Meiotic Studies in Seven Heteropteran Species

Vikas Suman and Harbhajan Kaur

Department of Zoology and Environmental Sciences, Punjabi University, Patiala-147 002, Punjab, India

Received March 1, 2012; accepted May 22, 2012

Summary In the present study, 5 species of Lygaeidae and 1 each of Berytidae and Malcidae have been cytologically investigated for the ﬁrst time; the diploid numbers in them vary from 12 to 20. Out of the 5 lygaeids, male diploid number is 12=8A+2m+XY in Dieuches leucoceras and Polycrates nexus while being 14=10A+2m+XY in Aphanus orientalis, Lanchnophorus sp. and Mizaldus sp. A pair of extremely large autosomes is found in both the species with 2n=12 while it may or may not present in species with 2n=14. One species each of Berytidae (Metacanthus pulchellus) and Malcidae (Malcus flavidipes) show 2n=20=18A+XY and 2n=16=12A+2m+XY respectively, and both lack an extremely large pair of autosomes. Microchromosomes are present in Lygaeidae (Rhyparochrominae) and Malcidae but absent in Berytidae. The sex determining mechanism in all the species of 3 families is XY/XX (♂/♀). Fragmentations seem to have played a piv- otal role in the origin of new species irrespective of family.

Key words Lygaeidae, Berytidae, Malcidae, Chromosomes, Meiosis.

In organisms with holocentric chromosomes, such as those of suborder Heteroptera, simple fusions and fragmentations readily lead to decreases and increases in chromosome number as the re- sulting chromosome elements retain the kinetic activity and thus can persist during dividing cycles (Heizer 1950, La Chance et al. 1970). Precisely for this reason, fragmentations and fusions have been considered to play the key role in evolution of heteropteran species (Ueshima and Ashlock 1980, Grozeva 1995). Members of Heteroptera are evolutionarily very successful because of their great array of feeding types and preferences which cover the entire range varying from phytophagous to zoopha- gous to hematophagous. Most of the heteropterans are herbivorous, piercing and sucking nutrients from plants, and thus are serious pests. Members of Lygaeidae (seed bugs) and Berytidae feed on reproductive parts such as ﬂowers, ovules and seeds of a wide variety of plants while members of Malcidae are found on wild vines. Lygaeidae, a large and diverse family, is paraphyletic with some of its subfamilies taken as sister taxons to members of other Heteropteran families such as Berytidae, Colobathristidae and Malcidae (Southwood and Leston 1959, Stys 1967). Thus, the taxonomic characterization of this family is difﬁcult and the complex relationship among its members is far from being established. Cytologically, Lygaeidae constitutes a heterogeneous group differing in chromosome number and types. The most common diploid chromosome number is 14 (Ueshima 1979, Ueshima and Ashlock 1980, Grozeva and Kuznetsova 1993, Souza et al. 2007, Kaur and Suman 2009). Berytidae is a small family comprising about 40 genera and more than 170 species grouped in 2 subfamilies, namely Metacanthinae and Berytinae. In this family, 2n=16 is the most common diploid chromosomal complement (Grozeva 1995). Malcidae consists of 2 subfamilies namely Malcinae and

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.77.311 312 V. Suman and H. Kaur Cytologia 77(3)

Table 1. Chromosomal complements of 7 studied species.

Species Location 2n EL

Lygaeidae Dieuches leucoceras Walker Punjab 12 8A+2m+XY + Polycrates nexus Stal* Punjab 12 8A+2m+XY + Aphanus orientalis Distant Punjab 14 10A+2m+XY － Lachnophorus sp. Reuter Punjab 14 10A+2m+XY + Mizaldus sp. Distant* Punjab 14 10A+2m+XY － Berytidae Metacanthus pulchellus Stal Punjab, Himachal Pradesh 20 18A+XY － Malcidae Malcus flavidipes Stal* Punjab, Himachal Pradesh 16 12A+2m+XY －

* First ever cytological report of the genus. EL=extremely large chromosome pair.

