Chemosterilization of Culex pipiens fatigans Wiedemann by exposure of aquatic stages

Bull Org.W ond.Santh 1972, 47, 305-308 Bull. Wid Hith Org. Chemosterilization of Culex pipiens fatigans Wiedemann by exposure of aquatic stages 3. Induction of dominant lethal mutations in the F1 generation by certain aziridines, phosphoramides, and s-triazines K. K. GROVER 1 & M. K. K. PILLAI ' In insects it is known that the chemosterilant treatment induces dominant lethal muta- tions which bring about embryonic death. The possibility of delayed expression of the chemosterilant-induced dominant lethals in F1 progeny of the tropical house mosquito, Culex pipiens fatigans Wiedemann has been investigated. The chemosterilants employed were 8 aziridinyl compounds, 3 phosphoramides, and 4 s-triazines. In general, all the chemosterilants tested were found to cause different degrees of mortality at different life stages of the F1 progeny. In this respect the aziridinyl compounds were more effective than the phosphoramides and s-triazines. This effect of the chemosterilants may be useful in the sterile-male technique. Several aziridines, phosphoramides, and s-triazines have been screened for their sterilization potential against the tropical house mosquito, Culex pipiens fatigans Wiedemann and many of them were found to induce high sterility (Grover et al., 1967; Pillai & Grover, 1969; Grover & Pillai, 1969). The mechan- ism of such induced sterility has been a topic of interest in the recent years. Both alkylating and nonalkylating chemosterilants have been found to cause cytogenetic damage in the somatic and repro- ductive tissues of this mosquito (Grover et al., 1971, 1972a, 1972b) as well as in other insect species (Rai, 1964a, 1964b; George & Brown, 1967; LaChance et al., 1968; LaChance et al., 1969). Such genetic damage is passed on to the embryo through the gametes and affected embryos usually die before the blastoderm stage is reached (Fahmy & Fahmy, 1954; LaChance & Riemann, 1964; LaChance et al., 1968; Grover et al. 1972b). However, in some organisms the lethal effect appears in the F1 generation (Lind- quist et al., 1964; Borkovec et al., 1967; Pillai & Grover, 1969). The present studies were, therefore, initiated to assess the ability of various aziridines, I Research Scholar, Department of Zoology, University of Delhi, Delhi-7, India. 'Reader in Zoology, Department of Zoology, University of Delhi, Delhi-7, India. phosphoramides, and s-triazines to induce dominant lethal mutations in the F1 generation of C.p. fatigans. MATERIALS AND METHODS The Delhi strain of C.p. fatigans mentioned in our earlier paper was used in the present investigation (Pillai & Grover, 1969). The chemosterilants em- ployed in the present studies may be grouped into 3 categories as follows: Aziridinyl compounds 2,2,4,4,6,6-hexakis(I-aziridinyl)-2,2,4,4,6,6-hexa- hydro-1,3,5,2,4,6-triazatriphosphorine (apholate) tris(I-aziridinyl)phosphine oxide (tepa) tris(2-methyl-1-aziridinyl)phosphine oxide (metepa) methyl bis(I-aziridinyl) phosphinate (ENT-61342) ethyl bis(l-aziridinyl) phosphinate (ENT-50761) propyl bis(l-aziridinyl) phosphinate (ENT-61343) isopropyl bis(l-aziridinyl) phosphinate (ENT-61355) butyl bis(I-aziridinyl) phosphinate (ENT-61354) Phosphoramides hexamethylphosphoric triamide (ENT-50882) N"-(2-methoxyethyl)-N,N,N',N,'-tetramethylphos- phonic triamide (ENT-60210) 2900 -305 K. K. GROVER & M. K. K. PILLAI N,N,N',N'-tetramethyl-P-piperidinophosphonic dia- mide (ENT-51007) s- Triazines hexamethylmelamine hydrochloride (ENT-50905) 2,4-diamino-6-morpholino-s-triazine hydrochloride (ENT-51 143) 2-amino-4,6-bis(dimethylamino)-s-triazine hydro- chloride (ENT-51146) 2,4-diamino-6-isopropyl-s-triazine monohydrochlo- ride (ENT-60433) The samples of chemosterilants were 95-99% pure and were received from Dr A. B. Borkovec, US Department of Agriculture, Md., USA. All the compounds are water soluble. Mosquitos were treated at the larval or pupal stages with the different chemosterilants at the doses shown in Tables 1 and 2. The methods of rearing the mosquitos and of treating them with the chemo- sterilants were those described by Pillai & Grover (1969) and Grover & Pillai (1969). More than 200 treated males were crossed with same number of treated females and were given blood meals on alter- nate days. Egg rafts laid during the first 