Astiotrema reniferum ( Looss, 1898 ) Looss, 1900

Karar, Yasser F. M., Blend, Charles K., Dronen, Norman O. & Adel, Asmaa, 2021, Towards resolving the problematic status of the digenean genus Astiotrema Looss 1900: An updated concept and revision of species composition for Astiotrema (sensu stricto), Zootaxa 4991 (1), pp. 36-72 : 38-40

Type-species: Astiotrema reniferum ( Looss, 1898) Looss, 1900

Diagnosis. Body large, elongate to elongate oval, spatulate. Tegument spinous. Forebody approximately one third of total body length. Suckers subglobular, unspecialized, with variable sucker ratio. Oral sucker subterminal. Ventral sucker in anterior third of body or at border between anterior and middle thirds. Prepharynx indistinct to absent. Pharynx well developed, smaller than suckers. Esophagus of varying length to indistinct, straight to sigmoid. Intestine bifurcates in posterior or anterior part of forebody. Cecal ends variable in extent, close to posterior extremity or some distance short of it in middle third of body. Testes two, intercecal, smooth to indented, separated by small inter-testicular space, elliptical to slightly irregular, tandem to oblique, rarely opposite. Cirrus-pouch clavate, submedian or diagonal to midline, extends distinctly into hindbody. Internal seminal vesicle large, thick-walled, saccular to elongate saccular, unipartite, occasionally slightly sinuous; pars prostatica and ejaculatory duct short, tubular, inconspicuous. Genital atrium short, inconspicuous. Genital pore immediately anterior to ventral sucker, postbifurcal to occasionally bifurcal, median to slightly submedian. Ovary globular to elliptical, intercecal, pre-equatorial, dextral to dextro-submedian, entire, at base of cirrus-pouch or lateral to it. Canalicular seminal receptacle distinct, post-ovarian, variable in size, elliptical to kidney-shaped. Vitellarium follicular, in extracecal fields and sometimes extends dorsal and ventral to ceca, extends longitudinally between pharynx and cecal extremities; anterior extent pre-bifurcal, bifurcal or post-bifurcal; posterior extent pre-testicular, testicular or post-testicular. Uterus inter- and post-cecal, occasionally slightly extracecal, fills inter-testicular space and most of hindbody posterior to ovary, extends to posterior extremity. Metraterm simple, originates alongside cirrus-pouch. Eggs operculated, elliptical, unfilamented, tanned, unembryonated in utero, numerous. Excretory vesicle Y-shaped; stem passes between testes and bifurcates in two short arms between ovary and anterior testis. Excretory pore terminal. In intestine of various freshwater fishes ( Bagridae , Cichlidae , Clariidae , Cyprinidae , Heteropneustidae & Tetraodontidae ), amphibians ( Bufonidae , Dicroglossidae & Hylidae ), lizards ( Varanidae ) and turtles ( Cheloniidae , Emydidae , Geoemydidae & Trionychidae ); cosmopolitan in Asia, Europe and Africa.

Remarks: Looss (1896) erected Distoma unicum Looss, 1896 for specimens collected from the intestine of the African or Nile soft-shelled turtle, Trionyx triunguis (Forsskål) (Syn. Trionyx niloticus Gray ) ( Testudines : Trionychidae ), in the River Nile of Egypt. Due to the homonym observed with Distomum unicum Molin, 1859 (see Molin 1859), Looss (1898) renamed his specimens Distomum reniferum Looss, 1898 . A year later, Looss (1899) considered D. reniferum as the type species of Astia Looss, 1899 . As Astia Koch, 1879 was already preoccupied (i.e., an arachnid genus for Australian jumping spiders—see Koch [1879]), Looss (1900) changed Astia to Astiotrema Looss, 1900 with type species, Astiotrema reniferum .

