Ilyocoris cimicoides, 2n = 51 (48A + 2m + X) ♂

The morphology of the testes of the examined males and the ovaries of the females matched the descriptions given by Papáček and Gelbič (1989) and Papáček et al. (1997), respectively.

Like all heteropteran species (Ueshima 1979, Papeschi and Bressa 2006, Kuznetsova et al. 2011), the chromosomes of this species are holokinetic – without localized centromere. In studied male and female nymphs, mitotic metaphases consisted of 48 autosomes (Figs 2 a, b, 3). Except the 48 autosomes, the karyotype included a pair of very small m-chromosomes (see below the description of the meiotic metaphase I), difficult for observation in mitotic cells. The X chromosomes were the largest chromosomes of the complement. At spermatogonial metaphase, the X chromosome displayed interstitial heterochromatin blocks after C-banding (Fig. 2b). In the early condensation stage (late meiotic prophase), we observed 24 bivalents consisting of two side-by-side aligned chromosomes without any sign of chiasmata between them, a pair of m-chromosome univalents, and the heteropycnotic X chromosome, which usually appeared close to the nucleolus (Fig. 4). At the late condensation stage, the X still tended to be close to the nucleolus (Fig. 5a, b). At metaphase I (MI), the autosomal bivalents were similar in size. Most of them formed a ring. The m-chromosome pair and some of the autosomal bivalents laid inside the ring (Fig. 6). The X chromosome was usually seen close to the periphery of the ring. The post-reduction for the sex chromosomes in male meiosis is another specific cytogenetic character of Heteroptera being typical for the majority of the studied species and higher taxa of this group (Ueshima 1979, Kuznetsova et al. 2011). Such was the case in I. cimicoides: the X was observed in all the examined anaphase I (AI) (Fig. 7 a–c), telophase I (TI) nuclei (Fig. 8) and in all daughter cells at metaphase II (MII) (Fig. 9). At AI, it was easy to distinguish the m-chromosomes, which were going ahead of the set (Figs 7, 8). At MII, only the large X could be recognized reliably (Fig. 9). At telophase II (TII), the second (equational for the autosomes but reductional for the sex chromosomes) division resulted in two types of daughter cells – with and without X chromosome (Fig. 10).

Figures 2–10.

Ilyocoris cimicoides (testis/ovary): Schiff-Giemsa (2a, 3–10), C-banding (2b) 2a, b spermatogonial metaphase (arrows indicate heterochromatin blocks) 3 oogonial metaphase 4–8 primary spermatocytes: 4 early condensation stage 5a, b late condensation stage 6 metaphase I 7 a–c anaphase I 8 telophase I 9, 10 secondary spermatocytes: 9 metaphase II 10 two telophases II – one with X, another without X. Scale bar:10 μm.

Figures 11–18.

Diplonychus rusticus (testis) 11 spermatogonial metaphase 12 spermatogonial anaphase 13–16 primary spermatocytes: 13 leptotene 14 pachytene 15 diffuse stage 16a, b metaphase I 17, 18 secondary spermatocytes: 17 a, b metaphase II 18 anaphase II. Scale bar:10 μm.

Cytogenetic data on I. cimicoides have been published by Divaz (1915) and Steopoe (1929). Divaz (1915) studied the spermatogenesis of I. cimicoides from Serbia, but he did not actually deal with the karyotype: he focused on the presence and behaviour of specific chromatophilic bodies (“corpuscules archoplasmiques”). Our results confirmed the chromosome formula of 2n = 51 (48A + 2m + X), and the chromosomes behaviour for male I. cimicoides reported by Steopoe (1929), based on specimens (without information of their developmental stage) collected north of the Danube River. These authors did not mention anything about the formation of chiasmata. In the present study, we provide the first report for achiasmate male meiosis in I. cimicoides and in the whole family Naucoridae. The achiasmate type of meiosis, called also “achiasmy” (Satomura et al. 2019), as a rule, is restricted to the heterogametic sex (White 1973). Hitherto, achiasmate male meiosis has been found in eight other families of three Heteroptera infraorders, namely Cimicomorpha: Anthocoridae, Microphysidae, Cimicidae, Miridae, and Nabidae; Leptopodomorpha: Saldidae; Nepomorpha: Micronectidae and Corixidae (Stoianova et al. 2015, more references in Kuznetsova et al. 2011). Most of the studied families seem to be homogenous in respect to the presence/absence of chiasmata. Based on the cited data it is suggested that in Heteroptera achiasmy is a stable cytogenetic characteristic at the family level (Grozeva et al. 2008, Kuznetsova et al. 2011). However, heterogeneity in respect to this character has been observed in family Corixidae (Nepomorpha) (Stoianova et al. 2015). Achiasmy is reported for two Cymatia species, while the rest examined species of the Corixidae display chiasmata. The achiasmy in I. cimicoides (present study) reveals the heterogeneity of the Naucoridae in respect to this character. Hitherto, cytogenetic studies have been published for eight other naucorid species (Papeschi 1992, for more references see Ueshima 1979). Two of them, Pelocoris lautus Berg, 1879 and P. binotulatus (Stål, 1862), were shown to display chiasmate meiosis in males (Papeschi 1992), while for another six species no information on the presence/absence of chiasmata has been provided (for references see Ueshima 1979). It is noteworthy that in another insect order (Diptera) heterogeneity in the type of male meiosis (achiasmate/chiasmate) has been reported even at genus level (see Satomura et al. 2019). Taking into consideration these new findings, more comprehensive cytogenetic studies in other species of the family Naucoridae are required to elucidate the type of meiosis (chiasmate/achiasmate) as a character in the systematics of the family.

