Hyaloklossia Labbé, 1896 (Alveolata: Apicomplexa) in frogs: Description of a new species and proposing a new subfamily to accommodate these enigmatic parasites

Abstract

Hyaloklossia Labbé ,1896 (Alveolata: Apicomplexa) is a monotypic genus of renal coccidia found in anurans, particularly in the edible frog Pelophylax kl. esculentus (Amphibia: Anura: Ranidae), distributed in different parts of Europe. Here we propose a new Hyaloklossia species from the Tokyo daruma pond frog, Pelophylax porosus porosus . The coccidium detected in the renal tissue of P. p. porosus shared some morphological characteristics with the type species, Hyaloklossia lieberkuehni (Labbé, 1894), reported from P. kl. esculentus . However, in addition to size differences in several oocyst and sporocyst features between these parasites, phylogenetic analysis of gene fragments from two nuclear ribosomal loci and the mitochondrial cytochrome c oxidase subunit 1, exposed distinct genetic differences between H. lieberkuehni and our new species. Although our analysis validated the monophyly of Hyaloklossia with some members of the Toxoplasmatinae Biocca, 1957, Cystoisosporinae Frenkel et al., 1987, and Eumonosporinae Chou et al., 2021 (Sarcocystidae Poche, 1913), comparison of genetic differences between Hyaloklossia species from P. p. porosus and H. lieberkuehni revealed the presence of a greater number of polymorphisms than that observed when comparing inter-species ( Heydornia spp., Besnoisita spp.) or inter-genus ( Toxoplasma vs . Neospora, Neospora vs. Hammondia, and Neospora vs . Heydornia ) variabilities among members of the Sarcocystidae. This indicates that Hyaloklossia , as re-erected and defined by Modrý et al. (2001, Int. J. Syst. Evol. Microbiol. 51, 767–772), with its homoxenous life cycle, requires placement in its own subfamily. Thus, we propose a new subfamily, Hyaloklossiinae n. subfam., to accommodate two species, H. lieberkuehni from Europe and Hyaloklossia kasumienesis n. sp. which we describe here from P. p. porosus in Japan.

Black-spotted pond frog Pelophylax nigromaculatus as a new host for the renal coccidian genus Hyaloklossia (Alveolata: Apicomplexa)

“…Based on the size of its sporocyst and SR the present species was identified as H. kasumiensis . The original description of H. kasumiensis oocysts was based on a small number obtained from tissue sections of the P. p. porosus kidney ( Tokiwa et al, 2021 ), and thus, the measurements were not accurate. This is the first detailed description of both the mature and immature oocysts of H. kasumiensis.…”

“…Abbreviations used throughout this article are standardized ( Wilber et al, 1998 ; Tokiwa et al, 2021 ); Oocyst characteristics: length (L), width (W), their ratio (L/W), micropyle (M), micropyle cap (MC), and oocyst residuum (OR); sporocyst characteristics: length (L), width (W), their ratio (L/W), Stieda body (SB), substieda body (SSB), sporocyst residuum (SR), and sporozoites (SZ).…”

“…(Anura: Ranidae) in Europe ( Modrý et al, 2001 ; Duszynski et al, 2007 ; Duszynski, 2021 ). Recently, a second species, Hyaloklossia kasumiensis Tokiwa, Chou, Tochigi, Katayama et Duszynski, 2021 , was described from Japan in East Asia ( Tokiwa et al, 2021 ). Genetically, Hyaloklossia belongs to the family Sarcocystidae and forms a sister group to other coccidia of medical and veterinary importance: Toxoplasma and Neospora in subfamily Toxoplasmatinae, Cystoisospora in subfamily Cystoisosporinae ( Modrý et al, 2001 ; Oborník et al, 2002 ; Šlapeta et al, 2003 ; Chou et al, 2020 , 2021 ; Tokiwa et al, 2021 ).…”

“…Recently, a second species, Hyaloklossia kasumiensis Tokiwa, Chou, Tochigi, Katayama et Duszynski, 2021 , was described from Japan in East Asia ( Tokiwa et al, 2021 ). Genetically, Hyaloklossia belongs to the family Sarcocystidae and forms a sister group to other coccidia of medical and veterinary importance: Toxoplasma and Neospora in subfamily Toxoplasmatinae, Cystoisospora in subfamily Cystoisosporinae ( Modrý et al, 2001 ; Oborník et al, 2002 ; Šlapeta et al, 2003 ; Chou et al, 2020 , 2021 ; Tokiwa et al, 2021 ). The morphological features of disporocystic and tetrasporozoic oocysts without Stieda bodies of Hyaloklossia are similar to those of other related species of the family Sarcocystidae with multi-host (i.e., indirect, heteroxenous, or facultatively heteroxonous) life cycle, with carnivorous animals as the definitive hosts.…”

“…However, it is believed that all Hyaloklossia species use only a single-host (homoxenous) life cycle, with anuran species as the definitive hosts ( Laveran and Mesnil, 1902 ; Duszynski et al, 2007 ). Hyaloklossia is thus a unique taxon with interesting aspects for studying the phylogenetics of Sarcocystidae; however, they are largely documented to be present in Pelophylax species in Europe, and have not been studied in endemic anurans other than P. p. porosus in Japan ( Tokiwa et al, 2021 ). In this study, coccidian parasites in the kidney tissues of anuran species in Japan were investigated to obtain basic knowledge about Hyaloklossia infection.…”