Research Article |
Corresponding author: Desislava Stoianova ( d.st.stoianova@gmail.com ) Academic editor: Seppo Nokkala
© 2020 Desislava Stoianova, Nikolay Simov, Manh Quang Vu, Duc Minh Nguyen, Snejana Grozeva.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Stoianova D, Simov N, Vu MQ, Nguyen DM, Grozeva S (2020) New data on karyotype, spermatogenesis and ovarian trophocyte ploidy in three aquatic bug species of the families Naucoridae, Notonectidae, and Belostomatidae (Nepomorpha, Heteroptera). Comparative Cytogenetics 14(1): 139-156. https://doi.org/10.3897/CompCytogen.v14i1.48709
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We report the karyotype, some aspects of spermatogenesis, and ovarian trophocytes ploidy in three aquatic bug species: Ilyocoris cimicoides (Linnaeus, 1758), Notonecta glauca Linnaeus, 1758, and Diplonychus rusticus Fabricius, 1871 from previously unexplored regions – South Europe (Bulgaria) and Southeast Asia (Vietnam). Our results add considerable support for the published karyotype data for these species. In I. cimicoides, we observed achiasmate male meiosis – the first report of achiasmy for the family Naucoridae. More comprehensive cytogenetic studies in other species of the Naucoridae are required to elucidate the role of achiasmy as a character in the systematics of the family.
Our observations on the association between phases of spermatogenesis and developmental stages in I. cimicoides and N. glauca differ from the previously published data. In these species, we assume that the spermatogenesis phases are not strongly associated with certain developmental stages. For further cytogenetic studies (on the Balkan Peninsula), we recommend July as the most appropriate month for collection of I. cimicoides and N. glauca.
In the ovaries of both species, we studied the level of ploidy in metaphase and interphase trophocytes. In I. cimicoides, diploid and tetraploid metaphase trophocytes were found. Heteropycnotic elements, observed in interphase trophocytes of this species, represented the X chromosomes. It allowed us to determine the trophocytes ploidy at interphase (2n was repeated up to 16 times). The situation with N. glauca was different. The metaphase trophocytes were diploid and we were not able to determine the ploidy of interphase trophocytes since such conspicuous heteropycnotic elements were not found. The scarce data available suggest a tendency for a low level of trophocyte ploidy in the basal infraorders (Nepomorpha and Gerromorpha) and for a high level in the more advanced Pentatomomorpha. Data about this character in species from other infraorders are needed to confirm that tendency.
achiasmate male meiosis (achiasmy), B-chromosomes, karyotype, nurse cells, South Europe and Southeast Asia, Diplonychus rusticus, Ilyocoris cimicoides, Notonecta glauca
Ilyocoris cimicoides (Linnaeus, 1758), Notonecta glauca Linnaeus, 1758, and Diplonychus rusticus Fabricius, 1871 are common predators in freshwater basins. The first two species are broadly distributed across the Palearctic region. Ilyocoris cimicoides (Naucoridae) inhabits most of Europe and Asia from Anatolia to Siberia and North China (
In many insect species, spermatogenesis completes at the final preimaginal developmental stage, so that the testes of adults contain only spermatids/spermatozoa (
A detailed cytogenetic analysis could elucidate the association of certain stages of meiosis with definite instars. Such data would be useful in further cytogenetic studies of these species (e.g. to collect the most appropriate developmental stage with meiotic or mitotic divisions).
In hemipteran species (incl. Heteroptera), the ovaries consist of meroistic telotrophic ovarioles, characterised by a tropharium in the apex and a vitellarium in the basal part (
The aim of the present cytogenetic study was to examine Ilyocoris cimicoides, Notonecta glauca, and Diplonychus rusticus originating from previously unexplored regions – South Europe (Bulgaria) and Southeast Asia (Vietnam), in order to 1) check cytogenetic differences between populations; 2) analyse the relationship between the developmental stages and the phases of spermatogenesis in testes for I. cimicoides and N. glauca; and 3) determine the ploidy level of trophocytes in ovaries on the example of I. cimicoides and N. glauca.
Specimens of Diplonychus rusticus (Belostomatidae) (4 males) were collected in September 2018 from Vietnam: Ca Mau Province, Tran Van Thoi District, Tran Hoi commune, U Minh Ha National Park, 09.22521N, 104.95898E (Fig.
Giemsa stained preparations were analysed under an Axio Scope A1 – Carl Zeiss Microscope) at 100× magnification and documented with a ProgResMFcool – Jenoptik AG digital camera. FISH preparations were analysed under a Leica DM 6000 B microscope and images were acquired using a Leica DFC 345 FX camera and Leica Application Suite 3.7 software with an Image Overlay module.
The specimens and the chromosome preparations used for this study are stored at the Lab of Cytotaxonomy and Evolution, Institute of Biodiversity and Ecosystem Research, BAS (Sofia, Bulgaria).
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
Like all heteropteran species (
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.
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
The internal reproductive system of the examined adult males confirmed the morphological descriptions given for Diplonychus rusticus by
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 (
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 (
The ovaria of the females and the testes of the males examined matched the morphological descriptions given by
In gonads of females and males, we found mitotic metaphase plates with 24 chromosomes including two sex chromosomes (Fig.
In adults collected in July, we found the advanced stages of spermatogenesis, from MI (Fig.
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.
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
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
Stages of spermatogenesis and spermiogenesis observed in the testes of IV and Vth instars and adult of I. cimicoides and N. glauca.
Developmental stage | Ilyocoris cimicoides | Notonecta glauca | ||
---|---|---|---|---|
After |
Present data | After |
Present data | |
Instar III | primary spermatocytes and as result of MI – secondary spermatocytes | spermatogonial stages | primary spermatocytes | spermatogonial stages |
Instar IV | primary spermatocytes and as result of MI – secondary spermatocytes | spermatogonial stages and meiotic prophase stages | primary spermatocytes and as result of MI – secondary spermatocytes | spermatogonial stages |
Instar V | collected in the end of the summer – bundles of spermatozoa | collected in July and August – from PMI to telophase II | disappearance “of the zone of spermatogonia” (the end of the spermatogonial divisions) | spermatogonial stages and meiotic prophase stages |
collected in September – spermatids/spermatozoa | ||||
Adult | mature spermatozoa | spermatids/spermatozoa | No information on the month of collection – spermatids/spermatozoa | collected in July – from MI to AII |
collected in September – spermatids/spermatozoa |
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.
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) (
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.
The collection of specimens used in this study was partly supported by Research project “Research on model construction of biodiversity conservation in village scale in Mui Ca Mau Biosphere reserve, Vietnam”; Code number: ĐTĐL.CN-26/17.
The cytogenetic analysis of Diplonychus rusticus was supported by the Bulgarian Ministry of Education and Science, grant KP-06-Russia-18/27.09.2019 of National Fund of Scientific Research.
The cytogenetic analysis of Ilyocoris cimicoides and Notonecta glauca was supported by the Bulgarian Ministry of Education and Science under the National Research Programme “Young scientists and postdoctoral students” approved by DCM # 577 / 17.08.2018.
We are grateful to M. Todorov for the help with collecting the Bulgarian material for this study.
We thank V. Kuznetsova and V. Evtimova for the useful help to improve the language and the text of the manuscript.
We kindly thank the subject editor S. Nokkala and the reviewers V. Lukhtanov, C. Nokkala and an anonymous reviewer for the valuable criticism and advice on how to improve the text and the illustrations.