Chemosterilization of the tropical house mosquito Culex pipiens fatigans Wied.: laboratory and field cage studies*

Bull. Org. mwnd. Sante 1973, 45-48Bull. Wid Hlth Org. I , 48, Chemosterilization of the tropical house mosquito Culex pipiens fatigans Wied.: laboratory and field cage studies * V. P. SHARMA,' R. S. PATTERSON,2 K. K. GROVER,8 & G. C. LaBRECQUE' Tris(I-aziridinyl)phosphine sulfide was found to be an excellent sterilant for male Culex pipiens fatigans. When male pupae were exposed to a 0.6% solution for 3 hours, the ensuing adults were rendered permanently sterile with no apparent reduction in their vigour or longevity based on laboratory andfield cage studies. Females, however, exposed to the same dosage for the same length of time were partially fertile. The common house mosquito Culex pipiens fati- gans is the principal vector of Wuchereria bancrofti and consequently considerable effort has been de- voted in recent years to developing methods to control this species. As the continued use of pesti- cides often leads to environmental contamination, as well as to the development of resistance, it has be- come imperative to explore and develop alternative methods of control. Since the successful eradication of the screw-worm fly from the south-east of the USA by the release of genetically impaired indivi- duals, there has been worldwide interest in the appli- cation of the " sterile-male technique " to control mosquitos (Knipling, 1959). Several small field studies based on this principle have been conducted against Aedes aegypti (Morlan et al., 1962), Anopheles quadri- maculatus (Weidhaas et al., 1962), and C. p. fatigans (Krishnamurthy et al., 1962; Laven, 1967; Patterson et al., 1970). In only two of these studies (both with Culex spp.) have the released males successfully competed in nature so that the indigenous population was suppressed. The first successful study was con- ducted by Laven (1967) in Burma, where males of a nonindigenous cytoplasmically incompatible strain of C. p. fatigans were released; within six genera- tions, no viable egg rafts were observed. In the second study, Patterson et al. (1970) released males * From the WHO/ICMR Research Unit on the Genetic Control of Mosquitos, Kilokri, New Delhi, India. Senior Scientist, ICMR. 'Entomologist, WHO. ' Research Assistant, ICMR. 'Project Leader. of C. p. fatigans that had been sterilized by tris(l- aziridinyl)phosphine sulfide (" thiotepa ") into a na- tural population on an isolated island: again within six generations this population was eliminated. These two preliminary trials have led to more intensive evaluation of the practicability of the sterile-male technique for mosquito control. In order to evaluate this technique for massive field releases certain preliminary experiments must be conducted and the object of this study was to evaluate thiotepa as a potential sterilant for male C. p. fatigans. First of all, the most efficient dosage and exposure period must be established for sterilizing the males with the minimum deleterious effect on their vigour. Secondly, since precise sexing in large releases is seldom achieved, the effect of the male sterilizing dose on female fecundity must also be evaluated. MATERIALS AND METHODS The strain of C. p. fatigans used in this study was originally collected in Delhi and has been under colonization for almost 2 years. The rearing proce- dures have been standardized at this laboratory. The chemosterilant thiotepa was selected for this study because of its unique quality of sterilizing the insects in the pupal stage (White, 1966; Patterson et al., 1970) and also because the adults 24-48 hours old carry no detectable residue of the chemical when released into the natural environment. (LaBrecque, personal communication). As this chemical is very stable when kept under refrigeration, but breaks down rapidly in an acid medium (Borkovec, 1966), 2977 -45- 4 V. P. SHARMA ET AL. all solutions for chemosterilization were buffered to pH 8. Each lot was often used several times with no reduction in its effectiveness as a sterilant. The pupae were placed in the desired concentration of thiotepa for fixed periods (as detailed later), then removed, rinsed twice in tap water, and placed in fresh dechlorinated water for eclosion in a standard aluminium frame cage (30 x 20 x 20 cm). Wooden framed field cages either 5 x 3.5 x2 m or 2 x 2 x2 m were used for outdoor studies. A suction tube