| \ 1 $ mm“ ! ‘ I \ H x l l I, V kl I -'l\]__s I\JO 0301\1 camosnmummu m: ovum MELANopg (L) wrm APHOLATE mo mammal; TiN HYDROXl-DE Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSETY M. ikechuku Ezueh 19616 fi‘HESlS *LIBRARY MIChlga ‘n'. 5 '3“ University ABSTRACT CHEMOSTERILIZATION 0F.0ULEMA MELANOPA (L.) WITH APHOLATE AND TRIPHENYL TIN HYDROXIDE by M. Ikechuku Ezueh Apholate (2,2,4,u,6,6, hexakis (l-aziridinyl) 2,2,4, 4,6,6, hexahydro 2,4,6,1,3,5, triphosphatriazarine) was an effective chemosterilant for the cereal leaf beetle, Oulema melanopa (L.). A 0.05% aqueous concentration, when given to both sexes, caused 100% sterility. When only males were treated a 0.1% solution was needed to effect complete ste- rility. Longevity of treated males was reduced at the sterilizing dosage, death occuring mostly 10 days after treatment. Treated males were found to reta in their normal mating behavior but early mortality seemed to reduce their mating competitiveness in comparison with the normal males. A ratio of treated males to untreated males of about 12:1 would be required to reduce egg viability to zero percent. The early mortality of the treated males would necessitate more frequent release of sterilized beetles. Histological sections revealed no damage to the testis of males treated with 0.1% apholate. Motile sperms were found in treated males 21 days after treatment. An exploratory study of the sterilizing properties of Dowco-186 (Triphenyl tin hydroxide) indicated that it M. Ikechuku Ezueh was most effective when both sexes were treated, and as long as the beetle fed on treated plants. Concentrations of 2.0% and 3.0% caused lethargy, manifested by inactivity and starvation. At lower concentrations, these effects were progressively less pronounced. CHEMOSTERILIZATION 0F OULEMA MELANOPA (L.) WITH APHOLATE AND TRIPHENYL TIN HYDROXIDE By M. Ikechuku Ezueh A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1966 ACKNOWLEDGMENTS The author wishes to show deep gratitude to Dr. R. A. Hoopingarner for suggesting this interesting project and for his guidance throughout the course of the investigation; to Mr. V. Connin and his team for kindly supplying the beetles; to Dr. E. C. Martin for his encouragements in the early stages of this work; and also to the Rockefeller Foundation for making this opportunity possible. The assistance of the A.R.S.-U.S.D.A. laboratory is gratefully acknowledged. This laboratory is supported under contract No. 12-14-10047726 (33) A.R.S.-U.S.D.A. Finally, much credit goes to my wife for her help and inspiration in the final preparation of this thesis. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . 11 LIST OF TABLES . . . . . . . . . . . . . . . . . . . v LIST OF GRAPHS . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . 4 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 10 Apholate treatment. . . . . . . . . . . . . . . 10 Mating competitiveness and longevity tests , , 11 Effect of early mortality on the mating compe- tition tests . . . . . . . . . . . . . . . . 12 Dowco-186 (Triphenyl tin hydroxide) , , , , , , 13 Cytological technique (Apholate treated males), 14 RESULTS . . . . . . . . . . . . . . . . . . . . . . . 15 Screening for dosage level . . . . . . . . . . 15 Male optimum sterilizing concentration , , , , 15 Mating competitiveness . . . . . . . . . . . . l7 Treated male longevity . , . . . . . . . . . . 18 Effect of early mortality on mating competition 20 Dowco-l86 trials . . . . . . . . . t . . . . . 21 Oral treatment . . . . . . . . . . . . . . . . 23" Cytological obserVations . . . . . . . . , . . 24 111 DISCUSSION SUMMARY . REFERENCES 9 iv Page 29 35 36 LIST OF TABLES Sterilizing effect of 30 seconds apholate dip on pre-diapause cereal leaf beetles . . . . Effect of 30 seconds single apholate dip on post-diapause cereal leaf beetles . . . . . Sterility of male cereal leaf beetles treated with 0.05% and 0.1% apholate concentrations Mating competitiveness of male cereal leaf beetles dipped once in 0.1% apholate . . . . The effect of 0.1% apholate on the longevity of male cereal leaf beetles dipped once for 30 seconds . . . . . . . . . . . . . . . . . . . The effect of early mortality of treated males on the mating competition tests . . . . . . . Effect of Dowco-186 on post-diapause cereal leaf beetles when males only were fed on treated plants for 7 days . . . . . . . . . . Effect of dipping males of post-diapause cereal leaf beetles in 0.1%. and 0.01% concentrations of Dowco- 186 . . . . . . . . . . . . Effect of feeding both sexes of cereal leaf bee- tles on plants treated with Dowco-l86 Page l6 l6 17 18 20 21 23 24 25 LIST OF GRAPHS Graph Page 1. Graph showing the relationship between increasing ratios of treated males and egg viabilities . . . . . . . . . . . . . . l9 2. Graph showing the effect of early mortality on the mating competitiveness of the treated males . . . . . . . . . . . . . . . . . 22 vi .,j-' ‘1'}IW" “,mcfi- .... t v. _ - CI .4: - u . _ ., '3. .a-LLLU ' 4'31- .¢.'.', LIST OF FIGURES Figure Page 1. Croes-section 0f the testis of male cereal leaf beetle treated with 0.1% apholate-—2700_X. . . 26 2. Cross-section of the testis of male cereal leaf beetle treated with 0.1% apholate--540 X . . . 27 3. Cross-section of the testis of normal male cereal leaf beetle . . . . . . . . . . . . . . 