© 2015 The Japan Mendel Society Cytologia 80(4): 405–413 C-Heterochromatin Distribution and Its Base Composition in Four Species of Mictini (Heteroptera, Coreidae, Coreinae) Nidhi Bansal and Harbhajan Kaur* Department of Zoology and Environmental Sciences, Punjabi University, Patiala 147002, Punjab, India Received December 29, 2014; accepted June 20, 2015 Summary The distribution and composition of C-heterochromatin in four species of Mictini (Coreinae), viz., Anoplocnemis compressa (Dallas, 1852), Anoplocnemis binotata (Distant, 1918), Ochrochira nigrorufa (Distant, 1889) and Prionolomia sp., have been analyzed by C-banding and DAPI/CMA3 sequence specific staining. Cytogenetically, the possession of holokinetic chromo- somes and a pre-reductional type of meiosis for sex chromosomes characterize these four species. The C-banding pattern has been found to be species-specific. In A. compressa, C-bands are thick and terminal, whereas in A. binotata, very thin C-bands are seen interspersed throughout the length of chromosomes. In Ochrochira nigrorufa, thick C-bands are present at terminal and interstitial regions. In Prionolomia sp., two conspicuous terminal C-bands are observed only on the largest autosomal pair while the rest of the complement is completely C-negative. This unique pattern can serve as a powerful cytological marker. Constitutive heterochromatin has been found to be rich in both AT and GC base pairs in all the studied species. Key words C-banding, DAPI/CMA3, Sequence-specific staining, Mictini. The family Coreidae, often called leaf-footed bugs, pod bugs or squash bugs, includes 2200 species belonging to 500 genera (Dursun and Fent 2009). Cytogenetic data pertains merely to 134 species (47 from India) referable to two subfamilies, Coreinae and Pseudophloeinae. Coreinae is divided into 31 tribes. Tribe Mictini, specifically found in the Eastern Hemisphere, is cytologically represented by 16 species belonging to seven genera. Cytogenetically, Coreidae is characterized by holocentric chromosomes, post-reductional division of sex chromosomes, a pair of microchro- mosomes and absence of Y chromosome. The most common diploid number of the subfamily is 21 observed in 48 species (Schuh and Slater 1995, Papeschi and Bressa 2006, Yang et al. 2012, Kaur and Bansal 2012, Bansal and Kaur 2013). Constitutive heterochromatin accumulation in the karyotype of a species is not a random pro- cess. Rather its acquisition and/or accumulation in different karyotypes is regulated by some con- straints. C-heterochromatin occurs either as repeated elements interspersed throughout the genome or as large arrays usually representing satellite DNA sequences (Brutlag 1980, Blanchelot 1991). In insect genomes, repeated DNA sequences have been found to be organized in different patterns. C-banding is one of the most used techniques for detecting heterochromatin that stains almost all constitutive heterochromatin segments. The base composition of heterochromatin (AT or GC rich) is revealed by fluorescent banding techniques. Chromosomal studies pertaining to constitutive heterochromatin in Coreidae are rather meager and account for only nine species worldwide (Dey and Wangdi 1990, Cattani et al. 2004, Bressa et al. 2005, Franco et al. 2006, Bressa et al. 2008) that include two species (Petillopsis patu- * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.80.405 406 N. Bansal and H. Kaur Cytologia 80(4) licollis and Ochrochira granulipes) from India (Dey and Wangdi 1990) while the information on sequence specificity of C heterochromatin is altogether lacking nationwide. In the present paper, four species of subfamily Coreinae, all belonging to the tribe Mictini, viz., Anoplocnemis compres- sa, Anoplocnemis binotata, Ochrochira nigrorufa and Prionolomia sp., have been described for the first time for the distribution of C-heterochromatin and its base specificity in terms of AT and GC rich regions. Materials and methods Adult male specimens, Anoplocnemis compressa, Anoplocnemis binotata, Ochrochira nigro- rufa and Prionolomia sp., were collected from regions falling in North India. Testes were dissected out in 0.67% saline water and were fixed in freshly prepared Carnoy’s fixative (3 : 1/absolute alco- hol : glacial acetic acid) for 15 min followed by a second change of the fixative. The fixed material was tapped on clean slides, air dried and stained. To perform C-banding, aged air-dried slides were stained with Giemsa as per the methodology suggested by Sumner (1972) with minor modifications. To reveal the AT-rich and GC-rich DNA, slides were treated with DAPI and CMA3 fluorescent dyes (Rebagliati et al. 2003). The slides were observed under a Nikon Optiphot Epifluorescence microscope and images were captured with a Nikon DXM 1200 C digital camera. Results Anoplocnemis compressa and Anoplocnemis binotata share a common diploid chromosome complement of 2n=15=14A+X0. Diploid chromosome complement of Ochrochira nigrorufa is 2n=21=18A+2m+X0 while that of Prionolomia sp. is 2n=27=24A+2m+X0. The general course of meiosis in these species has been described earlier (Kaur and