Research Article |
Corresponding author: Veronika Golygina ( nika@bionet.nsc.ru ) Academic editor: Igor Sharakhov
© 2015 Larisa Gunderina, Veronika Golygina, Andrey Broshkov.
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:
Gunderina L, Golygina V, Broshkov A (2015) Chromosomal organization of the ribosomal RNA genes in the genus Chironomus (Diptera, Chironomidae). Comparative Cytogenetics 9(2): 201-220. https://doi.org/10.3897/CompCytogen.v9i2.9055
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Chromosomal localization of ribosomal RNA coding genes has been studied by using FISH (fluorescence in situ hybridization) in 21 species from the genus Chironomus Meigen, 1803. Analysis of the data has shown intra- and interspecific variation in number and location of 5.8S rDNA hybridization sites in 17 species from the subgenus Chironomus and 4 species from the subgenus Camptochironomus Kieffer, 1914. In the majority of studied species the location of rDNA sites coincided with the sites where active NORs (nucleolus organizer regions) were found. The number of hybridization sites in karyotypes of studied chironomids varied from 1 to 6. More than half of the species possessed only one NOR (12 out of 21). Two rDNA hybridization sites were found in karyotypes of five species, three – in two species, and five and six sites – in one species each. NORs were found in all chromosomal arms of species from the subgenus Chironomus with one of them always located on arm G. On the other hand, no hybridization sites were found on arm G in four studied species from the subgenus Camptochironomus. Two species from the subgenus Chironomus – Ch. balatonicus Devai, Wuelker & Scholl, 1983 and Ch. “annularius” sensu Strenzke, 1959 – showed intraspecific variability in the number of hybridization signals. Possible mechanisms of origin of variability in number and location of rRNA genes in the karyotypes of species from the genus Chironomus are discussed.
Chironomus , 5.8S rDNA, ribosomal gene localization, polytene chromosomes, NOR, gene mapping, FISH
The ribosomal RNA genes in eukaryotic genomes are multiply repeated and form the family of ribosomal genes. They are arranged in clusters comprising hundreds of tandemly repeated units, each consisting of three genes – 18S, 5.8S, and 28S rRNA – separated by transcribed and untranscribed intergenic spacers (
The chromosome evolution of the species belonging to the genus Chironomus Meigen, 1803 has been studied in much more detail as compared with the other insect groups owing to the presence of polytene chromosomes with a distinct species-specific banding pattern in the nuclei of their salivary glands (
On the other hand, the information about the number and location of NORs in the chromosomes of Chironomus species is mainly based on a phase contrast analyses of acetorcein-stained chromosomes (
The goal of this work was to study the chromosomal localization of the rRNA locus in the genus Chironomus species by means of FISH. The DNA sequences of chironomid species from the rRNA locus carrying 5.8S rRNA gene (5.8S rDNA) and the internal transcribed spacer (ITS-1) separating 18S and 5.8S rRNA genes were selected as the probes.
The IV instar larvae of 21 Chironomus species belonging to the subgenera Chironomus and Camptochironomus Kieffer, 1914 sampled in aquatic bodies of the Novosibirsk region, Russia, were examined. The larvae of North-American species C. dilutus Shobanov, Kiknadze & Butler, 1999 were obtained from the laboratory culture maintained at the Institute of Biology of Inland Waters, Russian Academy of Sciences (Borok, Yaroslavl region, Russia). Seven examined species of the subgenus Chironomus belong to the group of Ch. plumosus sibling species, namely, Ch. agilis Schobanov & Djomin, 1988, Ch. balatonicus Devai, Wuelker & Scholl, 1983, Ch. borokensis Kerkis, Filippova, Shobanov, Gunderina & Kiknadze, 1988, Ch. entis Schobanov, 1989, Ch. muratensis Ryser, Scholl & Wuelker, 1983, Ch. nudiventris Ryser, Scholl & Wuelker, 1983, and Ch. plumosus (Linnaeus), 1758. These species as well as Ch. “annularius” sensu Strenzke, 1959, Ch. riparius Meigen, 1804, Ch. cingulatus Meigen, 1830, Ch. nuditarsis Keyl, 1961, and Ch. sororius Wuelker, 1973 belong to the «thummi» cytocomplex, characteristic of which is the arm combination AB CD EF G in the chromosomes of their karyotype. Ch. dorsalis Meigen, 1818, Ch. luridus Strenzke, 1959, Ch. melanescens Keyl, 1961, and Ch. pseudothummi Strenzke, 1959 (arm combination, AE BF CD G) belong to the pseudothummi cytocomplex and Ch. lacunarius Wuelker, 1973 (arm combination, AD BC EF G), to the lacunarius cytocomplex. The four species of the Camptochironomus subgenus – C. dilutus, C. pallidivittatus sensu Beermann, 1955, C. setivalva Shilova, 1957, and C. tentans Fabricius, 1805 – belong to the camptochironomus cytocomplex (arm combination, AB CF ED G).