28 vii INTRODUCTION Since its introduction into this country, the cereal leaf beetle, Oulema melanopa (L.) has spread over much of Michigan, seme parts of Ohio, Indiana and Illinois. This growing infestation has stimulated an active search for an effective means of control. Continuous efforts have been made in the past through research and field operations to achieve some control by conventional methods. Complete reliance on the use of insecticides for control or eradi~ cation programs has been on the wane due to the possible deveIOpment of insect resistance to insecticides and also due to the potential hazards to public health of accumulat- ing chemical residues in the natural environment. Research efforts have therefore, been directed to exploring other strategies for insect control, which would preclude undesir- able side-effects inherent in the widespread use of insecti- cides. The dramatic eradication of the screw-worm fly, 99gb; liomyia hominivorax (Coq.) from the island of Curacao and later from Florida and other southeastern states by sus- tained release of radiosterilized males, revolutionized the entire approach to insect control (Bushland and Hop- kins, 1951, 1953; Baumhover _e__1;__a_1_., 1955; Knipling, 1955). However, the deleterious effects of radiation on the long-’ evity and general vigor of insects, coupled with the l h) problems of mass-rearing and dissemination, often render this method impractical when extended to other pests. It is essential that the sterilized males remain in all bio- logical aspects fully competitive with the normal males (Chamberlain, 1962; Borkovec, 1964; Knipling, 1964). The discovery of radiomimetic chemicals which induced sterility without adversely affecting sexual behavior and life-span opened a new approach in the "sterile male" concept. Research entomologists are investigating the potentialities of chemosterilization in order to establish the theorized superiority of such chemicals over ionizing radiations and conventional insecticides. The most currently used chemosterilants are aziridine alkylating agents. Apholate, (2,2,4,4,6,6, hexakis (l-aziri- dinyD 2,2,4,4,6,6,hexahydro 2,4,6,l,3,5, triazatriphosphor- ine) was used in the present studies. Dowco 186 (Triphenyl tin hydroxide) represents a new class of chemosterilants that have been shown to produce sterility in the house fly, Musca domestica, the flour beetle Tribolium confusum Jac— quelin du Val, and the German cockroach Blatella germanica (L.) (Kenaga, 1965). Preliminary studies of its effects on the reproduction of the cereal leaf beetle were also car- ried out. The purpose of this work was to evaluate the possibil- ity of controlling the cereal leaf beetle by use of chemo— sterilized males. The main objectives were: 1. To determine the optimum dosage of apholate needed to sterilize the males and its effects on the long- evity and mating competitiveness of such sterilized males. 2. To evaluate some characteristics of Dowco 186 as a candidate chemosterilant for this pest. LITERATURE REVIEW The earliest recorded effects of radiation on insects was demonstrated on a coleopteran insect, the cigarette beetle,L'asiodermLserricorne (F.) (Runner, 1916). Exposing these beetles to Roentgen rays, he caused them to lay large numbers of infertile eggs. Mfiller (1927, 1928, l9h0) made a momentous discovery when he described the effects of X- rays on Drosophila melanogaster and used the term "dominant lethality" to account for the chromosome changes which led to the production of non-viable eggs. Hanson (1928) also reported the effects of X-rays on productivity and sex ratio in Drosophila melanogaster. Since then, extensive work on the effects of ionizing radiations has been done with a va- riety of.insects. Not until the work of Bushland and Hop- kins (1951, 1953) on the sterilization of the screw—worm flies was this ingenious discovery redeemed from the realm of scientific curiosity to the more practical. Knipling (1955) first proposed the idea of controlling insects by use of genetically deficient individuals of the same species. This principle was validated by the successful eradication of the screw-worm fly on the island of Curacao (Baumhover §£_g1., 1955). The sterilization of insects by chemicals is a recent corollary to the radiation-sterilization tech- nique. There are some classical references as to biological 4 effects of chemicals. Sternberg e£_al., (1958) cited the work of Gabriel and von Hirsch in 1896 as the first illus- tration of selective systemic damage by alkylating agents. In 1898, Paul Ehrlich recognized the extraordinary pharmo- cological properties of ethylene imine and ethylene oxide. However the idea that certain chemicals mimiced the biolog— ical effects of ionizing radiations deve10ped from the war time research on vesicant poison gases (Alexander, 1960). Mustard gas (2,2'-dichloro—diethy1 sulfide) was the first chemical found in these efforts to exhibit radiomimetic characteristics (Auerbach and Robson, 1943, Koller, 1947). Rapoport (1946) provided the final evidence along these lines when he described the mutagenic action of an alkyla- ting agent, diethyl sulfide. Various chemicals have been reported over many years as being capable of reducing the reproductive potential in insects and a list of these has been presented in the lit— erature review by Smith, LaBrecque and Borkovec (1964). The aziridine derivatives constitute the most important and extensively used group of chemosterilants. Their steri- lizing prOperty depends on the aziridine ring which attacks the genetic material or some other compound with biologi— cally significant functional groups, by a process of biolog- ical alkylation involving nucleOphilic substitution (Borko— vec 1962). Alexander (1960) pointed out that there were other potential nucleophilic centers in the biological sys- tem on which alkylation may occur such as proteins and vitamins. He however, observed that the consistency of the genetic effects of these compounds, suggested that they at- tacked the genetic material-DNA. Several theories have been put forward about the detailed cytological effects of azir- idine compounds. Among these, the cross-linking theory of Stacy g£_§1. (1958), had gained popular credence. LaBrecque g£_a1.