was normally used for transferring and sexing the mos- quitos. The adults were given 1 % glucose solution for 3 days, and this was replaced by water for 24 hours before a young chick was placed in the cage for the females to feed on. A 1 % fermented yeast infusion was used as an oviposition medium 3 days later. Each egg raft was checked for hatchability under a dissecting microscope. Inseminations were checked by dissecting out the spermathecae and examining for sperm under a compound microscope. All contaminated glassware and other equipment was soaked in a 10% acetic acid solution prior to washing to remove thiotepa residues. RESULTS Several studies were conducted to select the most efficient sterilizing dose ofthiotepa with the minimum exposure period so that there would be no adverse effect on the males' vigour or mating competitiveness when released in the field. Short exposures were investigated since an exposure of longer than 5 hours would have created problems in handling during regu- Table 1. Percentage sterility induced in male C.p. fatigans exposed as pupae to a range of thiotepa concentrations for different exposure periods (average of 4 replicates of 25 males and 25 females) Thiotepa Duration of exposure to sterilant (hours) concen- tration 0 1 2 3 4 5 0.1 1.3 - 22.8 - 25.8 - 0.5 3.5 58.8 94.0 96.8 98.8 99.7 0.6 1.8 82.7 98.4 99.7 99.7 99.5 0.7 1.6 84.2 97.9 99.9 99.9 100.0 0.75 1.8 96.7 99.9 100.0 100.0 100.0 0.8 1.0 93.3 98.7 99.8 100.0 100.0 1.0 3.6 91.7 99.0 100.0 100.0 100.0 Table 2. Percentage sterility induced in female C. p. fatigans exposed as pupae to a range of thiotepa con- centrations for different exposure periods (average of 2 replicates of 25 males and 25 females) Thiotepa Duration of exposure to sterilant (hours) concen- tration 0 1 2 3 4 5 0.5 1.1 18.5 19.7 31.9 39.3 51.2 0.6 0.7 22.7 35.8 38.1 48.8 57.3 0.7 1.3 26.8 36.3 43.3 45.4 50.7 0.8 4.7 18.9 42.0 46.3 55.9 60.2 1.0 6.4 - - 38.2 90.2 73.6 lar working hours. For evaluation, male and female pupae (normally 2-26 hours old) were held in diffe- rent concentrations of thiotepa for different expo- sure periods from 1 to 5 hours. After eclosion the treated males or females were placed in the same cage as normal individuals in equal proportions. The egg rafts were subsequently checked for per- centage hatch. The results showed that with different exposure periods males can be sterilized by a range of con- centrations of thiotepa (Tables 1 and 2). A concen- tration of0.6% with exposure for 3 hours consistently produced 99+1 % sterility in the males and appeared to be the most efficient. However, at this dosage the females were only 38% sterile. In studies to evaluate the vigour and mating compe- titiveness of males following chemosterilization, the pupae were less than 24 hours old when treated in one study and more than 24 hours old in another. Adult chemosterilized and normal male mosquitos were mixed in different proportions and were placed in the same cage as virgin females. The egg rafts were evaluated for sterility. The results of these experi- ments (Table 3) indicate that the chemosterilized males retained their mating competitiveness. In both studies the sterility obtained often exceeded the ex- pected sterility. The mating competitiveness of these mosquitos was further evaluated in two large field cages. Follow- ing chemosterilization in a 0.6% thiotepa solution for 3 hours the pupae were held in the laboratory for eclosion and then released as adults into the cages along with untreated individuals at the ratio of 9: 1. Young chicks were maintained within the cage to provide a regular blood source. Egg rafts were col- 46 CHEMOSTERILIZATION OF C. P. FATIGANS Table 3. Mating competitiveness of male C. p. fatigans exposed as pupae for 3 hours to a 0.6 % thiotepa solution (average of 4 replications of 50 normal males/ test) Sterility (%) Ratio Observed sterile fertile Expected Pupae Pupae <24 h old >24 h old 0:1 0 6.2 0 1:0 100 100 100 1:1 50 50.0 55.0 2:1 66.6 66.6 - 3:1 75 - 77.4 4:1 80 82.2 - 6:1 85.8 95.4 - 9:1 90.0 97.5 - lected daily for 20 days from the mud pots containing 1 % fermented yeast. Of the total of 9 000 rafts collected from the field cage, over 90% were sterile. Thus, thiotepa treatment did not adversely affect mating competitiveness under field cage conditions. The mating ability and longevity of males follow- ing sterilization as young pupae in 0.6% thiotepa solution for 3 hours