SEPTEMBER 1990 Wnrrn LlRvnr, MurlNt oF TX. gzLENDENS 447 A NEW MUTANT, WHITE LARVA, OF THE MOSQUITO T OXO RHY N CH I TES SP LE N DENS;GENETICS AND CANNIBALISM1 MASHIRO HORIO,, MASUHISA TSUKAMOTO, lNo ICHIRO MIYAGI3 ABSTRACT. A strain of a new body-color mutant, white larua (ul), was established from a freld- collectedwild-type strain of Toxorhynchites spl,endens.The mutant can be distinguished from the wild type in both the larval and pupal stages,but not in the adult. Crossingexperimenls confirmed its mode of inheritance to be a single recessivesystem, This is the first visible mutant found in Tx. splendens. Larvae ofthe rul phenotype seemto be recognizedas prey by other individuals in mass larvae iearing. INTRODUCTION Environmental Health, Japan. This paper pre- sents the results of genetic analysis of a new At present, 76 speciesand subspeciesof the mutant, white larua (ul), and the effect of larval genus ?ororhynchites Theobald are known body color on cannibalism. (Knight and Stone 1977, Steffan and Evenhuis 1985, Evenhuis and Steffan 1986, Tsukamoto 1989). Among them, Toxorhynchites splendens MATERIALS (Theobald) occurswidely in Asia from Kashmir AND METHODS to the South Pacific. This speciesis considered The following strains were used: 1) Wild type: to be one of the most useful agents for the Colonized in the laboratory from field collec- biological control of aedine vectors of various tions of ?r. splendensftom leaf axils of Colocasin diseasessuch as filariasis, dengue and yellow plants and ovitraps on Palawan Island, Philip- fever. pines, in October 1982.2) White lnrua (wI): A The benefits of Toxorhynchitesas a laboratory visible mutant strain isolated from the Palawan animal have also recently been recognized for strain in August 1983. use in the isolation of arboviruses and filariae. Maintenance of mosquitoes.:Immature and Comparative studies have demonstrated this adult mosquitoeswere maintained in an insec- procedureis more sensitivefor primary isolation tary at a temperature of.26 + 1"C, RH 75 + |Vo, of arboviruses than is the inoculation of new- and light:dark regime of 14:10h. born mice or mammalian cell culture (Rosen For rearing Tx. spbndens. about 50 adults 1981). Advantages of Toxorhynchitesadults lie were kept in each cage(20 x 20 x 30 cm) with in their safety (nonbiting) and large size (Eshita a stainlesswire frame enclosedwith white nylon et al. 1982). mesh with a sleeveat a lateral end. Adults were No geneticalinformation has been previously fed on an 8% sucrose solution, in a 150-ml available on Tx. splendensexcept for a report Ehrlenmeyer flask with a cellulose wick. For on its karyotypeby Bhat (1976). oviposition, a black plastic cup was provided During the laboratory colonization of a pop- containing distilled water. For crossing experi- ulation of Tx. splendenscollected from Palawan ments, larvae were reared individually in 100- Island, Philippines, a colorless larva was found ml plastic vials eachcontaining 50 ml of distillec among the usual dark-colored larvae. Isolation water. For routine maintenance,however, larvae of this mutant was attempted from the same were reared en massein a plastic tray (20 x B0 population in succeedinggenerations. After sev- x 6.5 cm) containing 2 liters of distilled water. eral generations of trials, this colorless larval They were fed on larvae of Aedesaegpti (Linn.) mutant was established as a fixed colony. The with a prey:predator ratio of 15:1. Pupae were mutant strain has been maintained for bB een- sexed and placed individually into small cap- erationsup to the present (August 1g89),In a sules. The mean duration from egg hatch to Iaboratory of the University of Occupationaland adult emergencewas 18.9 days with a range of 17.6-20.2days (Horio and Tsukamoto 198b). 1 Crossing experiments: Upon emergence, 20 This study was supported in part by Grants-in- virgin females and 20 males were put together Aid for OverseasScientific Survey in fSiSf-SZ (Nos. in each cage for the crossing experiments 56041048.57043043) from the as Minisrry ofEducation, shown in Table 1. Scienceand Culture, Government ofJapan. 