Research Article |
Corresponding author: Olesya Buleu ( buleu.olesya@mail.ru ) Academic editor: María José Bressa
© 2020 Olesya Buleu, Ilyas Jetybayev, Mohsen Mofidi-Neyestanak, Alexander Bugrov.
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:
Buleu O, Jetybayev I, Mofidi-Neyestanak M, Bugrov A (2020) Karyotypes diversity in some Iranian Pamphagidae grasshoppers (Orthoptera, Acridoidea, Pamphagidae): new insights on the evolution of the neo-XY sex chromosomes. Comparative Cytogenetics 14(4): 549-566. https://doi.org/10.3897/compcytogen.v14.i4.53688
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For the first time, cytogenetic features of grasshoppers from Iran have been studied. In this paper we conducted a comparative cytogenetic analysis of six species from the family Pamphagidae. The species studied belong to subfamilies Thrinchinae Stål, 1876 (Eremopeza bicoloripes (Moritz, 1928), E. saussurei (Uvarov, 1918)) and Pamphaginae (Saxetania paramonovi (Dirsh, 1927), Tropidauchen escalerai Bolívar, 1912, Tropidauchen sp., and Paranothrotes citimus Mistshenko, 1951). We report information about the chromosome number and morphology, C-banding patterns, and localization of ribosomal DNA clusters and telomeric (TTAGG)n repeats. Among these species, only S. paramonovi had an ancestral Pamphagidae karyotype (2n=18+X0♂; FN=19♂). The karyotypes of the remaining species differed from the ancestral karyotypes. The karyotypes of E. bicoloripes and E. saussurei, despite having the same chromosome number (2n=18+X0♂) had certain biarmed chromosomes (FN=20♂ and FN=34♂ respectively). The karyotypes of T. escalerai and Tropidauchen sp. consisted of eight pairs of acrocentric autosomes, one submetacentric neo-X chromosome and one acrocentric neo-Y chromosome in males (2n=16+neo-X neo-Y♂). The karyotype of P. citimus consisted of seven pairs of acrocentric autosomes, submetacentric the neo-X1 and neo-Y and acrocentric the neo-X2 chromosomes (2n=14+neo-X1 neo-X2 neo-Y♂). Comparative analysis of the localization and size of C-positive regions, the position of ribosomal clusters and the telomeric DNA motif in the chromosomes of the species studied, revealed early unknown features of their karyotype evolution. The data obtained has allowed us to hypothesize that the origin and early phase of evolution of the neo-Xneo-Y♂ sex chromosome in the subfamily Pamphaginae, are linked to the Iranian highlands.
C-banding, FISH, karyotypes, neo-sex chromosomes, Pamphagidae grasshoppers, ribosomal DNA repeats, telomeric repeat (TTAGG)n
Among Pamphagidae grasshoppers, over 300 species inhabit the desert, semidesert and mountainous landscapes of the Palaearctic Region. All of them belong to the subfamilies Thrinchinae and Pamphaginae (
Moreover, the neo-Y chromosomes found in previously studied Thrinchinae (Asiotmethis and Glyphotmethis genera) and Pamphaginae (Nocarodeini tribe) species varies in size and content of constitutive heterochromatin. In the karyotypes of some Glyphotmethis and Asiotmethis species, the neo-Y chromosome is similar in size to its homologous XR-arm of the neo-X chromosome. But unlike the XR-arm of the neo-X chromosome the neo-Y chromosome showed two small interstitial C-bands near the pericentromeric region. In the karyotypes of all Nocarodeini species, the neo-Y chromosome is smaller than the XR-arm of the neo-X chromosome. But unlike the XR-arm of the neo-X chromosome the neo-Y chromosome showed a large pericentromeric C-band and two or three large subproximal interstitial C-bands located close to each other (Bugrov and Grozeva 1998;
Analysis of the geographical distribution of Pamphagidae species with neo-sex chromosomes allowed the assumption that the origin of this type of sex chromosome system may occur in the Western Asian region (
The present study reports the results of our comparative analysis of the karyotypes, C-banding patterns, distribution of clusters of telomeric (TTAGG)n repeats and ribosomal DNA (rDNA) in the chromosomes of the species studied. We hope that this study will provide the motivation for further cytogenetic study of Iranian grasshoppers.