was evaluated. Male mosquitos, either sterilized or normal, were caged with virgin females at the ratio of 1: 6 for 5 days, after which Table 4. The mating ability (average of 5 replications of 10 males and 60 females/test) and longevity in the laboratory (average of 10 replications of 80 males/test) of chemosterilized male C. p. fatigans Duration Rate Percentae survival a of mating ofinsemination/male (days) control treated control treated 1-5 4.1 4.1 88 (5) 90 (5) 6-10 3.4 1.9 78 (10) 86 (10) 11-15 2.7 1.3 68 (15) 66 (15) 16-20 2.6 1.0 40 (20) 44 (20) 21-25 2.0 0.5 - - a The figure in parentheses indicates the day on which survival was evaluated. the females were checked for insemination. Four further lots of virgin females were caged with these males, each lot for 5 days. The mortality of the males was also recorded during the 25-day period. Both the sterile and normal males were able to inseminate an equal number of females in the first 5-day period (Table 4), but after this the chemosterilized males were not as effective. There was, however, no adverse effect on the life span of the chemosterilized mosqui- tos in the laboratory. In another study in which treated or normal males were kept in cages by them- selves and allowed to feed on 1 % glucose solution, there was little difference in the survival pattern of the chemosterilized males and the normal mosquitos. A second study showed that the presence of females reduced the life span of males (Table 5). Studies were carried out to determine whether chemosterilization affected monogamy in females, which is apparently governed by the male accessory gland fluid " matrone " (Craig, 1967). Virgin females were placed in the same cage as sterile or normal males for 72 hours. The treated males were then removed and replaced by untreated males and vice versa. Of the eggs laid by females that had mated initially with normal males, 99% were viable, where- as of the eggs of those initially crossed with sterile males only 2-3% hatched. Thiotepa as a chemo- sterilant did not appear to damage the " matrone" and the females remained monogamous. To check the permanency of sterility in females, chemosterilized males were placed in the same cage Table 5. Effect of caging with females on the per- centage cumulative mortality of males exposed as pupae to 0.6 % thiotepa (average of 10 replications of 80 or 100 sterile males/test) Malesalone Males caged withDays after Males alone females sterilization control treated control treated 1-5 8 8 4 4 6-10 14 13 26 26 11-15 17 15 46 48 16-20 22 22 81 86 21-25 31 43 100 100 26-30 54 77 - - 31-35 92 96 - - 36-40 97 100 - - 47 48 V. P. SHARMA ET AL. as normal females and the females were held for ovi- position. After the first period of oviposition the females were offered a second blood meal and held until they had oviposited for a second time. The level of sterility in the females remained at 99 %'. To determine the permanence of sterility in males that had been sterilized as 2-26-hour-old pupae in 0.6% thiotepa solution for 3 hours, such males were caged with virgin females. The females were removed and were replaced with new females after 14 days and again after 18 days. Each cross was held for 4 days. Egg rafts were checked and the levels of sterility for the three crosses were 99.3%, 98.7%, and 97.4%, respectively. R, SUM t CHIMIOSTJRILISATION DE CULEX PIPIENS FATIGANS WIED.: ETUDES AU LABORATOIRE ET SUR DES MOUSTIQUES ENCAGES Le sulfure de tri(aziridinyl-1) phosphine (thiot6pa) a fait preuve de remarquables propriet6s sterilisantes chez Culex pipiens fatigans male. Les nymphes d'insectes males exposees a une solution de thiotepa a 0,6% pendant 3 heures ont donne naissance A des adultes d6finitivement steriles mais n'ayant rien perdu de leur vitalit6, de leur long6vit6 et de leurs aptitudes sexuelles. Des nymphes femelles, trait&es de fa9on identique, n'ont donn6 nais- sance A des individus steriles que dans la proportion de 38,1 %. REFERENCES Borkovec, A. B. (1966) Advanc. Pest Control Res., 7,1-133 Craig, G. B. (1967) Science, 156, 1499-1501 Knipling, E. F. (1959) Science, 130, 902-904 Krishnamurthy B. S. et al. (1962) Indian J. Malar., 16, 365-373 Laven, H. (1967) Nature (Lond.), 216, 383-384 Morlan, H. B. et al. (1962) Mosquito News, 22, 295-300 Patterson, R. S. et al. (1970) Science, 163, 1368-1370 Weidhaas, D. E. et al. (1962) Mosquito News, 22, 283-291 White, G. B. (1966) Nature (Lond.), 210, 1372-1373