Chauliopinae, with modal number still not distinctly known. In the present study, 5 species of Lygaeidae (Rhyparochrominae) and 1 species each of Berytidae (Metacanthinae) and Malcidae (Malcinae) from India have been cytologically investigated for the ﬁrst time to describe their chromosomal complements and course of meiosis.

Materials and methods

Adult male specimens of Dieuches leucoceras, Polycrates nexus, Aphanus orientalis, Lachnophorus sp., Mizaldus sp. (Lygaeidae), Metacanthus pulchellus (Berytidae) and Malcus fal- vidipes (Malcidae) were collected from different regions of Punjab and Himachal Pradesh, India (Table 1). Testes were dissected out in 0.67% saline water and were fixed in freshly prepared Carnoyʼs fixative (3:1, absolute alcohol: glacial acetic acid) for 15 min followed by a second change of fresh Carnoyʼs fixative. The fixed material was tapped on clean slides and air dried slides were stained with carbol-fuschin stain for 4 h followed by differentiation in N-butanol. The slides were allowed to dry and were finally mounted in DPX.

Results

Dieuches leucoceras Walker The spermatogonial metaphase plate reveals 2n=12=8A+2m+XY (Fig. 1) One of the autosomal pairs is distinctly larger in size while the rest are almost identical in size. X is larger than Y. Microchromosomes are the smallest and lightest elements of the complement. The diffuse stage shows 2 heteropycnotic bipartite bodies, representing X and Y lying well apart against diffuse chromatin (Fig. 2). At early diplotene, 4 autosomal bivalents, 2 darkly stained unequal sex chromosomes, X and Y and 2 faintly stained m-chromosomes are observed. During late diplotene, autosomal bivalents show single terminal or sub-terminal chiasma. Micro- chromosomes come close to forming a bivalent (Figs. 3 and 4). At metaphase-I, autosomal bivalents along with sex chromosomes and m-chromosomes roughly form a circle (Fig. 5). During anaphase-I, m-bivalent and autosomal bivalents divide reductionally while sex chromosomes X and Y 2012 Meiosis in Heteropteran Species 313

Figs. 1–8. Dieuches leucoceras. (1) Spermatogonial plate along with karyotype, (2) diffuse stage, (3 and 4) diplotene, (5) Metaphase-I, (6) Anaphase-I, (7) Metaphase-II, (8) Telophase-II. Bar: 0.01 mm. divide equationally so that each pole receives 4 autosomes, 1 m-chromosome and 2 sex chromosomes, X and Y (Fig. 6). At metaphase-II, autosomes roughly form a circle while X and Y come close to forming a pseudobivalent that, along with the m-chromosome, lies inside the circle (Fig. 7). Metaphase-II is reductional for sex chromosomes and equational for autosomes and m-chromosome as a result of which, 2 types of telophase nuclei are formed, one with 4A+m+X and the other with 4A+m+Y (Fig. 8).

Polycrates nexus Stal It is the ﬁrst species of the genus to be examined kayrologically. The diploid chromosomal complement is 2n=12=8A+2m+XY (Fig. 9). One of the autosomal pairs is distinctly larger while the rest of the 3 autosomal pairs are almost identical in size. Sex chromosomes are smaller than autosomes, X being larger than Y. Microchromosomes are the smallest elements. At the diffuse stage, 2 elongated bipartite heteropycnotic bodies representing X and Y are observed (Fig. 10). At diplotene, single terminal/sub-terminal chiasma are seen in 3 of the autosomal 314 V. Suman and H. Kaur Cytologia 77(3)

Figs. 9–14. Polycrates nexus. (9) Spermatogonial plate along with karyotype, (10) Diffuse stage, (11) diplotene, (12) Metaphase-I, (13) Anaphase-I, (14) Metaphase-II. Bar: 0.01 mm. bivalents while 1 bivalent shows 2 terminal chiasmata. Autosomes are isopycnotic with X chromosome while Y is negatively heteropycnotic (Fig. 11). The metaphase-I plate shows one of the autosomal bivalents lying in the centre of the ring formed by rest of the chromosomes (Fig. 12). During anaphase-I, sex chromosomes divide equationally while autosomes and m-chromosomes divide reductionally so that each pole receives 4 autosomes, 2 sex chromosomes, X and Y and 1 m-chromosome (Fig. 13). The metaphase-II plate shows 4 autosomes, XY pseudobivalent and faint m-chromosome (Fig. 14).

