The study of an organism genome at the chromosomal level can be used in differentiating one species from another, i.e., the analysis of chromosome numbers, size, and centromere positions [11,12,13]. In fact, modern cytogenetic techniques have since mid-1990s been adopted for studies of Gastropoda. The subfamily Biomphalarinae has been known to be a conservative group in regard to haploid chromosome numbers 18 (Table 1). In this study, the karyotype of B. tenagophila was studied in order to analyze their genetic relationships.

The 9 specimens used in this study were collected in Museu de Arte Moderna (22°54'48.12"S, 43°10'19.17"W), Rio de Janeiro, Brazil, September 2012, and examined shortly after collection (Fig. 1). The chromosome preparations were made on gonad of the specimens by the usual air-drying method [14]. The prepared slides were observed under an Olympus (BX51) microscope. The identical specimens used for this study have been deposited at the Department of Environmental Medical Biology, Kwandong University College of Medicine, Korea.

A microphotograph of somatic metaphase chromosomes and the karyogram are shown in Fig. 2, and measurements of the chromosomes are presented in Table 2. Fig. 2B shows the karyotype of this species arranged by size. The present cytological preparations showed 10 well-spread mitotic cells on a slide. This species showed a diploid chromosome number of 2n=36, with 7 metacentric, 8 submetacentric, and 3 subtelocentric chromosome pairs. Observed chromosomes ranged from 2.4 to 6.4 µm in length. This species was no inter-specimen variability in chromosome counts. Also, sexual dimorphism of chromosomes was not found in this study.

Most species of the Biomphalaria have been investigated and at present, B. glabrata, B. pfeifferi, B. madagascariensis, B. sudanica, B. tanganyicensis, B. alexandrina, and B. truncatus have a haploid chromosome complement of 18 (Table 1). The chromosomes are relatively small and monomorphic. Conservation of chromosome number has been pointed out for many gastropoda groups [15]. Burch [16], in the 1960s, utilized preparations from the ovo-testis and reported a haploid complement of 18; however, these results were deducted from the meiotic stage, where it was often different to identify each and every chromosome. However, with the development of cytological methods such as hypotonic treatment of tissue samples and pre-treatment with colchicines (air drying technique), clearer deductions were possible on the chromosome number and morphology [11]. The morphology of mitotic metaphase chromosomes has been reported in 10 species of the subfamily Biomphalrinae with n=18 and 2n=36 (Table 1).

The present results revealed that the diploid chromosome number of B. tenagophila is 36. With regard to karyotype analysis in the genus Biomphalaria, only 4 paper about B. glabrata, B. truncates, and B. alexandria has been published [17,18,19]. Though the chromosome number is the same between B. glabrata, B. alexandria, B. truncates, and B. tenagophila, the karyotype is different. B. glabrata and B. alexandria have 10 pairs of metacentric, 4 pairs of submetacentric, 2 pairs of acrocentric, and 2 pairs of telocentric chromosomes, and B. truncatus has 10 metacentric, 4 submetacentric, 2 subtelocentric, and 2 telocentric chromosomes, whereas B. tenagophila has 7 metacentric, 8 submetacentric, and 3 subtelocentric chromosomes. The results of this study on the mitotic metaphase chromosomes provided a much more elucidated observation of the chromosomes of this species. Further studies are needed with various analyses for the investigation of the karyo-systematic evolution, and accumulation of karyological information is very much required in the subfamily Biomphalarinae.