Karyotype Studies in Oriental Anophelines I. T. T. Avirachan, P. L. Seetharam and B. N. Chowdaiah Department of Zoology, Bangalore University, India

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1 418 Cytologia 34 Karyotype Studies in Oriental Anophelines I Received August 11, 1968 T. T. Avirachan, P. L. Seetharam and B. N. Chowdaiah Department of Zoology, Bangalore University, India Mosquitoes are well-known as pests and disease vectors in many parts of the world. Although considerable amount of biological knowledge has accumulated concerning these species, relatively few are known cytologically. Among the important earlier contributions to this field are those of Kitzmiller and Frizzi (1954), Breland (1960, 1961), Rai and Craig (1961) and Rai (1961, 1963). However, the tropical mosquitoes, especially the oriental species are almost neglected cytogenetically. For a review of the pertinent literature on the genetics and cytogenetics of mosquitoes, refer Kitzmiller (1953, 1963, 1967). A few papers have appeared which reveal only a beginning in the field of cyto logical investigations of oriental anophelines (Rishikesh 1955, 1959, Chowdaiah et al. 1967, 1968). The present paper is an attempt to begin a comparative karyotype studies, describing the morphology of the mitotic chromosome comple ments and to evaluate their importance. Materials and methods Fourth instar female larvae of six Indian anopheline species belonging to two subgenera constituted the material for the present investigation. They are A. barbirostris and A. hyrcanus nigerrimus of the subgenus Anopheles; and A, fluviatilis, A. stephensi (type), A. vagus and A. subpictus of the subgenus Cellia. Of these species A. fluviatilis and A. stephensi are the vectors of malaria. The larvae used were all field collected except those of A. fluviatilis and A. stephensi which were used from the laboratory colonies. Chromosome preparations were made from brain tissue using the standard techniques for anophelines (French, Baker and Kitzmiller 1962). Pre-treatment with colchicine yielded good results. Temporary slides were ringed with wax and photo micrographs were taken using a 35mm Leica camera. Chromosome meas urements were made by the procedure suggested by Rai (1963). Observations Diploid chromosome number is six in all the species examined (Figs. 1-6). The normal karyotype of each of the species investigated is characterized by the presence of three pairs of chromosomes which are individually recognizable as in the case of other anopheline species thus far studied. As usual, the three pairs have been designated I, II and III based on their length and

2 1969 Karyotype Studies in Oriental Anophelines I 419 position of the centromere. The small pair which is considered to be the sex pair is designated as I and the remaining two longer pairs of mediocentric chromosomes as II and III. The latter two differ slightly in their length. The karyotype of A. barbirostris is a typical "maculipennis" type, consisting of a small pair of subtelocentric sex-chromosomes and two pairs of larger metacentric autosomes, varying slightly in size each with arms of ap- Figs Mitotic chromosomes of anopheline mosquitoes from brain cells. 1, A. barbiro stris. 2, A. hyrcanus nigerrimus. 3, A fluviatilis. 4, A. stephensi. 5, A. vagus. 6, A. subpictus. proximately equal length (Fig. 1). Although the karyotypes of all the remaining species studied are closely similar to that of A. barbirostris, they reveal some important cytological differences. The sex-pair in A. hyrcanus nigerrimus, A. fluviatilis, A. stephensi and A. vagus appear to be submetacentric with arms of slightly unequal length (Figs. 2, 3, 4 and 5). However, it is significant that the sex-pair in the case of A. subpictus is at least acrocentric, though not telocentric (Fig. 6). 28*

3 420 T. T. Avirachan, P. L. Seetharam and B. N. Chowdaiah Cytologia 34 In all the species studied, a conspicuous, heavily stained irregular chro matin mass is invariably encountered in the interphase nuclei (Fig. 7a, b); some giant nuclei are frequently met with (Fig. 7b); some of which are found at tetraploid level (Fig. 8). Discussion The diploid chromosome number of 6 has remained constant in all the species studied. However, the ratios in measurements of all the three pairs of chromosomes are variable, each measurement being species-specific (Table 1), and the comparative karyotype studies have revealed some differ ences, especially among the sex chromosome pairs (Fig. 9). Even though, there is not much size difference between the two longer Figs , two interphase nuclei; one diploid (a) and another polyploid (b), each with a chro matin mass. 8, metaphase chromosomes from a tetraploid nucleus. pairs of mediocentric chromo somes II and III, the smaller chromosome pair I presents an interesting variation; it is sub metacentric in A. hyrcanus nigerrimus, A. fluviatilis, A. stephensi and A. vagus; sub telocentric in A. barbirostris and acrocentric in A. subpictus. The sex-chromosomes in the female of A. subpictus appear to lack the distinct knob like termi - Table 1. Measurements of metaphase chromosomes in 6 species of Anopheline mosquitoes

