and Shenker (1965) reported this behavior also occurred when oothecal-bearing females were exposed to diazinon. Methods used in their study did allow for adequate comparisons between the effects of organophosphates and other groups of insecticides.

The use of organophosphates for the control of German cockroaches is widespread (Cornwell. 1976). Nevertheless, little is known about the effects of organophosphates on oothecal-bearing females. The present study addresses this problem. The objectives of the research reported here were to compare the effects of exposure to malathion and diazinon on susceptible and resistant females and their oothecae. It is also the first attempt to compare the effects of either diazinon and malathion on susceptible and resistant strains.

This study also provides a basis for comparison of the effects of two organophosphates (malathion and diazinon) to a carbamate insecticide (propoxur).

38 39

5.2 Materials and H~thods

Oothecal-bearing females for experiments with malathion were obtained from the VPI and Carver strains. Females obtained for experiments with di'azinon were from the VPI and

Lynn Haven strains. Culture and selection of oothecal- bearing females were described in section 3.3. Females bearing an ootheca in late stage XII of development were selected for testing with hatch expected in 48 to 72 h

(Tanaka. 1976).

Females were exposed to either 0.08 ml of technical grade malathion (0.356 ul/cm2 ) or 0.015 ml technical grade diazinon (0.067 ul/cm2 ) as described in sections 3.3.3 and

3.3.4.

Data recording• percentage calculations. and data analysis were as outlined in sections 3.4. 3.5. and 3.6.

Data on premature oothecal drop were recorded at two different times; during the exposure period and 24 h post­ exposure. Data were recorded at 24 h post-exposure because both malathion and diazinon undergo an activation reaction prior to exerting their toxic effects (Matsumura. 1985).

The 100 min exposure period was not have deemed sufficient for an accurate determination as to whether exposure would cause premature oothecal drop. 40

5.3 Result§

Table 5.1 shows the effects of malathion exposure on

female mortality and oothecal drop. The mean mortality of

VPI strain females was 56%. Mortality of females that prematurely dropped their oothecae was higher than that of

females that retained their oothecae. This may or may not represent a real difference because only 3 females prematurely dropped their oothecae.

Cockroaches in the resistant strain (Carver) showed a significantly (P

VPI strain. Mortality of females that prematurely dropped their oothecae was higher than that of females that retained

their oothecae. but the mortality est~mate for females without oothecae was based on a very low number ( 4) of

females.

When the mortality of all females the prematurely dropped their ootheca were combined across the strains. mortality was significantly (P

females retaining their oothecae.

By the end of a period equivalent to 24 h post-exposure

(as with the treated females). oothecae of untreated females had begun to drop and hatch (Table 5~1). In the VPI strain.

the percentage drop was increased significantly (P>O.OS) by exposure to malathion. Drop by treated Carver strain

females was less than that of the controls. Apparently. 41

malathion caused some pr&mature oothecal drop by VPI strain females, but not by Carver strain females.

Table 5.2 shows the effect of malathion on oothecal hatch and nymphal survival. Hitch time of oothecae manually detached from untreated VPI females did not differ significantly from that of oothecae that hatched naturally.

Although manually detached oothecae of Carver strain females hatched more quickly than oothecae ·retained by untreated females, it is doubtful this was due to the detachment procedure. It may be related to an unexpectedly long hatch time for oothecae retained by Carver strain females, since all other data on untreated females were similar.

The time required for hatch of oothecae retained by malathion treated Carv~r and VPr strain females was similar to that of the controls (Table 5.2). Exposure to a malathion treated surf ace re su 1 ted in a reduct ion in hatch time. The decrease was avident among prematurely dropped as well as oothecae that were detached, even though the number of prematurely ·dropped oothecae was low in both strains.

Slight differences in selection·· of oothecae or in their development could accotint for some variation in hatch time, but the consistency of the reduction among oothecae on the treated surface leaves liti~e doubt that this was an effect of the malathi~n treatment.

Hatch of oothecae of untreated VPI str~in females

(controls) was not affected by manual detachment. Hatch of 42

manually detached oothecae in the Carver strain showed a slight. but significant reduction (Table 5.2). Hatch of oothecae was lower among treated VPI females that retained their oothecae than in the VPI strain control group. A similar tendency occurred among Carver strairi females. but the difference fr~m the controls was not statistically significant (P>0.05). Overall. malathion had little effect on the number of oothec•e that hatched. although exposure of

VPI females may have resulted in ~ slight reduction of oothecal hatch.

