_??_1992 by Cytologia, Tokyo Cytologia 57: 143 -147 , 1992

Chromosomes of a Freshwater Gastropod, Iuteola Lamarck (: )

R. C. Choudhury, R. K. Pandit and T. Sahu

Department of Zoology, Berhampur University, Berhampur-760 007, Orissa, India

Accepted October 17, 1991

Although study on the chromosomes of molluscs dates back from the last part of the 19th century, not more than 0.5% of the present day form of molluscs are known karyologically (Patterson 1969). Moreover, because of inferior optical instrumentation and the then crude technique of paraffin sectioning for chromosome studies, the earlier reports are found inac curate and unreliable. Even the aceto-orcein squash preparations for molluscan chromosomes , which started since the late 1960s, are also mainly elucidating only the number of chromosomes excepting a few sporadic reports on the size and morphology of the chromosomes by Patterson (1971), Babrakzai et al. (1974, 1975), Goldman et al. (1980), Bottke (1982), Kobayashi (1986), Boato (1986), Chambers (1987), Vitturi et al. (1987), Vitturi and Catalano (1988), Page (1988). However, the fragmentary work, which have been done during the last three decades, on the chromosomes of molluscs and their bearings on speciation, systematics and evolution have paved the way for important synthetic studies on the phylum discerning many controversial taxonomic problems, especially, at the lower taxon level. The selection of a tissue for chromosome preparations in molluscs is problemetical because of the very low mitotic index. Further, the presence of larger number of very small chro mosomes in them is also a hurdle on the way of progerss of cytological studies in this group of . So molluscan is lagging behind the rapid technological progress in the cytogenetics of other groups of organisms. However, the recent employment of air drying-Giemsa technique for chromosome preparations of molluscs by Prasad and Das (1978) and Goldman et al. (1980) are worthly rewarding. So, besides the detail study on the chromosomes of new , the reinvestigation of old data are warranted.

Material and methods

Specimens for the present study were collected from freshwater ponds from the nearby villages of the university campus and were identified by the Zoological Survey of India, Calcutta as Lymnaea Iuteola Lamarck. Chromosome preparations were made by the colchicine-acetic methanol-air drying-Giemsa technique of Prasad and Das (1978) with slight modification as per the following brief procedure. Each living specimen was injected with about 0.1 to 0.2ml (according to the size of the specimen) of 0.025% colchicine solution by making a small puncture on the second whorl. After three to four hours of colchicine injection, the specimens were sacrificed and their ovo testes were homogenized with distilled water, flushed thoroughly by a pasteur pipette and left for 45min. Then the cell suspension was centrifuged at 2000rpm for 5min. The supernatant was discarded and a little amount of freshly prepared fixative (glacial acetic acid and methanol 1:3 volume/volume) was added and flushed. The fixed cell suspension was recentrifuged and a desired volume of fresh fixative was added. Usually after 24hr. of fixation, the cell suspension was dropped on clean, grease free, prechilled slides in 50% ethyl 144 R. C. Choudhury, R. K. Pandit and T. Sahu Cytologia 57 alcohol and immediately shown to flame for burning. Then the slides were left overnight for natural drying and then stained with 10% Giemsa (diluted in Sorenson's buffer, pH 6.8) for one hour and washed thoroughly. After natural drying, on the next day the slides were ready for observation under microscope.

Fig. 1. A) spermatogonial metaphase, B) zygotene stage, C) pachytene stage, D) diplotene stage, E) diakinesis stage, F) metaphase I.

Observations

Mitotic chromosomes The diploid number 2n=34 has been confirmed from 15 well spread spermatogonial metaphases (Fig. 1A) in all the seven specimens of the present study. The chromosomes are nearly homogenously stained, randomly scattered and individuated clearly showing the arms and constrictions. All of them are biarmed and monocentrics with median or submedian position of their centromeres. So the number of fundamental arms (FN) is 68. 1992 Chromosomes of Lymnaea luteola 145