Tremiorchis Mehra & Negi, 1926 was erected for the type-species Tremiorchis ranarum Mehra & Negi, 1926 collected from the small intestine and duodenum of the Indian bullfrog, Hoplobatrachus tigerinus (Daudin) (Syn. Rana tigrina [Duadin]) ( Anura : Dicroglossidae ), in Northern India ( Mehra & Negi 1926). That same year, from the intestine of the same host, H. tigerinus , from Allahabad and Nagpur, India, Bhalerao (1926) proposed Centrovitus Bhalerao, 1926 for its type species Centrovitus pentadelphi Bhalerao, 1926 . Ejsmont (1928) presented a detailed comparison between T. ranarum and C. pentadelphi and, consequently, C. pentadelphi was synonymized with T. ranarum , and it followed that Centrovitus was considered a synonym of Tremiorchis . Verma (1930) supported the conclusions of Ejsmont (1928), discussed the systematic position of Tremiorchis , modified the generic diagnosis of Tremiorchis based on describing another species, Tremiorchis varanum Verma, 1930 , and revised its taxonomic position clarifying the high importance of Tremiorchis because of possessing common features with several genera of the Plagiorchiinae Pratt, 1902 (= Plagiorchiidae Lühe, 1901 ) and probably some genera of the Brachycoeliinae Looss, 1899 (currently considered a synonym of the Brachycoeliidae Looss, 1899 ). Mehra (1931a) transferred Tremiorchis to the Lepodermatinae Looss, 1899 from the Lepodermatidae Looss, 1899 . Thus far, most of the dispute involving the taxonomic status of Tremiorchis has involved whether this genus belongs to the Plagiorchiidae ( Bhalerao 1926; Mehra & Negi 1926; Mehra 1931a; Bhardwaj 1962; Rai 1962; Gupta, 1964; Skrjabin et al. 1964; Agrawal 1968; Ali & Karyakarte 1970; Mukherjee & Ghosh 1972; Singh 1977; Hafeezullah 1989; Rajendran & Janardanan 1993; Bhutia 2006; Pandey & Agrawal 2013) or the Brachycoeliidae ( Dollfus 1930; Kalyanker & Palladwar 1978; Hafeezulla & Dutta 1981; Ghosh & Chakraborty 1999; Ghosh & Srivastava 1999; Bilqees & Khan 2003; Pojmańska 2008; Ghosh & Chakrabarti 2013; Keloth & Kandambeth 2018). Bhardwaj (1962) erected the Tremiorchinae Bhardwaj, 1962 within the Plagiorchiidae to accommodate Tremiorchis based on (i) the plagiorchiid nature of Tremiorchis despite its considerable divergence from this line of evolution, (ii) the convergence of Tremiorchis with the Dicrocoeliidae Odhner, 1910 in regard to the shortening of the ceca and the limited extent and size of the vitellarium, (iii) the potential evolution of Brachycoelium Dujardin, 1845 from Tremiorchis reflected in maintaining the Brachycoelinae Looss, 1899 within the Plagiorchiidae (see Mehra 1931a), and (iv) the assignment of the Brachycoelinae among the dicrocoeliids. Fotedar (1971) compared the generic characteristics of Tremiorchis with Astiotrema , synonymized both genera, and considered T. ranarum as Astiotrema ranarum ( Mehra & Negi, 1926) Fotedar, 1971 . Rajendran & Janardanan (1993) apparently did not recognize the synonymy of Tremiorchis with Astiotrema and presented a detailed life-cycle of T. ranarum revealing that the first intermediate host was the amphibious apple snail, Pila virens (Lamarck) ( Gastropoda: Ampullariidae ), and that the second intermediate hosts were tadpoles of the Indian bullfrog, H. tigerinus , and the Indian skittering frog, Euphlyctis cyanophlyctis (Schneider) (Syn. Rana cyanophlyctis [Schneider]) ( Anura : Dicroglossidae ), which within the former sporocysts produce xiphidiocercariae. Pojmańska (2008) and Pojmańska et al. (2008) considered Tremiorchis and Astiotrema separate genera; Pojmańska et al. (2008) concluded Astiotrema a genus incertae sedis in the Plagiorchioidea (sensu lato) Pojmańska, 2008 whilst Pojmańska (2008) reassigned Tremiorchis to the Brachycoeliidae , distinguishing it from other brachycoeliids by an elongate body, ceca that terminate anterior to or in the testicular zone, ceca and vitellarium that reach to the level of the testis, and a large distance between the ovary and testes. In addition, T. ranarum and T. varanum were considered as members of Tremiorchis (Pojmańska 2008, figs 12.1, 12.2).