Percentage emergence per ootheca was affected only when oothecae were manually detached and hatched on a malathion treated surface. The reduction in the VPI strain did not differ significantly from that in the Carver strain.

A slight reduction in survival of newly emerged nymphs recorded 24 h post-emergence occurred when VPI strain oothecae were manually detached and placed .on the malathion treated surface (Table 5.2). The lack of a similar difference in emergence from prematurely dropped oothecae may have been due to low nu~bers of oothecae. However. very few VPI nymphs that hatched on a treated surface were able to move about freely. The percentage of VPI nymphs able to move about freely at 24 h post-emergence was much less than that of Carver strain nymphs. although movement of Carver s t r a i xi nymphs was reduced ( Tab 1 e 5 • 2) • The rest ric t ion of movement reflects the inability of many nymphs to free 43

themselves from the embryonic cuticle. Nymphs that were a 1 i ve and free of the embryonic cuticle were not knocked down.

Table 5.3 shows the effect of diazinon on female mortality and oothecal drop. Mean mortality for VPI strain females was 65%. Exposure of Lynn Haven females produced no mortality. although one untreated Lynn Haven female died.

One treated Lynn Haven strain female prematurely dropped her ootheca during the exposure period. This ootheca hatched during the exposure period suggesting inadvertent inclusion of a Stage XIII oothecae. Otherwise. no premature oothecal drop occurred during the exposure period (Table 5.3). Percentage oothecal drop 24 h post- exposure showed a considerable di£ference between treatment groups. while values for untreated VPI and Lynn Haven females were not significantly (P>0.05) different (15 and

17%. respectively). Treated VPI strain females dropved 26% of their oothecae 24 hours post-exposure as compared to 15% in the controls and only 8% for treated Lynn Haven females.

Table 5.4 shows the effects of diazinon on oothecal hatch and nymphal survival. Times from exposure to hatch of untreated VPr strain oothecae retained by females (attached) did not differ significantly from oothecae that were manually detached. Time to hatch of untreated VPI strain oothecae (attached and manually detached) was significantly less than that of untreated Lynn Haven oothecae (attached 44

and manually detached). suggesting a possible strain

difference. Hatch time of oothecae retained by treated VPI

strain females was the longest of all treatment groups (3.1

days).

Percentage hatch of oothecae retained by treated Lynn

Haven and VPI strain females was significantly (P<0.05) less

than in the controls (Table 5.4). Percentage emergence

(Table 5.4) was similar among the control oothecae except

for a slight reduction in emergence from manually detached

Lynn Raven oothecae. Emergence from oothecae retained by

treated females of both strains and from manually detached

VPI strain oothecae that hatched on the treated surface was

less than in the controls. It was of some interest that

percentage emergence from detached oothecae was the same in

both controls and treated Lynn Haven strain oothecae.

A significant reduction in the survival of newly emerged nymphs recorded 24 h post-emergence occurred when

VPI strain oothecae were manually detached and placed on the diazinon treated surface (87%) (Table 5.4). In contrast.

the survival of nymphs from detached oothecae of Lynn Haven

strain females was not affected. A wider difference was observed between the percentage of VPI and Lynn Haven nymphs able to move about freely 24 hours post-emergence (Table

5.4). Nearly all VPI strain nymphs were entangled in the embryonic cuticle and were unable to move about freely.

Entanglement was prevalent among Lynn Haven and Carver 45

strain nymphs. but not to the extent seen in the VPI strain.

All nymphs that emerged onto the insecticide treated surface were pigmented.

5.4 Discussioa

The higher adult female mortality of VPI strain females as compared to Carver strain females confirmed malathion resistance in the field strain. As reported in the preceeding chapter. this has also been shown for propoxur.

Very few females of both strains exposed to malathion dropped their oothecae during the exposure period.

Significant (P<0.05) differences arose when premature oothecal drop was assessed 24 h post-exposure. The Carver strain had a lower percentage drop than the VPI strain

(Table 5.1). The delay in drop may have been due to the slower action of malathion (Matsumura. 1985). thus it did not cause immediate premature oothecal drop upon initial exposure. as was evident with propoxur.