The internal reproductive system of the examined adult males confirmed the morphological descriptions given for Diplonychus rusticus by Pendergrast (1957: as Sphaerodema rusticum Fabricius, 1871). Every colorless testis consisted of one more or less spherical follicle, decreasing in diameter from the apex to the vas deferens, which expanded to a vesicula seminalis. Such structure of the male reproductive system was described and illustrated well in another Belostomatidae species – Lethocerus patruelis (Stål, 1854) (Grozeva et al. 2013).

Chromosome complement in males of D. rusticus (as S. rusticum) from India was published as 2n = 28 (24A + 2m + XY), together with drawings of the chromosomes at different stages of spermatogenesis (Bawa 1953, Jande 1959). These authors claim a symmetric karyotype and describe in detail all stages of spermatogenesis: spermatogonial mitosis and the behaviour of the chromosomes during both meiotic divisions.

We studied males of this species from Vietnam, collected in U Minh Ha National Park. Spermatogonial metaphases resembled those of D. rusticus, D. annulatus (Fabricius, 1781) and D. molestus (Dufour, 1863) (as D. subrhombeus (Mayr, 1871)) studied from India (Bawa 1953, Jande 1959). They consisted of 28 chromosomes (Fig. 11) but it was difficult to identify individual chromosomes in the set. At early spermatogonial anaphase, the chromosomes were split in chromatids, lying in parallel (Fig. 12), which is the case for holokinetic chromosomes (for instance, Lukhtanov et al. 2019) and one could recognize the X, Y and m-chromosomes. At leptotene (Fig. 13), long and thin chromosomes began to thicken, the sister chromatids were not visible as separate entities. At pachytene (Fig. 14), one or two heteropycnotic bodies of the sex chromosome heterochromatin could be observed. During the diffuse stage, the sex chromosomes were more often associated with each other (Fig. 15). At MI, 12 bivalents, two sex chromosomes as univalents, and a pseudobivalent of the m-chromosomes could be seen (Fig. 16 a, b). At MII, every plate consisted of 12 autosomes, a pseudobivalent of the sex chromosomes, and one m-chromosome (Fig. 17 a, b). The first meiotic division was thus reductional for the autosomes and equational for the sex chromosomes. At AII, the sex chromosomes were going ahead to the poles (Fig. 18). The study of males from Vietnam fully confirmed the observations and description on the spermatogenesis of this species from India (Bawa 1953, Jande 1959). Here, we provide for the first time, photographs of the spermatogenesis stages for D. rusticus.

The ovaria of the females and the testes of the males examined matched the morphological descriptions given by Papáček and Soldán (1987, 1992, respectively).

In gonads of females and males, we found mitotic metaphase plates with 24 chromosomes including two sex chromosomes (Fig. 19 a, c). As an exception, only two mitotic metaphase plates with 26 elements were found: one in a ovariole (Fig. 19 b), in which we found mitotic metaphase plates with 24 chromosomes and one in a testis (Fig. 19 d), in which we found mitotic metaphase plates with 24 chromosomes.

In adults collected in July, we found the advanced stages of spermatogenesis, from MI (Fig. 20) to AII (Fig. 23). At MI, 10 autosomal bivalents and the X and Y chromosomes formed a ring with a pseudobivalent of m-chromosomes in it (Fig. 20). It was difficult to distinguish the sex chromosomes in the majority of the meiotic stages. At all the examined AI nuclei, 13 chromosome elements could be counted with the m-chromosomes going ahead of the set (Fig. 21). At MII, a pseudobivalent of the sex chromosomes was placed inside a ring formed by the autosomes; the m-chromosome was indistinguishable (Fig. 22).

Figures 19.

Notonecta glauca (testis/ovary) a, b mitotic metaphase in a ovariole: a with 24 chromosome elements b with 26 chromosome elements c, d mitotic metaphase in a testis: c with 24 chromosome elements d with 26 chromosome elements. Scale bar:10 μm.

Figures 20–23.

Notonecta glauca (testis/ovary) 20, 21 primary spermatocytes: 20 metaphase I 21 anaphase I 22, 23 secondary spermatocytes: 22 metaphase II with 13 chromosome elements 23 anaphase II. Scale bar:10 μm.