Meiotic chromosomes The zygotene stage (Fig. 1B) shows the lengthwise pairing of homologous chromosomes which are shortened and thickened. Peculiarly, here an asynchronous pairing of homologues is marked. While synapsis has completed among some homologues, it has not started among others. So here in the zygotene stage some 11 bivalents and rest univalents are seen (Fig. 1B). In addition, synapsis is probably taking place part by part lengthwise, so that loop like portions are seen in some bivalents. The pachytene stage (Fig. 1C) shows the completion of synapsis. Here the homologues are found closely intertwined with each other followed by longitudinal contraction. Some of such elements show clear open ends indicating the beginning of separation of homologues which is to be followed immediately. The presence of deeply stained dot like portions on the pachytene elements, some with more than one, indicates the allocycly or heteropycnocity of their heterochromatin portions. In the diplotene stage (Fig. 1D) the bivalents are found further thickened and shortened. Complete terminalization of chiasmata are seen in some of the bivalents but the homologues are still in contact with each other by terminal chiasmata. The diplotene elements are not uniformly stained. Six of them are found more condensed and intensely stained. This unequal staining might be due to their asynchronous condensation. Very short, thick and intensely stained diakinetic elements are seen with smooth outlines (Fig. 1E). Ring and cross shaped elements are found with two terminal and two subterminal chiasmata respectively. Some of the bivalents appear so contracted that it is not possible to surmise on the presence and position of chiasma. The haploid number n=17 has been con firmed from diakinesis stages. The diakinetic elements are also found unequally stained. In metaphase-I the bivalents are so much condensed that all of them appear as smooth round dots from the polar view and more or less dumb-bell shaped from the side view (Fig. IF). Most of the elements show complete terminalization of chiasmata and a few with ter minal chiasma. The degree of terminalization is found greater in between diakinesis and metaphase-I than in between diplotene and diakinesis.

Discussion

The diploid and haploid numbers of chromosomes of Lymnaea luteola as 34 and 17, obtained from the spermatogonial and meiotic metaphases respectively, confirm to that of the earlier report of Natarajan (1960) on the same species. Except for the diploid number, there is a considerable variation in our observation from that of Natarajan who has reported the presence of 17 homologus pairs of chromosomes with no apparent constrtictions, of which 3 pairs larger (size markers) and oblong shaped and the rest 14 pairs smaller and dot shaped. Inaba (1953) and Inaba and Tanaka (1953) have reported the cccurrences of only rounded or oblong shaped chromosomes in other basommatophoran . So Natarajan (1960) gen eralized the basommatophoran snalis with unclear morphology of their chromosomes. But in contrast, in our present study, the clear morphology of the chromosomes have been recorded showing all the 34 chromosomes as biarmed with median or submedian position of their cen tromeres (metacentrics and submetacentrics) (Fig. 1A) so that the number of fundamental arms (FN) is 68. Besides, he has reported the presence of 3 pairs of size markers in L. luteola. But no such size markers have been observed in the present study. The differences in the two sets of observations in respect to the chromosome morphology and presence of marker pairs are might be due to his erratic observations and/or the then crude techniques. Conservativeness with regard to chromosomal change is evident in most mollusc groups. Many higher taxa have characteristic chromosome numbers with only a few species having 146 R. C. Choudhury, R. K. Pandit and T. Sahu Cytologia 57 wide deviations from the basic haploid set. So most variations within a taxon is seldom great er than 1 or 2 bivalents (Patterson 1969). For the order Basommatophora the available information indicate the range of haploid numbers from 15 to 72 (Table 1). But among its nonpolyploid members the haploid chromosome number varies from 15 to 19 and predominate ly it is 18. The families Ancylidae and indicate clearly the existence of polyploidy with the haploid numbers of 30, 60 and 36, 54, 72 respectively. So it seems justified in dividing the order Basommatophora into two suborders keeping the families Siphonariidae, Ellobiidae, Amphibolidae, Chilinidae, Latiidae and Acroloxidae as primitive basommatophorans under the suborder Archaeopulmonata and the families Ancylidae, Planorbidae, Lymmaeidae and Physidae with more specialized limnic basommatophorans under the suborder Branchiopul monata following the suggestions of Morton.

Table 1. Haploid chromosome number of different families of the order Basommatophora

Out of the 45 species and subspecies studied so far from the family Lymnaeidae, 29 of them show n=18. So n=18 is the predominant number of the family. But its widely dis tributed genus show n=17 and three other species of the family are known to have n= 16. So Patterson (1969) considered that the original lymmnaeid ancestors were with n=16 which gave rise to Radix and subsequently to the other lymnaeids with n=18 by adding one or two bivalents. But these mere sepeculations can be strengthened only after obtaining the detail karyotype of these species. Further more, except Lymnaea luteola the other four species of the genus, so far studied, L. stagnalis (Burch 1965), L. lacustris (Perrot 1930), L. jugularis (Burch 1960) show n=18. That is why perhaps, Patterson (1969) considered Lymnaea luteola of Natarajan (1960) with n=17 as . However, the present study has confirmed the report of Natarajan on L. luteola for the number of chromosomes. So it can be said that L. luteola with n=17 might be primitive than the other species of Lymnaea with n=18. And it can be speculated that the Lymnaea with n=18 might have been derived from Radix (n=17) through L. luteola (n=17), or the congeneric variation in chromosome number of Lymnaea might be due to some other mechanism (s) like Robertsonian fusion or fission in between one or two pairs of chromosomes without causing any genetic loss to the species. But these views can only be strengthened from the karyotypic analysis and banding pattern studies on the chro mosomes of these species.

