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Chelatrematidae n. fam., a new family of digenetic trematodes from the South Western Ghats, India, erected on the basis of morphological and molecular studies

Published online by Cambridge University Press:  14 July 2022

P.J. Jithila Open the ORCID record for P.J. Jithila [Opens in a new window] ,
D.M. Atopkin  and
P.K. Prasadan Open the ORCID record for P.K. Prasadan [Opens in a new window]
P.J. Jithila
Affiliation:
Ecological Parasitology and Tropical Biodiversity Laboratory, Department of Zoology, Kannur University, Mananthavady Campus, Wayanad – 670645, Kerala, India
D.M. Atopkin
Affiliation:
Federal Scientific Center of East Asian Terrestrial Biodiversity, Far Eastern Branch of Russian Academy of Sciences, Vladivostok, Russia Institute of the World Ocean, Far Eastern Federal University, Vladivostok, Russia
P.K. Prasadan*
Affiliation:
Ecological Parasitology and Tropical Biodiversity Laboratory, Department of Zoology, Kannur University, Mananthavady Campus, Wayanad – 670645, Kerala, India
*
Author for correspondence: P.K. Prasadan, E-mail: prasadanpk@kannuruniv.ac.in
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Abstract

On the basis of the morphological characterization of Chelatrema neilgherriensis Manjula & Janardanan, 2006 recovered from the freshwater fish Barilius gatensis (Valenciennes, 1844) in the Wayanad region of the Western Ghats, the diagnostic features of the genus Chelatrema Gupta & Kumari, 1973 have been modified. Based on the phylogenetic analysis of C. neilgherriensis and comparative morphology studies relative to members of other families of Gorgoderoidea Looss, 1901, this genus is placed in a new family Chelatrematidae n. fam. The studies revealed the molecular and morphological closeness of Chelatrema with Paracreptatrematina limi Amin & Myer, 1982, and the latter is transferred to this new family. Hence the new family Chelatrematidae n. fam. comprises the genera Chelatrema and Paracreptatrematina.

Keywords

ChelatremaChelatrematidaephylogenetic analysis28s rdnaIndia
Type
Research Paper
Information
Journal of Helminthology , Volume 96 , 2022 , e47
Copyright
Copyright © The Author(s), 2022. Published by Cambridge University Press

Introduction

The composition of the superfamily Gorgoderoidea Looss, 1899 is always contentious. According to Bray & Blair (Reference Bray, Blair, Bray, Gibson and Jones2008) there are no outstanding morphological autapomorphies for this superfamily. The hosts and sites of infection vary greatly within this group and it can be found virtually in all vertebrate groups from elasmobranchs to mammals.

According to Odening (Reference Odening1974) this superfamily contained only a single family, Gorgoderidae Looss, 1899. Later the molecular phylogenetic analyses of Trematoda by Olson et al. (Reference Olson, Cribb, Tkach, Bray and Littlewood2003) added seven more families to the superfamily Gorgoderoidea: Callodistomidae Odhner, 1910; Dicrocoeliidae Looss, 1899; Encyclometridae Mehra, 1931; Haploporidae Nicoll, 1914 (with Atractotrematidae, Yamaguti, 1931 nested within it); Orchipedidae Skrjabin, 1913; Paragonimidae Dollfus, 1939; and Troglotrematidae Odhner, 1914. Curran et al. (Reference Curran, Tkach and Overstreet2006) opined that the superfamily Allocreadioidea Looss, 1902 should be treated as a junior synonym of Gorgoderoidea and Allocreadiidae Looss, 1902 should, therefore, come under Gorgoderoidea. At the same time the families Haploporidae and Atractotrematidae which were included earlier (Olson et al., Reference Olson, Cribb, Tkach, Bray and Littlewood2003) under Gorgoderoidea should be recognized in a separate superfamily Haploporoidea. Choudhury et al. (Reference Choudhury, Valdez, Johnson, Hoffmann and Pérez-Ponce de León2007) pointed out that Allocreadiidae is closely associated with Callodistomidae and Gorgoderidae. Bray & Blair (Reference Bray, Blair, Bray, Gibson and Jones2008) followed the classification proposed by Olson et al. (Reference Olson, Cribb, Tkach, Bray and Littlewood2003) but for ‘convenience of identification’ several other families, Anchitrematidae Mehra, 1935, Brachycoeliidae Looss, 1899, Braunotrematidae Yamaguti, 1958, Collyriclidae Ward, 1917, Cortrematidae Yamaguti, 1958, Mesocoeliidae Dollfus, 1929 and Prouterinidae Foreyt, Schell & Beyer, 1996, were added to Gorgoderoidea on the basis of morphological similarities to the families recognized through molecular studies. Later, Heneberg & Literák (Reference Heneberg and Literák2013) transferred the family Collyriclidae to the superfamily Microphalloidea Ward, 1901 based on phylogenetic analyses of 18S and 28S rDNA sequences. According to Kanarek et al. (Reference Kanarek, Zaleśny, Sitko and Tkach2014) the family Cortrematidae should be considered among the synonyms of Pleurogenidae Looss, 1899. Tkach et al. (Reference Tkach, Achatz, Hilderband and Greiman2018) synonymized the family Anenterotrematidae Yamaguti, 1958 with Dicrocoeliidae. More molecular studies are required to understand and establish the accurate taxonomic positions of these parasite groups.