The absence of mortality among Lynn Haven females. when exp.osed to a dosage of diazinon that killed 63% of the VPI females. indicates a level of resistance in the Lynn Haven strain. Some premature oothecal drop occurred in both strains during the exposure period. but was not significant.

A delay in premature drop may have been due to the time required for the activation reaction of diazinon to diazoxon. the toxic form of this compound (Matsumura. 1985). 46

The 100 min exposure period was evidently not sufficiently

long to allow for the diazinon to be activated. exert its

toxic affect on the females. and cause premature oothecal

drop during the exposure period.

The extent of premature drop that occurred 24 h after

exposure confirmed that dia~inon does cause premature

oothecal drop. as reported by Van den Heuve 1 and Shenker

(1965). The higher percentage of drop by the VPI strain

females compared to the Lynn Haven females rshowed that

-retention of the oothecae by resistant females was higher

than that of suseeptible strain females. These results are

similar to those reported in the experiments with propoxur.

Ootheca from untreated Lynn Haven females took longer

to hatch than the corresponding oothecae from VPI strain

females suggesting a possible strain difference. Exposure

of VPI females to dia•inon apparently prolonged hatch time

of the oothecae. but this was not apparent with Lynn Haven

females. Hatch time of oothecae retained by VPI females

exposed to diazinon was lnnger than in either of the

equivalent groups of Lynn Haven oothecae or the control

groups. Perhaps treatm~nt of VPI strain females resulted in

an increase in retention of the oothecae and the time these

oothecae took to hatch. Van den Heuvel and Shenker (1965)

tested 12-day old oothecae on diazinon and reported the age

of the oothecae when hatching occurred was 29-30 days. three 47

days longer than the average 26 days reported by Tanaka

(1976).

The only effect of diazinon on hatch of oothecae was a reduction among oothecae that were retained by treated females. This was apparently due to treatment of the females since a similar reduction did not occur among oothecae from unexposed £~males that hatched on the diazinon treated surface.

Resistance in first instar nymphs was shown in the percentage survival and ·percentage of freely moving Lynn

Haven nymphs 24 h post-emergence as compared to VPI nymphs.

This documents the occurence of resistance in first stage nymphs of the Lynn Haven strain.

Exposure to malathion and diazinon caused premature oothecal drop for both strains. This drop occurred within

24 h after exposure to malathion or diazinon. due to the time needed for the activation reactions of the -insecticides

(Matsumura. 1985). During the 24 h after exposure. premature drop from VPI females exposed to malathion was essentially identical to that from females exposed to diazinon. This suggests a relationship between mortality due to the insecticide exposure and premature oothecal drop since dosages of each chemical were selected to produce similar mortality. PerhapB it will be possible to predict the percentage premature oothecal drop on the basis of the 48

mortality produced by a given dose of an organophosphate

insecticide.

The Lynn Haven strain was more resistant to diazinon

treatment than the Carver strain was to malathion. as shown by female mortalities. This could account for the lesser

premature oothecal drop among Lynn Haven females exposed to diazinon than among Carver females exposed to malathion.

There also seems to be a relationship between the degree of susceptibility and the percentage oothecal.drop as observed

in the experiments with propoxur. In the malathion tests. mortality was higher among females that dropped their

oothecae during the exposure period than among those that retained their oothecae. although the number of oothecae dropped was very low.

Killough (1958) tested malathion. diazinon. chlordane.

lindane. dieldrin. and DDT on oothecae of the American cockroach. Periplaneta americana... He reported all

insecticides tested except DDT produced nymphs unable to

escape the embryonic cuticle upon hatch. He hypothesized

that presence of the 'chemicals in the keels of treated

oothecae was responsible for this response in emerging nymphs. He also reported diazinon treatment seemed to prevent embryonic development through deep penetration of

the chemical through the keel of treated oothecae a few days before hatch. Diazinon treatment also produced higher proportions of abnormal nymphs as the concentration and/or 49

length of exposure were increased. This may also be a

reason for the reduction in hatch and emergence in

experiments with diazinon in this study.

