Introduction

In the fish family Gobiidae, there are around 1809 species belonging to 258 genera and the genus Glossogobius of this family acknowledge around 46 known species till date (www.fishbase.org/ ver. 06/2018). The tank goby, G. giuris, is habitant to freshwater as well as saltwater. It is widely distributed in freshwater bodies in India. It grows to a much larger size in brackish water than in freshwater. It has been categorized as Least Concern ver. 3.1 under IUCN Red List of Threatened Species (IUCN, 2019; https://www.iucnredlist.org/species/166533/19011337) and the population trend of the species is not known.

The cytogenetic information is useful in characterization of the species and is one of the reliable taxonomic criteria for few organisms. The cytogenetic applications have also concentrated on understanding the distribution pattern and species evolution, which seems very promising [1]. Considering the large number of species found in this family, the cytogenetic analyses are still scarce. There are many studies where basic cytogenetic investigations were undertaken in this species, but the molecular cytogenetic information is lacking in this species.

The present study is undertaken to provide information on basic karyomorphology of G. giuris, mainly for further validating the diploid chromosome number (2n) and fundamental arm number (FN) through construction of karyotype or ideograms of the goby fish. Silver nitrate (AgNO3)- and chromomycin-A3 (CMA3)-stained positive nucleolar organizer region (NOR) site at metaphase spread and the description of the localization of 18S rDNA on metaphase chromosome were investigated as cytotaxonomic reliable tools [2] for identification of this fish species along with putative hybrids. Few classical cytogenetic markers were earlier used for species identification as well as phylogenetic relationship and to resolve taxonomic ambiguity among the related species through karyomorphology and other staining techniques, like Giemsa, AgNO3 and CMA3 [3].

Material and Methods

Fish sample collection and identification

Live specimens (n = 25) of tank goby, G. giuris, were collected from Loktak lake, near Imphal, Manipur in India with the help of fisherman using cast net and were transported to the laboratory in a plastic container with proper oxygenation. Taxonomic identification of the individuals was done using the keys developed by Talwar and Jhingran [4].

Chromosome preparation and staining

The metaphase chromosomes complements of G. giuris were prepared from anterior kidney cells using standard hypotonic treatment (0.56% KCl), methanol/acetic acid (in 3:1 ratio) fixation and flame-drying technique [5]. The dried chromosomes spread on slides was stained with 6% Giemsa in phosphate-buffered saline (KH2PO4 and Na2HPO4 with pH 6.8) for 20 min at room temperature. Around 75 good-quality metaphase chromosome spreads possessing characteristic morphology were screened and used for karyotype preparation. The homologous chromosome pairs were arranged according to their morphology as well as the centromeric positions in the decreasing order of the size or length of the chromosomes with the help of Lieca CW4000Karyo software.

The averages of the homologous chromosomes were taken for estimating the length of long (q) and short (p) arms, arm ratio, centromeric index and the relative length (%) of the chromosome. The ratio of q and p arms (q/p) was used to classify the chromosomes’ morphology as metacentric (m), submetacentric (sm), subtelocentric (st), and telocentric (t), as proposed by Levan et al. [6], for making karyogram, i.e., built according to relative length and centromeric index. The authors measured the chromosomes in terms of centromeric position and relative size differences between the homologous pairs for overall symmetry and asymmetry. The NOR-staining of chromosomes with AgNO3 and CMA3 was done according to the methods of Howell and Black [7] and Ueda et al. [8], respectively. A particular band pattern of Giemsa, AgNO3 staining and CMA3 positive site was determined for each specimen by analyzing more than 50 metaphase spreads in light microscope under 100X magnification with oil immersion.

DNA extraction and amplification

The genomic DNA was extracted from muscle tissue using the standard phenol–chloroform–isoamyl alcohol method [9]. The PCR amplification of 18S rDNA was done in 50 µl reaction mixture using 10 × buffer (having 15 mM MgCl2), 10 mM dNTPs mix, 10 pmol of primers (forward: 5′-TTGGTGACTCTCGATAACCTC-3′ and reverse: 5′-CCTTGTTACGACTTTTACTTCCTC-3′), 5U Taq DNA polymerase and 200 ng template DNA under thermal cycling condition of: initial denaturation at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and primer extension at 72 °C for 3 min, with final extension at 72 °C for 10 min. The quality of amplified product was checked in 1% agarose gel with 1 kb DNA ladder (Fermentas).

Probe labeling and hybridizations

The PCR product of 18S rDNA was purified and labeled with rhodamine 12-dUTP (Roche) by nick translation for probe construction. Single-color FISH was performed to determine the localization of 18S rDNA on the metaphase chromosome complements. Approximately, 2–3 day-aged chromosome containing slides were baked at 94 °C for 1 h and the fluorescence in situ hybridization (FISH) of rDNA probe on metaphase chromosomes were performed using protocol of Winterfeld and Roser [10], with slight modification at post-hybridization washing at 45 °C.

The metaphase chromosome slides hybridized with DNA probe were counterstained with DAPI staining and mounted with Vectrashield mounting medium (Vector Labs). The mounted slides were observed under fluorescence microscope (Leica) with two band filter for simultaneous visualization of the two colors, i.e, DAPI filter for chromosomes visualization in blue color and rhodamine filter for labeled probes visualization in red color. Two images were superimposed to visualize the exact location of 18S rDNA on the metaphase complements.