The larvae were fixed with 96% ethanol (for further DNA extraction) or 3: 1 v/v of 96% ethanol and glacial acetic acid (for making preparations of salivary gland polytene chromosomes for FISH hybridization) and stored at –20 °C. Species were identified according to morphological characteristics of larvae and by cytogenetic analysis of banding patterns of polytene chromosomes from salivary glands (
Genomic DNA was isolated from individual larvae using a DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer’s protocol. DNA probes were produced by polymerase chain reaction (PCR) with the primers 5’-GTAACAAGGTTTCCGTAGG-3’ (chir5F) and 5’-CGACACTCAACCATATGTACC-3’ (chir5R) (
Species | DNA probe | GenBank accession number | |
---|---|---|---|
1 | Ch. agilis | ITS-1 + 5.8S_agi | GU053584 |
2 | Ch. “annularius” | ITS-1 + 5.8S_ann | HQ656600 |
3 | Ch. balatonicus | ITS-1 + 5.8S_bal | GU053586 |
4 | C. dilutus | ITS-1 + 5.8S_dil | KP985232 |
5 | Ch. dorsalis | ITS-1 + 5.8S_dor | GU053590 |
6 | Ch. muratensis | ITS-1 + 5.8S_mur | GU053605 |
7 | C. pallidivittatus | ITS-1 + 5.8S_pal | KP985231 |
8 | Ch. plumosus | ITS-1 + 5.8S_plu | GU053597 |
9 | Ch. riparius | ITS-1 + 5.8S_rip | GU053603 |
10 | C. setivalva | ITS-1 + 5.8S_set | - |
11 | C. tentans | ITS-1 + 5.8S_ten | KP985230 |
For FISH, the polytene chromosomes were prepared from the larvae fixed with 3 : 1 v/v of 96% ethanol and glacial acetic acid according to the following procedure. A larva was placed into 70% ethanol to extract its salivary glands and transfer them onto a glass slide into a drop of 45% acetic acid. The cells were separated from secretion by removing it from the glass, gently covered with a cover glass, and squashed, removing excess acid with filter paper. The ready preparation was placed for 10–15 min onto a metal table cooled with liquid nitrogen to remove the cover glass; the slide was then kept for 5 min at a room temperature, 5 min in 70% ethanol, and air-dried for 1 week.
FISH was conducted according to the following protocol. The preparations were air-dried for one week. They were then incubated with RNase A (100 mg/ml in 2× SSC) for 1 h at 37 °C, washed at a room temperature for 5 min with 2× SSC, dehydrated with alcohols (70, 90, and 96% ethanol, 5 min in each), and air-dried for 10 min. Then the slide was incubated with 0.02% pepsin in 10 mM HCl for 6 min at 37 °C, washed with a series of phosphate buffers (5 min in PBS, 5 min in PBS with 50 mM MgCl2, 10 min in PBS with 50 mM MgCl2 and 1% formaldehyde, and again in PBS and PBS with 50 mM MgCl2, 5 min each) at a room temperature, and dehydrated in alcohols as described above. DNA probe (dissolved in 20 µl of 2× SSC with 50% deionized formamide for 1 h at 37 °C in a thermoshaker at 800 rpm) was applied to dry slide, covered with a cover glass, and incubated for 12–15 h at 37 °C in a humid chamber. The slides were then washed in a shaker (100 rpm, 37 °C) two times for 10 min in 2× SSC with 50% deionized formamide and 0.1% NP40, two times for 5 min in 2× SSC, two times for 5 min in 0.2× SSC, and one time in 4× SSC with 3% BSA; then antibody solution (20 µl) was added, the slide was covered with a cover glass and incubated in a humid chamber at 37 °C for 40 min. The DNA probes labeled with biotin or digoxigenin were detected using the antibodies labeled with the fluorochromes avidin-Alexa fluor®488 or Cy3, respectively. The antibodies were diluted with 4× SSC containing 3% BSA (1–2 µl antibodies per 100 µl reaction mixture) and dissolved for 1 h in a thermoshaker (800 rpm, 37 °C) in parallel with washings after the hybridization with DNA probes. On completion of the incubation with antibodies, the slides were washed in a shaker (110 rpm, 37 °C) three times, 5 min each, in 4× SSC with 0.1% NP40; dehydrated with alcohols; air-dried for 15 min; mounted in a DAPI-containing antifade; and covered with a cover glass. Homologous DNA probes (the karyotype and DNA probe belongs to the same species) and heterologous DNA probes (the karyotype and DNA probe belongs to different species) were used for FISH.