I Table 5.1. Effect of malathion on mortality and oothecal drop in female ~ germanica carrying Stage XII. oothecae (oot).a

%mortality ~ oothecal drop Strain Treatment N · % mortalityb females w/oot females w/o ootc during exposure 24 h post N % N %

VPI untreated 27 0 A 0 Aa --- 0 0 a 4 15 a 55 Cc 66 Cb J J a 28 28 c V1 malathion 100 56 c 0

Carver untreated J4 J A J Aa --- 0 0 a 7 21 c malathion 102 24 B 23 Bb 50 Ca 4 4a 17 17 b

~eans in rows with the same upper case letters do not differ significantly (P) 0.05); means in columns with the same lower case letters do not differ significantly (P) 0.05). b72 h after exposure to untreated or malathion-treated filter paper. cDropped during exposure peri 6d. Table .5.2. Effects of malathion on hatch.of and nymphs from Stage XII: ~ ~ermanica oothecae (oot).

Experimental Time to Ootb . Nymphs from ha~hed oot.b . conditions hatch (days)a N %hatched % emergence % survival % freely moving~

Untreated females

aot attached VPI 1.9b 27 tOO a 96 a 100 a 100 a Carver 2.7 c 32 100 a 96 a 100 a 100 a oot detached VPI 2.1 b 36 92 a 9.5 a 100 a 100 a Carver 2.3 b 29 8.5 b 90 b 100 a 100 a

Treated females e V1 ...... oot attached VPI 1.9b 84 86 b 94 a 100 a 100 a

Carver 2.3 b 9.5 94 a 91 ah 100 a 100 a oot dropped VPI 0.3 a 3 100 a 92 a 97a 2 d Carver LOa 1 100 a 93 a 100 a .54 b oot d.etached VPI 1..5a 101 9a a 90 b 94 b 0 d Carver 1.8a 89 94 a 8J.b 95 al:> 2J c

~rom end ·of exposure period (2.5 h and 40 min after oot stage identification) to hatch. bMeans with same letters within coluums not significantly different (P) 0 .0.5). cNymphs that emerged and were alive at 24 h after emergence. See text. ~ymphs that emerged, ~d were alive and able to move about 24 h after emergence. See text. e Oot attached and dropped from treated females; oot detached from untreated females. Table 5.3. Effect of diazinon on mortality and oothecal drop in female~ germanica carrying Stage XII oothe_cae ( oot) • a

b /co mortality % oothecal drop Strain Treatment N %mortality females w oot females w/o ootc during exposure 24 h post N % N %

VPI untreated 27 0 A 0 Aa --- 0 0 a 4 15 b V1 diazinon 100 6J B 6J Bb --- 0 0 a 26 26 c ~ Lynn Haven untreated JO J A J Aa --- 0 0 a 5 17 b ' d.iazinon 48 0 A 0 Aa 0 Aa 1 2 a 4 8 a

~eans in rows with the same upper.case letters do not differ significantly (P) 0.05); means in columns with the same lower case letters do not differ significantly (P) 0.05). · b72 h after exposure to untreated or diazinon-treated filter paper. c . Dropped during exposure period. Table 5.4. Effects of diazinon on hatch of and nymphs from Stage XII ~ germanica oothecae (oot).

b Experimental Time to Ootb Nymphs from hatch8d oot, d conditioos hatch (days)a N ~{, hatched % emergence % survival %freely moving

Untreated females

oot attached VPI 1.9a 27 100 a 96 a 100 a 100 a Lynn Haven 2.5 b 30 100 a 95 a 99 a 100 a oot detached VPI 2.1 a 36 92 a 95 a 100 a 100 a Lynn Haven 2.2 b JO 100 a 89 b 100 a 100 a

U1 Treated females (.,.)

oot attached VPI 3.1 c 80 80 b 83 b 100 a 100 a Lynn Haven 2.5 b 4o 85 b 80 b 100 a 100 ,a oot dropped VPI --- 0 Lynn Havene ___ 1

oot detached VPI 1.5a 102 97a 83 b 87 b 0 d

Lynn Haven 2.3 b 46 93 a 89 b 99 a 18 c

~rom end of exposure period (25 h and 4o min after oot stage identification) to hatch. bN~ans with same letters within columns not significantly different (P) 0.05), cNymphs that emerged and were alive at 24 h after emergence. See text.