(l960) were the first to show the sterilizing prOperty of apholate on house fly reproduction. Later, the screw-worm fly was sterilized with the same com— pound, by dipping larvae and prepupae, by dusting prepaupae and adults and by feeding adults 0.5% and 1.0% (Chamber- lain). Similar tests were done by Crystal (1963), Crystal and LaChance (1963) on the house fly with thiotepa, treta- mine, apholate and other aziridine derivatives. Lindquist §£_31. (1964) reported that out of 50 potential sterilants, only apholate consistently sterilized the boll weevil. One of the acclaimed advantages of Chemosterilants over high energy radiation was that they induced sterility without significantly reducing the longevity and sexual vigor of the treated insects. It is however, noteworthy that the screw-worm flies which were used in the classical demonstration of the sterile—male principle manifested no loss of sexual vigor or vitality when irradiated as pupae (Bushland and Hopkins, 1953). This response has been asso— ciated with several other members of the Diptera, irradiated in the pupal stage. Henneberry and McGovern (1963) using Drosaphila melanegaster and Steiner and Christenson (1956) using Oriental fruit fly, Dacus dorsalis Hendel observed that the irradiated pupae gave rise to adults which were quite competitive. Earlier work on Coleoptera indicated a high rate of survival when these insects were treated with gamma-radiation. Jaynes and Godwin (1957) found that 5,000-10,000 roentgen (r) caused sterility in the pine weevil, Pisodes Strobi without reduction in longevity. Cork(1957) also stated that gamma-irradiated flour beetles lived longer when given several small daily doses. These accounts would appear to challenge the theoretical advan- tages of chemosterilants over ionizing radiations, but comparative and singular studies have clearly shown that chemosterilants are the better choice for the sterilization of many insects. Weidhass and Schmidt (1963) irradiated Aedes aegypti (L.) and observed a reduction in mating be— havior, but chemically sterilized males retained normal sexual vigor. Davis et_a1. (1959) studied the effects of gamma-radiation on mating behavior of the common malaria mosquito, Anopheles quadrimaculatus Say and reported a remarkable loss in mating competitiveness by the treated males. The comparative studies of Schmidt et_§l. (1964) very strongly stressed the merits of chemosterilization. They treated house fly pupae 31-5Ahr. prior to eclosion with 2850r and fed 1% apholate to l-day-old adults. Mixed ratios of treated and untreated males were allowed to com- pete for virgin females in several cages. Chemosterilized males either equalled or surpassed the radiosterilized ones and besides greater permanency was exhibited in chemi- cally treated house flies than in the irradiated ones. Apholate sterilized adults of Culex pipiens quinquefasciatus Say were highly competitive (Dame and Ford 1964). LaBrecque, Meifert and Smith (1962) sterilized male house flies on diet containing 1% apholate, and these were found to be as successful as normal males in competition for mates. Adult males of green sheep blow fly, Lucilia sericata Meigen treated topically with apholate were reported to be equally competitive as the untreated males (Miller, 1965). Among the Coleoptera, efforts to sterilize the boll weevil, Antho- nomus grandis Boheman with gamma-irradiation revealed that the sterilizing dose reduced longevity drastically and ster— ility was not permanent. Mayer and Brazzel (1966) irra- diated 12- and 36-hr. old adult boll weevils with 8,000r and found that although they mated normally for 10 days, longevity was highly reduced. Lindquist §t_§1, (196A) conducted sterility studies on the boll weevil with apholate concentrations of 0.5%, 1.0% and 2.0%. Mortality of the treated males was high particularly at 11 and 20 days after treatment and they were not as competitive as normal males. Lindquist (unpublished data, 1965) observed how- ever, that apholate sterilization was less deleterious to the insect than radiation. Male azuki bean weevils steri- lized by apholate topically applied in acetone were as com- petitive as normal males in mating (Nagasawa and Shinohara, 1965). The triphenyl tin compounds have only recently been evaluated by Kenaga(1965) and he showed that these chemié cals sterilized at dosages which did not affect the biolog- ical functions of the insect adversely. Japanese workers, Nagasawa, Shinohara and Shiba studied the sterilizing effect of Dowco-186 on Callosobruchus chinensis but did not report mating or longevity observations. Hoopingarner et_a1. (1965) indicated that X-rays were detrimental to adult cereal leaf beetle. No mating tests were undertaken in these studies as there was no known sex- ing method. The high rate of mortality reported, above 90% in 14 days at 5,000r, necessitated the present studies with chemosterilants in the hope of finding a more prac- tical sterilizing technique for this pest. MATERIALS AND METHODS The beetles used in these studies were either field- collected or laboratory-reared adults. Pre-diapause bee- tles were obtained from wheat and bat farms as summer adults. Post-diapause beetles were obtained as spring adults or by cold-treating laboratory-reared adults at 40°F for at least 8 weeks. Post-diapause beetles were stored in plastic containers at 40°F until needed, while pre- diapause beetles were maintained on young wheat plants in cages of about l5"x17”xl8". Apholate Treatment A fresh stock of 2% aqueous solution of apholate (technical) w/v was made up for each testing period. 0.2% of Triton X-100 was added to the solution as a wetting agent. Series of desired dilutions were made from this stock prep- aration. The beetles were anaesthesized with ether and treated by a modified dipping technique of Sawicki and Farn- ham (1964). The apparatus consisted of a sintered glass Buchner funnel fitted to l-litre Buchner flask.. Suction pressure was provided by a vacuum tap.) Twenty—five ran— domly selected beetles were used in the dosage assay because a reliable sexing method was not initially available. _These were tipped into the funnel after anaesthesia and the appro— priate apholate concentration was immediately added. Each 10 11 treatment lasted for 30 seconds. Solutions of 2% to 0.001% were screened for the optimum sterilizing dosage. The bee- tles were reared on young potted wheat plants in glass chim- ney cages kept in a Sherer controlled environment chamber at 80°F, relative humidity of about 50-60% and 16-hour photo- period. Temperature and humidity of chamber were recorded by means of a Friez hygro-thermograph model 594. Mortality was checked daily and dead beetles were removed. Fresh plants were supplied when necessary and all transfer of beetles was done with an aspirator. Eggs were kept in cov- ered petri dishes layered with wet filter papers and allowed 4—7 days