SCIENTIFIC CORRESPONDENCE

(Dinophysis spp.), which form a compo- 8. Hansen, P. J., Nielsen, L. T., Johnson, thanks DST, New Delhi for providing nent of harmful algal blooms. M., Berge, T. and Flynn, K. J., Harmful INSPIRE fellowship. Algae, 2013, 28, 126–139. 9. Jankowski, A. W., In Materials II. All- 1. Mohanty, A. K., Satpathy, K. K., Sahu, Union Conference on Protozoology. Part Received 25 February 2015; revised accepted G., Sasmal, S. K., Sahu, B. K. and Pani- 1, General Protozoology (eds Markevich, 20 October 2015 grahy, R. C., Curr. Sci., 2007, 93(5), A. P. and Yu, I.), Naukova Dumka, Kiev, 1976, pp. 167–168. 616–618. 1, 2. Sasamal, S. K., Panigrahy, R. C. and 10. Garcia-Cuetos, L., Moestrup, O. and BIRAJA KUMAR SAHU * 1 Misra, S., Int. J. Remote Sensing, 2005, Hansen, P. J., J. Eukaryot. Microbiol., R. C. PANIGRAHY 2 26(17), 3853–3858. 2012, 59(4), 374–400. S. K. BALIARSINGH 1 3. Taylor, F. J. R., Blackbourn, D. J. and 11. Lindholm, T., Lindroos, P. and Mork, C. PARIDA 1 Blackbourn, J., J. Fish. Res. Board Can., A. C., Biosystems, 1988, 21, 141–149. K. C. SAHU 2 1971, 28, 391–407. 12. Peterson, T. D. et al., Aquat. Microb. ANEESH A. LOTLIKER 4. Montagnes, D. J. S. and Lynn, D. H., J. Ecol., 2013, 68, 117–130. 13. Park, M. G. et al., Aquat. Microb. Ecol., Plankton. Res., 1989, 11, 193–201. 1Department of Marine Sciences, 2006, 45, 101–106. 5. Lindholm, T., Adv. Aquat. Microbiol., Berhampur University, 1985, 3, 1–48. Berhampur 760 007, 6. Crawford, D. W., Mar. Ecol. Prog. Ser., 2 1989, 58, 161–174. ACKNOWLEDGEMENTS. We thank the Indian National Centre for Ocean 7. Crawford, D. W., Purdie, D. A., Lock- Head, Department of Marine Sciences, Ber- Information Services, wood, A. P. M. and Weissman, P., hampur University for providing the neces- Hyderabad 500 090, India Estuarine Coastal Shelf Sci., 1997, 45, sary facilities and INCOIS, Hyderabad for *For correspondence. 799–812. sponsoring the SATCORE project. B.K.S. e-mail: [email protected]

Karyotype of the Indian giant (Ratufa indica)

The Indian Giant squirrel syn. Malabar within a very short duration. Other tissue heparin is suitable in terms of handling, giant squirrel (Ratufa indica) is an upper- samples have to be transported without transport and maintenance of sterility, canopy dwelling , endemic to exposure to extremes of temperature and but collecting fresh blood from wild spe- southwestern, central and eastern penin- under sterile conditions4. Whole blood in cies is impractical. The procedures are sular India, found specifically in the , Satpuras and . It is seen at elevations of 180– 2300 m and is widely distributed1. The species has been classified under the Class, Mammalia; Order, Rodentia; Sub- order, ; family, Sciuridae; subfamily, Ratufinae; Genus, Ratufa and species Ratufa indica2. According to the IUCN, ver 3.1, R. indica is classified un- der the ‘Least Concern’ category, but its population shows a decreasing trend due to habitat loss3. It is also listed under the Schedule II of the Indian Wildlife (Pro- tection) act, 1972. Karyotyping is an important source of genomic information from a species. Karyotype studies in wild species are confronted with difficulties. First, the habitats of wild are remote and away from lab facilities. Bone marrow cells and tissue samples for in vitro cul- ture can be used only if available from fresh carcasses. Most often, the carcasses in the wild are noticed late after death and putrefied. Even if fresh, the cells extracted from bone marrow have to be Figure 1. a, Carcass of Ratufa indica; b, Bleeding from mouth and nostrils; c, d, Arrows indi- processed immediately or transported cating male genitalia; e, Unclotted blood in thoracic cavity.