~Jymphs that emerged, and were alive and able to move about freely 24 h after emereence. See text. eOot hatched during exposure period, 6. DISCUSSIOB ABD COBCLUSIOBS

Exposure of VPI (susceptible) strain German cockroach females to selected dosages of propoxur. malathion. and diazinon produced approximately 6~% mortality in each case.

Very little mortality (24%) occurred in Carver strain females except when exposed to malathion. There was no mortality in Lynn Haven strain females when exposed to diazinon. This indicates a higher level of resistance in the Lynn Haven strain to diazinon as compared to resistance in Carver strain females to malathion. Carver and VPI strain females without oothecae had higher percentage mortality 72 h post-exposure to propoxur than those females retaining oot:_hecae.

Most Carver and VPI ~train females prematurely dropped their oothecae during exposure to propoxur than after exposure. Exposure to diazinon or malathion did not cause substantial oothecal drop during the exposure period. Drop after exposure to the organophosphates was observed 24 h post-exposure. but a larger increase in premature oothecal drop was observed in the control groups of both strains.

This delay may be due to the differences in action of the. carbamate and organophosphates. Exposure to propoxur caused greater premature oothecal drop during the exposure period in the VPI females than in Carver females. A similar

54 55

difference between the susceptible and resistant strains was shown in the delayed oothecal drop after organophosphate exposure.

Several aspects of the ·data suggest a relationship between the frequency of premature oothecal drop and insecticide resistance. Mortality was higher among females that dropped their oothecae than among females that retained them. When mortalities were- similar after exposure to the organophosphates • the frequency of premature oothecal drop was similar. The mean premature oothecal drop was lower among resistant than susceptible strain females. regardless of whether the strain was resistant to propoxur. malathion. or diazinon.

Oothecae from Lynn Haven control females took longer to hatch than VPI strain control oothecae. possibly indicating a difference in the oothecal developmental time of the two strains. Oothecae retained by propoxur- or diazinon-treated females took longer to hatch than either control. prematurely dropped. or detached oothecae. This indicates that these insecticides caused oothecae to hatch later than if prematurely dropped or detached. Oothecae hatched on a malathion-treated surface (prematurely dropped and detached) took less time to hatch than if retained by malathion­ treated females. In the diazinon experiments. only a slight tendency towards a reduced time to hatch was evident in the

VPI strain oothecae. Treatment of Lynn Haven females did 56

not have as significant an effect on the period required for

oothecal hatch.

The percentage of oothecae that hatched from Carver

strain females prematurely dropped on the propoxur-treated

surface was the only reduction in oothecal hatch in the

propoxur experiment. The results were aberrent since any

effect of propoxur on oothecal hatch should have occurred

among detached as well as prematurely dropped oothecae.

Otherwise. no significant reduction occurred. although there

was a tendency towards a reduced hatch in the VPI strain.

Malathion treatment affected oothecal hatch only in VPI

strain females. The higher hatch of oothecae of resistant

females. coupled with less premature oothecae drop. suggests

a significant advantage in a resistant population treated

/with malathion. Oothecae of VPI and Lynn Haven strains

retained by diazinon treated females had a lower percentage

of oothecae that hatched than the controls or those dropped

prematurely. ,Toxic effects of diazinon on females were

apparently passed on to the embryos developing in some of

the oothecae. In this case. resistance did not increase

hatch over that of oothecae of the susceptible strain

females in contrast to the results obtained when females

were treated with malathion. Lawson (1951) repor~ted that

the percentage of oothecae that hatched was similar if

dropped or retained after treatment of females with benzene

hexachloride. toxaphene. or pyrethrins; oothecae of treated 57

females generally had a decreased percentage of oothecae that hatched.

Oothecae hatched on the propoxur-treated surface had a lower percentage emergence per ootheca than both the controls and oothecae retained by propoxur-treated females of both strains. A larger percentage reduction in emergence of oothecae of the VPI strain females compared to that of

Carver strain females suggests a higher pre-hatch mortality among the susceptible than resistant strain nymphs.

Malathion had less effect on nymphal emergence than propoxur. Emergence from detached oothecae of malathion- treated VPI strain females. but not from Carver strain females was less than in the controls. This suggests that the resistant strain may have had a slightly decreased pre­ hatch mortality. The most severe effects on emergence occurred in the diazinon experiments. Oothecae retained by diazinon-treated females of both strains (VPI and Lynn

Haven) had the greatest reduction in the percentage e'mergence from the three insecticide~.