Males of the Eremopeza saussurei (Uvarov, 1918), E. bicoloripes (Moritz, 1928) belonging to the Thrinchinae, and Saxetania paramonovi (Dirsh, 1927), Tropidauchen escalerai Bolívar, 1912, Tropidauchen sp. (Tropidauchenini), Paranothrotes citimus Mistshenko, 1951 (Nocarodeini) from the subfamily Pamphaginae were collected in the early summer season (1st to 12th June, 2018) in mountain and semidesert landscapes in Iran (Table
List of species, collection locations and number of specimens of the studied Pamphagidae species.
Taxa | Species | Location | Number of males |
---|---|---|---|
Thrinchinae Thrinchini | Eremopeza saussurei (Uvarov, 1918) | Iran, Fars Prov., Zagros Range, 1433 m. asl. 29°25’54.9"N, 052°46’20.0"E | 7 |
Eremopeza bicoloripes (Moritz, 1928) | Iran, Khorosan-e Razavi Prov., 60 km, N. of Mashhad, Ferizi vil. vicinities, ~1800 m. asl. | 5 | |
Pamphaginae Nocarodeini | Paranothrotes citimus Mistshenko, 1951 | Iran, Qazvin Prov., Alborz Range, Qazvin town vicinities, 2380 m. asl. 36°7’29.0"N, 50°40’ 25"E | 1 |
Pamphaginae Tropidauchenini | Saxetania paramonovi (Dirsh, 1927) | Iran, Khorosan-e Razavi Prov., 60 km, N. of Mashhad, Ferizi vil. vicinities, ~1800 m. asl. | 10 |
Tropidauchen escalerai Bolívar, 1912 | Iran, Fars Prov., Zagros Range, Estahban, Runiz town vicinities, 1800 m. asl. | 1 | |
Tropidauchen sp. | Iran, Fars Prov., Zagros Range, 2800 – 3200 m. asl. 30°23’10.1"N, 51°55’35.2"E | 7 |
The 0.1% colchicine solution was injected into the abdomens of collected males. After 1.5–2 hours, their testes were dissected and placed into a 0.9% solution of sodium citrate for 20 minutes. Then the testes were fixed in 3:1 (ethanol : glacial acetic acid) for 15 minutes. Thereafter, fixed testes were stored in 70% ethanol in a refrigerator at 4 °C until used. Air-dried chromosome preparations were made by squashing testis follicles in 45% acetic acid and subsequently freezing them in dry ice.
The constitutive heterochromatin was identified by C-banding, using the technique described by Sumner (1972) with minor modifications. Slides were treated with 0.2 N HCl for 15–20 minutes at room temperature then incubated in a saturated solution of Ba(OH)2 at 61°C for three to five minutes, rinsed in tap water and incubated in 2×SSC at 61 °C for 60 minutes. After washing in distilled water, the slides were stained with 2% Giemsa solution on Sorensen’s phosphate buffer for 30 to 60 minutes.
Fluorescence in situ hybridization (FISH) with telomeric (TTAGG)n DNA probes and rDNA genes on meiotic chromosomes was carried out according to the protocol by Pinkel (1986) with modifications as described in previous studies (
Microscopic analysis was performed at the Centre for Microscopy of Biological Objects of SB RAS (Novosibirsk, Russia). Chromosomes were studied with an AxioImager M1 (Zeiss, Germany) fluorescence microscope equipped with filter sets #49, #46HE, #43HE and a ProgRes MF (MetaSystems GmbH, Germany) CCD camera. The ISIS5 software package was used for image capture and analysis.
The karyotype of E. bicoloripes consisted of nine pairs of acrocentric autosomes and one subacrocentric X chromosome in males (2n♂=19; 18AA+X) (Fig.
C-banded metaphase I of Eremopeza bicoloripes. Arrow indicate the telomeric C-bands in M6 autosome bivalent. Scale bar: 10 µm.
Distinct pericentromeric C-bands were revealed in all chromosomes of the complement (Fig.
Telomeric DNA repeats were hybridized at the terminal region of all chromosomes (Fig.
FISH with telomeric (TTAGG)n probe (red) in male meiotic chromosomes of Eremopeza bicoloripes A metaphase I B metaphase II. Chromosomes were counterstained with DAPI (blue). Arrows indicate the telomere hybridization signals at the terminal and pericentromeric regions of X chromosome
The clusters of rDNA genes were located at pericentromeric regions in most autosome bivalents, except the L4 and S9 autosome bivalents and the X chromosome (Fig.
The karyotype of E. saussurei consisted of nine pairs of autosomes and the X chromosome in males (2n♂=19; 18AA+X). Four autosome bivalents were large (L1–L4), three were medium (M5–M7) and two were small (S8–S9). The X chromosome was of medium-sized. All large autosome bivalents, the M5, the M7, and the X chromosome were subacrocentric. The M6 autosome bivalent was submetacentric. Small (S8–S9) autosome bivalents were acrocentric (Fig.