15 days were collected and allowed to hatch in vials. Random samples of 100-200 larvae were collected from each concentration and were reared up to the adult stage. The mortality at the different stages (larvae, pupae, and adults up to 1 day after emergence) of develop- ment of the F1 generation was recorded. Parallel control experiments on the F, progeny of untreated mosquitos were carried out and mortality was recorded. The mortality was corrected by means of Abbot's formula. RESULTS The effects of different chemosterilant treatments, given at the larval and pupal stages, on the F1 gene- ration of C.p. fatigans are shown in Tables 1 and 2. All the treatments with aziridines caused some mor- tality in the F1 progeny. Among the polyaziridinyl compounds apholate induced more mortality than did either metepa or tepa. Among the diaziridniyl compounds used at the larval stage, ENT-50761 was most effective, causing mortality as high as 70% at a concentration of 10 ,ug/g. The same treatment with ENT-61343 caused only 53% mortality. Of the substances used to treat pupae, ENT-50761 was most effective, causing 82% mortality at a concen- tration of 1 %. The treatment of larvae with phosphoramides at a concentration of 250 ,ug/g caused 20-33% morta- lity. Similarly, s-triazines caused significant mor- tality in the F1 generation and the treatment of lar- vae with ENT-51146 at a concentration of 50 ,ug/g induced mortality as high as 69%. However, the treatment of pupae with 1% ENT-50882 caused only 9% mortality in the F1 progeny, while the same dose of ENT-50905 caused 32% mortality. Thus all the compounds screened induced some mortality in the F, generation. Table 1. Effect of certain aziridines on the Fi genera- tion of C.p. fatigans adults exposed as early 2nd-instar larvae and newly emerged pupaea Percentage chemsterlantConcentra - Pecnaeof adults Chemosterilant tion Percentage surviving Percentage(ppm) pupation up tol1 daY mortality bafter emergence Treatment of larvae apholate 5 75 68 20 1 0 79 55 35 metepa 5 92 84 1 10 85 75 12 tepa 5 83 73 14 10 74 63 26 ENT-61342 5 82 76 10 10 75 66 22 ENT-50761 5 80 64 25 10 25 25 70 ENT-61343 5 90 77 9 10 50 40 53 ENT-61355 5 83 74 13 10 75 64 25 ENT-61354 5 75 73 14 10 61 55 35 control - 93 85 - Treatment of pupae apholate 10 000 65 60 37 metepa 10 000 71 65 32 tepa 10 000 no egg laying & hence no hatch ENT-61342 10 000 77 75 21 ENT-50761 10 000 20 17 82 ENT-61343 10 000 83 78 18 ENT-61355 10 000 65 50 47 ENT-61354 10 000 81 79 17 control - 96 95 - a Larvae exposed continuously until pupation and pupae exposed for 28 hours. b Using Abbot's formula. Includes mortality at larval, pupal, and adult stages (adults up to 1 day after emergence). 306 CHEMOSTERILIZATION OF C. P. FATIGANs. i. Table 2. Effect of certain phosphoramides and s-triazi- nes on the Fl generation of C.p. fatigans adults expo- sed as early 2nd-instar larvae and newly emerged pupae a Percentage Concentra- of adults Chemosterilant tion Percentage surviving Percentage (p) pupation up to I day mortality b after emergence Treatment of larvae control - 98 98 - phosphoramides ENT-50882 100 90 88 10 250 80 70 28 ENT-51007 100 80 80 18 250 70 66 33 ENT-60210 100 80 80 18 250 80 78 20 500 70 70 28 s-triazines ENT-50905 10 84 79 19 25 61 55 44 ENT-60433 10 90 90 8 25 86 86 1 2 ENT-51146 10 86 82 16 25 69 67 32 50 40 30 69 ENT-51143 0.5 86 78 20 1.0 85 75 23 Treatment of pupae control - 95 95 - ENT-50882 10 000 90 89 9 ENT-50905 10 000 80 68 32 a Larvae exposed continuously until pupation and pupae exposed for 28 hours. b Using Abbot's formula. Includes mortality at larval, pupal, and adult stages (adults up to 1 day after emergence). DISCUSSION Fahmy & Fahmy (1954) suggested that although dominant lethal mutations are usually expressed prior to hatch and thus result in nonviable eggs, less critical chromosomal changes sometimes permit the embryos to complete their development and the eggs to hatch. The genetic complement of such individuals may carry gene mutations that are lethal, or that lead to morphological deformities in the progeny. Pillai & Grover (1969) found frequent morphological deformities in the eggs and embryos of C.p. fatigans treated with different aziridines. In the present study, significant mortality in the F1 generation was observed with all the aziridinyl com- pounds screened, indicating