to hatch. Tests lasted from 9-25 days. All counts of eggs and larvae-were made with a manual tally counter. Viability was determined on the basis of egg hatchability. A control cage was set up for each test. Mortality data was corrected by a modification of Abbott's formula (Hinman, 1947). Mating Competitiveness and Longevity Tests At this stage a reasonably reliable sexing method was found, based on the morphological character of the inverted "V"-shaped intercoxal portion of the first abdominal ster- nite.. Only post-diapause beetles were used in these tests. Four proportions of treated and untreated males were used _ in the ratios of treated males:to normal males:to normal females of 2:1:1, 4:1:1, 6:1:1 and 9:1:1. The sterilizing dosage used was 0.1% apholate as this was found to induce 100% dominant lethals in males after a single dipping 12 treatment. Four cages were set up in the usual manner and in addition, one cage consisting of treated males only and virgin females was used as check on the adequacy of the treatment. A control cage was used in order to estimate the natural egg viability. Normal males were differentiated from treated males by clipping off the elytron at the pos- terior margin with very fine scissors. This facilitated the recOrding of mortality data and did not hinder the normal activities of the beetles so deformed. Observations were made daily for dead beetles and eggs were incubated as described before. Ratios were maintained as much as pos- sible by replenishing from separate colonies of each cat— egory. Mortality data were statistically checked by the Fourfold Contingency tests for unequal samples at the 5% significance level (Mainland and Murray, 1952). Effect of Earlngortality on the Mating Competition Tests Four replications of the 2:1:1 ratio of treated males, normal males, and normal females were established and daily mortality recorded of both treated and normal males. Two control cages were used with these tests. Eggs were har- vested every 48 hours and incubated as described above for 5 days. Percent viability of each egg batch was noted in order to establish a trend in viability over an entire test period of 21 days and to relate this to the mortality rates of both treated and normal males. 13 Dowco-186 (Triphenyl Tin_Hygroxide) (a) Dipping method: 0.1% w/v solution of the chem- ical was made up With 70% v/v mixture of acetone and water. The beetles were slowed down with ice and then treated with 0.1% and 0.01% concentrations of the chemical by the dip- ping technique used in previous tests. Treatment was for one minute and rearing was carried out as before in a Sherer controlled chamber. (b) Oral treatment-—ma1es only: The chemical was administered orally by treating wheat plants on which the beetles fed. 3%-O.1% concentrations w/w were applied by. Spraying, using Bentonite as the carrier. The sterilant was first dissolved in acetone and then mixed with a known weight of Bentonite. The solvent was driven off with an air- stream in an evaporating dish placed in a ventilating hood. Each preparation was emulsified with 2 ml. of Triton X*100 by Lourdes Instrument model mm-l using approximately 3 times its own weight of water. These were stored in the refrig- erator until required for application. An improvised sprayer was used in the spraying of the plants. A11 Spray- ing was'done in'protected areas. Male beetles were allowed to feed on treated plants for 7 days after which females were introduced. Rearing and observations were carried out routinely. The test lasted for 18 days. (0) Oral treatment--both sexes: 3.0%—0,1% concentra- tions of the triphehyl tin compound were used. Ten males and ten females were introduced on the treated wheat plants 14 and allowed to feed for 10 days. The plants were changed every two days because of the phytotoxicity of the chemical. They were subSequently transfered to untreated plants for mating and oviposition. Egg batches were collected While they fed on treated plants and after transfer to the un» treated plants.. The harvested eggs were incubated and checked for viability. Cytological Technique (Apholate Treated Males) Feulgen-fast green staining of lOu sections of testes from treated males were made for cytological observations. Microphotographs were taken with Kodak pony II camera and Wild Heerburg phase microscope. The final photographs rep- resent 2700 X, 1100 X, and 540 X magnifications. Panatomic X high contrast film was used. Microscopic examination of spermatozoa from treated males was also carried out 21 days after treatment. Carlson's Drosophila Sperm media, with the omission of penicillin, was prepared for this work. All disections were done in the sperm media and the testis was carefully teased out in a drOp of the preparation for Sperm motility observations. Testes sizes were visually estimated for gross comparisons. RESULTS Screening for Dosage Level Preliminary assay for optimum dosage level was carried out with randomly chosen beetles. Results obtained with treated pre-diapause beetles were quite inconsistent and so most of the tests were done with post-diapause beetles only. Apholate concentrations of 0.1%, 0.25%, and 0.5% sup- pressed oviposition and caused high mortality. There was apparently some difference in survival between treated pre- diapause and post-diapause beetles (see Tables 1 and 2). In the pre-diapause beetles there was over 80% mortality at the above concentrations in 12 days. At corresponding dosage levels, post-diapause beetles ranged from 48-76% in mortality. Apholate was therefore more toxic to pre- diapause than post-diapause beetles; 0.05% concentration induced complete sterility but at lower concentrations, adult survival and percent hatch of eggs were as high as in the controls (Table 3). 