C-banded diakinesis of Eremopeza saussurei. Arrow indicates the telomeric C-band in L2 autosome bivalent. Scale bar: 10 µm.
Distinct pericentromeric C-bands were revealed in all chromosomes of the complement (Fig.
Telomeric DNA repeats were observed at terminal regions of all chromosomes (Fig.
FISH with telomeric (TTAGG)n probe (red) (A) and rDNA genes (green) (B) in cell at diakinesis of Eremopeza saussurei. Arrows indicate the telomere hybridization signals at pericentromeric regions of L2 and L3 bivalents and X chromosome. Chromosomes were counterstained with DAPI (blue). Scale bar: 10 µm.
The karyotype of S. paramonovi consisted of nine pairs of acrocentric autosomes and an acrocentric X chromosome in males (2n♂=19; 18AA+X). The male meiotic karyotype was represented by four large (L1–L4), three medium (M5–M7) and two small (S8–S9) autosome bivalents. The X chromosome was of medium-sized (Fig.
Saxetania paramonovi A C-banded metaphase I B FISH with telomeric (TTAGG)n probe (red) and rDNA genes (green) in cell at diakinesis. Chromosomes were counterstained with DAPI (blue) (B). Scale bar: 10 µm.
Pericentromeric C-bands were revealed in all autosome bivalents and the X chromosome (Fig.
Telomeric DNA repeats were only observed in the terminal regions of all chromosomes (Fig.
The karyotype of T. escalerai consisted of 18 acrocentric chromosomes (2n=16+neo-Xneo-Y♂): four large (L1–L4), two medium (M5, M6) and two small sized (S7, S8) autosome bivalents (Fig.
Tropidauchen escalerai A C-banded metaphase I. The inset in the right upper corner shows the neo-X chromosome in meiotic metaphase II. Arrows indicate the C-bands in the XR-arm of the neo-X and neo-Y chromosomes B FISH with telomeric (TTAGG)n probe (red) and rDNA genes (green) in cell at metaphase I. Chromosomes were counterstained with DAPI (blue) (B). Scale bar: 10 µm.
Distinct pericentromeric C-bands were found in all autosome bivalents and in the neo-X chromosome (Fig.
Telomeric DNA repeats were located only at terminal regions of all chromosomes (Fig.
The karyotype of the Tropidauchen sp. consisted of 18 chromosomes (2n=16+neo-Xneo-Y♂): three large (L1, L2, L3), two medium (M5, M6) and two small (S7, S8) acrocentric autosome bivalents (Fig.
Tropidauchen sp. A C-banded metaphase I. The inset in the lower left corner shows the L4 chromosome in meiotic metaphase II. Arrows indicate the interstitial C-band in the S8 autosome bivalent; B FISH with rDNA (green) and telomeric (TTAGG)n (red) probes in cell at metaphase I. Arrows indicate the cluster of rDNA genes in the S8 autosome bivalent. Chromosomes were counterstained with DAPI (blue) (B). Scale bar: 10 µm.
Pericentromeric C-bands were detected in all chromosomes (Fig.
Telomeric DNA repeats in Tropidauchen sp. were localized only at terminal regions of the all autosomes (Fig.
The karyotype of P. citimus consisted of 14 autosomes and three neo-sex chromosomes (2n=14+neo-X1neo-X2neo-Y♂). The karyotype structure was represented by two large (L1–L2), four medium (M3–M6) and one small (S7) acrocentric autosome bivalents and three neo-sex chromosomes (Fig.
Paranothrotes citimus A C-banded chromosome in metaphase I B FISH with rDNA (green) and telomeric (TTAGG)n (red) probes in cell at metaphase I. The inset in the right upper corner shows the L2 chromosome in meiotic metaphase II. Chromosomes were counterstained with DAPI (blue) (B). Scale bar: 10 µm.
Distinct pericentromeric C-positive blocks were observed in all chromosomes. The YL-arm of the neo-Y chromosome was completely C-positive (Fig.
FISH signals of telomeric DNA probe were observed in the terminal regions of all chromosomes (Fig.
The chromosome number, morphology, sex chromosome system, distribution of heterochromatin (C-bands) and location of rDNA and tDNA genes in the studied Pamphagidae species presented in Table
The chromosome number, chromosomal morphology, sex chromosome system, distribution of constitutive heterochromatin (C-bands) and location of rDNA and tDNA genes in the studied Pamphagidae species.