2 Scoring for the white Department of Medical Zoology,School of Medi- or dark larval pheno- cine, University of Occupational and Environmental types was done at the 4th instar, and sex deter- Health, Kitakyushu 80?, Japan. mination was done in pupae 2- to B-days-old " Laboratory of Medical Zoology,School of Health under a binocular microscope. Divergence of Science,Faculty of Medicine, and ResearchCenter of data from the expected segregation ratios for C_omprehensiveMcdicine; University of the Ryukyus. sexesand mutant phenotypeswas examined by Nishiharamachi, Okinawa g0B-01,Japan. chi-squaretests. SEPTEMBER1990 Wruro Lenver,Muretr op T5<.spnnxostts 443 survivors betweenthe mutant tlpe and the wild type, whereas only the alimentary canal and typg by either the individual or mass rearing tracheal tubes can be seen through the trans- method? If the difference is significant among parent skin in the white larval mutant (Fig. 1, these conditions, the results may provide an left). answer to the question of whether or not the This pigment is not produced in the white dark body color contributes to protection from larval mutant even after the eating of prey. In attacks by other individuals. the wild type larvae the chitinous plate is ini- tially colorlessjust after ecdysis,but it becomes RESULTS AND DISCUSSION brown within several hours. In contrast. the dark color of the larval skin can even be ob- Description of the mutanif..The normal color servedimmediately after ecdysis. of larval skin inTx. splcndensis dark mahogany- The normal pupal color in Tx. splendensis red on the dorsal half with a grayish-white ven- entirely dark brown in the wild phenotype type tral half. The head capsule,siphon, saddle and (Fig.2, right). Pupaeof the mutant showa dark sclerotizedsetal plates are dark chitinous brown color but can easily be distinguished from the (Fig. 1, right). Pigments of dark color are depos- wild type pupae because their intersegmental ited as particles in the fat bodies of the wild membranesare still grayish-white (Fig. 2, left). 100 70 :o 9oo €) .a.) gso _ 1OO .: leo a 80 't2 .t4 8 10 16 $ 20 o 2 4 6 810 12 14 16't820 Time after hatch (days) Fig' 3. Survival rate after mass (A and B) and individual (C and D) rearings from 100 of each larval type. Open circles indicate white larua and solid circles wild type. SEPTEMBER1990 WHttn LlRvel MurlNt op Tx. spttttoeus prey of both dark and white larvae even though Eshita, Y., T. Kurihara, T. Ogawaand A. Oya. 1982. the foodprovided (Ae. aegypti)was adequate.In Studies on the susceptibility of mosquito dengue other words, white larvae may not be recognized virus. II. Improved methods for the laboratory rear- as companions but attacked as prey either by ing on Toxorhynchitesamboinensis (Doleschall), as laboratory another white larva or by a dark larva (Fig. 5). hosts of the virus. Jpn. J. Sanit. Zool. 33:65-70. It is frequently found that in trees holes many Evenhuis,N. L. and W. A. Steffan.1986. Classification larvae of Orthopodom.yiaarcpheloide,s Theobald of the subgents Toxorhynchites (Diptera: Culici- (the body color is dark reddish-brown) can sur- dae). II. Revision of the Toxorhynchitesacaudatus vive with larvae of Toxorhynchite,s,whereas lar- group.J. Med. Entomol. 33:538-574. vae of other speciesare not found (Tanaka et Hartberg, W. K. 1978. White larva, a mutant of the al. 1979). mosquito Toxorhynchites breuipalpis Theobald. Mosq.News 38:563-565. Horio, M. and M. Tsukamoto. 1985.Successful labo- ratory colonization of 3 Japanesespecies of ?oro- ACKNOWLEDGMENTS rhynchites mosquitoes.Jpn. J. Sanit. Zool.36:87- 93. We would like to expressour sincere thanks Knight, K. L. and A. Stone. L977. A catalog of the to D. G. Rivera, the former director of the Ma- mosquitoesof the world (Diptera: Culicidae),vol. 6. laria Eradication Service, Ministry of Health, Thomas Say Found., Entomol. Soc. Am. Philippines; to Motoyoshi Mogi, Saga Medical Rosen,L. 1981.The tse of Toxorhynchltesmosquitoes School, for his collaboration in collecting mos- to detect and propagate dengue and other arbovi- quitoes on Palawan Island, Philippines; and we ruses.Am. J. Trop. Med. Hyg. 30:177-178. would like to acknowledgecooperation of the Steffan, W. A. and N. L. Evenhuis. 1985,Classification late E. C. Rausa, Superintendent of Iwahig of the subgenus Toxorhynchites (Diptera: Culici- Prison and Penal Farm, Palawan, Philippines. dae). I. Australian, easternPalaearctic and Oriental species-groups.J. Med. Entomol. We are also grateful to Kyoko Masuda and 22:421-446. Tanaka, K., K. Mizusawa and E. S. Saugstad.1979. Nobuko Fuji for their help A in rearing mosqui- revision ofthe adult and larval mosquitoesofJapan toes. (including the Ryukyu Archipelago and the Ogasa- wara Islands) and Korea (Diptera: Culicidae). Con- trib. Am. Entomol. Inst. (Ann Arbor) 16:41-987. REFERENCES CITED Tsukamoto, M. 1989.Two new mosquito speciesfrom a pitcher plant of Mt. Kinabal, Sabah, Malaysia: Bhat, U. K. M. 1976.Karyotypes of three speciesof Culexrajah and.Toxorhynchites roToh (Diptera: Cul- mosquitoesfrom India.Current Sci. 4E:266-267. icidae). Japan. J. Trop. Med. Hyg. 77:213-2t8..