0.05% was the lowest concentration that caused sterility to both sexes. Male Optimum Sterilizing CCncentration After a suitable sexing technique became available -and the technique was perfected, male beetles were assayed for the required sterilizing concentration using 0.1% and 0.05% levels. Eggs resulting from the matings of 0.1% 15 TABLE 1--Sterilizing effect of 30 sec. apholate dip on cereal leaf beetles (pre-diapause adults)--two replications. ==gz _g_ ‘2: ._ % Concentration Average % Mortality Eggs Percent Apholate in 12 daysa Laid Hatch 1.0 96.7 None -—-- 0.5 80.0 None ---- 0.25 80.0 None -—-- Control 0 425 58.5 TABLE 2—-Effects of 30 sec. diapause cereal leaf beetles--2 replications. single apholate dip on post— % Concentration a Number of Percent Apholate % Mortality Eggs Hatch 1.0 76* —--- ---- 0.5 72* ---- -—-- 0.25 52* ---- ---- 0.1 48* -—-- ---- Control 0* 310 56.0 0.05 36.4** 98 0 0.025 4.5** 120 30.8 0.01 4.5** 180 43.3 0.001 0**. 182 68.7 Control 0** 328 57 aCorrected by Hinman's (1947) modification of Abbott's formula. *In twenty-one days. **In eighteen days. 17 TABLE 3--Sterility of male cereal leaf beetles treated (by 30 sec. dipping) at 0.05% and 0.1% apholate concentrations. Concentration No. of Beetles Average 0f Treated Eggs/' % Hatch Apholate (%) Males Females Female 0.1 I 25 25 14.4 0 II 15 15 13.9 0 0.05 (once) 25 25 9.0 20.5 Control 25 25 26.6 49.8 treated males did not hatch. Some egg hatch resulted from the matings of beetles treated with 0.05% concentration. It would appear that 0.1% was an effective sterilizing dosage for the male cereal leaf beetle, since subsequent trials produced consistent effects. This concentration was used in subsequent tests. In some of the later tests how- ever, about 5—10% viability was observed at this dosage level, but this was attributed to the chemical which had degenerated in storage over a long period of time. Mating Competitiveness In these tests, percent viability conformed very closely with theoretical expectations based on the ratios of treated and untreated males. Chi2 probabilities in all ratios except the 931:1 ratio indicated no significant vari- ation between observed and expected results (Table 4). At this ratio probability was between .05 and .02. Mating activity appeared quite normal in all treatments and there 18 was no reduction in fecundity when results from the control were compared. These results indicated that the treated males were at least as competitive as the normal males as expected viabilities were observed in most cases. The graph (No. l) pictorially relates these data. TABLE 4--Mating Competitiveness of male cereal leaf beetles dipped once in 0.1% apholate. ___._ 1.. Sex Ratio8 Number of % Viability T. M.:N. M.:N. F. Eggs Laid Expected Observed 9 1 1 86 6.3 3.5 6 l 1 180 8.6 7.2 4 1 l 173 12.0 10.9 2 1 1 222 20.1 15.3 1 O l 607 O O O 1 l 325 ——-- 60.3 T M. = treated males N. M. = normal males N. F. = normal females aMultiples of 4 and 8. Chi2 probabilities for all ratios were greater than 0.2 except in the 9:1:1 ratio. In this ratio P = 0.02-0.05. Significance was calculated at the .05 and .01 fidicual limits. Treated Male Longevity Normal males lived longer than apholate-treated males (see Table 5), as a 0.1% concentration caused about 90% mortality in 28 days. Significant mortality occurred at about 10 days after treatment (Analysed by the Fourfold 19 25)- I5" Em. 3 > 3' 5- l J J l l l l 1 2 4 6 8 9 l2 l4 TREATED MALE RATIOS Graph No. l.-—Graph showing the relationship between increas- ing ratios of treated males and egg viabilities. 20 TABLE 5--The effect of 0.1% apholate on longevity of male cereal leaf beetle dipped once for 30 seconds. r 7.7 r Percent Mortality Av . of 3 Re licates Treatment Mean No. of ( g p ) Beetles/Treatment Days After Treatment 7 10. 14 21 28 Apholate 0.1% 28 14 43 57 82 9O 16 12.5 37.5 69 75 88 Control 15 O O 7 2O 2O Contingency tests of Mainland and Murray, 1952). Normal males averaged 20-28% mortality in 28 days, with little or no deaths occurring in the first 10 days after treatment. Apholate reduced male longevity in the cereal leaf beetle. Effect of Early Mortality on Mating Competitiveness of Treated Male Cereal Leaf Beetles Actual figures obtained from four replications were somewhat variable, but a general trend was established. In all cases, there was initial reduction in egg viability very close to that expected from the ratio studies of treated males. The expected viability was 20 percent for the 2:1:1 ratio used in this study. Viability progres- sively increased with time as the number of competing sterile males was decreased by death. Data presented (Table 6) represents the average results obtained from the repli- cations (see graph No. 2). Early mortality of treated males clearly affected the egg viability. Since more than 21 TABLE 6--The effect of early mortality of treated males on the mating competition tests. Avg. No. of Avg. No. of Days After Males Alive Eggs Per Avg. % Hatch Range Treatment Treated Normal Treatment 0 l6 8 7-8 12 7 71 17.9* 15.2-20.0 9-10 8 7 86.5 30.2 13.7-38.0 11-12 4 6 140.8 30.7 21.2-40.5 13-14 4 6 179.8 28.7 21.9-41.2 15-16 3 5 158.5 34.9 25.0-50 0 17-18 3 5 116.5 42.3 30.0-59.8 19-20 3 5 133.0 43.5 35.0-59.2 *Expected egg viability — 20%. 50% of treated males were dead by the 10th day at which time oviposition generally began, this factor weighted the ratios in favor of the untreated males. Dowco-186 Trials (Oral treatment—-males only) The results were astonishing in that there was no indication of any reproduction control (see Kenaga, 1965). Egg viabilities in all treatments were either normal or surpassedthat of the control (Table 7). Mortality was quite low throughout the entire l8—day test period. .Since only one test was done these results cannot be conclusive, however, there was an indication that this chemical was not a male sterilant for cereal leaf beetles. 22 PERCENT l l l i 1 l J l 4 8 l2 l6 20 24 DAYS AFTER TREATMENT one. Maddy momma mom. Unca’ 'I a uIMIIMI u H' E99 viability. Graph No. 2.--Graph showing the effect of early mortality on the mating competitiveness of the treated males. 23 TABLE 7--Effect of Dowco-186 on post—diapause cereal leaf beetles when males only were fed on treated plants for 7 days (15 males x 15 females). %Concentration No. of Males Alive Eggs % Hatch 3.0 14 62 85.5 2.0 15 139 68.5 1.0 14 19O 61.0 0.1 13 136 56.6 Control 14 212 58.5 Dowco-186 (Dipping test-males only) , In the dipping test, 0.1% Dowco-186 in 70% aqueous acetone caused 100% mortality of the beetles in 24 hours. 