New Evidence for the Presence of the Telomere Motif (TTAGG)N in the Family Reduviidae and Its Absence in the Families Nabidae
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COMPARATIVE A peer-reviewed open-access journal CompCytogen 13(3): 283–295 (2019)Telomere motif (TTAGG ) in Cimicomorpha 283 doi: 10.3897/CompCytogen.v13i3.36676 RESEARCH ARTICLEn Cytogenetics http://compcytogen.pensoft.net International Journal of Plant & Animal Cytogenetics, Karyosystematics, and Molecular Systematics New evidence for the presence of the telomere motif (TTAGG) n in the family Reduviidae and its absence in the families Nabidae and Miridae (Hemiptera, Cimicomorpha) Snejana Grozeva1, Boris A. Anokhin2, Nikolay Simov3, Valentina G. Kuznetsova2 1 Cytotaxonomy and Evolution Research Group, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia 1000, 1 Tsar Osvoboditel, Bulgaria2 Department of Karyosystematics, Zoological Institute, Russian Academy of Sciences, St. Petersburg 199034, Universitetskaya nab., 1, Russia 3 National Museum of Natural History, Bulgarian Academy of Sciences, Sofia 1000, 1 Tsar Osvoboditel, Bulgaria Corresponding author: Snejana Grozeva ([email protected]) Academic editor: M. José Bressa | Received 31 May 2019 | Accepted 29 August 2019 | Published 20 September 2019 http://zoobank.org/9305DF0F-0D1D-44FE-B72F-FD235ADE796C Citation: Grozeva S, Anokhin BA, Simov N, Kuznetsova VG (2019) New evidence for the presence of the telomere motif (TTAGG)n in the family Reduviidae and its absence in the families Nabidae and Miridae (Hemiptera, Cimicomorpha). Comparative Cytogenetics 13(3): 283–295. https://doi.org/10.3897/CompCytogen.v13i3.36676 Abstract Male karyotype and meiosis in four true bug species belonging to the families Reduviidae, Nabidae, and Miridae (Cimicomorpha) were studied for the first time using Giemsa staining and FISH with 18S ribo- somal DNA and telomeric (TTAGG)n probes. We found that Rhynocoris punctiventris (Herrich-Schäffer, 1846) and R. iracundus (Poda, 1761) (Reduviidae: Harpactorinae) had 2n = 28 (24 + X1X2X3Y), whereas Nabis sareptanus Dohrn, 1862 (Nabidae) and Horistus orientalis (Gmelin, 1790) (Miridae) had 2n = 34 (32 + XY) and 2n = 32 (30 + XY), respectively. FISH for 18S rDNA revealed hybridization signals on a sex chromosome, the X or the Y, in H. orientalis, on both X and Y chromosomes in N. sareptanus, and on two of the four sex chromosomes, Y and one of the Xs, in both species of Rhynocoris Hahn, 1834. The results of FISH with telomeric probes support with confidence the absence of the “insect” telomere motif (TTAGG)n in the families Nabidae and Miridae and its presence in both species of genus Rhynocoris of the Reduviidae, considered as a basal family of Cimicomorpha. Increasing evidence reinforces the hypothesis of the loss of the canonical “insect” telomere motif (TTAGG)n by at least four cimicomorphan families, Nabidae, Miridae, Tingidae, and Cimicidae, for which data are currently available. Keywords Heteroptera, Rhynocoris punctiventris, R. iracundus, Nabis sareptanus, Horistus orientalis, TTAGG-FISH Copyright Snejana Grozeva et al. 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. 284 Snejana Grozeva et al. / Comparative Cytogenetics 13(3): 283–295 (2019) Introduction The true bugs (Hemiptera: Heteroptera), with almost 45,000 described species dis- tributed into 91 families and seven infraorders (Henry 2017), are one of the largest and most diverse groups of non-holometabolous insects. Overall, 40 species, 27 gen- era and 10 families have been studied in respect to the telomere structure (Okazaki et al. 1993, Sahara et al. 1999, Grozeva et al. 2011, Golub et al. 2015, 2017, 2018, Pita et al. 2016, Chirino et al. 2017, Angus et al. 2017). The species studied belong to three largest infraorders, including a more basal infraorder Nepomorpha and the evolutionary derived sister infraorders Pentatomomorpha and Cimicomorpha. The “insect” telomere motif (TTAGG)n was found in all studied species of the families Belostomatidae (Kuznetsova et al. 2012, Chirino et al. 2017) and Nepidae (Angus et al. 2017) from the Nepomorpha. Likewise, this motif was reported for the suborder Coleorrhyncha, a sister group to the Heteroptera (Kuznetsova et al. 2015). These facts indicate that it is most likely the ancestral telomeric repeat sequence of the Heterop- tera in general. In contrast, all studied species of the families Lygaeidae s.l., Pentato- midae, and Pyrrhocoridae from the Pentatomomorpha, as well as those of the families Nabidae, Tingidae, Cimicidae, and Miridae from the Cimicomorpha were shown to lack this motif (Okazaki et al. 1993, Sahara et al. 1999, Grozeva et al. 2011, Golub et al. 2015, 2017, 2018). Based on this evidence, a hypothesis was advanced that the ancestral telomeric repeat TTAGG was lost at the base of the clade Pentatomomor- pha + Cimicomorpha (= the Geocorisae sensu Schuh et al. 2009) being secondarily replaced by another yet unknown motif or an alternative telomerase-independent mechanism of telomere