that all these com- pounds induce dominant lethal mutations in F1 generation. Lindquist et al. (1964) showed that egg hatch is not an accurate indication of sterility following treatment with apholate. Their data showed that about 91 % of the F1 larvae carry domi- nant lethal mutations that are expressed prior to emergence of the adults. It is interesting to note that the sterilizing potency of apholate was better than that of metepa or tepa in C.p. fatigans larvae (Pillai & Grover, 1969). The present finding indicates that apholate-induced lethal mutations find expression not only at the embryo stage but also at various other stages of the F1 generation. The substituted diaziridinyl compounds tested caused delayed expression of dominant lethal mutations more fre- quently than did tepa. It is worth mentioning that the treatment of larvae with some of these com- pounds induced higher levels of sterility than did tepa in C.p. fatigans (Pillai & Grover, 1969). The phosphoramides and s-triazines also caused significant mortality in the F1 progeny of C.p. fatigans and thus it is clear that they also induce dominant lethal mutations that are expressed in the F1 progeny. Borkovec et al. (1967) showed that the production of mortality in the F1 generation was characteristic of all s-triazines. This ability of the chemosterilants to induce mortality in the F1 gen- eration is an additive effect and would be advan- tageous in sterile-male release programmes. In the present study, although no attempt was made to investigate death caused by sex-linked lethal mutations, no significant difference in the sex ratio was apparent. Murray & Bickley (1964) also found no evidence of sex-linked recessive lethal mutations in C.p. quinquefasciatus (= C.p. fatigans) after treatment with apholate. ACKNOWLEDGEMENTS rhe authors are grateful to Dr A. B. Borkovec of the US Department of Agriculture, Md., USA for providing the samples of chemosterilants used in the present study. 307 308 K. K. GROVER & M. K. K. PILLAI RtSUMt CHIMIOSTERILISATION DE CULEX PIPIENS FA TIGANS WIEDEMANN PAR EXPOSITION DES STADES AQUATIQUES: 3. INDUCTION DE MUTATIONS LETALES DOMINANTES DANS LA GENERATION F1 PAR CERTAINS AZIRIDINES, PHOSPHORAMIDES ET TRIAZINES-S On a recherche dans quelle mesure certains chimio- sterilisants pouvaient induire des mutations letales domi- nantes dans la g6n6ration F1 chez le moustique Culex pipiens fatigans Wiedemann. Les essais ont port6 sur 8 aziridines, 3 phosphoramides et 4 triazines-s. Les insectes ont ete trait6s aux stades larvaire et nymphal. Les femelles et les males ont ete ensuite accoupl6s et les larves survivantes de la generation F1 ont et6 6lev6es jusqu'au stade de l'adulte afin de d6terminer la mortalite. Le traitement par les chimiost6rilisants a et6 a l'origine, a un degr6 variable suivant les comopses, de mortalite a difftrents stades evolutifs de la g6neration F1. Les aziridines, et notamment l'apholate, se sont montrees particuli&rement efficaces. La mortalite 6tait de toute 6vidence due a l'expression retard& de mutations l6tales dominantes. Cet effet complementaire pourrait 'etre avantageusement mis a profit lors des applications de la technique des males st6riles. REFERENCES Borkovec, A. B. et al. (1967) J. econ. Ent., 60, 893-894 Fahmy, 0. G. & Fahmy, M. J. (1954) J. Genetics, 53, 602-613 George, J. A. & Brown, A. W. A. (1967) J. econ. Ent., 60, 974-978 Grover, K. K. & Pillai, M. K. K. (1969) Bull. Wld Hlth Org., 41, 929-936 Grover, K. K. et al. (1967) Curr. Sci., 36, 625-627 Grover, K. K. et al. (1971) Indian J. exp. Biol., 9, 150- 152 Grover K. K. et al. (1972a) Cytologia, (In press) Grover, K. K. et al. (1972b) J. med. Ent., 9, 451-460 LaChance, L. E. et al. (1964) Mutation Res., 1, 318-333 LaChance, L. E. et al. (1969) Mutation Res., 7, 63-74 LaChance, L. E. et al. (1968) Cytogenetic and cellular basis of chemically induced sterility in insects. In: La Breque, G. C. & Smith, C. N., ed., Principles of insect chemosterilization, New York, Appleton-Century- Crofts, pp. 99-157. Lindquist, D. A. et al. (1964) J. econ. Ent., 57, 745-750 Murray, W. S. & Bickley, W. E. (1964) Univ. Maryland Agric. Exp. Sta. Bull., No. 134, pp. 1-37 Pillai, M. K. K. & Grover, K. K. (1969) Bull. Wld Hlth Org., 40, 229-233 Rai, K. S. (1964a) Cytologia, 29, 346-353 Rai, K. S. (1964b) Biol. Bull., 127, 119-131.