0.01% had no effect on either survival or reproduction. Viability of eggs was close to that of the control. The dipping technique does not appear to be suitable with this chemical for the cereal leaf beetles. Oral Treatment--both sexes Feeding was allowed on the treated plants for about 10 days in order to ensure that sufficient Dowco-186 was obtained by the beetles. Only at 0.1% treatment was there normal feeding and some oviposition while on the treated plants. Some degree of reproduction control was obtained at this level during this peroid. A complete recovery of fertility followed as soon as the beetles were transfered to untreated plants. 24 TABLE 8--Effect of dipping males of post-diapause cereal leaf beetles in 0.1% and 0.01% concentrations of Dowco-186 for 1 minute. L % Mort. % Mort. % Concentration Males Females at at gafgfi 24 hrs. 9 days 0.1 15 15 100 100 ---- 0-01 15 15 ——— 27.0 53.3 Control 15 15 —-- 20.0 55.0 At 2.0% and 3.0% levels most of the beetles were either dead or extremely lethargic. No mating or oviposi- tion occurred at these concentrations before and after the introduction of untreated plants (see Table 9-B). 1.0% caused some lethargy but some of the beetles regained activity as soon as a fresh untreated plant was introduced. Initial oviposition was low and viability was only 4.0%. The second batch of eggs increased in number considerably and hatchability was in the normal range. Continuous feeding on treated plants appeared necessary before any sterility could be achieved. 2.0% and 3.0% were probably above sterilizing dose. Cytological Observations (0n apholate treatment) Gross comparison of testes from treated males with athose from normal males showed no difference in size. There appeared to be no regression due to the effect of the chemical at the 0.1% level. lOu sections stained in Feulgen-fast green, showed no tissue degeneration. At the 25 TABLE 9--Effect of feeding both sexes of cereal leaf beetle on plants treated with Dowco-186 (10 days duration). Dowco-186 Treated Plants % Mort. lst Egg Batch 2nd Egg Batch at % Concn. Eggs % Hatch Eggs % Hatch 20 Days 0.1 41 14.6 52 38 1.0 -- ---- -- -- 2.0 -- ---— -- -- 3.0 -- ---- -- -- Control 37 51:4 52 69 Untreated Plants 3rd Egg Batch 4th Egg Batch 0.1 104 63.5 5§__ 40.7 50 1.0 46 4.0 242 56.2 60 2.0 --— ---- --- ---- 75 3.0 —-- -—-- --- ---- 8O Contr 01 48 41.7 --- ---- 36 cellular level nuclei appeared normal and a few cells caught at the prOphase stage portrayed no abnormalities (see figure 1). Testis was filled with spermatozoa which were morpho— logically as normal as those of untreated males (see figures 2 and 3). Motile spermatozoa were found in treated males 21 days after treatment. Sperm motility was therefore not hampered by the treatment. 26 Figure l.--Cross-section of testis of male cereal leaf beetle treated with 0.1% apholate, showing spermatozoa and some nuclei in prophase. No abnormalties noticed. Stain-—Feulgen—fast green; Magnification........27OO X. 27 Figure 2.--Part of the cross—section of the testis of male cereal leaf beetle treated with 0.1% apholate, showing abundant spermatozoa in normal conditions. Stain--Feulgen-fast green; Magnification........540 X. 28 Figure 3.--Cross-section of testis of normal male post- diapause cereal leaf beetle. Stain--Feulgen-fast green; Magnification ........ 1100 X. DISCUSSION Apholate successfully sterilized adult cereal leaf beetles under laboratory conditions. It does not seem however, that it is a very practical chemosterilant for this pest in terms of actual field Operations. In order to realize the full potentials of the sterile-male tech- nique in insect control, the survival of sterilized males must be high enough to sustain the desired competition in the natural pOpulation. In present investigation apholate significantly reduced the life-span of the male cereal leaf beetle at the dosage required for sterilization. Mor- tality was found to be high particularly at 10 days after treatment. Hoopingarner g£_al. (1965) found that radio- sterilized adults suffered a mortality of over 90% in 14 days. Mbrtality of apholate-treated adults for a corres— ponding period of time was found to be about 36%. Treated male mortality ranged from 60—70%. These figures indicate _ a greater degree of survival in apholate-treated beetles than in the irradiated ones. However, since mortality of apholateésterilized males exceeded that of normal males (0-7% in 14 days) the use of this chemical for the steri- lization of the cereal leaf beetle promises no clear ad-. vantage over irradiation. Apholate-sterilized cereal leaf beetle males were as competitive as normal males (see Table 4), but the occurence 29 3O of early mortality among the treated males obscured the in- fluence of this factor in actual results. It is suspected that in this insect continual insemination might be essen- tial for adequate production of eggs. If this is so, the advantage to normal males of this early mortality is very evident. For, if the normal males continued to mate after the reduction in the number of the competing sterile males, the situation would allow for a preponderance of normal spermatozoa over spermatozoa with dominant lethals in the Spermatheca. In a strictly monogamous species or even in a polygamous species in which only the first few matings are relevant, sterile male mortality might not be very in- fluential in mating competitiveness since all the dominant lethal spermatozoa required to compete would have been in- troduced before any significant death occurred, provided the sterile males were competitive. Murray and Bickley (1964) reported that in Culex pipiens quiquefasciatus the sperm of the first mating was responsible for zygote for- mation. ILaBrecque(196l) also found that in the house fly, the first male to copulate with the female influenced egg viability. In these cases, early mortality would not upset the expectations to a great extent. But, in Drosophila melanogaster Henneberry (1963) reported that females which mated first with treated males produced sterile eggs, but when subsequently mated with untreated males they produced viable eggs. Similar findings were