Aphanus orientalis Distant The chromosomal complement is 2n=14=10+2m+XY. At the diffuse stage, 2 unequal elements representing X and Y associated with the nucleolar cloud are seen (Fig. 15). At the diplotene stage, 4 autosomal bivalents show single terminal/ sub-terminal chiasma each while 1 bivalent shows 2 terminal chiasmata (Figs. 16 and 17). At metaphase-I, autosomal bivalents roughly form a ring while the m-bivalent (very faint) and sex chromosomes arrange themselves in the centre (Fig. 18). At metaphase-II, X and Y come close to forming a pseudobivalent (Fig. 19). At telophase-II, 2 types of nuclei are formed, 1 with n=5A+m+X and the other with n=5A+m+Y (Fig. 20).

Lachnophorus sp. Reuter The chromosomal complement is 2n=10A+2m+XY (Fig. 21). One pair of autosomes is distinctly larger in size while remaining 4 pairs show gradation in size. X is bigger than Y. Microchromosomes are the smallest elements in the complement. At the diffuse stage, unequal X and Y appear as well separated bipartite heteropycnotic bodies 2012 Meiosis in Heteropteran Species 315

Figs. 15–20. Aphanus orientalis. (15) Diffuse stage, (16 and 17) diplotene stages, (18) Metaphase-I, (19) Mataphase-II, (20) Telophase-II (arrowheads show m-chromosomes). Bar: 0.01 mm.

(Fig. 22). At diplotene, all the autosomal bivalents show single terminal or sub-terminal chiasma each. Sex chromosomes lie well separated and 2 very faintly stained m-chromosomes lie close to each other (Fig. 23). At metaphase-I, autosomal bivalents roughly form a ring and X and Y along with the m-bivalent, lie in the centre of the ring (Fig. 24). During anaphase-I, autosomes along with m-chromosomes divide reductionally while sex chromosomes X and Y divide equationally (Fig. 25). At telophase-I, 5 autosomes are placed in a ring while X and Y which associate closely, lie in the centre. Microchromosomes are not traceable (Fig. 26). At metaphase-II, sex chromosomes X and Y join end to end to form a pseudobivalent (Fig. 27). During anaphase-II, autosomes and m- chromosome divide equationally and sex chromosomes divide reductionally (Fig. 28).

Mizaldus sp. Distant It is the ﬁrst species of the genus to be examined kayrologically. The chromosomal complement is 2n=14=10A+2m+XY. At the diffuse stage, nuclei show 2 dark unequal heterochromatic elements representing X and Y (Fig. 29). At the diakinesis stage, 5 autosomal bivalents, 2 m-chromosomes and bipartite sex chromosomes X and Y are seen. Micro-chromosomes appear as a faint streak (Fig. 30). At metaphase-I, 5 autosomal bivalents roughly form a ring with sex chromosomes X and Y in the centre (Figs. 31 and 32). During anaphase-I, autosomes and m-chromosomes divide reductionally while sex chromosomes divide equationally (Fig. 33). At metaphase-II, autosomes roughly form a ring and the pseudobivalent XY lies inside the ring. However, the m-chromosome is not traceable (Fig. 34). During anaphase-II, autosomes and the m-chromosome divide equationally while sex chromosomes divide reductionally as a result of which 2 types of nuclei are formed, 1 with n=5A+m+X and the other with n=5A+m+Y (Figs. 35 and 36). 316 V. Suman and H. Kaur Cytologia 77(3)

Figs. 21–28. Lachnophorus sp. (21) Spermatogonial plate along with karyotype, (22) diffuse stage, (23) diplotene, (24) Metaphase-I, (25) Anaphase-I, (26) Telophase-I, (27) Metaphase-II, (28) Anaphase-II. Bar: 0.01 mm.