Besprozvannykh et al. (2015) established a genetic characterization and a detailed description of a life-cycle for Astiotrema odhneri Bhalerao, 1936 as well as discussed previously reported life-cycles for species of Astiotrema . This revealed some points of high phylogenetic and ontogenetic importance: (i) the first intermediate host of A. odhneri is a planorbid snail, Anisus (Gyraulus) centrifugops Prozorova & Starobogatov ( Gastropoda: Planorbidae ), while the second intermediate hosts include the ramhorn snail, Helicorbis sujfunensis Starobogatov ( Gastropoda: Planorbidae ), and A. centrifugops , tadpoles of the Dybowski’s frog, Rana dybowskii Günther ( Anura : Ranidae ), and the Chinese sleeper fish, Perccottus glenii Dybowski ( Perciformes : Odontobutidae ); (ii) sporocysts develop in xiphidiocercariae; and (iii) A. odhneri clustered with other 28S rRNA gene sequences of Astiotrema forming a monophyletic clade closer and in a basal position to the Opisthorchioidea, obviously distant and separated from all members of the Plagiorchioidea . Furthermore, Besprozvannykh et al. (2015) disagreed with Shevchenko & Vergun (1960) regarding the interpretation of the life-cycle of Astiotrema monticellii Stossich, 1904 ; the analysis by Besprozvannykh et al. (2015) indicated that Shevchenko & Vergun (1960) most likely described larvae belonging to a member of the Opisthorchioidea rather than a species of Astiotrema . Thus, in their view, the prosobranch (gilled) snail Bithynia leachii (Sheppard) ( Gastropoda: Bithyniidae ) did not represent a first intermediate host for A. monticellii and the development of this parasite includes neither formation of rediae nor cercariae from the Pleurolophocerca group. Regarding the criteria and diagnostic features of Tremiorchis proposed by Pojmańska (2008) (see above), we conclude that these characters are identical with published diagnoses of Astiotrema (sensu stricto); therefore, we do not support placing Tremiorchis within the Brachycoeliidae . Based on the similarities between Tremiorchis and Astiotrema (sensu stricto), particularly in morphology and life history (i.e., observed intermediate hosts and development of a xiphidiocercaria within a sporocyst), we again consider Tremiorchis a synonym of Astiotrema (sensu stricto), consequently validating A. ranarum within Astiotrema (sensu stricto).