CURRENT SCIENCE, VOL. 110, NO. 6, 25 MARCH 2016 983 SCIENTIFIC CORRESPONDENCE not only difficult, but also bound by legal Institute, Namakkal, the nearest facility The carcass of the squirrel was fresh implications. A thorough search of the available for culture and karyotyping. with unclotted blood available in the tho- literature indicated absence of information The chromosome spreads were pre- racic cavity, which was successfully used on karyotype of R. indica. The karyotype pared from leucocyte cultures by stan- for chromosome preparation. Figure 2 of the Indian Giant squirrel (R. indica) dard procedure5. Cultures were set up in shows the karyotype of the male Indian was studied by chromosome preparation RPMI 1640 culture medium (8 ml) with giant squirrel obtained in the present of leukocyte culture from unclotted Pokeweed (0.1 ml) as mitogen. Autolo- study. From the spreads analysed, it was blood collected during necropsy. gous plasma (1.5 ml) and buffy coat found that the diploid number of chro- A male Indian Giant squirrel (Figure (0.2 ml) from the centrifuged sample mosomes in R. indica was 42 (2n), com- 1 c, d) was found dead under a cyprus were added to cultures. The cultures prising 20 pairs of autosomes and a pair tree in Cairn hill Reserve Forest, Udha- were incubated at 37C for three cell of sex chromosomes. As there was no gamandalam, in the Nilgiri Biosphere cycles (72 h). Ninety minutes prior to reference karyotype available for the Reserve, and its necropsy was conducted. harvest 0.01% colchicine was added to species, chromosomes were arranged The carcass was fresh and death might the cultures and the incubation was con- following standard procedure based on have occurred a short while ago, as its tinued. The cultures were subjected to morphology and decreasing size. Rela- body was warm, rigor mortis was absent hypotonic treatment (0.075 M KCl) and tive length of autosomes varied from and fresh bleeding noticed from the nos- fixed in Cornoy’s fixative (methanol – 3 2.09% to 6.96% (Figure 3). All the 20 trils and mouth (Figure 1 b). A large parts: acetic acid – 1 part). The slides pairs of autosomes were biarmed with blood clot was found adhering to the were prepared and stained with 4% metacentric and submetacentric positions lung. About 20 ml of unclotted blood Giemsa. Non-overlapping metaphase of centromere. The Centromeric Index was found in the thoracic cavity (Figure spreads with full chromosome (2n) com- (CI) varied from 0.53 to 0.75. The sex 1 e). The had probably died of plement were chosen and examined chromosomes showed marked difference shock due to pulmonary rupture. Ten ml under Leica model DM 2500 microscope in size and the relative length of X and Y of unclotted blood from the thoracic at 1000 magnification. chromosomes was 5.04 and 2.42 respec- cavity was collected in heparinized Photomicrographs were obtained for tively. The sex chromosomes were meta- vacutainer and transported in ice to preparation of karyotypes and assessing centric with CI values of 0.56 and 0.62 Veterinary College and Research morphometric characteristics. Each chro- respectively. mosome was cut out separately and Literature survey indicated absence of matching pairs were arranged according information on the karyotype of R. to decreasing size with small arm upwards. indica. Family Sciuridae includes 273 The sex chromosomes were placed in the species6, in which 10 species studied end. The length of each chromosome was cytogenetically were characterized by a measured from tip to tip using a vernier stable diploid number of 2n = 40, and caliper with an accuracy of 0.05 mm. little variation in fundamental number Relative length was calculated as the (FN) = 72 or 76. The X chromosome was ratio of individual chromosome length to always metacentric or submetacentric. that of total haploid genome length (length Among other arboreal in the of autosomes + X + Y chromosome). The family Sciuridae reviewed for chromo- centromeric index was calculated as the some studies, chromosome number ratio of short arm (p) length to total ranged from 38 to 62 with majority of length (p + q) of the chromosome. species having 40 chromosomes7. The

Figure 2. Karyotype of Ratufa indica. Figure 3. Relative length of 20 autosomes, X and Y chromosome in R. indica.