The genus Chelatrema Gupta & Kumari, Reference Gupta and Kumari1973 was erected for Chelatrema smythi Gupta & Kumari, Reference Gupta and Kumari1973 from the freshwater fish, Chela bacala (Hamilton, 1822) of Ropar, India. Gupta & Kumari (Reference Gupta and Kumari1973) described this genus as a member of the family Hemiuridae Looss, 1899, as it possesses a number of characters relevant to the family. Later, Manjula & Janardanan (Reference Manjula and Janardanan2006) described a new species, Chelatrema neilgherriensis Manjula & Janardanan, Reference Manjula and Janardanan2006, from freshwater fishes of Noolpuzha river, Kerala, India. Campbell (Reference Campbell, Bray, Gibson and Jones2008) stated that Chelatrema appears to be a member of the family Gorgoderidae Looss, 1899, but should be considered as genus Inquirendum, of Gorgoderidae, pending further study. In the present study we provide the first molecular data for a member of Chelatrema – C. neilgherriensis from Barilius gatensis (Valenciennes, 1844) in the Wayanad region of the Western Ghats, India and the results of the studies on phylogenetic relationships of this genus with other members of the superfamily Gorgoderoidea Looss, 1901. On this basis, a new family, Chelatrematidae n. fam., is proposed to accommodate the genus Chelatrema.

Material and methods

Isolation and study of parasite

Live specimens of B. gatensis were collected from water bodies of Wayanad (between North 11′27′ and 15′58′ and East 75′47′ and 70′27′), Kerala, India. The specimens were dissected in physiological saline (0.75% sodium chloride solution) under a Labomed (Luxeo 4Z) stereozoom microscope. Parasites were transferred to a Petri dish containing saline, and live parasites (both unstained and neutral red-stained) were observed under a Nikon ECLIPSE Ni–U phase contrast Research Microscope (Japan) to study the morphological characteristics. Permanent whole mounts were prepared by fixing them in 5% formalin under slight cover glass pressure and staining with acetocarmine following Cantwell (Reference Cantwell and Clark1981). The infection parameters such as prevalence and mean intensity were calculated following Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997). Photographs were taken with Nikon Y-TV55 camera and Nikon NIS Elements imaging software attached to the microscope. Figures were drawn with Nikon Y-IDT drawing tube and measurements (in μm) with mean in parentheses were taken with Nikon NIS Elements imaging software.