4 1969 Karyotype Studies in Oriental Anophelines I 421 nal portions of the "maculipennis" type and most of the preparations reveal a very minute second arm, which otherwise is too small to be detected easily. Anyhow, this pair may well be compared with the 'dot-like' heterosomes of A. claviger and A. quadrimaculatus (Frizzi 1953). But unlike A. claviger, where a larger portion which is heterochromatic is missing, in the case of A. subpictus it is in all probability the same larger portion that is retained along with the minute second arm. Thus, the karyotype of A. subpictus might form another new type in addition to the already existing ones for anophelines. The vali dity of this interpretation could be checked in meiosis and in the salivary gland chromosomes of this species. A standard chro mosome map is prepared for A. barbirostris (Chowdaiah et al. 1968) and similar maps for other species are being prepared in this laboratory. The heavily stained distinct mass observed in the interphase nuclei of all the species studied probably represents the positively heteropycnotic sex chromosomes. And it is most unlikely, that this is similar to the two Feulgen positive particles reported by Rai (1963) in the resting nuclei of Aedes atropalpus. Further studies of chromo somal polymorphism and com- Fig. 9. Diagramatic representation of karyotypes of 6 species of anophelines examined. parison of karyotypes of large number of species would contribute to an understanding of the evolution and speciation of the subgenus Anopheles as a whole. Summary A comparative study of the karyotypes of six species of oriental anophelines including two vector species belonging to two subgenera has been made. The diploid chromosome number is constant (2n=6) in all the species studied. The ratios in measurements of the three pairs of chromosomes of these species reveal some differences especially of the sex-chromosome pairs.

5 422 T. T. Avirachan, P. L. Seetharam and B. N. Chowdaiah Cytologia 34 Acknowledgment This study has been supported by a grant from the World Health Organi zation. Literature Breland, O. P Studies of the chromosomes of mosquitoes. Anat. Rec. 137: Studies on the chromosomes of mosquitoes. Ann. Ent. Soc. Amer. 54: Chowdaiah, B. N., Avirachan, T. T. and Seetharam, P. L Salivary gland chromo somes in four species of tropical anophelines: A. subpictus, A. vagus, A. hyrcanus nigerrimuc and A. barbirostris. Proc. Ind. Acad. Sci. 65: , - and Chromosome studies of oriental anophelines I. The salivary gland chromosome map of Anopheles barbirostris. Caryologia (in press). French, W. L., Baker, R. H. and Kitzmiller, J. B Preparation of mosquito chromo somes. Mosq. News. 22: Frizzi, G Extension of the salivary chromosome method to Anopheles claviger, quadrimaculatus and aquasalis. Nature 171: Kitzmiller, J. B. and Frizzi, G A survey of the chromosomal complements in several species of mosquitoes (Diptera: Culicidae). In: Atti del IX congresso internazionale di Genetica, Caryologia, Suppl. 2: Mosquito genetics and cytogenetics. Rev. Bras. Malar. 5: cited Mosquito cytogenetics. Bull. Wld. Hlth. Org. 29: Mosquito cytogenetics. In: Genetics of Insect Vectors of Disease. Eds. Wright, J. W. and Pal, R. Elsevier Pub. Co., Amsterdam: Rishikesh, N The larval salivary glands of Anopheles stephensi Liston. Curr. Sci. 24: Chromosome behavior during spermatogenesis of Anopheles stephensi sensu stricto. Cytologia 24: Rai, K. S Distinctive karyotypes in some genera of mosquitoes. Bull. Ent. Soc. Amer. 7: A comparative study of mosquito karyotypes. Ann. Ent. Soc. Amer. 56: and Craig, G. B A study of the karyotypes of some mosquitoes. Genetics 46: 891.