Summary

Mitotic and meiotic chromosomes from the gonads of the freshwater gastropod, Lymnaea luteola Lamarck, family Lymnaeidae have been studied. The 2n=34 and n=17 were con firmed from the spermatogonial and meiotic metaphases respectively. All the chromosomes 1992 Chromosomes of Lymnaea luteola 147

were found biarmed with median or submedian position of their centromeres. Allocycly has been observed among the chromosomes at different meiotic stages . The differences in the morphology of the chromosomes of the same species reported earlier have been pointed out . Chromosome number conservatism at the confamilial and congeneric level of the order Basom matophora with the tendency towards an increase in the chromosome number have been discussed.

Acknowledgments

We are deeply indebted to Professor B. K. Patnaik and Professor G. P. Verma for provid ing laboratory facilities. We are also grateful to Dr. N. V. Subba Rao, Zoological Survey of India, Calcutta for the identification of the specimens.

References

Babrakzai, N., Miller, W. B. and Ward, O. G. 1974. Cytotaxonomy of some Arizona Oreohelicidae (Gastro poda: ). Am. Malacol. Union Inc. Bull.: 4-11. - , Ward, O. G. and Miller, W. B. 1975. The introduction of giemsa and centromeric banding techniques of chromosomes to molluscan cytotaxonomy. Am. Malacol. Union Inc. Bull.: 67 (Abstra.) Boato, A. 1986. A preliminary karyological analysis of five species of Solatopupa (Pulmonata, Chondrinidae) . Boll. Zool. 53: 15-22. Bottke, W. 1982. Heterochromatin in a pulmonate , Planorbarius corneus L. Caryologia 35: 443-448. Burch, J. B. 1960. Chromosome studies of aquatic pulmonate snails. Nucleus 3: 177-208. - 1965. Chromosome numbers and systematics in ehthyneuran snails. Proc. first Europ. Malacol. Congr. 1962: 215-241. Chambers, S. M. 1987. Rates of evolutionary change in chromosome numbers in snails and vertebrates. Evolution 41: 166-175. Goldman, M. A., LoVerde, P. T. and Chrisman, C. L. 1980. Comparative karyology of the freshwater snails Bulinus tropicus and B. natalensis. Can. J. Genet. Cytol. 22: 361-367. Inaba, A. 1953. Cytological studies in Molluscs. I. Chromosomes in Basommatophoric pulmonates. J. Sc. Hiroshima Univ. Ser. B. 14: 221. - and Tanaka, H. 1953. Studies on the chromosome numbers of some freshwater gastropods. Ibid. 14: 213. Kobayashi, T. 1986. Karyotype of four species of the genus Semisulcospira in Japan. Venus Jpn. J. Malacol. 45: 127-137. Natarajan, R. 1960. Further cytological studies in Pulmonata (: ). J. Zoo. Soc. India 12: 69-79. Page, C. 1988. The chromosome complement of Nucella lapillus (L.) Mollusca: Gastropoda: Prosobranchia. Caryologia 41: 79-91. Patterson, C. M. 1969. Chromosomes of Molluscs. Proc. Symp. Moll. Mar. Biol. Ass. India 2: 635-686. - 1971. A karyotype technique using embryos. Malacol. Rev. 4: 27. Perrot, J. L. 1930. Chromosomes et heterochromosomes Chez les gasteropdes pulmones. Re. Suisso zool. 37: 397-434. Prasad, R. and Das, C. C. 1978. Air Drying Giemsa Technique for Gastropod Chromosomes. The Veliger 20: 386-387. Vitturi, R. and Catalano, E. 1988. A male XO Sex-determining mechanism in Theodoxus meridionalis (Neriti dae) (Prosobranchia, Archaeogastropoda). Cytologia 53: 131-138. -, -, Macaluso, M. and Maiorca, A. 1987. Spermatocyte chromosomes in six species of Neogastropoda. Biol Zentral B1. 106: 81-88.