Taxa | 2n♂; FN | SD | KS | CM | C-bands | rDNA | tDNA |
Thrinchinae, Thrinchini | |||||||
Eremopeza bicoloripes | 19; 20 | X0 | L1–L4, M5-M7, S8–S9, X | all a | p all; | p1,2,3*,4,5, | d all |
XX | X sm | t 1,4, | 6,7*,8 | dpd X | |||
5,6,9 | |||||||
Eremopeza saussurei | 19; 34 | X0 | L1–L4, M5–M7, S8–S9, X | 1-4,5,7, X sa; | p all; | p1-4,5,6,7,X | d all |
XX | 6 sm; | t 2 | dpd 2,3,X | ||||
8,9 a | |||||||
Pamphaginae, Nocarodeini | |||||||
Paranothrotes citimus | 14;18 | neo-X1X2Y | L1–L2, M3–M6 S7 | all a | p all; | p5; d2; X1L | d all |
neo-X1X1X2X2 | neo-X1 | neo-X1 sm | t X1L | ||||
neo-X2 | neo-X2 a | ||||||
neo-Y | neo-Y sm | ||||||
Pamphaginae, Tropidauchenini | |||||||
Saxetania paramonovi | 19;19 | X0 | L1–L4, M5-M7, S8–S9 X | all a | p all; | p2,4;i3 | d all |
XX | t 7,9,8 | ||||||
Tropidauchen escalerai | 18;19 | neo-XY | L1–L4, M5,M6 S7, S8 | all a | p all; | p2i2; p3;i6 | d all |
neo-XX | neo-X | neo-X m | i XR, neo-Y; | ||||
neo-Y | neo-Y a | t 1,2,4, neo-X | |||||
Tropidauchen sp. | 18;19 | neo-XY | L1–L4, M5,M6 S7, S8 | 1-3,5,6,7,8 a, | p all; | ip2, id2; i7; 8* | d all |
neo-XX | neo-X | 4 sa | i 5,8; | dpd neo-X | |||
neo-Y | neo-X sm neo-Y a | t 6,7 |
A comparative cytogenetic analysis of Iranian Pamphagidae provides new information about the karyotype evolution in this group of grasshoppers. Two species from the Eremopeza Saussure, 1888 genus (Thrinchinae) have the fundamental chromosome number of the Pamphagidae karyotype (2n=19♂). However, unlike the standard Pamphagidae karyotype, in which all chromosomes are acrocentric, in Eremopeza subacrocentric chromosomes were found. Early, biarmed chromosomes were found in Eremopeza festiva (Saussure, 1888) from Armenia (
The discovery of some Pamphagidae species with neo-sex chromosome systems supports our hypothesis that the origin of this unusual sex chromosome system is the West Asian region (
The fluorescence in situ hybridization (FISH) with telomeric probe and rDNA genes is a very useful tool for comparative analysis of karyotype in Orthoptera insects (
Saxetania cultricollis: FISH with telomeric repeats (red) in cells at diakinesis. Chromosomes were counterstained with DAPI (blue). Scale bar: 10 µm.
The distribution of rDNA clusters in the chromosomes of the Saxetania and Tropidauchen species was similar to the distribution of rDNA in the chromosomes of previously studied Pamphaginae species (
The neo sex chromosome systems were observed in two subfamilies (Thrinchinae and Pamphaginae) of the Pamphagidae grasshoppers. Based on the analysis of the chromosome features (karyotype, C-banding, telomeric (TTAGG)n and rDNA genes) we see that the neo-sex chromosome system in the genera Saxetania and Tropidauchen in the subfamily Pamphaginae is at a similar level of chromosome evolution to the neo-sex chromosomes in the genera Glyphotmethis and Asiotmethis of the subfamily Thrinchinae (
Analysis of the geographic distribution of Pamphaginae grasshoppers with different types of the sex chromosome systems (
This work was partly supported by research grants from the Russian Foundation for Basic Research #15-04-04816-a, and the Federal Fundamental Scientific Research Program for 2013–2020 (project no. AAAA‐A16‐116121410121‐7).
The authors are grateful to Aleksejs Černihs for comprehensive assistance during field studies in Iran. We also would like to thank reviewers Prof. Elżbieta Warchałowska-Śliwa and Prof. Elio Rodrigo Castillo and subject editor Dr. María José Bressa for their valuable comments and helpful suggestions for the manuscript.