Research Article |
Corresponding author: Valentina G. Kuznetsova ( valentina_kuznetsova@yahoo.com ) Academic editor: Ilya Gavrilov-Zimin
© 2016 Valentina G. Kuznetsova, Gadzhimurad N. Khabiev, Boris A. Anokhin.
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:
Kuznetsova VG, Khabiev GN, Anokhin BA (2016) Cytogenetic study on antlions (Neuroptera, Myrmeleontidae): first data on telomere structure and rDNA location. Comparative Cytogenetics 10(4): 647-656. https://doi.org/10.3897/CompCytogen.v10i4.10775
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Myrmeleontidae, commonly known as “antlions”, are the most diverse family of the insect order Neuroptera, with over 1700 described species (in 191 genera) of which 37 species (in 21 genera) have so far been studied in respect to standard karyotypes. In the present paper we provide first data on the occurrence of the “insect-type” telomeric repeat (TTAGG)n and location of 18S rDNA clusters in the antlion karyotypes studied using fluorescence in situ hybridization (FISH). We show that males of Palpares libelluloides (Linnaeus, 1764) (Palparinae), Acanthaclisis occitanica (Villers, 1789) (Acanthaclisinae) and Distoleon tetragrammicus (Fabricius, 1798) (Nemoleontinae) have rDNA clusters on a large bivalent, two last species having an additional rDNA cluster on one of the sex chromosomes, most probably the X. (TTAGG)n - containing telomeres are clearly characteristic of P. libelluloides and A. occitanica; the presence of this telomeric motif in D. tetragrammicus is questionable. In addition, we detected the presence of the (TTAGG)n telomeric repeat in Libelloides macaronius (Scopoli, 1763) from the family Ascalaphidae (owlflies), a sister group to the Myrmeleontidae. We presume that the “insect” motif (TTAGG)n was present in a common ancestor of the families Ascalaphidae and Myrmeleontidae within the neuropteran suborder Myrmeleontiformia.
TTAGG, rDNA, fluorescence in situ hybridization, Palpares libelluloides , Acanthaclisis occitanica , Distoleon tetragrammicus , Libelloides macaronius , Myrmeleontidae , Ascalaphidae , Neuroptera
The ends of eukaryotic chromosomes are capped with complex nucleoprotein structures, the telomeres, which preclude fusion, recombination and degradation of the chromosome ends and thus maintain the genome integrity. In a large number of organisms, telomeric DNA consists of highly repetitive short sequences. Although telomeres are diversified in their DNA sequence composition in different eukaryotic groups, several phylogenetic lineages demonstrate highly conserved motifs. In animals, three main types of telomeric repeats are known: TTAGGG, TTAGGC, and TTAGG. Motif (TTAGGG)n prevails in the multicellular animals, except roundworms and arthropods, and is probably ancestral for all Metazoa; motif (TTAGGC)n is characteristic of nematodes; motif (TTAGG)n, which is a derivative of (TTAGGG)n, occurs in all arthropod groups (sea spiders, chelicerates, myriapods, crustaceans, and hexapods), supporting their origin from a common ancestor (
Neuroptera, also known as Planipennia, are a highly heterogeneous insect order, with 5803 species described in 16 families (
Within Neuroptera, the family Myrmeleontidae, commonly known as “antlions”, due to the fiercely predatory habits of their larvae, is the most diverse group having worldwide distribution. This family is considered monophyletic, with over 1700 extant species in 191 genera. In Myrmeleontidae, as many as 12 subfamilies, among them Palparinae, Pseudimarinae, Stilbopteryginae, Dimarinae, Echthromyrmicinae, Dendroleontinae, Nemoleontinae, Glenurinae, Myrmecaelurinae, Acanthaclisinae, Brachynemurinae, and Myrmeleontinae (
Until now, the cytogenetic studies in the Myrmeleontidae have been carried out on 37 species from 21 genera, and were focused exclusively on the basic features of the karyotypes such as chromosome numbers and sex determination systems (reviewed in
The aim of the present study is to further characterize chromosomes of antlions and to study their evolution by exploring the telomere structure and chromosomal location of the major ribosomal RNA (rRNA) genes using fluorescence in situ hybridization (FISH). The FISH technique was applied for the first time in the family Myrmeleontidae.
We examined the presence/absence of TTAGG telomeric repeats and location of the rDNA clusters in Palpares libelluloides (Linnaeus, 1764), Distoleon tetragrammicus (Fabricius, 1798) and Acanthaclisis occitanica (Villers, 1789) from the family Myrmeleontidae. In addition, we studied telomere structure in Libelloides macaronius (Scopoli, 1763) belonging to the sister family Ascalaphidae.
Three antlion species, involving three different genera from three subfamilies (sensu
Examined material and main karyotypic features obtained during the present study.
Taxon | Sampling locality and month and year of collection | Number of studied males | Diploid karyotype | Telomeric sequence | 18S rDNA clusters location |
---|---|---|---|---|---|
Myrmeleontidae | |||||
Palparinae | |||||
Palpares libelluloides (Linnaeus, 1764) | Russia, Dagestan, near Makhachkala 42°59'59.6"N 47°13'33.0"E, VI.2015 |
2 | 24 + XY | (TTAGG)n | AA* |
Nemoleontinae | |||||
Distoleon tetragrammicus (Fabricius, 1798) | Russia, Dagestan, near Makhachkala 43°00'28.7"N 47°14'51.3"E, VII.2015 |
2 | 14 + XY | ?** | AA + X |
Acanthaclisinae | |||||
Acanthaclisis occitanica (Villers, 1789) | Russia, Dagestan, near Makhachkala 43°00'28.7"N 47°14'51.3"E, VII.2015 |
2 | 16 + XY | (TTAGG)n | AA + X |
Ascalaphidae | |||||
Ascalaphinae | |||||
Libelloides macaronius (Scopoli, 1763) | Russia, Dagestan, near Gelinbatan village 41°56"50"N, 48°10"2"E, VII.2015 |
1 | -*** | (TTAGG)n | -*** |
Chromosome preparations were obtained from male gonads. Testes were dissected in a drop of 45% acetic acid and squashed. The coverslips were removed using dry ice. Prior to FISH treatment, the preparations were examined using phase contrast microscopy.