Metacanthus pulchellus Stal The chromosomal complement is 2n=20=18A+XY (Figs. 37 and 38). At the diffuse stage, 2 rounded well separated unequal bipartite heteropycnotic bodies representing X and Y are seen (Fig. 39). During diplotene/diakinesis, all the autosomal bivalents show a single terminal chiasma (Fig. 40). The sex chromosomes X and Y appear as condensed and constricted bodies which remain well separated. At metaphase-I, autosomal bivalents roughly form a ring and sex chromosomes X and Y lie in the centre of the ring (Fig. 41). During metaphase-II, 9 autosomes and closely placed X and Y are randomly arranged. The latter do not form a pseudobivalent, instead they lie close to each other as a distant pair (Fig. 42).

Malcus ﬂavidipes Stal It is the ﬁrst species of the genus to be examined kayrologically. The chromosomal complement is 2n=16=12A+2m+XY (Fig. 43). Microchromosomes are the smallest and lightest elements 2012 Meiosis in Heteropteran Species 317

Figs. 29–36. Mizladus sp. (29) Diffuse stage, (30) diakinesis (faint m-chromosome shown by arrow), (31 and 32) Metaphase-I, (33) Anaphase-I, (34) Metaphase-II, (35) Anaphase-II, (36) Telophase-II. Bar: 0.01 mm. of the complement. At the diffuse stage, 2 heteropycnotic bodies of unequal size lying at the periphery are clearly observed representing sex chromosomes X and Y (Fig. 44). During diplotene, autosomal bivalents show single terminal-/sub-terminal chiasma each. Sex chromosomes X and Y lie separated (Fig. 45). During metaphase-I, 6 autosomal bivalents and sex chromosomes X and Y form a ring and the m-chromosomes lie in the centre (Fig. 46). During metaphase-II, autosomes along the with m-chromosome form a ring and the XY pseudobivalent lies inside the ring (Fig. 47).

Discussion

In the present study, 7 Indian heteropterans belonging to 3 families, namely Lygaeidae, Berytidae and Malcidae have been introduced to the cytological world for the ﬁrst time. Lygaeidae, a large and diverse family, constitutes a heterogeneous group differing in chromosome number and types of chromosomes. The modal diploid number for the family is 14. Cytological data compiled 318 V. Suman and H. Kaur Cytologia 77(3)

Figs. 37–42. Metacanthus pulchellus. (37) Prometaphase stage, (38) spermatogonial plate along with karyotype, (39) diffuse stage, (40) diplotene, (41) Metaphase-I, (42) Metaphase-II. Bar: 0.01 mm. by Ueshima and Ashlock (1980) and Grozeva and Kuznetsova (1993) reveal all the species to be characterized by the presence of microchromosomes and XY/XX (♂/♀) sex determining system with few exceptions. The 5 species of Lygaeidae investigated in the present study belong to Rhyparochrominae which is the most heterogeneous subfamily with respect to chromosome number. Diploid chromosomal complements of 12, 14, 16 18, 20, 22 and 24 are prevalent, out of which, 12, 14 and 16 are the most common (Ueshima 1979, Ueshima and Ashlock 1980, Grozeva and Kuznetsova 1993, Kaur and Suman 2009). Out of the 5 studied species, 2 species (Dieuches leucoceras and Polycrates nexus) have 2n=12=8A+2m+XY while 3 (Aphanus orientalis, Lachnophorus sp. and Mizaldus sp.) have 2n=14=10A+2m+XY. In the former 2 species, 1 pair of autosomes is extremely large as has earlier been observed in all the Rhyparochrominae species with 2n=12 such as Dieuches uniguttatus (Manna 1951), Dieuches sp. (Parshad 1957a, Grozeva and Kuznetsova 1993), Dieuches insignis (Kaur and Suman 2009), Lachnophorus singalensis (Parshad 1957b), Aphanus sordidus (Parshad 1957a), Aphanus sp. (Manna and Deb Mallick 1981) and Paromius gracilis (Malipatil 1979). In the presently studied species with 2n=14, an extremely large pair of autosomes is present only in Lachnophorus sp. while is absent in the other 2 species. In Rhyparochrominae, a pair of extremely large chromosomes may or may not be present in species with 2n=14. Orsillinae and Blissinae, too, show a diploid number of 14 but these subfamilies differ from Rhyparochrominae in always having the extremely large pair of autosomes. Chromosomal behavior during meiosis in Rhyparochrominae is quite heterogeneous. In Lygaeidae, so far, the most common arrangement of chromosomes at metaphase-I and II is the one 2012 Meiosis in Heteropteran Species 319