DNA extraction, amplification and sequencing

A single adult specimen preserved in 96% ethanol was used for molecular analysis (table 1). Total DNA was extracted from the individual fluke using a ‘hot shot’ technique (Truett, Reference Truett and Kieleczawa2006). Ribosomal 28S rRNA gene fragment was amplified with the primers 28S_A (5′-TCG ATT CGA GCG TGA WTA CCC GC-3′) and 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) (Tkach et al., Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003; Matejusova & Cunningham, Reference Matejusova and Cunningham2004). Initial polymerase chain reaction (PCR) was performed in a total volume of 25 μl containing 0.25 mm of each primer pair, 25 mg of total DNA in water, 12.5 μl GoTaq Green Master mix (Promega, Madison, Wisconsin, USA). Amplification of a 1200-base pairs (bp) fragment of 28S rRNA gene was performed in a GeneAmp 9700, Applied Biosystems (Waltham, Massachusetts, USA), with a 5-min denaturation at 96 °C, 35 cycles of 1 min at 96 °C, 20 s at 55 °C and 2 min 30 s at 72 °C, and a 7-min extension at 72 °C. Negative and positive controls using both primers were used. PCR products were directly sequenced using an ABI Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, Massachusetts, USA), as recommended by the manufacturer, with the internal sequencing primers described by Tkach et al. (Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003). PCR product sequences were analysed using a GA3500 (Applied Biosystems, Waltham, Massachusetts, USA) genetic analyser at the Federal Scientific Center of the East Asia Terrestrial Biodiversity FEB RAS. The sequence was submitted to the GenBank database (ON493538).

Table 1. List of taxa, incorporated into 28S rDNA based molecular analysis (n – number of sequences).

Alignments and phylogenetic analysis

Ribosomal DNA sequences were assembled with SeqScape v.2.6 software, provided by Applied Biosystems (Waltham, Massachusetts, USA). Alignments and estimations of the number of variable sites and sequence differences were performed using the MEGA 7.1 software (Kumar et al., Reference Kumar, Stecher and Tamura2016). Phylogenetic analysis was performed using the Bayesian algorithm with the MrBayes v. 3.1.2 software (Huelsenbeck et al., Reference Huelsenbeck, Ronquist, Nielsen and Bollback2001). The best nucleotide substitution model, TVM + I+G (Posada, Reference Posada2003) was estimated with jModeltest v. 2.1.5 software (Darriba et al., Reference Darriba, Taboada, Doallo and Posada2012). Bayesian analysis was performed using 10,000,000 generations with two independent runs. Summary parameters and the phylogenetic tree were calculated with a burn-in of 25% of generations. The significance of the phylogenetic relationships was estimated using posterior probabilities (Huelsenbeck et al., Reference Huelsenbeck, Ronquist, Nielsen and Bollback2001). GenBank sequence data for representatives of Gorgoderoidea Looss, 1901 and out group taxa used in molecular analysis, including references and accession numbers are given in table 1.

Results

Chelatrema neilgherriensis Manjula & Janardanan, Reference Manjula and Janardanan2006 (fig. 1)

Type host: Devario neilgherriensis (Day, 1867), Cyprinidae Ranifesque, 1815.

Fig. 1. Chelatrema neilgherriensis Manjula & Janardanan, Reference Manjula and Janardanan2006.

Other hosts: Labeo rohita (Hamilton, 1822), B. gatensis, Cyprinidae.

Site of infection: intestine.

Locality: Niravilpuzha, Varadimoola, Valavayal, Thirunelli, Periya and Makkimala of Wayanad region.

Period of collection: March – April 2019, July 2019 and November 2019.

Prevalence of infection: 12 of 48 (25.00%) B. gatensis examined.

Mean intensity of infection: 1.66 (20 parasites from 12 infected fishes)

Adult worm (based on 18 specimens): elongate body, aspinose, slightly pink, 1375–4976 × 583–2140 (2792 × 1074). Diffused eye spot pigments present in immature specimens, lateral to oesophagus. Oral sucker subterminal, round 234–527 × 217–504 (364 × 359). Ventral sucker round, larger than oral sucker, 228–897 × 237–868 (502 × 489), 354–1289 (797) from oral sucker. Pharynx muscular, 65–153 × 65–153 (102 × 98). Oesophagus 74–322 × 15–134 (164 × 74). Intestinal bifurcation anterior to ventral sucker; caeca terminate near posterior extremity, 1055–3709 × 50–254 (1896 × 109). Excretory bladder I-shaped, extends to level of testes. Two testes: left testis 49–276 × 33–262 (150 × 117) and right testis 45–261 × 31–294 (134 × 113). Cirrus-sac anterior to ventral sucker, post-bifurcal, medially placed; containing bipartite seminal vesicle and ejaculatory duct; 70–191 × 37–107 (144 × 80). Genital pore lateral. Ovary posterior to ventral sucker 66–218 × 61–235 (120 × 107). Uterine seminal receptacle round, lateral to ovary. Vitellerium single compact mass. Uterus fills testicular region, extends extra-caecal up to level of intestinal bifurcation. Metraterm opens at genital pore. Eggs numerous, round to oval, embryonated, 11–63 × 6–44 (34 × 22).