FISH with (TTAGG)n and 18S rDNA probes was applied as previously reported for some other insects (
Chromosomes were mounted in antifade medium (ProLong Gold antifade reagent with DAPI; Invitrogen) and covered with a glass coverslip. Chromosome slides were analyzed under a Leica DM 6000 B microscope. Images were taken with a Leica DFC 345 FX camera using Leica Application Suite 3.7 software with an Image Overlay module.
In male P. libelluloides, we found 12 autosomal bivalents and X and Y univalent chromosomes (Fig.
Different stages of spermatogenesis in antlion species Palpares libelluloides (1–3), Acanthaclisis occitanica (4), Distoleon tetragrammicus (5–8) and owlfly species Libelloides macaronius (9–10) after standard staining (1) and FISH with the 18S rDNA and telomeric (TTAGG)n probes (2–10). 1–3 MI, n = 12 + X + Y 4 diakinesis/MI transition, n = 8 + X + Y 5, 6 MI, n = 7 + X + Y 7 pachytene 8 spermatids 9 mitotic metaphase 10 spermatids. Asterisks mark sex chromosomes; red signals identify the (TTAGG)n - positive telomeres (arrowed); green signals identify rDNA clusters. Scale bar on Fig. 2 also applies to Figs 3–10.
In male A. occitanica, we found 8 bivalents of various sizes and the X and Y univalent chromosomes, suggesting 2n = 18 (16 + XY). This species is cytogenetically studied for the first time. Its karyotype includes one exceptionally large pair of bi-armed autosomes, another pair of large bi-armed autosomes and the two sex chromosomes, with the longer metacentric chromosome presumably being the X, while the other metacentric chromosome − the Y; the detailed morphology of other chromosomes remained unknown. The majority of bivalents and sex chromosomes displayed strong signals of the telomere probe. As for the heterogeneity for presence/absence and intensity of the (TTAGG)n signals, it could be explained by technical artifacts. Apart from a few scattered signals, the ribosomal probe detected a considerable accumulation of the 18S rDNA sequence on the second large pair of bi-armed autosomes and, additionally, on the putative X chromosome (Fig.
In male D. tetragrammicus, we found 7 autosomal bivalents and X and Y univalent chromosomes (Figs
In male L. macaronius, the karyotype remained unknown. However the (TTAGG)n -positive signals could be clearly observed in some cells including spermatids (Figs
As summarized recently (
In our previous paper (
The karyotype of A. occitanica differs by having both very large autosomal bivalent and sex chromosome (supposedly the X). To our knowledge, this karyotype structure was never reported for the Myrmeleontidae. However, it is worth noting that antlion karyotypes were almost exclusively illustrated with drawings in the past (as opposed to photos) with no significant details of the chromosome structure and size reported.
The data obtained in the present study demonstrate for the first time the presence of the insect-type telomeric repeat (TTAGG)n in antlions. We have reliably shown that this motif is characteristic of P. libelluloides and A. occitanica. The third examined antlion species, D. tetragrammicus, in which only rare TTAGG-positive signals of uncertain location were detected at best, most likely does not have the canonical (TTAGG)n insect telomeric motif. However, the TTAGG sequence could actually be present in the telomeres but in very low copy numbers, making it difficult to detect this sequence by FISH. Consequently, we consider the data on D. tetragrammicus as preliminary and therefore deserving further clarification. We also showed that the (TTAGG)n repeat was present in telomeres of L. macaronius (Ascalaphidae). Earlier, this telomeric motif was recorded for another owlfly species, Protidricerus japonicas, by
At present, the only other neuropteran species with known telomere structure is Chrysoperla carneas. lato belonging to the large worldwide family Chrysopidae (green lacewings). Based on the Southern hybridization results,
Ribosomal gene markers have provided useful information regarding chromosome evolution in different groups of insects. In some groups, the number and chromosomal localization of rDNA clusters, usually located in the nucleolus organizing regions (NORs), serve as additional markers to characterize species and higher taxa (
Our study contributes to the current knowledge of cytogenetics of the neuropteran family Myrmeleontidae. The principal outcomes of this study are: (1) the discovery of one or two major rDNA clusters per haploid karyotype; the clusters are located either only on a pair of autosomes in a particular species or, additionally, on one of the sex chromosomes in another two studied species and (2) the discovery of the insect-type (TTAGG)n telomeric sequence at least in two of the three studied species. Because the (TTAGG)n sequence is likewise found in the two studied owlfly species, we suggest that this telomere structure was inherent in the last common ancestor of the phylogenetic lineage Myrmeleontidae + Ascalaphidae.
The complete financial support for this study was provided by the grant from the Russian Science Foundation no. 14-14-00541 to the Zoological Institute of the Russian Academy of Sciences. We thank Dr. E.V. Ilyina (Dagestan Scientific Center RAS, Makhachkala) for help in collection and Dr. V.A. Krivokhatsky (Zoological Institute RAS, St. Petersburg) for help in identification of species, studied in the present paper.