Figs. 43–47. Malcus flavidipes. (43) Spermatogonial plate along with karyotype, (44) diffuse stage, (45) diplotene, (46) Metaphase-I, (47), Metaphase-II. Bar: 0.01 mm. in which, autosomes form a ring while sex chromosomes and m-chromosomes lie in the centre at metaphase-I as well as metaphase-II (Manna 1951, Parshad 1957a, Ueshima 1979, Ueshima and Ashlock 1980, Kaur and Suman 2009). This condition has been observed only in Mizaldus sp. In Dieuches leucoceras, all the chromosomes are peripheral at metaphase-I while at metaphase-II, sex chromosomes and m-chromosome lie in the centre of the ring formed by autosomes, a condition re- corded for the first time in Lygaeidae. In Aphanus orientalis and Lachnophorus sp., sex chromosomes and m-chromosomes lie inside the autosomal ring at metaphase-I. In Polycrates nexus, metaphase-I shows one of the autosomal bivalents lying in the centre of the ring formed by rest of the chromosomes. Unfortunately, a polar view of metaphase-II for these 3 species could not be found. In the side view of metaphase-II, sex chromosomes are seen to form a pseudobivalent. Berytidae is a small family comprising about 40 genera and more than 170 species grouped in 2 subfamilies, namely Metacanthinae and Berytinae. From India, only 3 species are reported (Distant 1904). No subsequent data is available and cytological work, too, is lacking. The present study reports, for the first time, the chromosome number and meiotic details of 1 species of Berytidae from India, Metacanthus pulchellus (Metacanthinae). The male diploid chromosomal complement is 2n=20=18A+XY. Three earlier studied species of Metacanthus viz., M. meridiona- lis, M. tenellus and M. exilis from Bulgaria, Cuba and Korea respectively possess 2n=16=14A+XY with 1 pair of distinctly large autosomes in their complements (Grozeva 1995). This pair is not present in Metacanthus pulchellus suggesting its complement of 2n=20 to have originated from 2n=16 through fragmentation of autosomes including that of the large pair of autosomes. Grozeva (1995), too, considered 2n=16 as the basic number for the family and higher numbers to have been 320 V. Suman and H. Kaur Cytologia 77(3) derived from it by fragmentation. In Metacanthus pulchellus, 2 unequal well separated bipartite heteropycnotic bodies representing X and Y are seen at the diffuse stage. At metaphase-I, X and Y lie inside the ring formed by autosomal bivalents. Similar observations have been reported by Grozeva (1995) in Metacanthus me- ridionalis, M. tenellus, Apoplymus pectoralis and Neides afghanus. However, in Metatropis rufescens, the non-radial arrangement of 9 autosomal bivalents has been observed by Nokkala and Grozeva (1997). At metaphase-II, no regular arrangement is observed in Metacanthus pulchellus. However, in Neides afghanus, autosomes form a ring and XY lie in the centre, while in Apoplymus pectoralis, autosomes and one of the sex chromosomes form a circle and the other sex chromosome lies in the centre (Grozeva 1995). In Berytidae, sex chromosomes X and Y are seen to show variable association during metaphase-II. They may join to form a pseudobivalent as in Metatropis rufescens (Nokkal and Grozeva 1997) or just associate terminally as in Neides afghanus and Gampsocoris punctipes or may not associate at all and lie separately forming a distant bivalent on the metaphase plate as is observed in Apoplymus pectoralis (Grozeva 1995) and in Metacanthus pulchellus (present study). Berytidae was placed close to Cyminae of Lygaeidae on the basis of chromosome number by Southwood and Leston (1959). However, Hamid (1975) disagreed and refused to consider chromosome number to be evidential of this relationship as there was much variability in both groups. Grozeva (1995), too, suggested Berytidae to be distant from Cyminae, Colobathristidae and Malcidae, and placed it closer to Lygaeinae. From the chromosomal data available so far, Berytidae seems to have been derived from the subfamily Lygaeinae (2n=14) of Lygaeidae as both have XY sex mechanism and lack m-chromosomes. All the species with 2n=16 in Berytidae retain 1 large chromosome suggesting that they have been derived by fragmentation of any chromosome other than that of the large chromosome of Lygaeinae. The family Malcidae consists of 2 subfamilies namely Malcinae and Chauliopinae. Malcus flavidipes is the first species of the monogeneric subfamily Malcinae to be cytologically described. It has a complement of 2n=16=12A+2m+XY. Six pairs of autosomes show gradation in size. Two species of Chauliopinae which have been studied previously, namely Chauliopinae fallax and Chauliopinae bisontula, also, possess 2n=16=12A+2m+XY (Ueshima and Ashlock 1980). However, Malcinae differs from Chauliopinae in lacking 1 distinctly larger pair of autosomes and in the meiotic behavior of chromosomes (Table 1). In Malcus flavidipes, autosomes form a ring at metaphase-I and metaphase-II while X and Y lie on the periphery and the m-chromosomes lie in the centre at metaphase-I and XY pseudo-bivalent occupies the centre and m-chromosome lies on the periphery at metaphase-II. Similar meiotic behavior is observed in Ischnorhynchinae (Lygaeidae) (Table 2). In Chauliopinae, on the other hand, autosomes form a ring while X, Y and