reported with fruit flies (Steiner and Christenson, 1956). In these examples, 31 early death of the sterile males would favor normal males. Multiple matings in this instance poses a problem. If the sterile-male method were to be used for this pest, it would entail more frequent releases in order to maintain the de- sired ratio of untreated to treated males. This would of course involve large-scale rearing procedures which might be unattainable if research resources were inadequate. A graph relating egg viabilities with ratios of treated males was presented earlier (see Graph No. l) on which a few theoretical inferences could be based. The graph shows that a ratio of treated males to untreated males of about 12:1 would be required to reduce egg viability to zero percent. This would of course entail raising a large number of insects for treatment and release. Judging from the difficulties at the moment of maintaining a large col- ony of these beetles in the laboratory, a twelve-fold re- lease seems quite unattainable and therefore unadvisable. In the mating competitiveness studies reported elsewhere in this work, a 4:1 ratio was found to have resulted in an egg hatch of 10%, which corresponds to about 50% reduction in normal egg viability. If this technique were to be employed for the eradication of the cereal leaf beetle, this ratio would be sufficient to initiate an effective downward trend in the natural population. Moreover, from a practical point of view, it is.a more attainable ratio even with the limited facilities that are now existing. Although apholate-sterilization of the cereal leaf beetle appears to have poor prospects for use in the 32 sterile-male release method, the search should continue for a chemical with a wider margin of safety. Chamberlain (1962) remarked that a potential chemosterilant should not be dis- carded merely on the basis of some toxicity. If rearing methods are improved sufficiently to ensure a large produc- tion of insects, it might be possible to adopt frequent re- leases in order to off-set the problems of early mortality of treated males. If a chemical were found that could be applied as a contact residue baited with some specific at— tractant in the natural pOpulation, male mortality might not be a major problem if the males retained their normal mating behavior after autosterilizing themselves. In this case male and female sterilization might occur which should facilitate realization of the desired effect. Preliminary trials with Dowco-186 produced no con- clusive results but a few observations appear to be quite relevant for future work with it. It was found that this compound may not be a male sterilant since females mated with treated males produced viable eggs at all concentra~' tions assayed. It might be necessary therefore to treat both sexes in order to produce sterility. Kenaga (1965). in his pioneer reports on the triphenyl tins pointed out that they were less effective on males than on females of those insects studied. Concentrations of 0.1 and 1.0% did not seriously affect the feeding behavior and general vigor of the beetles. At these two concentrations, some temporary reproduction control was obtained which was lost 33 as soon as the insects were transfered to untreated plants. It does seem that feeding on the sterilant must not be broken in order to ensure complete sterility. High mor- tality observed at concentrations of 2% and 3% resulted mainly from starvation, because the beetles were very lethargic and could not carry out normal functions. Flint (1965) described similar conditions in the eye gnats which were treated with high doses of radiation. Grosch (1956) had earlier described an identical occurrence as a ”radi— ation induced lethargy." The potentialities of Dowco-186 as a chemosterilant for this pest cannot be fully appraised until a reliable method of administering it to the insect is found. The method of application used in these studies posed feeding problems and retarded progress with the inves- tigation. A Histological sections of the testis of apholate- treated males showed no tissue damage or cellular disrup- tion. The testis was full of spermatozoa which appeared morphologically normal. Motile sperms were observed in the testis of males up to 21 days after treatment. Damage to reproductive tissue has been reported in the boll weevil Anthonomus grandis treated with apholate at 15 ppm. (Lind- quist et al., 1964). Rai (1964) also stated that at 0.05% concentration, apholate caused degeneration of the testis in Drosophila melanogaster. vThese authors treated younger stages of the insects and it is probable that in such early formative stages the reproductive organs might be more 34 susceptible to damage. The male post—diapause cereal leaf beetles were at a mature stage and any damage would have been less easily discernible. SUMMARY 1. Apholate sterilized post—diapause adults of the cereal leaf beetle. Both sexes were sterilized by 0.05% while 0.1% sterilized the males. 2. Treated males did not live as long as normal males, and mortality was high from 10 days after treatment. 3. Mating behavior of treated males was not affected by the sterilizing dosage. 4. A 12:1 ratio of treated males to untreated males would be required to reduce egg viability to zero percent. The early mortality of the treated males would necessitate more frequent release of sterilized beetles. 5. Dowco-186 produced some sterility while both sexes fed on treated plants. This sterility was lost as soon as the beetles were returned to untreated plants. 6. High doses of Dowco—186 caused lethargy which eventually led to death. 7. No tissue damage or cellular disruption occured in the reproductive organs of post-diapause males treated with 0.1% apholate. Sperm motility was not affected by this dosage level. 35 REFERENCES Auerbach, C. and Robson, J. M. 1947. The production of mutations by chemical substances. Proc. Roy. Soc. Edinburgh. B62:271. Baumhover, A. H. 1958. Florida Screw-worm control program Vet. Met. 53:214-219. , Graham, A. J., Bitter, B. A., Hopkins, D. E., New, W. D., Dudley, F. J., and Bushland, R. C. 1955. Screw-worm control through release of sterilized males. Science 122(3163):287-288. Borkovec, A. B. 1962. Sexual sterilization of insects by chemicals. Science 137(3535):1034-1037. Bushland, R. C., and Hopkins, D. E. 1953. Sterilization of Screw-worm flies with x-ray and gamma rays. J. Econ. Entomol. 