The slides were examined using the equipment of the Joint Access Center for Microscopy of Biological Objects with the Siberian Branch of the Russian Academy of Sciences, namely, AxioPlan2 Imaging microscope and Axio Cam HRc CCD camera with the help of Isis 4 software package (Zeiss, Germany).
Mapping of polytene chromosomes in arms A, C, D, E and F was done according to Keyl–Devai system (
Karyotypes of most Chironomus species studied in this work have four polytene chromosomes, which corresponds to the haploid chromosome set n = 4 (Figs
FISH of homologous (a) and heterologous (b–d) rDNA probes on the polytene chromosomes of C. tentans. aITS-1 + 5.8S_ten (Cy3) bITS-1 + 5.8S_pal (Cy3) cITS-1 + 5.8S_dil (Cy3) dITS-1 + 5.8S_set (Cy3). Letters designate chromosomal arms. Bar = 10 µm.
FISH of rDNA probes on polytene chromosomes of species from the subgenus Chironomus with one NOR in karyotype. a Ch. borokensis b Ch. dorsalis c Ch. entis d Ch. lacunarius e Ch. luridus f Ch. nuditarsis g Ch. melanescens h Ch. plumosus i Ch. sororius j Ch. riparius. Letters designate chromosomal arms. Bar = 10 µm.
FISH of rDNA probes on polytene chromosomes of species from the subgenus Chironomus with multiple localization of hybridization sites. a Ch. agilis b Ch. cingulatus c Ch. muratensis d Ch. nudiventris e Ch. pseudothummi fCh. “annularius”g Ch. balatonicus with one NOR h Ch. balatonicus with additional NOR in arm D. Letters designate chromosomal arms. Green arrows show sites of weak hybridizations signals. Bar = 10 µm.
FISH of rDNA probes on polytene chromosomes of species from the subgenus Camptochironomus. a–c C. tentans, where b and c specimens with heterozygous inversions in arm B that change the position of NOR in one of the homologues d C. dilutus; e C. pallidivittatus f C. setivalva. Letters designate chromosomal arms. Bar = 10 µm.
DNA-probe used for FISH analysis consists of two main components: gene coding 5,8S rRNA and internal transcribed spacer (ITS1). The percent of identity between ITS-1 sequences of the Chironomus species used for producing DNA probes is considerably lower as compared with the percent of identity between their 5.8S rDNA sequences (Table
The percent of identity between 5.8S rDNA nucleotide sequences (above) and ITS-1 (below) in Chironomus species.
Ch. agilis | Ch. balatonicus | Ch. muratensis | Ch. plumosus | Ch. “annularius” | Ch. riparius | Ch. dorsalis | C. dilutus | C. pallidivittatus | C. tentans | |
---|---|---|---|---|---|---|---|---|---|---|
Ch. agilis | 100 | 100 | 100 | 99 | 99 | 100 | 100 | 100 | 100 | |
Ch. balatonicus | 92 | 100 | 100 | 99 | 99 | 100 | 100 | 100 | 100 | |
Ch. muratensis | 94 | 95 | 100 | 99 | 99 | 100 | 100 | 100 | 100 | |
Ch. plumosus | 91 | 94 | 96 | 99 | 99 | 100 | 100 | 100 | 100 | |
Ch. “annularius” | 87 | 90 | 87 | 87 | 98 | 100 | 100 | 100 | 100 | |
Ch. riparius | 75 | 76 | 76 | 77 | 76 | 100 | 100 | 100 | 100 | |
Ch. dorsalis | 74 | 78 | 76 | 76 | 73 | 80 | 100 | 100 | 100 | |
C. dilutus | 76 | 79 | 77 | 78 | 79 | 73 | 73 | 100 | 100 | |
C. pallidivittatus | 77 | 79 | 78 | 78 | 79 | 73 | 73 | 96 | 100 | |
C. tentans | 77 | 80 | 79 | 78 | 78 | 74 | 73 | 95 | 98 |
The number of NORs in the studied Chironomus species is different (Figs
The number of chromosome pairs in karyotype, combinations of arms in chromosomes, and number and locations of nucleolus organizer regions (NORs) in Chironomus species.