Remarks: Chelatrema neilgherriensis was first described by Manjula & Janardanan (Reference Manjula and Janardanan2006) from freshwater fishes, Danio neilgherriensis (Day, 1867) and L. rohita (Hamilton, 1822), of Wayanad as the second species of the previously monotypic Chelatrema. In the present study, adult worms were collected from a new host, B. gatensis, from the same region. The specimens agree well with C. neilgherriensis in the presence of uterine coils up to the level of caecal bifurcation, presence of diffused eye spot pigments, sucker length ratio and shape of eggs. At the same time, there are discrepancies in morphometric parameters of our worms and C. neilgherriensis from the first description (table 2). Nevertheless, based on location and high morphological similarities we considered the worms from our study to be C. neilgherriensis.

Table 2. Morphological and morphometric comparison of original description of Chelatrema neilgherriensis Manjula & Janardanan, Reference Manjula and Janardanan2006 with worms obtained in the present study.

Genus Chelatrema Gupta & Kumari, Reference Gupta and Kumari1973

Diagnosis: elongate body, medium to large size; tegument thick and smooth; diffused eye spot pigments present or absent. Oral sucker subterminal. Ventral sucker larger than oral sucker, pre-equatorial. Pharynx well developed. Oesophagus short. Caeca terminating near posterior extremity. Two testes, diagonal to symmetrical, in hindbody, separated by uterus. Cirrus-sac short, median, postbifurcal, anterior to ventral sucker, enclosing seminal vesicle and cirrus. Genital pore postbifurcal, submedian, slightly anterior to ventral sucker. Ovary pre-testicular, submedian. Uterine seminal receptacle present. Uterus strongly convoluted in intercaecal and extracaecal areas and extends to posterior extremity; sometimes extracaecal up to caecal bifurcation. Vitellarium single, compact, slightly lobed mass, submedian, lateral to ovary. Eggs small, round to oval, numerous, embryonated. Excretory vesicle I-shaped; pore terminal. Intestinal parasites of freshwater fishes, India. Type-species Chelatrema smythi Gupta & Kumari, Reference Gupta and Kumari1973.

Remarks: the genus Chelatrema was proposed in the family Hemiuridae Looss, 1899. Campbell (Reference Campbell, Bray, Gibson and Jones2008) stated that it should be considered as a gorgoderid genus inquirendum until it is re-examined. The study of Manjula & Janardanan (Reference Manjula and Janardanan2006) pointed out similarities and differences of C. neilgherriensis with C. smythi. The notable morphological differences are the presence of diffuse eye spots and extent of uterine coils (which extend extracaecally up to the level of caecal bifurcation) in C. neilgherriensis. Therefore, the diagnosis of Chelatrema was corrected and presented here.