Bansal 2012, Bansal and Kaur 2013). C-banding In Anoplocnemis compressa, at diffuse stage, besides positively heterochromatic X chromo- some, five to six C-positive regions in the chromatin are visible (Fig. 1). At diplotene, terminal bands are seen on all the autosomal bivalents but no localized C-band is seen on X chromosome. As condensation proceeds, X chromosome appears positively heterochromatic (Figs. 2, 3). In A. binotata, very thin C-bands are seen throughout the length in six autosomal bivalents and the X chromosome at diplotene. One autosomal bivalent appears C-heterochromatic throughout (Figs. 8, 9). In Ochrochira nigrorufa, the X chromosome is C-positive at diffuse stage as well as diplo- tene. At diplotene, all autosomal bivalents show thick terminal and interstitial C-bands and micro- chromosomes show heavy C-bands (Figs. 16–18). In Prionolomia, at diffuse stage, two conspicuous C-positive regions are visible (Fig. 23) which correspond to terminal C-bands of one autosomal bivalent in diplotene which are observed at diakinesis and metaphase I too. Rest of the 11 autosomal bivalents and microchromosomes are completely C-negative (Figs. 24–26). Sequence-specific staining In Anoplocnemis compressa, X is DAPI and CMA3 bright at diffuse stage as well as diplotene. All the C-positive regions appear DAPI and CMA3 bright (Figs. 4–7). In A. binotata, at diffuse stage, X chromosome is positive to both DAPI and CMA3 (Figs. 10, 11). At diplotene, C-bands of autosomal bivalents appear positive for both the stains while X chro- mosome is negative to both DAPI and CMA3 (Figs. 12–15). 2015 C-Heterochromatin Distribution and Its Base Composition in Four Species of Mictini 407 Figs. 1–7. Anoplocnemis compressa, Figs. 1–3. C-banding, Fig. 1. Diffuse stage showing positively heterochromatic X chromosome and five to six C-positive regions in the chromatin. Figs. 2, 3. Diplotene and diakinesis showing terminal C-bands on all autosomes and positively het- erochromatic X chromosome. Figs. 4–7. Sequence-specific staining, Figs. 4, 5. Diffuse stage showing DAPI and CMA3 bright X chromosome. Figs. 6, 7. Diplotene showing all C-positive regions to be DAPI and CMA3 bright. Arrowheads indicate X chromosome. Bar=0.01 mm. In Ochrochira nigrorufa, all the C-bands present on autosomal bivalents, X chromosome and microchromosomes appear positive for both DAPI and CMA3 (Figs. 19–22). In Prionolomia sp., two bright DAPI and CMA3 signals are seen against the diffuse chroma- tin and a few small CMA3 signals (Figs. 27, 28). At diplotene and metaphase I, bright DAPI and CMA3 signals appear on the terminal ends of the largest autosomal bivalent while X is negative to both the stains (Figs. 29, 30). 408 N. Bansal and H. Kaur Cytologia 80(4) Figs. 8–15. Anoplocnemis binotata, Figs. 8, 9. C-banding, Fig. 8. Diffuse stage showing positively het- erochromatic X chromosome. Fig. 9. Diplotene showing very thin C-bands throughout the length in six autosomal bivalents and the X chromosome. One autosomal bivalent appears positively C-heterochromatic. Figs. 10–15. Sequence-specific staining, Figs. 10, 11. Dif- fuse stage showing DAPI and CMA3 bright X chromosome Figs. 12–15. Diplotene showing all C-positive regions to be DAPI and CMA3 bright while X chromosome negative to both the stains. Arrowheads indicate X chromosome. Empty arrowhead indicates completely C- heterochromatic autosome. Bar=0.01 mm. Discussion In the present paper, four species belonging to the tribe Mictini, viz., Anoplocnemis compressa, Anoplocnemis binotata, Ochrochira nigrorufa and Prionolomia sp., have been analysed for the first time to study the distribution pattern of C-heterochromatin and its base specificity. C-banding The C-banding pattern has been found to be different in all the four studied species of Mic- 2015 C-Heterochromatin Distribution and Its Base Composition in Four Species of Mictini 409 Figs. 16–22. Ochrochira nigrorufa, Figs. 16–18. C-banding, Fig. 16. Diffuse stage showing positively heterochromatic X chromosome. Figs. 17, 18. Diplotene showing heavy terminal and inter- stitial C-bands in all autosomal bivalents while microchromosomes show heavy C-bands. Figs. 19–22. Sequence-specific staining, Figs. 19, 20. Diffuse stage showing DAPI and CMA3 bright X chromosome. Figs. 21, 22. Diplotene showing all C-positive regions to be DAPI and CMA3 bright. Arrowheads indicate X chromosome. Arrows point to microchro- mosomes. Bar=0.01 mm. tini. In A. compressa, C-bands are thick and positioned at terminal regions whereas in A. binotata, C-bands are very thin and are interspersed throughout the length of chromosomes. In Ochrochira nigrorufa, thick C-bands are present at terminal and interstitial regions. In Coreidae, terminal C- bands on autosomes have been earlier reported in Camptischium clavipes, Ochrochira granulipes, Leptocorisa acuta, Leptoglossus impictus and Phthia picta (Dey and Wangdi 1990, Cattani et al. 2004, Bressa et al. 2005) while interstitial bands on one or two chromosomes have been reported in Petillopsis patulicollis, Spartocera batatas and Holhymenia rubiginosa (Dey and Wangdi 1990, Franco et al.