Research Article |
Corresponding author: Svetlana V. Pavlova ( swpavlova@mail.ru ) Academic editor: Jan Zima
© 2018 Svetlana V. Pavlova, Larisa S. Biltueva, Svetlana A. Romanenko, Natalya A. Lemskaya, Anton V. Shchinov, Alexei V. Abramov, Viatcheslav V. Rozhnov.
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:
Pavlova SV, Biltueva LS, Romanenko SA, Lemskaya NA, Shchinov AV, Abramov AV, Rozhnov VV (2018) First cytogenetic analysis of lesser gymnures (Mammalia, Galericidae, Hylomys) from Vietnam. Comparative Cytogenetics 12(3): 361-372. https://doi.org/10.3897/CompCytogen.v12i3.27207
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Gymnures are an ancient group of small insectivorous mammals and are characterized by a controversial taxonomic status and the lack of a description of karyotypes for certain species. In this study, conventional cytogenetic techniques (Giemsa, CBG- and GTG-banding, Ag-NOR), CMA3-DAPI staining, and fluorescent in situ hybridization (FISH) with telomeric DNA probes were used to examine for the first time the karyotypes of lesser gymnures of group Hylomys suillus Müller, 1840 from northern and southern Vietnam. All studied specimens had karyotypes with 2n=48, NFa=64. C-positive heterochromatic blocks existed in centromeric regions of 7 bi-armed autosomes and the submetacentric X chromosome. The Y chromosome is a C-positive and dot-like. The nucleolus organizer regions resided terminally on the short arms of 2 small bi-armed pairs. Positive signals at the telomeres of all chromosomes were revealed by FISH. CMA3-positive blocks were localized on the telomeric and pericentric regions of most bi-armed and acrocentric chromosomes. Despite the large genetic distances between Hylomys Müller, 1840, lesser gymnures from H. suillus-group from northern and southern Vietnam have similar karyotypic characteristics.
cell culture, cryoconservation, FISH, insectivorous mammals, karyotype, telomeric sequence
The order Erinaceomorpha is a diverse group of small insectivorous mammals that are widely distributed throughout Africa, Europe, and Asia. According to most current taxonomic systems, this order contains the single family Erinaceidae with 2 subfamilies: Erinaceinae (hedgehogs) and Galericinae (gymnures) (
The family Galericidae comprises 6 recent genera, with 6–12 species in total (
Lesser gymnures Hylomys spp. inhabit the Greater Sunda Islands, Indochina, and southern China (
In this report, we characterized for the first time the karyotypes of lesser gymnures of Hylomys suillus-group from northern and southern Vietnam using a set of cytogenetic tools. Prior to comprehensive taxonomic revision of the group H. suillus, we use the name Hylomys suillus microtinus for the gymnures from northern Vietnam and Hylomys sp. for those from southern Vietnam (see
Gymnures were collected during biodiversity surveys carried out by the Joint Vietnam-Russian Tropical Research and Technological Centre in 2013–2014. Voucher specimens are deposited in the Zoological Museum of Moscow State University (
Primary fibroblast cell cultures that were derived from tail biopsies of individuals from northern Vietnam were established and subsequently deposited to the cell banks of 2 cytogenetic laboratories (in Moscow and Novosibirsk, Russian Academy of Sciences). The cell cultures were established in parallel to prevent the loss of valuable material. As a result, the cell culture from a female (
Each lab modified the standard cell culture protocol (
Metaphase chromosome preparations from primary fibroblast cultures were made following the standard technique (
The standard field procedure for bone marrow cultures was used to obtain chromosome preparations for a female (
Air-dried chromosome spreads of all specimens were stained conventionally with 2% Giemsa for 4-5 minutes and then submitted to differential staining.
To determine the location of heterochromatin, C-banding was performed per the standard technique (
The fluorochromes chromomycin A3 (CMA3) and 4,6-diamidino-2-phenylindole (DAPI) were applied to identify GC- and AT-rich heterochromatic regions, respectively (Lemskaya et al. unpubl.).
The standard trypsin-Giemsa staining technique (
Nuclear organizer regions (NORs) were detected by silver nitrate staining following
To detect telomeric repeats, the G-banded metaphase chromosomes of a female from northern Vietnam was hybridized in situ with a fluorescein-conjugated peptide nucleic acid (PNA) probe from the Telomere PNA FISH Kit/FITC (K5325 from Dako, Glostrup, Denmark) following the manufacturer’s instructions.