46(4):648-656. , 1951. Experiments with screw—worm flies steri— lized by x-rays. J. Econ. Entomol. 44(5):725—73l. Chamberlain, W. F. 1962. Chemical sterilization of the Screw-worm, Jour. Econ. Entomol. 55(2):240-248. Cork, J. M. 1957. Gamma radiation and longevity of the flour beetle, Radiation Research. 7:551-557. ' Crystal, M. M.- 1963. The induction of sexual sterility in the screw—worm fly (Cochliomyia hominivorax) by anti— metabolites and alkylating agents. J. Econ. Entomol. 56(4):468-473. , and La Chance, L. E. 1963. The modification of reproduction in insects treated with alkylating agents. I. Inhibition of ovarian growth and egg production and hatchability. Dame, D. A., Ford, H. R. 1964. Chemosterilization and its permanency in mosquitoes. Nature, 201(4920):733—734. Davis, A. N., Gahan, J. B., Weidahass, D. E., and Smith, C. N. 1959. Exploratory studies on gamma radiation for the sterilization and control of Anopheles quadrimacu- latus. J. Econ. Entomol. 52(5):868—870. 36 37 Davich, T. B. and Lindquist, D. A. 1962. Exploratory studies on gamma radiation for the sterilization of the boll weevil. J. Econ. Entomol. 55(2):104-l67. Flint, H. M. The effects of gamma radiation on the fertil- ity and longevity of Hippelates pusio. Jour. Econ. Ent. 58(B):550-559. Guyer, G. 1962. The bionomics of Oulema melanopa. (L.) on small grains in Michigan. Abst. in Bul. Ent. Soc. Amer. 8(3):162. Hanson, F. B. 1929. The effects of x—rays on productivity and sex ratio in D. melanogaster Amer. Nat. 62:352. Henneberry, T. J. and McGovern, w. L. 1963. Some effects of gamma radiation on mating competitiveness and be— havior of Drosophila melanogaster males. J. Econ. Entomol. 56(6Iz739-341. Hoopingarner, R. A., Kumararaj S. and French, A. L. 1965. Gametogenesis and radiation effects in the cereal leaf beetle, Oulema melanopa (L.) Ann. Ent. Soc. America 58(6):777-781} Jaynes, H. A., and Godwin, P. A. 1957. Sterilization of the white-pine weevil with gamma radiation. J. Econ. Entomol. 50:393-5. Knipling, E. F. 1955. Possibilities of insect control or eradication through the use of sexual sterile males. J. Econ. Entomol. 48(4):459-462. ' . 1960. Use of insects for their own destruction. J. Econ. Entomol. 53(3) 415-420. . 1959. Sterile male method population control. Science 139(3380):902—904. Koller, P. C. 1954. Comparative effects of alkylating agents on cellular morphology. Ann. N. Y. Acad. Sci. 68:783-801. LaBrecque, G. 0-, Meifert, D. N., and Smith, C. N. 1962. Mating competitiveness of Chemosterilized and nor— mal male house flies. Science l36(3514):388-389. , Adcock, P. H., and Smith, C. N. 1960. Tests with compounds affecting house fly metabolism. J. Econ. Entomol. 56(4):476. 38 Lindquist, D. A., Garzycki, L. J., Mayer, M. S., Scales, A. L. and Davich, T. B. 1964. Laboratory studies on sterilization of the boll weevil with apholate. J. Econ. Entomol. 57(5):745-750. Mainland, D., and Murray, I. M. 1952. Tables for use in Fourfold Contingency tests. Science ll6:59(-594). MUller, H. J. 1927. Artificial transmution of the gene. Science 66:84-47. . 1928. The problem of genetic modification (Ver— hand der. V. Kongf. Vereb, 1927). Proc. 5th Inter- nat, cong. gent. Berlin, 1927. Sup. Bd. I:234-260. . 1940. An analysis of the process of structural change in the chromosomes of DrOSOphila, Jour. Gen— etics, 40:1-66. Murrax W. S., and Bickey, W. E. 1964. Effect of Apholate on the Southern House Mosquito Culex pipiens quinque- fasciatus Say: Bull. A-l34 Agr. Expt. Station Univer- sity of Maryland. Nagasawa, S., Shinohara, H., and Shiba, M. 1965. Chemo- sterilants of insects. VI. Sterilizing effect of Dowco-186 on Callosobruchus chinensis, with special reference to the hatchability-of the eggs deposited by treated adults. Bochy-Kagaku 30.(3):91-95. , and Shinohara, H. 1965. Mating competition be- tween apholate-sterilized and normal males of Azuki been weevil. Callosobruchus chinensis L. Jap. J. Appl. Ent. Zool. 9T4):271-274. Rai, K. S. 1964. Cytogenetic effects of chemosterilants in mosquitoes. I. Apholate—induced aberrations in the somatic chromosomes of Acdes aegypti L. Cyto- logia. 29:(3), 364-53. Rapoport, I. A. 1947. Derivatives of Carbamic acid and mutations. Byul Eksperin. Biol. 1 Med., 23:198-201. Rogers, W. I., and Hichey, J. D. 1960. Studies on the post-irradiation feeding activity of Tribolium castanum. J. Econ. Entomol. 53:584-90. Runner, G. A. 1916. Effect of roentgen rays on the to- bacco or cigarette beetle and the results of ex— periments with a new form of roentgen tubes, Jour. Agr. Res. 6:383-388. 39 Sawicki, R. M., and Farnham, A. W._ 1964. A dipping tech- nique for selecting house-flies, Musca domestica L., for resistance to insecticides. Bulletin of Ento- mological Research 55(3):54l-546. Schmidt, C. H., Dame, D. A., and Weidhass, D. E. 1964. Radiosterilizatlon vs Chemosterilization in house flies and mosquitoes. J. of Econ. Entomol. 57(5): 615-796. Schwartz, P. H., Jr. 1965. Effects of apholate, metepa, and TEPA on reproductive tissues of Hippelates pusio. J. Invertebrate Pathol. 7(2):l48—151. Smith, N. C., LaBrecque, G. C., and Borkovec, A. B. 1964. Insect chemosterilants. Ann. Rev. Entomol. 9:269-284. . 1964. Insect chemosterilants. In_Annual Review of Entomology, Vol. 9, pp. 269-284, Annual Reviews, Inc. Palo Alto, Calif. Stacey, K. A., Cobb, M., Couseno, S. F., and Alexander, P. 1958. The reactions of the ”radiomimetic” alky- lating agents with macromolecules in vitro. Ann. N. Y. Acad. Sci. 68:682-701. Steiner, L. F., and Christenson, L. D. 1956. Potential usefulness of the sterilenmleNrelease method in fruit fly eradication. Sternberg, S. 8., Philips, F. S., and Scheller, J. 1958, Pharmacological and pathological effects of alky-- lating agents. Ann. N. Y. Acad. Sci. 68:811-25. Yeoman, G. H., and Warren, B. C. 1965. Chemosterilization of the sheep blowfly, Lucilia Sericata with apholate. Vet. Record 77(32):922¥8. l I ll III I