Results and Discussion

Chromosome number and morphology

The specimens of G. giuris revealed 2n = 46 and FN = 46. All the chromosomes were telocentric in morphology showing karyotype formula of 46t. All mono-armed telocentric chromosomes are represented in karyotype as well as in ideogram (Fig. 1) and by their statistics (Table 1). Here, the authors find 2n = 46 with all telocentric morphology in around 80% of the cells, whereas 20% of the cells showed 2n = 43, 44, 45 and 47. This variation in the number of chromosomes may be due to handling of the chromosomes. The present study was in agreement with the finding of the earlier reports [11,12,13,14,15,16,17] of 2n = 46 in G. giuris, except that of Prasad [18], who reported same 2n = 46 but with karyotype formula of 4st + 42A and FN = 46. The relative length, based on the chromosome morphology of the metaphase spreads, ranged from 5.816 to 2.816 in the present study.

Fig. 1
figure 1

a Giemsa-stained metaphase spread, b karyotype, and c ideogram of G. giuris chromosomes

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Table 1 Chromosomes statistics of G. giuris
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In the fish family Gobiidae, around 80 fish species are cytogenetically investigated (www.fishbase.org/ ver. 06/2018) and their 2n ranged from 30 (Mesogobius batrachocephalus, subfamily: Gobiinae) to 52 (Gobius niger, subfamily: Gobiinae), with most frequent 2n = 44, 46 and 48. The species look-alike another gobies and eleotrids revealed the variability of diploid chromosomes from 43 to 62, along with most of the 2n = 44, 46 and 48. The variability in 2n counts leads to the generalization of the certain hypothesis of changes in chromosomal number due to centromeric fusions/fission and translocations.

NOR Localization

The impregnation of AgNO3 revealed localization of one pair of NOR at centromeric position of 12th pair chromosomes in 90% of the metaphase complements of G. giuris (Fig. 2a). The GC-rich and transcriptionally active rDNA in many vertebrates, including fishes [19], has been identified by CMA3 staining, which are useful in detecting many clusters of implicit rDNA [20]. In the present study, the authors found that 85% metaphase spreads showed the presence of GC-rich NORs at one pair of homologous chromosomes (Fig. 2b), whereas 15% showed NORs at only one chromosome. Thus, the NOR number is reported in G. giuris as one pair located by AgNO3 and CMA3 staining at telocentric chromosomes.

Fig. 2
figure 2

a Homologous chromosomes showing AgNO3-stained NORs, b CMA3 positive sites, and c metaphase chromosomes complements showing 18S rDNA FISH signals in G. giuris (bar size: 5 microns)

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The numbers of AgNO3 stained and CMA3 positive NOR sites were found at similar location in many fish species; however, it may be sometimes different also which may be due to visualization of transcriptionally active site of rDNA. The AgNO3 stains those NORs that expressed themselves during preceding interphase by attaching to a composite of acidic proteins affiliated with nucleolus and pre-RNA [21]. Further, the AgNO3 stains essential heterochromatin, in addition to heterochromatin associated with NORs [22]. Single NOR pair is common feature of several fishes; however, some fishes showed more than one NOR pairs on chromosomes, which indicates polymorphisms related to transcriptionally inactive NORs that have long been described in many organisms, but the etiology of such type of variation is not very clear. Such NORs type information is very useful for detecting inter- as well as intra-specific differences, which may serve for demonstrating the taxonomical position of species [23].

Localization of 18S rDNA

The repetitive DNA is used for chromosomal mapping for the characterization of species, comparative genomics and karyo-evolutions. In the present study, the FISH signals of 18S rDNA were clearly located in most of metaphase spreads of G. giuris on the arms of one pair of telocentric chromosomes. The FISH signals are very strong indicating the presence of highly repeated rRNA genes on these chromosomes (Fig. 2c). Localization of the rDNA on the metaphase chromosomes has a more substantial impact on the tempo of concerted evolution than the number of loci [24]. The physical mapping of chromosomes by FISH showed new type of potential chromosomal information; this type of mapping mainly focuses on highly repetitive DNA or multi-gene families because of the technical difficulties found for mapping low-copy genes [25].

The valuable information generated in the study is the similar position of the presence of the signals of 18S rDNA using FISH mapping, silver nitrate staining of NORs and CMA3 positive sites. This type of correlations between active rRNA genes and CMA3 positive sites were also reported earlier in few coregonid fishes [19]. Das and Khuda-Bukhsh [26] also reported this type of association between silver-stained NORs and GC-rich active rRNA genes in fishes. Such type of co-localization also reported in O. belangeri [27]. Pattern of similar localization of AgNOR, CMA3 and labeled 18S rDNA confirmed the activeness of single pair NORs in this Glossogobius species.

The study of the localization of rDNA gene and their activities have been given importance in broad range of organism for phylogenetic as well as evolutionary relationships [28] and for characterization of species as well as population stocks in several fish species, like Cyprinidae. The variability in number and localization of rDNA loci on metaphase complement in earlier studies provided species-specific pattern in their distribution. The distribution pattern of rDNA in the present study may be a valuable genetic marker for the evolutionary studies and genetic characterization of this and other related species for effective molecular taxonomy. It is also useful in population genetics for identification and characterization of stocks and population of similar or different species [29]. Presently, these markers are mostly used for the identification and characterization of the species/stocks, but they will also be useful for enabling the identification of the hybrids and confirming the paternity in future [30].

Conclusion

The present study updates the data of the previous karyomorphological studies performed using silver nitrate and chromomycin A3 staining on chromosomes of G. giuris. It provides new molecular cytogenetic information on the tank goby fish using single-color FISH with labeled 18S rDNA. Hence, the data is useful for future studies related to cytotaxonomic identification, phylogenetic/evolutionary relationships, population genetic studies, genetic improvement program and conservation as well as management of this species.