maintenance (Frydrychová et al. 2004, Mason et al. 2016). However, a recent research of Pita et al. (2016) discovered the putative ancestral “in- sect” motif in the cimicomorphan family Reduviidae (the assassin bugs), namely in the comparatively young (24–38 Ma, after Hwang and Weirauch 2012) hematopha- gous subfamily Triatominae. Due to this finding, the validity of the above hypothesis was questioned. Moreover, the postulated lack of the (TTAGG)n detection, at least in the families of Cimicomorpha, was suggested to be “due to a methodological problem of the telomeric probe rather than a loss process during their evolution” (Pita et al. 2016). Primarily to address this issue, we did a (TTAGG)n FISH experiment involving four species of the Cimicomorpha, which have not previously been studied in respect to telomere composition. These areNabis (Halonabis) sareptanus Dohrn, 1862 from the family Nabidae; Horistus orientalis (Gmelin, 1790) from the family Miridae; Rhynocoris punctiventris (Herrich-Schäffer, 1846) andR. iracundus (Poda, 1761) from the family Reduviidae, the subfamily Harpactorinae. Specifically, we looked for a strong evidence of the absence of the (TTAGG)n telomere motif in Nabidae and Miridae as well as an additional evidence of the presence of this motif in the family Reduviidae. In addition, we aimed to detect the 18S rDNA loci in the above species. Finally, we characterized, for the first time, the karyotype and meiotic pattern of spermatogenesis in each of the species under study. Telomere motif (TTAGG )n in Cimicomorpha 285 Material and methods Taxon sampling, fixation and slide preparation The true bug specimens were collected in May-June 2018, in Bulgaria. The localities from which the bugs were collected and the number of males and mitotic/meiotic preparations studied are given in Table 1. The insects were brought to the lab and fixed alive in a fixative consisting of 3 parts of 95% ethanol and 1 part of glacial acetic acid. Chromosome preparations were made from the male gonads. The testes were extracted from the abdomen, placed on a slide in a drop of 45% acetic acid, and squashed. The coverslips were removed with a razor blade after freezing with dry ice, and the slides were, then, dehydrated in fresh fixative (3 : 1) and air dried. Routine staining For this staining, we followed the Schiff-Giemsa method described by Grozeva and Nokkala (1996). Fluorescence in situ hybridization (FISH) Probes for 18S rDNA and (TTAGG)n were prepared and FISH was performed according to Grozeva et al. (2015) with some modifications. For primer information, see Grozeva et al. (2011). The telomere probe (TTAGG)n was amplified by PCR and labelled with rhodamine-5-dUTP (GeneCraft, Köln, Germany). An initial denaturation period of 3 min at 94 °C was followed by 30 cycles of 45 s at 94 °C, annealing for 30 s at 50 °C and extension for 50 s at 72 °C, with a final extension step of 3 min at 72 °C. The 18S rDNA probe was amplified by PCR and labelled with biotin-11-dUTP (Fermentas, Vilnius, Lithuania) using genomic DNA of the true bug Pyrrhocoris apterus (Linnaeus, 1758). An initial denaturation period of 3 min at 94 °C was followed by 33 cycles of 30 s at 94 °C, annealing for 30 s at 50 °C and extension for 1.5 min at 72 °C, with a final extension step of 3 min at 72 °C. The chromosome preparations were treated with 100 μg/ml RNase A and 5 mg/ml pepsin solution to remove excess RNA and proteins. Chromosomes were denatured in the hybridization mixture containing labelled 18S rDNA and (TTAGG)n probes (80–100 ng per slide) with an addition of salmon sperm blocking reagent and then hybridized for 42 h at 37 °C. 18S rDNA probes were detected with NeutrAvidin-Fluores- cein conjugate (Invitrogen, Karlsbad, CA, USA). The chromosomes were mounted in an antifade medium (ProLong Gold antifade reagent with DAPI, Invitrogen) and covered with a glass coverslip. The number of males involved in the study ranged from three to one (Table 1), and the number of preparations examined ranged from one (Horistus ori- entalis) to 11 (Rhynocoris iracundus) and the number of prophase/metaphase plates exam- ined ranged from a few (H. orientalis) to several dozen (N. sareptanus and Rhynocoris spp). 286 Snejana Grozeva et al. / Comparative Cytogenetics 13(3): 283–295 (2019) Table 1. Material studied. Number Number of males/ of males/ Date of preparations Species Locality preparations collection analysed by Shiff- analysed by Giemsa staining FISH Rhynocoris punctiventris (Herrich-Schäffer, 1846) 23 May 2018 1/6 1/3 Bulgaria, Kresna Gorge Rhynocoris iracundus (Poda, 1761) 23 May 2018 3/11 2/5 41.762378N, 23.169228E Horistus orientalis (Gmelin, 1790) 23 May 2018 1/1 1/2 Bulgaria, Pomorie Lake Nabis (Halonabis) sareptanus Dohrn, 1862 07 June 2018 3/5 1/1 42.565609N, 27.630627E As a control for the efficacy of our (TTAGG)n FISH experiments, we used chro- mosome preparations from Scarlupella discolor (Germar, 1821) (Hemiptera: Auchen- orrhyncha) known to be (TTAGG)n -positive (Maryańska-Nadachowska et al.