Molecular characterization and confocal laser scanning microscopic study of Pygidiopsis macrostomum (Trematoda: Heterophyidae) parasites of guppies Poecilia vivipara

Abstract

Pygidiopsis macrostomum and Ascocotyle (Phagicola) pindoramensis (Digenea: Heterophyidae) parasitize guppies as intermediate hosts and, respectively, fish‐eating mammals or birds as definitive hosts. Heterophyids have zoonotic potential, and molecular studies associated with morphological and ecological aspects have helped to clarify their taxonomy and phylogeny. Poecilia vivipara naturally parasitized by metacercariae of both species (100% prevalence) exhibit no external signs of parasitism. In this work, four new sequences of P. macrostomum (18S rDNA, 28S rDNA and ITS2 rDNA) and one new sequence of A. (P.) pindoramensis (mtDNA cox‐1) are presented. Phylogeny reconstructions linked P. macrostomum to other heterophyids, but the separation of the Heterophyidae and Opisthorchiidae remains unclear. Additionally, we used indirect immunocytochemistry and the phalloidin‐fluorescence techniques allied with confocal laser scanning microscopy to describe muscular and neuronal structures of P. macrostomum. A complex arrangement of muscular fibres is associated with the tegument, suckers, gut and reproductive system. Radial fibres around the ventral sucker are thick, branched and extend to the body wall. High‐resolution confocal imaging revealed a typical digenean muscular arrangement and important heterophyid morphological traits. These data will support future control measures to reduce the parasitism in guppies reared in fish farming systems, especially for aquarium and experimental purposes.

A molecular phylogenetic appraisal of the acanthostominesAcanthostomumandTimoniellaand their position within Cryptogonimidae (Trematoda: Opisthorchioidea)

“…loossi, and illustrate different intergeneric relationships among cryptogonimids. Phylogenetic analyses show that the Heterophyidae and Opisthorchiidae are paraphyletic as previously reported ( Thaenkham et al, 2011 ; Thaenkham et al, 2012 ; Fraija-Fernández et al, 2015 ; Stoyanov et al, 2015 ; Borges et al, 2016 ), and that the family Cryptogonimidae appears to have arisen from the paraphyletic Heterophyidae/Opistorchiidae. This phylogenetic inference is based on a dataset of 51 taxa of Cryptogonimidae that included 24 genera.…”

Current Knowledge of Small Flukes (Digenea: Heterophyidae) from South America

“…Sequences of the 18S, 28S, and ITS2 rDNA regions of Pygidiopsis macrostomum and mtDNA cox-1 sequences of Ascocotyle pindoramensis have already been described [8]. Molecular phylogeny linked P. macrostomum and A. pindoramensis with other heterophyids but, in agreement with Thaenkham et al [10], the distinction of the Heterophyidae and Opisthorchiidae remained unclear.…”

“…Pygidiopsis macrostomum was originally described based on a single specimen obtained from the intestine of Rattus norvegicus (Erxleben, 1777) in Brazil [37], but was subsequently reported from the intestine of the bulldog-bat Noctilio leporinus mastivus Ribeiro, 1914 in Cuba [38,39]. This parasite was redescribed using morphological, ultrastructural and molecular data [8,40], and its life cycle was completed using naturally infected snails and fish, and hamsters as experimental definitive hosts [4] [4]. Indirect immunocytochemistry and phalloidin-fluorescence techniques, allied with confocal laser scanning microscopy, were also used by Borges et al [8] to describe the muscular and neuronal structures of Pygidiopsis macrostomum, revealing the complex arrangement of muscular fibers and ganglia within the body.…”

“…According to Chai and Jung [ 7 ], 30 species are able to infect humans worldwide, with a global level of infection of around 7 million people. Integrative studies, including molecular data associated with structural, morphological and ecological aspects, have helped elucidate the taxonomy and phylogeny of the group [ 6 ], although, based on limited molecular evidence, Thaenkham et al [ 8 , 9 ] have questioned the recognition of this family as distinct from the Opisthorchiidae. It is clear that further taxonomic studies of the Heterophyidae are required, since recognition is the basis for prevention and control programs for these emerging fish-borne trematodoses [ 10 ].…”

Novel luminescent dyes for confocal laser scanning microscopy used in parasite Trematoda diagnostics*

“…At present, CLSM is widely used to study various species' morphological and physiological structures in fixed trematode specimens (Jurberg et al, 2008;Borges et al, 2017). Souza and coworkers (Souza et al, 2011) utilized hydrochloric carmine to observe trematode reproductive system by CLSM.…”