Species | Number of chromosome pairs in karyotype | Arm combination in chromosomes | Number of NORs | NOR location | |
---|---|---|---|---|---|
Chromosome arm | Chromosome region | ||||
Subgenus Chironomus | |||||
1. Ch. agilis | 4 | AB CD EF G | 2 | G | 1a, 1bc |
2. Ch. balatonicus | 4 | –//– | 2 | G D |
1 18fg |
3. Ch. borokensis | 4 | –//– | 1 | G | 1 |
4. Ch. entis | 4 | –//– | 1 | G | 1 |
5. Ch. muratensis | 4 | –//– | 6 | B C D F G |
24i-j 16 2h, 2d 10c 1 |
6. Ch. plumosus | 4 | –//– | 1 | G | 1 |
7. Ch. nudiventris | 3 | AB CD GEF | 3 | G D |
1 2h, 2d |
8. Ch. “annularius” | 4 | AB CD EF G | 5 | A C E G |
3g 15c–17b 3a–4h, 9–10b not mapped |
9. Ch. riparius | 4 | –//– | 1 | G | 3 |
10. Ch. cingulatus | 4 | –//– | 2 | B G |
not mapped not mapped |
11. Ch. nuditarsis | 4 | –//– | 1 | G | not mapped |
12. Ch. sororius | 4 | –//– | 1 | G | not mapped |
13. Ch. lacunarius | 4 | AD BC EF G | 1 | G | not mapped |
14. Ch. dorsalis | 4 | AE BF CD G | 1 | G | not mapped |
15. Ch. luridus | 4 | –//– | 1 | G | not mapped |
16. Ch. melanescens | 4 | –//– | 1 | G | not mapped |
17. Ch. pseudothummi | 4 | –//– | 3 | G C G |
not mapped 4d not mapped |
Subgenus Camptochironomus | |||||
18. C. dilutus | 4 | AB CF ED G | 2 | B C |
9 10 |
19. C. pallidivittatus | 4 | –//– | 1 | A | 12 |
20. C. setivalva | 4 | –//– | 1 | B | 9 |
21. C. tentans | 4 | –//– | 2 | B D |
9a–b 9b |
The species belonging to the subgenera Chironomus and Camptochironomus are similar in the number of rDNA loci in their karyotypes (one or two NORs) but differ considerably in their chromosomal positions. In species from the subgenus Chironomus NORs have been found in all chromosomal arms, whereas in species from the subgenus Camptochironomus NORs have been detected in arms A, B, C and D only. Unlike species belonging to the subgenus Chironomus with obligatory presence of one of the NORs in arm G (Figs
Seven species from the subgenus Chironomus carried rDNA hybridization sites in other chromosomal arms besides the NOR in arm G. These species can be divided into three groups according to the hybridization pattern of DNA probes.
Two NORs are always observed in the karyotypes of the first group (Ch. agilis and Ch. cingulatus) and the hybridization sites of DNA probes are similar in the intensity of hybridization and completely coincide with the localized NORs. Both NORs of Ch. agilis are located in arm G (one in the centromeric and the other in the telomeric regions); as for the Ch. cingulatus NORs, they are located on arms B and G (Fig.
The second group includes species with the number of hybridization sites for DNA probes exceeding the number of cytologically identifiable NORs and with the intensity of hybridization varying between hybridization sites (Ch. muratensis, Ch. nudiventris, and Ch. pseudothummi). In the karyotype of Ch. muratensis, two strong rDNA hybridization signals are always detected in regions developing NORs – one in arm G and the other in arm C, and in addition, weak hybridization signals varying in their intensity and number are detected in arms B, C, D, and F in the regions, where a developed nucleolus has never been observed (Fig.
The number of NORs in the karyotypes of the third group of species (Ch. balatonicus and Ch. “annularius”) may vary, however the hybridization sites of DNA probes always coincide with the active NORs (Fig.
The karyotype of Ch. “annularius” has either four or five NORs. Four NORs are found in all studied specimens (two NORs in arm E and one in each of arms C, and G). An intraspecific NOR polymorphism has been observed in Ch. “annularius” arm A, region 3g (Fig.
