Endolimax piscium sp. nov. (Amoebozoa), causative agent of systemic granulomatous disease of cultured sole, Solea senegalensis Kaup

Abstract

A new amoeba species pathogenic for Senegalese sole is described based on ultrastructural analysis and SSU rDNA phylogenetic inference. The parasite presents round to ovoid trophozoites (<5 μm) with a high degree of intracellular simplification. No mitochondria were observed, but mitosome‐like organelles were present. No cysts could be detected. Phylogenetic analysis confirmed the Senegalese sole parasite as an amitochondriate Archamoeba related to Endolimax nana and Iodamoeba spp., and we tentatively describe it as a new species in the genus Endolimax, Endolimax piscium. However, the genetic distance with E. nana is quite large, with only 60% pairwise identity between both SSU rDNA genotypes. Although the overall topology of the Archamoebae cladograms containing E. piscium was consistent, the support for the branching of Endolimax spp. relative to its closest neighbours was variable, being higher with distance or parsimony‐based inference methods than with ML or Bayesian trees. The use of stringent alignment sampling masks also caused instability and reduced support for some branches, including the monophyly of Endolimax spp. in the most conservative data sets. The characterization of other Archamoebae parasitizing fish could help to clarify the status of E. piscium and to interpret the large genetic distance observed between Endolimax species.

Development of different diagnostic techniques for Endolimax piscium (archamoebae) and their applicability in Solea senegalensis clinical samples

“…In contrast, the probes designed in this study are species specific targeting variable regions of E. piscium SSU rDNA, in which even related arachamoebae show strikingly large genetic distance (Stensvold, Lebbad & Clark ; Constenla et al . ). This fact guarantees significantly enhanced specificity levels in a ‘blind’ diagnostic context.…”

“…In the course of parasitological surveys conducted between 2011 and 2012 at different Senegalese sole farms located in NW Spain, a total of 92 fish displaying signs of presumptive E. piscium infection in skeletal muscle (symptomatic fish) were sampled. These signs include macroscopic lumps in the muscle noticeable at the skin surface as described previously (Constenla et al 2014). In addition, 118 normal, asymptomatic sole were randomly sampled from fish farms in the same geographic area.…”

“…These signs include macroscopic lumps in the muscle noticeable at the skin surface as described previously (Constenla et al . ). In addition, 118 normal, asymptomatic sole were randomly sampled from fish farms in the same geographic area.…”

“…In situ hybridization technique (ISH). Oligonucleotide probes were designed to specifically hybridize the negative strand of E. piscium small subunit ribosomal RNA gene (SSU rDNA) (Constenla et al 2014). Choice of probes was aided by software packages OLIGO-7 (Rychlik 2007) and PRIMER PREMIER (Premier Biosoft).…”

“…Endolimax piscium is the causative agent of systemic amoebiasis in Senegalese sole Solea senegalensis (Kaup) (Constenla, Padr os & Palenzuela 2014). This archamoeba may cause conspicuous lumps in the muscle and granulomatous inflammatory reaction in different organs of infected sole (Constenla & Padr os 2010).…”