Genetic characterization and phylogenetic relationships of Phyllodistomum parasites in Indian subcontinent: insights from freshwater fish and shrimp hosts

Phyllodistomum is the large digenean group of fish parasites, with 25 species described so far in the Indian subcontinent. Here, we redescribed two adult species of Phyllodistomum (P. srivastava Rai 1964 and P. parorchium Jaiswal 1957) collected from freshwater fish Heteropneustes fossilis Bloch, 1974 and Glossogobius giuris Ham, 1822, respectively, and an unknown Phyllodistomum metacercaria from shrimp (Macrobrachium dayanum Henderson, 1893). These parasites were genetically characterized using 28S and first and second internal transcribed spacers (ITS1 and ITS2) regions of the nuclear ribosomal DNA and CoxI region of the mitochondrial (mt) DNA to establish the link between metacercaria and adult. Morphologically, both the unknown metacercaria in shrimp and adult Phyllodistomum srivastava in fish, resembled in terms of crenulated margin of hind body, arrangement of diagonal testes, bipartite seminal vesicle, and compact paired vitelline masses. The two adult parasite species, P. srivastava from P. parorchium, were different in terms of shape and size of the body, ratio of suckers, the absence of crenulated margin of hind body, a single chambered seminal vesicle, and deeply lobed paired vitelline masses, in the former species. Comparison of the 28S, ITS, and mtCoxI sequence data suggested P. srivastava and Phyllodistomum metacercaria belong to the same species, and supported the distinction between P. srivastava and P. parorchium. Exploring the potential impact of Phyllodistomum infection on host behaviour and health would be prospective areas for future research. Supplementary Information The online version contains supplementary material available at 10.1007/s00436-023-07930-3.


Introduction
The genus Phyllodistomum Braun 1899 is a large group of digenean fish parasites, comprising 120 species, under the family Gorgoderidae, Looss 1901. Phyllodistomum spp. are known to parasitize both marine and freshwater fishes, with occasional reports of infestations in amphibians (Campbell 2008). They have global distribution. The genus Phyllodistomum is characterized by broad, tapering fore body and foliate hind body with more or less crenulated body margin (Campbell 2008). Of the 120 species belonging to the genus Phyllodistomum, 25 nominal species, distinguished by conventional morphological characters, are so far reported from freshwater fish in the Indian subcontinent (Table 1) (Jaiswal 1957;Rai 1964;Sarwat 2011;Naz and Siddique 2012;Sen 2014). In previous studies, Pandey (1970) and Rai (1964) collected phyllodistome metacercariae encysted in the hepatopancreas of crustaceans, Macrobrachium dayanum Henderson (1893) and obtained their adults, by performing feeding experiments in fish, Heteropneustes fossilis. They identified these phyllodistomes as P. srivastava and P. lucknowensis.
Digeneans commonly undergo a series of developmental stages involving different hosts, making their life cycles intricate and challenging to study solely through traditional methods (Aghlmandi et al. 2018). Sequence data have proven to be invaluable in elucidating the life cycles of digeneans, which typically exhibit an indirect life cycle. The use of genetic information, such as DNA sequencing of specific regions like 28S rRNA, ITS1, and CoxI, has provided crucial insights into the complex life cycles of these parasitic flatworms (Huston et al. 2018;Rochat et al. 2020;Shamsi et al. 2021a;Shamsi et al. 2021b;Shamsi et al. 2023, Barton et al. 2022Choudhary et al. 2022). Cribb (1987) highlighted that the proper identification of Phyllodistomum spp. is challenging due to significant intra-specific morphological variations and inadequate descriptions of several species. However, recent advances in molecular biology, particularly PCR and sequencing-based molecular techniques, have proven effective in identifying and distinguishing digenean parasites (Blair et al. 1996;Hust et al. 2004;Goswami et al. 2009). These molecular techniques have been particularly useful in elucidating the taxonomic status of Phyllodistomum species from various parts of the world. For instance, molecular studies have successfully identified and distinguished species such as P. centropomi, P. lacustri, P. inecoli, P. cribbi, P. wallacei, and P. spinopapillatum from Mexico; P. kanae, P. parasiluri, and P. elongatum from Japan; P. folium from Spain; P. pseudofolium and P. angulatum from Lithuania and Russia; P. magnificum and P. symmetrochis from Australia; and P. staffordi  Cutmore et al. 2013;Razo-Mendivil et al. 2013;de León et al. 2015a;de León et al. 2015b;Nakao 2015;Urabe et al. 2015;Stunžėnas et al. 2017). However, the molecular study of Phyllodistomum species from India remains entirely unexplored. The ribosomal DNA (rDNA) is helpful in resolving the phylogeny because it is universal and composed of highly conserved as well as variable domains (Tkach et al. 2000;Mwita and Nkwengulila 2010). The internal transcribed spacer regions (ITS1 and ITS2) also include a high degree of inter-and intra-specific genetic variations (Nolan and Cribb 2005;Littlewood 2008;Choudhary et al. 2015). Additionally, the mitochondrial cytochrome oxidase 1 (Cox1) has also been used for distinguishing digenean species and inferring phylogenies (Georgieva et al. 2013). Therefore, by coupling molecular and morphological approaches, uncertainties of existing species could be resolved. Also, the preliminary identification and molecular data allow establishing a link between metacercaria found in shrimp and adult in freshwater fish, not yet elucidated in India.
We hereby present first molecular data to (1) distinguish and identify two Phyllodistomum species and (2) correlate one with its metacercaria, in combination with sequences, and (3) to determine the systematic position.