Our observations confirm the chromosome formula of 2n = 24 (20A + 2m + XY) and post-reduction of the sex chromosomes reported by Browne (1916) and Halkka (1956) for males of N. glauca, collected in England and Finland, respectively. Angus et al. (2004) studied the karyotype of N. glauca in midgut cells from both male and female specimens collected in England. He found some specimens with 2n = 26 along with the regular 2n = 24. Our observations of mitotic metaphase plates with 26 chromosomes confirm the occasional occurrence of an extra pair of chromosomes, which was interpreted by Angus et al. (2004) as that of the B-chromosomes. In the present study, C-banding technique was performed but it did not provide additional information.

Our observations of the association between phases of spermatogenesis and developmental stages in I. cimicoides and N. glauca differ from those published for these species by Papáček and Gelbič (1989) and Papáček and Soldán (1992), respectively (see Table 1). In I. cimicoides, we observed meiotic divisions in instar V, while Papáček and Gelbič (1989) reported the instar V to have spermatids and spermatozoa only. In adults of N. glauca, we observed meiotic divisions, while Papáček and Soldán (1992) found only spermatids and spermatozoa. According to our observations, in both I. cimicoides and N. glauca, males of the same stage of development show different stages of spermatogenesis if they are collected in different seasons (Table 1). For these species, we assume thus that the spermatogenesis phases are not strongly associated with certain developmental stages. It could be speculated that spermatogenesis phases in these species follow the seasonal changes of factors such as temperature and/or photoperiod. There are no publications about the influence of temperature and/or photoperiod on spermatogenesis in heteropteran species. Nevertheless, the influence of these factors on the development and reproduction of Heteroptera has been well documented (Dunbar and Bacon 1972, Ali and Ewiess 1977, Spence et al. 1980, Braman and Pendley 1993, Nagai and Yano 1999, Niva and Takeda 2003, Zerbino et al. 2013, Gusev and Lopatina 2018, Santos et al. 2018). Temperature effect on the development of subspecies Ilyocoris cimicoides exclamationis (Scott, 1874) from Japan has also been reported (Kaneda and Yoshiyasu 2007). We have no data on the environmental factors in the collection sites; therefore, the present study cannot contribute to understanding the influence of environmental factors on the spermatogenesis in the studied species. Nevertheless, for practical purposes, we assume that in Bulgaria (in regions at less than 800 m a.s.l.) and probably on the Balkan Peninsula, July is the most appropriate month for collecting I. cimicoides and N. glauca for cytogenetic studies (Table 1).

In the ovaries of I. cimicoides and N. glauca, we studied the level of ploidy both in mitotic (metaphases) and in interphase trophocytes (nurse cells). Among the trophocytes of I. cimicoides, only diploid and tetraploid metaphases were found (Fig. 24). In I. cimicoides, we observed interphase trophocytes with conspicuous heteropycnotic elements, which varied in number from 2 to 32 (Figs 24, 25), almost always an even number. In cells with a higher (8–32) number of elements, the exact counting was often impeded by the clumping of the elements. In ovarian mitosis of I. cimicoides, hybridization signals after FISH for 18S rDNA were found in the telomeric region of the sex chromosomes (Fig. 26). The same hybridization signals were likewise found in heteropycnotic elements of trophocytes (Fig. 27 a, b). This suggests that the heteropycnotic elements found in the trophocytes represent the sex chromosomes. Thus, in I. cimicoides the interphase trophocytes with high number of heteropycnotic elements, i.e. X chromosomes, were highly polyploid (2n was repeated up to 16 times). The situation with N. glauca was different. We did not find interphase trophocytes with such conspicuous heteropycnotic bodies (Fig. 28) and we were able to determine the ploidy (2n) only in mitotic trophocytes (Figs 19, 28).

The level of trophocyte ploidy in the ovarioles of I. cimicoides (Nepomorpha) (present study) is the same (16 times increase) as reported for Gerris najas (Gerromorpha) (Choi and Nagl 1977). Much higher level of trophocyte ploidy has been reported for the two hitherto studied species of infraorder Pentatomomorpha: Oncopeltus fasciatus – 128 times increase (Cave 1975); Dysdercus intermedins – 124 times increase (Dittmann et al. 1984). The scarce data presently available suggest a tendency for a low level of trophocyte ploidy in basal infraorders (Nepomorpha and Gerromorpha) and a high such level in the more advanced Pentatomomorpha. Data on additional species of the same and other infraorders are needed to confirm this tendency.

Figures 24–28.

Ilyocoris cimicoides and Notonecta glauca (ovarioles): Schiff-Giemsa (24, 25, 28) and FISH with 18S rDNA (26, 27) 24–27 Ilyocoris cimicoides: 24 interphase trophocytes with 2 and 4 heteropycnotic elements, and a metaphase tetraploid trophocyte 25 interphase trophocytes with 16 and about 32 heteropycnotic elements 26 18S rDNA signals on the sex chromosomes in a oogonial diploid metaphase plate 27 a, b 18S rDNA signals on the heteropycnotic elements in interphase octoploid trophocytes 28 Notonecta glauca: interphase trophocytes and a mitotic metaphase diploid trophocyte. Scale bars: 10 μm.