Table 2. Arrangement of chromosomes at metaphase I and II in different subfamilies of Lygaeidae and Malcidae.

Metaphase-I Metaphase-II 2n E. L. A X Y m A X Y m

Lygaeidae Orsillinae 14 P C C C P C C C + Blissinae 14 P C C C P C C C + Ischnorhynchinae 16 P P P C P C C P + Malcidae Chauliopinae 16 P C C C P C C C + Malcinae 16 P P P C P C C P －

A: autosomes, X, Y: sex chromosomes, m: microchromosomes-chromosomes, P: peripheral, C: central 2012 Meiosis in Heteropteran Species 321 m-chromosomes lie in the centre at metaphase-I as well as metaphase-II, which is similar to the pattern found in Orsillinae and Blissinae (Lygaeidae) (Ueshima and Ashlock 1980). These observations point towards the possibility of different lines of origin for the 2 subfamilies of Malcidae. Malcinae seems to have originated from Ischnorhynchinae by fragmentation of the large autosomal pair of species having 2n=14 and sharing the same meiotic behavior as that of Malcinae. Chauliopinae, on the other hand, resembles Orsillinae in possessing the extremely large pair of autosomes and showing similar meiotic behavior of chromosomes. Earlier, Chauliops was included in Heterogastrinae and Malcus in Colobothristinae of the family Lygaeidae (Distant 1904). These were later placed in newly created subfamilies Chauliopinae and Malcinae respectively, grouped together in Malcidae (Schuh and Slater 1995). So far as chromosome cytology is concerned, Malcinae seems not to be close to Chauliopinae and their grouping together in the family Malcidae is not supported by present cytological observations. However, to arrive at any deﬁnite conclusion, the cytological data of more species of Malcinae is needed.

Acknowledgements

The authors are thankful to the Department of Science and Technology, New Delhi, India for providing ﬁnancial support (Grant No. 96915) to carry out the present study.

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