The genus Chironomus comprises over 150 species (
NORs are additional markers in polytene chromosomes. Nucleoli are actively transcribed regions of chromosomes, visible on chromosomes as giant puffs. Phase contrast microscopy of orcein-stained chromosomes allows them to be distinguished from any other functionally active chromosome regions (
In some cases the number of NORs detected by FISH or AgNO3 staining does not match to number of NORs detected by phase contrast analysis. In particular, staining with AgNO3 detected six NORs in the polytene chromosomes of Ch. duplex Walker, 1856 salivary gland, but only one NOR in interphase ganglion cells and none in the meiotic late prophase and metaphase I as well as mitotic chromosomes. The authors assume that the observed differences are determined by tissue-specific features in the NOR function, namely, fusion of nucleoli in ganglion cells and a decrease in the NOR transcription activity after the pachytene in meiosis (
One of the possible reasons underlying the variation in NOR activity in chironomids is a change in the number of transcriptionally active copies of ribosomal genes. A special study into the chromatin structure of Ch. riparius ribosomal genes has shown that not all these copies are equally active in transcribing rRNA. Along with transcriptionally active copies of ribosomal genes, free of nucleosomes, populations of these genes also contain transcriptionally inactive copies displaying nucleosome organization. The share of transcriptionally active copies in the population of ribosomal genes is tissue-specific, amounting to 80% in the fat body cells, to 50% in the salivary glands, and only 20% in the Malpighian tube cells (
Variability in activity of NORs might be also determined by such characteristics of this locus as multiple copies of rRNA genes and a presence of transposable elements (TE) (
If this mechanism is involved, the variability in intensity of hybridization signals of rDNA on chromosomes of Ch. muratensis, Ch. nudiventris and Ch. pseudothummi might be determined by the difference in the number of gene copies presented in each NOR. Thus, intense hybridization signals were detected in regions with active NORs while weak signals occurred in regions with no visible NOR activity. A similar pattern has been observed in wheat (
The analysis involving FISH and silver staining has shown a considerable diversity in the NOR number and locations in the chromosomes constituting karyotypes of 32 Palearctic and Australian Chironomus species (
NORs can be located on all seven chromosome arms of the chironomid karyotypes; however, none of the NORs have been detected on the same chromosome arm in all 32 species of the genus Chironomus. Most frequently, NOR is located on arm G (in 25 species out of 32), but none of the species belonging to the subgenus Camptochironomus had NOR on this arm. The locations of NORs is also different in species from the subgenus Chironomus inhabiting remote geographic regions: species from Western Siberia may carry NOR in all chromosome arms (Table
Along with the interspecific variation in the NOR number and location, chironomids also display intraspecific variation in these characteristics. Three species (Ch. balatonicus, Ch. “annularius”, and Ch. tepperi) may carry different numbers of NORs in individual karyotypes. In situ hybridization of Ch. tepperi chromosomes with 28S rRNA (
Variety in number and locations of NOR on chromosomes in karyotypes of species from the genus Chironomus can occur due to several reasons: as a result of chromosomal rearrangemens, mainly inversions and translocations that are widespread in chironomids (
Transposable elements (TE) can also cause considerable changes in organization of NORs in karyotypes of species from the genus Chironomus. They can change activity of NORs or cause their complete inactivation. Several types of TE were found in the genus Chironomus. Common features for all of them are the presence in genome of multiple copies of each element, multiple location sites, species-specific but demonstrate intraspecific, intra- and interpopulations variability (
The obtained results allowed us to characterize chromosomal organization and evolution of rRNA genes family in the genus Chironomus. The tree of phylogenetic relationships between species from the genus Chironomus constructed on the basis of comparison sequences of ITS1 and 5,8S rDNA shows that species groups into tree distinct clusters that coincide with cytocomplexes that differ from each other by arm combinations in chromosomes (Fig.
NJ tree based on maximum likelihood distances for ITS1 and 5,8S rDNA sequences from the genus Chironomus species. Drosophila melanogaster is used as an outgroup species. Maximum likelihood bootstrap values (1000 replicates) (> 50%) are shown next to the nodes. NORs chromosomal arms location, arm combinations and name of cytocomplexes are listed at the right.
Addition of data on the number and chromosomal positions of NORs to the phylogenetic tree of studied chironomid species shows that there is no correlation between evolution of nucleotide sequences of ribosomal genes and chromosomal organization of NORs in the karyotypes of species (Fig.
At the same time the combined data allow us to suggest a hypothesis about location of NOR in the karyotype of an ancestor species of the genus Chironomus. As all species from both “thummi” and “pseudothummi” cytocomplexes always have one NOR in arm G it is possible to suppose that an ancestor chironomid species had a NOR in this arm. And the absence of NOR in the arm G of species from the “camptochironomus” cytocomplex is probably caused by its loss in the ancestor species of this cytocomplex after its separation from “thummi” cytocomplex.
The work is financially supported by the grant from Russian Foundation for Basic Research #14-04-01126 and basic project VI.53.1.4. Authors are grateful to the Joint Access Center for Microscopy of Biological Objects SB RAS and SB RAS Genomics Core Facility for providing equipment and service support for this research and to G. Chirikova for translation of the manuscript from Russian into English.