Phylogenetic analysis

A 1261 bp fragment of the 28S rRNA gene was successfully generated for a single specimen of C. neilgherriensis. Alignment of all available 28S rDNA sequences of Gorgoderoidea allows a 933 bp fragment for phylogenetic analysis. Results of Bayesian phylogenetic analysis showed clustering of trematodes according to familial membership within a monophyletic Gorgoderoidea (fig. 2). Chelatrema neilgherriensis was closely related to Paracreptatrematina limi Amin & Myer, Reference Amin and Myer1982; these two species formed a clade, sister to Dicrocoeliidae and Encyclometridae. The genetic P-distance value between C. neilgherriensis and P. limi was 8.67 ± 0.84% which corresponds to internal mean values for most gorgoderoid families, calculated on the basis of the available data set: 6.73 ± 0.44% for Allocreadiidae; 7.52 ± 0.47% for Dicrocoeliidae; and 8.75 ± 0.7% for Encyclometridae Mehra, 1931. An extreme minimum mean value was observed for Callodistomidae Odhner, 1910 (2.64 ± 0.41%); we omit these data because of lack of representative molecular data for this family. Genetic P-distances between different families within Gorgoderoidea from our study ranged from 12.15 ± 0.88% (Allocreadiidae/Callodistomidae) to 20.45 ± 1.05% (Gorgoderidae/Callodistomidae), considerably higher than the P-distance value between C. neilgherriensis and P. limi. Within Gorgoderidae, mean P-distance value by means of 28S rDNA sequence data was 13.52 ± 0.6%, corresponding to interfamilial divergence level. Accepting that the Gorgoderidae clade showed internal differentiation into four highly supported and divergent subclades, this family represents a gorgoderoid group requiring comprehensive studies taxonomically and phylogenetically. On the basis of our molecular results, we propose that C. neilgherriensis and P. limi represent members of a new family of Gorgoderoidea.

Fig. 2. Bayesian phylogenetic tree of Gorgoderoidea reconstructed on the basis of 28S rDNA sequences. Original sequences are in boldface type. Nodal numbers – a posteriori probability values (only significant values presented).

Family Chelatrematidae n. fam.

Elongate body, fusiform, medium to large; tegument thick, smooth; eye spot pigments present or absent. Suckers well developed. Oral sucker subterminal, with or without lobes. Ventral sucker equal or larger than oral sucker, pre-equatorial. Pharynx well developed. Oesophagus short. Caeca extend to near posterior extremity. Testes diagonal, symmetrical, in hindbody, separated by uterus. Cirrus-sac small, postbifurcal, median or lateral to ventral sucker, enclosing seminal vesicle and cirrus. Genital pore median or lateral, slightly anterior to ventral sucker. Ovary submedian, pre-testicular, posterior to ventral sucker. Seminal receptacle present. Uterus extends to posterior extremity, intercaecal or extracaecal; sometimes extracaecal up to caecal bifurcation. Vitellarium variable, single compact mass or small follicles in lateral fields or clearly identifiable vitellaria absent. Eggs small, round to oval, numerous, embryonated. Excretory vesicle I-shaped; pore terminal. In intestine of freshwater fishes. Type genus Chelatrema Gupta & Kumari, Reference Gupta and Kumari1973.

Key to genera

1a. Oral sucker without lobes, smaller than ventral sucker; uterus intercaecal and extracaecal up to posterior extremity; sometimes extracaecal up to caecal bifurcation; vitellarium a single compact mass Chelatrema Gupta & Kumari, Reference Gupta and Kumari1973

1b. Oral sucker with lobes, equal to or slightly larger than ventral sucker; uterus intercaecal up to posterior extremity; vitellarium variable, small follicles in lateral fields or lacking of clearly identifiable vitellaria Paracreptatrematina Amin & Myer, Reference Amin and Myer1982

Discussion

The genus Chelatrema was described in the family Hemiuridae by Gupta & Kumari (Reference Gupta and Kumari1973). Gibson (Reference Gibson, Bray, Gibson and Jones2002) transferred it to family Gorgoderidae Looss, 1899. Later, Campbell (Reference Campbell, Bray, Gibson and Jones2008) stated that it should be considered as gorgoderid genus inquirendum pending for the study. The genus Paracreptatrematina Amin & Myer, Reference Amin and Myer1982, from freshwater fish of the United States, was described in the family Allocreadiidae. According to Platta & Choudhury (Reference Platta and Choudhury2006) P. limi has unique oral muscular papillae (compared with other papillose allocreadiids) and absence of clearly identifiable vitellaria. Curran et al. (Reference Curran, Tkach and Overstreet2011) concluded that P. limi does not belong to Allocreadiidae on the basis of molecular analysis. Members of the new family differ from each other mainly in the presence of a ventrolateral pair of triangular lobes in P. limi that are absent in Chelatrema. Curran et al. (Reference Curran, Tkach and Overstreet2011) did not include P. limi under any named family nor its own family due to shortage of data related to the life history and genera that are closely related to this species.

