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 chromosomes 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 microchromosomes 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 compressa, 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 nigrorufa 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 chromosome, 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 diplotene. At diplotene, all autosomal bivalents show thick terminal and interstitial C-bands and microchromosomes 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 chromosome 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 heterochromatic 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 chromatin 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 heterochromatic 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 interstitial 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 microchromosomes. 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. 2006, Bressa et al. 2008). In other families of Heteroptera, too, most commonly, C- 410 N. Bansal and H. Kaur Cytologia 80(4)

Figs. 23–30. Prionolomia sp., Figs. 23–26. C-banding, Fig. 23. Diffuse stage showing negatively heterochromatic X chromosome and two conspicuous C-positive regions in the chromatin. Figs. 24–26. Diplotene showing terminal C-bands in the largest autosome and C-negative X chromosome. Figs. 27–30. Sequence-speciﬁc staining, Figs. 27, 28. Diffuse stage showing

two bright DAPI and CMA3 signals. Figs. 29, 30. Diplotene and diakinesis showing bright DAPI and CMA3 signals on the terminal ends of the largest autosomal bivalent while X negative to both the stains. Arrowheads indicate X chromosome. Arrows point to microchromosomes. Empty arrowhead indicates terminal C-heterochromatic regions in the largest autosome. Bar=0.01 mm. heterochromatin has been found to be telomeric in position (Papeschi 1988, 1991, 1995, Panzera et al. 1992, Perez et al. 1997, 2000, Grozeva and Nokkala 2001, Angus et al. 2004, Grozeva et al. 2004, 2008, Waller and Angus 2005, Lanzone and Souza 2006, Kaur et al. 2012). Less commonly, one or two chromosomes have been observed with interstitial bands (Muramoto 1978, Camacho et al. 1985, Panzera et al. 2000, Grozeva and Nokkala 2001, Ituarte and Papeschi 2004, Grozeva et al. 2004, Waller and Angus 2005, Grozeva et al. 2006, Kuznetsova et al. 2007). 2015 C-Heterochromatin Distribution and Its Base Composition in Four Species of Mictini 411

In A. binotata, very thin C-bands are present all along the length of chromosome which is similar to the pattern reported in Oxycarenus hyalinipennis (Lygaeidae), Physopelta quadriguttata (Largidae) and Dysdercus evanescens (Pyrrhocoridae) (Suman 2010, Suman et al. 2012). In Core- idae, intercalary location of C-heterochromatin has earlier been reported in Holhymenia historia (Bardella et al. 2014). In Prionolomia sp., two conspicuous terminal C-positive bands are observed only on the largest autosomal pair while the rest of the complement is completely C-negative. This pattern is unique and has not been recorded as yet in the entire Heteroptera group, although completely C-negative complements have been reported in Athaumastus haematicus (Coreidae) by Bressa et al. (2005), in Acalyptya carinata and Lasiacantha capucina (Tingidae) by Grozeva and Nokkala (2001), in Prostemma gutala (Nabidae), in Corixa dentipes, Corixa iberica and Corixa punctata (Corixidae) by (Waller and Angus 2005), in Macrolophus geranii and Macrolophus pygmaeus (Miridae) by Grozeva et al. (2005, 2007), in Neophysopelta schlanbushi by Suman et al. (2012) and in Piezodorus rubrofasciatus, Carbula humerigera (Pentatomidae) by Muramoto (1978) and Kerisew (2011). The unique C-banding pattern may serve as a powerful cytological marker. Microchromosomes are C-negative in Anoplocnemis compressa and Prionolomia sp. How- ever, in Ochrochira nigrorufa, interstitial C-bands are seen on microchromosomes which might be because of exceptionally large size of chromosomes. Usually microchromosomes are very small and appear C-negative.

Sequence-speciﬁc staining There is still little information about heterochromatin base composition in Heteroptera. Most reports referring to heterochromatin characterization describe C-bands as DAPI-bright/CMA3-dull (Bressa et al. 1999, 2005, Perez et al. 2000, Papeschi and Bressa 2002, 2006, Papeschi et al. 2003, Rebagliati et al. 2003, Ituarte and Papeschi 2004, Bressa et al. 2005). However, in some species of Pentatomidae, Micronectidae, Miridae and Cimicidae, GC-rich clusters are dispersed within AT- rich repeats (Rebagliati et al. 2003, Ituarte and Papeschi 2004, Grozeva et al. 2006, Grozeva and Nokkala 2002). In all the species of the present study, C-heterochromatin on the autosomes and sex chromosomes show overlapping staining pattern with DAPI-CMA3 dyes showing it to be rich in both AT and GC bases, which disagrees with previous reports on Coreidae (Holhymenia rubiginosa, Spartocera batatas, Athaumastus haematicus, Leptoglossus impictus and Phthia picta) depicting C-heterochromatin to be only AT-rich (Bressa et al. 2005, 2008, Franco et al. 2006).

A few CMA3 positive signals observed at diffuse stage in Prionolomia sp. may correspond to NOR’s as reported earlier in some heteropterans (Gonzalez-García et al. 1996, Papeschi and Bressa 2002, Papeschi et al. 2003, Rebagliati et al. 2003, Cattani et al. 2004, Ituarte and Papeschi 2004, Bressa et al. 2005). To conclude, the distribution of constitutive heterochromatin has been found to be species- speciﬁc. In Prionolomia sp., distinct terminal C-bands on one of the autosomal pair may serve as a strong cytological marker. C-heterochromatin has been found to be rich in both AT and GC base pairs in all the species which is in contrast to the previous reports on Coreidae.

Acknowledgements

The authors are thankful to the Department of Zoology and Environmental Sciences, Punjabi University, Patiala for providing the necessary laboratory facilities. 412 N. Bansal and H. Kaur Cytologia 80(4)

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