Images were captured with a ProgRes CCD (Jenoptic) camera mounted on an Axioscope 2 plus (Zeiss) microscope with filter sets for DAPI, FITC, and rhodamine, using VideoTesT-FISH 2.0 and VideoTesT-Karyo 3.1. (VideoTesT, Saint Petersburg, Russia) software. A Leica DFC-295 CCD camera mounted on a DM1000 (Leica) or Metasystems CCD (Zeiss) camera mounted on an Axioscope 2 (Zeiss) microscope were used to capture all other non-fluorescence images using a Metasystems Ikaros ver.5.3 and Leica Application ver.3.2 softwares, respectively.
The diploid chromosome number of the male and female karyotypes was 2n=48, NFa=64 (Fig.
A female karyotype of the lesser gymnure H. suillus microtinus from northern Vietnam: conventional staining (a) and C-banding (b). 2n=48, NFa=64. XX – the female sex chromosomes. C-banded sex chromosomes of a male (XY) are given in a frame.
G-banded female (a) and male (b) karyotypes of the lesser gymnure H. suillus microtinus from northern Vietnam.
C-heterochromatic blocks were revealed in the pericentric regions of 7 bi-armed autosomes (№ 2, 4–9) and the X chromosomes. The autosome 6 has the largest C-block (Fig.
By silver nitrate staining the terminal localization of NORs was revealed on p-arms of 2 pairs of small autosomes in the female karyotype (Fig.
CMA3-positive blocks were seen in the telomeric and pericentric regions of most bi-armed and acrocentric chromosomes (Fig.
The hybridization with the telomeric DNA (telDNA) probe revealed distinct signals at the telomeres of all chromosomes in the female karyotype (Fig.
Ag-stained female karyotype of the lesser gymnure H. suillus microtinus from northern Vietnam. Black arrows indicate the localizations of NORs. Bar = 10 µm.
CMA3-DAPI stained male karyotype of the lesser gymnure H. suillus microtinus from northern Vietnam: inverted DAPI(a) and CMA3-DAPI staining (b). X and Y indicate the sex chromosomes. Bar = 10 µm.
The karyotype of the female had the same number of chromosomes and chromosome arms as the karyotypes above: 2n=48, NFa=64 (Fig.
Karyotypes of several hundred mammalian species have been described in the several decades since the development of various methods for characterizing the chromosome sets (
The karyotypes of lesser gymnures from northern and southern Vietnam that we studied had 2n=48 and NFa=64. These data were first reported for the genus Hylomys and H. suillus-group. The karyotype structure and chromosome number differed from those of 3 karyotyped gymnure species: P. truei, N. sinensis, and N. hainanensis (
The sex chromosomes of all studied gymnure species, including Hylomys, had similar morphologies – the X chromosome is a mid-sized submetacentric, and the Y chromosome is the smallest acrocentric. However, notably, the Y chromosome in N. hainanensis is a small metacentric.
Based on an unpublished mtDNA analysis (Bannikova et al. in prep.), our specimens from northern Vietnam (
The diploid number in all spiny hedgehogs (Erinaceidae) studied so far appears to be 2n=48 (
In conclusion, here we have provided the first karyotype description of 2 potential species – Hylomys suillus microtinus and Hylomys sp.–which are distributed throughout the northern and southern parts of Vietnam, respectively. A detailed characterization of the karyotype of Hylomys sp. from southern Vietnam by different chromosome staining is needed to provide a comprehensive comparison between these 2 forms.
We are grateful to the administration of the Xuan Son and Chu Yang Sin national parks for providing us with an opportunity to carry out field surveys. We thank A.N. Kuznetsov and Nguyen Dang Hoi (Joint Vietnam-Russian Tropical Research and Technological Centre, Hanoi) who made considerable efforts in preparing for the field works. We would like to express our sincere thanks to Drs Y.M. Kowalskaya, M.N. Erofeeva, I.V. Lukyanova, M.V. Rutovskaya, and V.M. Aniskin (all from the A.N. Severtsov IEE, Moscow) for their assistance in this study. Especial thanks to Dr. Eugeny Yu. Krysanov (A.N. Severtsov IEE, Moscow) who provided a telomeric probe, and gave us the permission for using the lab equipment for FISH analysis. Also we are very thankful to Dr. Anna A. Bannikova (Moscow State University) for her helpful advices on the manuscript. The Blue Pencil Science Company corrected English.
This study was funded in part by the Presidium of the Russian Academy of Sciences, Program № 41 «Biodiversity of natural systems and biological resources of Russia» and RSF № 16-14-10009.