Bilqees et al. (2002) created Astioglossimetra for its type species Astioglossimetra karachiensis Bilqees, Khatoon & Khan, 2002 , collected from the intestine of the green sea turtle, Chelonia mydas (Linnaeus) ( Testudines : Cheloniidae ), off the coast of Karachi, Pakistan. Bilqees et al. (2002) differentiated Astioglossimetra from two closely related genera, Astiotrema and Glossimetra Mehra, 1937 , based on the shorter length of the vitelline fields and ceca in the former. Pojmańska et al. (2008) discussed the status of Astioglossimetra and pointed out its resemblance to members of both Astiotrema and Neoastiotrema Tkach, 2008 ; however, the problematic taxonomic status of Astioglossimetra resulted in it being declared temporarily a genus incertae sedis based on the fact that (i) both Astiotrema and Neoastiotrema are known only from freshwater habitats and hosts ( Astioglossimetra was reported from a marine turtle in the northern Arabian Sea), and (ii) the description of the seminal vesicle in A. karachiensis as “smooth or bipartite” is ambiguous in that it may potentially put this genus into two families or even superfamilies. Bilqees et al. (2002) described A. karachiensis based on three specimens from a single host, two of which were fully illustrated with their genital systems in addition to the genital system only of the third specimen (see Bilqees et al. 2002, figs. 1–3). The seminal vesicle in the holotype and one paratype is unipartite (see Bilqees et al. 2002, figs. 1 & 3); whereas, this feature appears to have a slightly sinuous wall (i.e., a somewhat “pinched” appearance) but not a distinct constriction dividing the seminal vesicle into two parts that is more indicative of a bipartite structure in the third figure (see Bilqees et al. 2002, fig. 2). There have been similar cases observed in other species of Astiotrema (see Mehra 1931b, fig. 2; Bhalerao 1936, fig. 4; Tang 1941, fig. 19; Gupta 1954, fig. 5; Dollfus & Simha 1964, fig. 1; Wang 1987, fig. 2) strongly indicating that the seminal vesicle in Astioglossimetra is unipartite. In addition, it has been our experience that the seminal vesicle of many flukes develops by the spiraling or twisting of a more tubular singular structure, where depending on the number of resultant twists, there may be a main chamber (unipartite), two main chambers (bipartite) or three main chambers (tripartite). This development may confuse the issue of its actual structure based on the age of the specimens and how far along the development of this feature may be. The illustrated holotype ( Bilqees et al. 2002, fig. 1) appears to be an early mature specimen whereas the illustrated paratype (fig. 3) represents a fully gravid specimen and both have a unipartite seminal vesicle, thus, the unipartite structure of the seminal vesicle in Astioglossimetra is confirmed. Accordingly, Astioglossimetra appears more closely related to Astiotrema than Neoastiotrema (i.e., unipartite seminal vesicle in the former vs a bipartite one in the latter). As for the recording of Astioglossimetra from the marine turtle C. mydas and in contrast to records of Astiotrema from freshwater habitats and hosts, C. mydas has been known to enter rivers and feed for reasonably long periods of time ( Cammarata & Dronen 2020); consequently, potential infection of C. mydas with a species of Astiotrema is possible. Furthermore, C. mydas belongs to a large, widely distributed and variant group, the Testudines , which represent one of the most common hosts for species of Astiotrema (see Discussion below); therefore, we cannot exclude the presence of marine species of Astiotrema in marine turtles. Bilqees et al. (2002) stated that Astioglossimetra is characterized by having a relatively short longitudinal distribution of the vitellarium from the intestinal bifurcation to a level anterior to the posterior testis and by having short ceca that terminate anterior to or at the level of the posterior testis. Our research clarified that species of Astiotrema exhibit a wide spectrum in the vitelline field length (between pharynx and cecal extremities) and in the location of the cecal ends (from level of anterior testis to near posterior extremity). Thus, the criteria used by Bilqees et al. (2002) for differentiating Astioglossimetra fall within those of Astiotrema and appear to be effective for differentiating species, but not at the generic level. Based on the identical morphology, the sharing of the same host group ( Testudines ), and the closely related localities ( Pakistan & India) reported between Astioglossimetra and Astiotrema , we hereby consider Astioglossimetra to be another synonym of Astiotrema .

Pojmańska et al. (2008) included some vague and problematic genera within the Plagiorchioidea (sensu lato) as genera incertae sedis. As stated earlier, the ambiguous state of Astiotrema is attributed to its closer phylogenetic affinity to opisthorchioids and their positioning together in one cluster distinctly separated from plagiorchioids (see Tkach et al. 2001; Olson et al. 2003; Pojmańska et al. 2008; Tkach 2008; Besprozvannykh et al. 2015; de León & Hernández-Mena 2019) despite Astiotrema resembling the latter in its morphology and life-cycle patterns (see Pojmańska et al. 2008; Tkach 2008; Besprozvannykh et al. 2015). For interpreting the ambiguous phylogenetic status of Astiotrema, Tkach et al. (2001) suggested two variants: either Astiotrema is unrelated to the Plagiorchiidae or it represents a heterogeneous group with polyphyletic origin where some of its members may belong not only to different genera, but even to different families; Galaktionov & Dobrovolskij (2003) adopted the latter variant. Pojmańska et al. (2008) considered Astiotrema as incertae sedis within the Plagiorchioidea (sensu lato). Besprozvannykh et al. (2015) revealed through molecular studies that Astiotrema is phylogenetically closer to opisthorchioids and separate from plagiorchioids (as stated earlier by Pojmańska et al. 2008); however, they considered Astiotrema as incertae sedis within the Opisthorchioidea. Due to the potential polyphyletic origin of Astiotrema and its closely resembling the Plagiorchioidea in morphology and life-cycle patterns, we prefer to consider this genus within the expanded concept of the Plagiorchioidea ( Pojmańska et al. 2008) until such a time as the actual systematic position of Astiotrema can be determined.