Molecular cytogenetic analysis of a triploid population of the human broad tapeworm, Dibothriocephalus latus (Diphyllobothriidea)

Abstract

The large-sized tapeworm Dibothriocephalus latus is known as the broad or fish-borne cestode of mammals that is capable to infect humans and cause diphyllobothriosis. Recently, molecular data on D. latus has been accumulating in the literature and a complete genome sequence has been published; however, little is known about the karyotype and chromosome architecture. In this study, an in-depth karyological analysis of 2 D. latus specimens was carried out. The plerocercoids originated from a perch caught in subalpine Lake Iseo (Italy) and the tapeworms were reared in hamsters. Both specimens contained cells with a highly variable number of chromosomes ranging from18 to 27. Nevertheless, the largest portion of mitotic figures (47%) showed a number corresponding to the triploid set, 3n = 27. Accordingly, the karyotype of the analyzed specimens consisted of 9 triplets of metacentric chromosomes. Fluorescence in situ hybridization (FISH) with the 18S rDNA probe clearly demonstrated the presence of 3 clusters of hybridization signals on the triplet of chromosome 7, thus confirming the triploid status of the specimens. FISH with a telomeric (TTAGGG)n probe confined hybridization signals exclusively to the terminal chromosomal regions, supporting the earlier findings that this repetitive motif is a conserved feature of tapeworm telomeres.

Keywords: 18S rDNA; parthenogenesis; polyploidization; telomeres; triploid.

Graphical abstract

Fig. 1.

Metaphase chromosomes of Dibothriocephalus latus. (A) Giemsa and (B) DAPI staining (3n = 27; scale bar = 10 μm).

Fig. 2.

Karyotype derived from mitotic metaphase cells of Dibothriocephalus latus (3n = 27 m). (A) Giemsa staining. (B) DAPI staining showing AT-rich bands. The small inset shows a detail of chromosomes of the triplet No. 7 with distinct 18S rDNA [nucleolar organizer region (NOR)] loci located on each individual chromosome (scale bar = 10 μm).

Fig. 3.

Chromosomes of Dibothriocephalus latus stained with DAPI (blue) and 18S rDNA fluorescence in situ hybridization probe (red). (A) An interphase nucleus with 3 18S rDNA clusters. (B, C) Mitotic metaphases showing 3 chromosomes with hybridization signals. (D) Early mitotic anaphase showing early segregation of chromatids to opposite poles (scale bar = 10 μm).

Fig. 4.

Distribution of telomeric sequence (TTAGGG)_n after FISH in Dibothriocephalus latus showing an exclusively telomeric pattern. (A) Pachytene nucleus and (C) mitotic metaphase after FISH with biotin-labeled telomeric probe. (B) Pachytene nucleus and (D) mitotic metaphase after TSA-FISH (scale bar = 10 μm). Abbreviations: FISH, fluorescence in situ hybridization; TSA-FISH, FISH with tyramide signal amplification.

Fig. 5.

Visualization of nucleoli (N) in meiotic spermatocytes of Dibothriocephalus latus after AgNO₃ staining (scale bar = 10 μm).

Fig. 6.

The course of meiotic division in Dibothriocephalus latus after FISH with 18S rDNA probe (red) counterstained with DAPI (blue). (A) Interphase nucleus with a large nucleolus and 3 18S rDNA clusters. (B) Leptotene. (C–G) Pachytene nuclei showing irregular pairing of spermatocyte chromosomes carrying rDNA genes; (C, D) pachytene with NOR-bearing triplet No. 7 in form of 1 univalent and 1 bivalent or (E–G) 3 univalents. (H) Metaphase II. (I) Secondary spermatocyte, the early stage of spermiogenesis (scale bar = 10 μm). Abbreviations: FISH, fluorescence in situ hybridization; NOR, nucleolar organizer region

Fig. 7.

Summary of available data on the diploid/triploid chromosome number (2n/3n) and chromosome morphology in karyologically studied diphyllobothridean species along with their latest phylogenetic relationships based on Waeschenbach et al. (2017).