Diazinon possibly affects eggs and/or nymphs while the ootheca is still attached to the female. Detached Lynn

Haven oothecae hatched on the diazinon treated surface had a slightly higher percentage emergence than detached VPI strain oothecae. suggesting an effect of diazinon resistance. The percentage emergence of detached Lynn Haven strain oot:hecae was higher than that of detached Carver 58

strain oothecae that were hatched on a malathion treated surface. Thus. the two experiments show that where diazinon resistance was higher than malathion resistance. the percentage emergence was lik~wise higher and. as noted earlier. the percentage oothecal drop was less.

A marked reduction in the percentage survival of nymphs emerging onto the propoxur-treated surface was evident.

There was a lack of difference between the strains (VPI and

Carver) when oothecae were prematurely dropped and hatched on the propoxur-treated surface. This was probably due to low numbers of resistant strain females that dropped their oothecae prematurely. Survival of Carver strain nymphs was much higher than that of VPI strain nymphs that hatched from detached oothecae on the propoxur-treated surface. Nymphal survival 24 h post-emergence onto a treated surface was much less in experiments with the organophosphates than with propoxur. Perhaps this difference may be due in part to the greater delay in the action of the organophosphates. A difference in time of death was already evident in that nymphs exposed to propoxur died before pigmentation was I complete whereas dead nymphs in the organophosphate experiments were fully pigmented. The ultimate mortality from the organophosphates would also be less than from propoxur. as discussed below. Therefore. it appears that similarity in adult female mortality from exposure to propoxur to that from exposure to organophosphates does not 59

mean that mortalitites among newly-emerged nymphs will be similar.

Nymphs from oothecae retained by malathion- or diazinon-treated females but that hatched on an untreated surface were able to move about freely 24 h post-emergence.

Evidently. treatment of the females did not result in the inability of the nymphs to free themselves from the embryonic cuticle. Some nymphs in the propoxur experiments were able to free themselves from the •mbryonic cuticle. but all were knocked down during the exposure. Thus. at the dosage used. all would have eventually died. Carver strain oothecae that hatched on the malathion-treated surface had a consider~bly larger percentage 6f nymphs able to move about freely 24 h post -emergence as compared to nymphs from VPI strain oothecae. A similar strain difference was seen in the diazinon experiments between the VPI and Lynn Haven strains. Thus. some of the nymphs from the organophosphate resistant strains had the ability to move away from the treated surface. but this was not the case in the propoxur experiments.

Lawson (1949) reported that German cockroach nymphs were unable to free themselves from the embryonic cuticle and/or completely pi~~ent themselves after exposure to benzene hexachloride • toxaphene • or pyrethrins. Killough

(1958) reported topical treatment of oothecae of the

American cockroach with diazinon or malathion produced 60

nymphs unable to free themselves from the embryonic cuticle.

More of these abnormal nymphs were produced if the oothecae were treated on the keel than on the side. bottom. or end of the oothecae. Lawson (1949) 'and Killough (1958) concluded the insecticides were able to move into the suture and/or tubes of the keel. The nymphs were thought to have received a toxic dose of insecticide as they pressed against the keel during emergence. Killough (1958) reported diazinon caused the highest number of dead and/or abnormal nymphs as compared to malathion because of actual penetration into the keel of the ootheca.

In conclusion. the effects on oothecae-bearing females should be considered when selecting an insecticide for

German cockroach control. The capability of a population for regrowth would be decreased if treatment caused extensive and rapid oothecal drop. However. the deleterious effects of female productivity need to be evaluated according to the characteristics of the target population.

Field populations vary in their resistance and these variations ·may be accompanied by differences in their response to various insecticides. The present experiments showed differences between susceptible and resistant strains and different insecticides with regard to the frequency of premature oothecal drop. successful emergence onto an insecticide-treated surface. nymphal survival on an insecticide-treated surface. and the amount of nymphal 61

entanglement in the embryonic cuticle. Future research on this topic should investigate the variation in premature oothecal drop in response to insecticides. using a variety of insecticides and German cockroach strains. Further research should also include the use of oothecae in earlier stages of development. LITERATURE CITED

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