984 CURRENT SCIENCE, VOL. 110, NO. 6, 25 MARCH 2016 SCIENTIFIC CORRESPONDENCE

Venezuelan species Sciurus granetensis shop report, Zoo Outreach Organisation/ 10. Li, T., Wang, J., Su, W., Nie, W. and had a similar number to R. indica with 42 CBSG-South Asia, Coimbatore, India, Yang, F., Cytogenet. Genome Res., 2006, chromosomes. However, the FN in S. 2005, p. 618. 112, 270–276. granetensis was 78 with 19 pairs of 2. Erxleben, J. C. P. (in Latin), Systema biarmed and one pair of acrocentric auto- regni animalis per classes, ordines, gen- ACKNOWLEDGEMENTS. We acknowledge era, species, varietates cum synonymia et the assistance rendered by staff of office of the some. Karyotype obtained for R. indica historia animalium. Classis I. Mam- Ranger, Nilgiris South Division. We also thank in the present study indicates FN of 80 as malia, Leipzig, Germany: Impensis the Dean, Veterinary College and Research In- all autosomes were biarmed. The Iranian Weygandianis, 1777, 42; http://www. stitute, Namakkal, for laboratory facilities. arboreal species S. anomalus had 40 worldcat.org/oclc/14843832 chromosomes with FN of 76, as the 19th 3. Rajamani, N., Molur, S. and Nameer, P. Received 11 November 2015; revised accep- acrocentric autosome was absent8. Cross- O., IUCN Red List of Threatened Species, ted 26 December 2015 Version 2011.2, 2010; www.iucnredlist. species studies on squirrels, not inclusive org of R. indica show that karyotypes of 9,10 4. Bulatova, N. S., Searle, J. B., Nadjafova, 1, squirrels are highly conserved . R. VENKATARAMANAN * The diploid number of wild arboreal R. S., Pavlova, S. V. and Bystrakova, 2 N. V., Comp. Cytogenet., 2009, 3(1), 57– N. MURALI squirrel, R. indica endemic to South, 1 62. C. SREEKUMAR 1 Central and Eastern Peninsular India is 5. Halnan, C. R. E., Res. Vet. Sci., 1977, 22, K. V. GOWRIMANOKARI confirmed through karyotype obtained 40–43. from lymphocyte culture using unclotted 6. Fernando de, J., Lima, S. and Langguth, 1Postgraduate Research Institute in blood available from fresh carcass. A., Folia Zool., 2002, 51, 201–204. Animal Sciences, 7. Fagundes, V., Christoff, A. U., Amaro- Tanuvas, Ghilard, R. C., Scheibler, D. R. and Yo- Kancheepuram 603 203, India

nenaga-Yassuda, Y., Gen. Mol. Biol., 2Veterinary College and Research 1. Molur, S., Srinivasulu, C., Srinivasulu, 2003, 26(3), 253–257. Institute, B., Walker, S., Nameer, P. O. and Ravi- 8. Nadler, C. F. and Hoffmann, R. S., Ex- kumar, L., Status of South Asian non- perientia, 1970, 26, 1383–1386. Namakkal 637 001, India volant small : Conservation 9. Li, T. et al., Chromosome Res., 2004, 12, *For correspondence. assessment and management plan, Work- 317–335. e-mail: [email protected]

Breeding tubercles in scales of male Barilius bendelisis (Hamilton, 1807) identified as sexual dimorphic character

Sexual dimorphism in scales has been adult male and female fishes was ana- an interval of 15 min and finally storing reported in some cyprinids like Chon- lysed by both light microscopy and scan- in the same buffer until processed fur- drostoma nasus1, where breeding tuber- ning electron microscopy (SEM) to ther. Scales were gradually dehydrated in cles are present on the scales of both confirm the presence of breeding tuber- acetone grades (30–100%) and then males and females, but are smaller and cles and study the structures in detail. treated with tetramethylsilane (TMS)7. inconspicuous in females. The presence Hill stream fish B. bendelisis were Clean and dried samples were gold- of breeding tubercles in fishes was first captured from Basistha (260538.54N, coated for 10 min before viewing in reported by Vladykov et al.2 in species of 914657.27E), the only hill stream in SEM (model JSM-6360 JEOL) at 20 kV. genus Gadus3 which were used in taxo- the heart of Guwahati city, Assam, India The study revealed that surface topo- nomic identification, since both male and during February 2014 to January 2015. graphy of scales in both sexes differed in female fish possess this character. There Adult fishes were in the range 107– B. bendelisis. Light microscopic studies have been studies on breeding tubercles 124 mm total length and 13–26 g body revealed that in the male fish the exposed in scales suggesting a correlation to the weight. Juvenile as well as adult fishes area (ExA) of scales is studded with pre-spawning behaviour in males1. The were studied throughout the seasons. tubercles (Figure 1 a and b). SEM images role and evolution of such tubercles are Sexes of the fishes were confirmed revealed that tubercles contained numer- however not known, they are believed to through dissection and scales were care- ous elongated spine-like structures called be used for conspecific recognition2,4. It fully taken out from near the head region unculi (Figure 2 b and c). These were was also suggested that the morphology above the lateral line and also from other observed to be smooth surfaces with of such tubercles may vary among differ- areas for microscopic observations. pointed distal ends and broad proximal ent species. Although there are reports Scales were superficially cleaned with edges. When observed under the light on the use of scales as a key character for 70% alcohol using a fine brush to re- microscope (Figure 1 c and d) as well as analysing age and growth in Barilius move any attached debris and immersed in SEM (Figure 2 d), scales of adult bendelisis5,6 there is no report on sexual in 3% glutaraldehyde solution for fixa- female fish were devoid of tubercles. dimorphism in this fish using scales as tion. Buffer treatment consisted of wash- However, the exposed area of scales key structures. Thus in the present study, ing the scales three times in 0.1 M revealed numerous mucous pores with the surface topography of scales from solution of sodium cacodylate buffer at depressions (Figure 2 e).

CURRENT SCIENCE, VOL. 110, NO. 6, 25 MARCH 2016 985