Molecular cytogenetic analysis and the establishment of a cell culture in the fish species Hollandichthys multifasciatus (Eigenmann & Norris, 1900) (Characiformes, Characidae)

Abstract

Hollandichthys is a fish genus of the family Characidae that was until recently considered to be monotypic, with cytogenetic, morphological, and molecular data being restricted to a few local populations. In the present study, the karyotype of a population of Hollandichthys multifasciatus was analyzed using classical and molecular cytogenetic approaches for the investigation of potential markers that could provide new perspectives on the cytotaxonomy. H. multifasciatus presented a diploid number of 2n=50 chromosomes and a karyotype formula of 8m+10sm+32st. A single pair of chromosomes presented Ag-NORs signals, which coincided with the 18S rDNA sites visualized by FISH, whilst the 5S rDNA sequences were mapped in two chromosome pairs. The distribution of the U snRNA genes was mapped on the Hollandichthys chromosomes for the first time, with the probes revealing the presence of the U1 snDNA on the chromosomes of pair 20, U2 on pairs 6 and 19, U4 on pair 16, and U6 on the chromosomes of pair 11. The results of the present study indicated karyotypic differences in comparison with the other populations of H. multifasciatus studied previously, reinforcing the need for further research to identify isolated populations or the potential existence of cryptic Hollandichthys species.

Figure 1 -. Primary cell culture following the enzymatic digestion of the fin tissue of Hollandichthys multifasciatus and the cell confluence after four days (a). Cell population in the second cultivation (b). Scale bar = 100 µm.

Figure 2 -. Karyotype of Hollandichthys multifasciatus (2n=50 chromosomes) analyzed in the present study by conventional Giemsa staining (a). The nucleolus organizing regions are located in the interstitial region of the long arms of chromosomes pair 4 (box). (b) C-banded karyotype. Scale bar 10 µm.

Figure 3 -. Karyotypes of males of Hollandichthys multifasciatus arranged after FISH mapping of (TTAGGG)n sequences (red signals) (a); (b) dual-color FISH with 5S (green signals) and 18S rDNA (red signals) sequences; (c) dual-color FISH with U1 (green signals) and U2 snDNA (red signals) probes and (d) dual-color FISH with U6 (green signals) and U4 snDNA (red signals) probes. The chromosomes were counterstained with DAPI (blue). Scale bar = 10 µm.