C-Banding and Ag-NOR Staining on Neurobasis Chinensis Chinensis (Linnaeus) of Family Calopterygidae from Himachal Pradesh, India (Odonata: Zygoptera)

C-Banding and Ag-NOR Staining on Neurobasis chinensis chinensis (Linnaeus) of Family Calopterygidae from Himachal Pradesh, India (Odonata: Zygoptera)

Gurinder Kaur Walia*, Jaspreet Kaur Gill and Harkiran Kaur Hallan

Department of Zoology and Environmental Sciences, Punjabi University, Patiala-147002, India

Received June 1, 2015; accepted April 1, 2016

Summary Neurobasis chinensis chinensis of the family Calopterygidae is the only species of this genus present in India. This species was collected from Andretta and Mcleodganj areas of Himachal Pradesh, India. The species possesses 2n=23 which is less than the type number (2n=25) of the family. The chromosome complement shows the presence of large pair of autosomes originated by the fusion of two autosome pairs and is responsible for the reduction in the chromosome number. This fusion has been conﬁrmed by C-banding with the presence of two interstitial and terminal C-bands on the largest autosomal bivalent having two chiasmata while remaining autosomal bivalents possess terminal C-bands and single chiasma which is the characteristic feature of Odonata. Ag- NOR staining shows the presence of terminal NOR bands in seven autosomal bivalents and the X chromosome is rich in NORs. C-banding and Ag-NOR staining have been performed for the ﬁrst time on this species.

Key words Autosomal fusion, Chromosome number, C-banding, Ag-NOR staining, Calopterygidae, Zygoptera, Odonata.

The damselflies of the family Calopterygidae are were found flying just above the water or seeking resi- commonly known as broad-winged damselflies. Taxo- dence in areas of vegetation and debris. nomically, this family includes 21 genera and 185 spe- Live male specimens were dissected in the field in cies all over the world while six genera and nine species 0.67% saline solution. Testes were kept in sodium citrate are present in India (Subramanian 2014). So far, 20 spe- (0.9%) for 45 min then fixed in Carnoy’s fixative. The cies of genera Calopteryx, Mnais, Epallaga, Neurobasis, testes were teased on clean slides, air dried and stored Phaon and Vestalis have been studied cytologically. The in a refrigerator. Slides were further treated for cytoge- majority of the species possess n=13 which is the type netical investigations by conventional staining (Carr and number of the family with XO-XX sex determining Walker 1961), C-banding (Sumner 1972) and Ag-NOR mechanism (Carnoy 1885, Oguma 1930, Makalowskaja staining (Howell and Black 1980). The relevant stages 1940, Omura 1957, Dasgupta 1957, Hirai 1956, Kiauta were microphotographed to study the structure and be- 1967a, 1970b, 1971a, 1972d, 1973 and Walia and Kaur haviour of chromosomes during meiosis, sex determina- 2011). The only exception is Calopteryx virgo meridi- tion, localization of constitutive heterochromatin and onalis, possesses n=13m, 14m, 15m as haploid chromo- nucleolar organiser regions. some numbers and which originated due to the precocious segregation of autosomal bivalents (Kiauta 1971a). Results In the present study, Nuerobasis chinensis chinensis possesses 2n=23 with XO-XX type sex determination. Conventional staining In the spermatogonial metaphase plate, 23 elements Materials and methods including 22 autosomes and one darkly stained smallest X element are present. The chromosome complement The damselfly is beautifully coloured and commonly possesses one pair of large chromosomes originated by called as stream glory with brown coloured hindwings the fusion of two pairs of auotosomes. (Fig. 1A). Dur- and bright iridescent green coloured fore wings. The ing diakinesis, 12 elements are visible. Among these, specimens for this study were collected from the run- 11 are autosomal bivalents which also include darkly ning water of permanent streams in the Mcleodganj and stained largest autosomal bivalent and one X chromo- Andretta areas of Himachal Pradesh. The damselflies some, occupies the peripheral position (Fig. 1B). In the metaphase I, all the autosomal bivalents including * Corresponding author, e-mail: [email protected] largest bivalent are rod shaped due to condensation and DOI: 10.1508/cytologia.81.175 terminalisation of chaismata while the X chromosome 176 G. K. Walia et al. Cytologia 81(2)

Fig. 1. A) Spermatogonial metaphase. B) Diakinesis. C) Metaphase I. D) Prophase-II. E, F) C-Banded Diakinesis. G) Ag-NOR stained Interphase. H) Ag-NOR stained Pachytene. I) Ag-NOR stained Diakinesis. J) Ag-NOR stained Prophase-II. Largest autosomal bivalent (shown with arrow head) in all the stages. Bar=0.01 mm. is mostly present at the periphery (Fig. 1C). During pro- However, the largest autosomal bivalent possesses two phase II, all the chromosomes show ‘€’ shaped structures chiasmata with two interstitial and terminal C-bands which is the characterstic feature of the odonate chromo- which conﬁrms the fusion of two autosome pairs, other- somes. The X chromosome is darkly stained and lies at wise in Odonata, a single chiasma per bivalent is present the outer position (Fig. 1D). (Fig. 1E, F).

C-banding Ag-NOR staining In the diakinesis out of 11 autosomal bivalents, eight The X chromosome is darkly stained and present in bivalents show terminal C-bands while three bivalents interphase as well as pachytene stages which indicate are C-negative. The X chromosome is lightly stained, the presence of nucleolar organizer regions (Fig. 1G, having less amount of constitutive heterochromatin. H). During diakinesis, seven autosomal bivalents show 2016 C-banding and Ag-NOR Staining on Neurobasis chinensis chinenesis 177 terminal NOR bands while remaining bivalents includ- References ing largest autosomal bivalent are NOR-negative. The X chromosome is rich in NORs and darkly stained (Fig. Carnoy, B. J. 1885. La cytodierese chez les arthropods IV, Pseudo- 1I). At prophase II, seven chromosomes have terminal Neuropteres. Cellule 1: 279–282. Carr, D. H. and Walker, J. E. 1961. Carbol-fuchsin as a stain for Hu- NOR bands while the remaining four are NOR-negative man chromosomes. 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