Comparison of morphological characters of the proposed new family with those of other families under Gorgoderoidea as given by Bray & Blair (Reference Bray, Blair, Bray, Gibson and Jones2008) showed some major difference in shape and position of testes and ovary, position of uterus, distribution of vitellarium and presence or absence of cirrus-sac. Members of the new family lack any papillae or spines on the tegument, which were reported in several members of Gorgoderidae, Brachycoeliidae Looss, 1899, Dicrocoeliidae, Mesocoeliidae, Paragonimidae, Prouterinidae and Troglotrematidae. The vitellarium in members of Dicrocoeliidae and Encyclometridae is usually limited in extent, forms two lateral bands or clusters, or rarely one band (Gupta & Mehrotra, Reference Gupta and Mehrotra1977; Tkach et al., Reference Tkach, Achatz, Hilderband and Greiman2018). Members of Chelatrema usually possess a single vitellarium, while P. limi has small follicles in lateral fields. Platta & Choudhury (Reference Platta and Choudhury2006) reported the absence of clearly identifiable vitellaria in P. limi.

In the present study, close phylogenetic relationships between C. neilgherriensis and P. limi have been revealed. Alongside with genetic-P distance analysis, these two species can be recognized as representatives of a new family within the Gorgoderoidea. Paracreptatrematina limi was described for the first time as a member of a new genus of Allocreadiidae from the mud-minnow Uumbra limi (Kirtland, 1840) from three states of the United States (Amin & Myer, Reference Amin and Myer1982). The first representative taxonomic and phylogenetic studies on this species with a molecular tool were made by Curran et al. (Reference Curran, Tkach and Overstreet2011), who showed an independent phylogenetic position of P. limi relative to Allocreadiidae and stated that this species was not an allocreadiid, but, probably, a member of its own family. Our results confirm this view, demonstrating Chelatrema from India as a species closely related to P. limi. Cases of close relationships of trematodes from Eurasian and North American continents have been observed for gorgoderoids. For example, detailed phylogenetic analysis of Allocreadiidae indicated such relationships, where North American species belong to a terminal clade, sister to Asian and European species within this family (Soldánová et al., Reference Soldánová, Georgieva, Roháčová, Knudsen, Kuhn and Henriksen2017; Petkevičiūtė et al., Reference Petkevičiūtė, Stunžėnas, Zhokhov, Poddubnaya and Stanevičiūtė2018; Atopkin et al., Reference Atopkin, Sokolov, Vainutis, Voropaeva, Shedko and Choudhury2020; Faltýnkova et al., Reference Faltýnkova, Pantoja, Skírnisson and Kuldai2020; Vainutis et al., Reference Vainutis, Voronova and Urabe2021; Bogatov & Vainutis, Reference Bogatov and Vainutis2022). However, lack of molecular data on species, closely related to C. neilgherriensis and P. limi as well as for type-species of Chelatrema is a barrier to clarify these relationships. Moreover, accepting these two species as representatives of a separate new family, presence and origin of considerable morphological differences of its members should be further considered. Representative data on closely related trematodes for this new family are strongly needed to address these questions.

Financial support

This work was supported by Kerala State Council for Science, Technology and Environment (KSCSTE) as a research fellowship for one of the authors (P.J. Jithila) (KSCSTE/972/2018-FSHP-MAIN Dated 23/01/2019) and is a part of the state-supported studies in the Federal Scientific Center of the East Asia Terrestrial Biodiversity of FEB RAS (Project No. 121031000154-4).

Conflict of interests

None.

Ethical standards

All procedures performed in the study involving animals were in accordance with the ethical standards of the institution or practice at which the study was conducted.

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Figure 0

Table 1. List of taxa, incorporated into 28S rDNA based molecular analysis (n – number of sequences).

Figure 1

Fig. 1. Chelatrema neilgherriensis Manjula & Janardanan, 2006.

Figure 2

Table 2. Morphological and morphometric comparison of original description of Chelatrema neilgherriensis Manjula & Janardanan, 2006 with worms obtained in the present study.

Figure 3

Fig. 2. Bayesian phylogenetic tree of Gorgoderoidea reconstructed on the basis of 28S rDNA sequences. Original sequences are in boldface type. Nodal numbers – a posteriori probability values (only significant values presented).