Parasite collection and identification
During winter months (November to January 2016), 65 live specimens of freshwater fish and shrimp were captured from the Gomti River, Lucknow, India (Fig. 1). To detect adult Phyllodistomum infection, the fish were euthanized through spinal severance and then examined under a stereomicroscope. In addition, numerous specimens of naturally infected Phyllodistome metacercariae from the hepatopancreatic tissue of the freshwater shrimp were collected. This work was conducted under animal ethic approval 20/I/2023/IAEC/LU.

Morphological study
The recovered worms were examined under a light microscope and identified using references from the literature (Yamaguti 1934;Pandey and Agrawal 2013;Pandey and Agrawal 2018). Once identified, they were fixed in 70% ethanol for whole mount preparations, and a small piece was preserved in absolute ethanol for DNA extraction. Permanent mounts were prepared by staining the fixed whole mounts in aceto-alum carmine. Subsequently, the specimens were dehydrated through a graded ethanol series (50%, 70%, 90%, and absolute ethanol), cleared in clove oil, and then mounted on glass slides using DPX mounting medium. Figures were illustrated by drawing tube, attached to a phasecontrast light microscope (Olympus BX-51). All morphometric measurements (in millimeters) were taken with the aid of ocular micrometer. Voucher specimens were deposited at Helminthological Collection of the Zoological Survey of India, Kolkata.

Molecular study
DNA was extracted using Qiagen's DNeasy Blood and Tissue Kit according to the manufacturer protocols. Three nuclear ribosomal loci (28S, ITS1, ITS2) and one mitochondrial cytochrome oxidase subunit I (CoxI) were amplified using the following primer sets: 28S ( (Bowles et al. 1993;Mollaret et al. 2000;Prasad et al. 2008). Each PCR amplification reaction is performed in a final volume of 12.5 μl, containing 10X buffer (100 mM Tris, pH 9.0), 50 mM KCl and 15mM MgCl2, 2.5 U Taq polymerase enzyme, 10 mM of each deoxynucleotide triphosphates (dNTPs), and 3μl DNA. The PCR conditions are as follows: initial denaturation at 94 °C for 5 min, annealing for 28S at 54 °C (1 min), ITS-1 at 54 °C (1.10 min), ITS-2 at 57 °C (1.10 min), mt CoxI at 56 °C (1.10 min), and final extension at 72 °C for 10 min. PCR products are checked on 2% agarose gel in TAE buffer, stained with ethidium bromide (EtBr) and visualized under UV light. The acquired PCR products were purified and subjected to Sanger sequencing in the forward direction, using an ABI3730xl DNA Analyzer (Applied Biosystems, Foster City, CA).
Phylogenetic trees were constructed (Table 2) using MEGA 11 (Tamura et al. 2013). Neighbor-joining (NJ) and maximum likelihood (ML) analysis were performed for each data set (28S, ITS1, ITS2, and mtCoxI). The best nucleotide substitution modal was estimated "Kimura 2-parameter model" for NJ tree and "General time reversible model" for ML trees with a gamma distribution of rates and proportion of invariant sites (GTR+G+I). The reliability of internal branches in all trees was evaluated by using the bootstrap method, 1000 replicates.

Results
Based on their morphological features, the specimens of Phyllodistomum were identified as P. srivastava, P. parorchium, and one unknown Phyllodistomum metacercaria (Fig. 2). Details of the parasites found in the present study are provided in Table 3.

Morphological redescriptions
Measurements of the specimens found in the present study (n = 10) are given in millimeters in Table 4. P. srivastava Rai, 1964 (Fig. 1a) Description: Body aspinose, elongated, pyriform, hind body with more or less crenated margin. Oral sucker globular, subterminal or terminal, pharynx absent, esophagus straight, thin, long, bifurcates into two intestinal ceca, extending up to hind end of the body. Ventral sucker larger then oral sucker situated in the middle of the body. Testes deeply lobed, oblique, intercecal, post-equatorial, tandem, vas deferens leading to bipartite vesicula seminalis, ejaculatory duct short, inconspicuous, opening into the genital atrium, cirrus lacking. Ovary subglobular or lobed, intercecal, anterior to left testis. Vitellaria paired compact masses, slightly lobed, round or oval, between ventral sucker and ovary. Laurer's canal not observed. Mehlis' gland small, between vitelline masses. Uterus extensively coiled, occupying entire hind body, intercecal or extracecal, occupying posterior half of hind body, uterine coils extending posteriorly beyond ceca, filled with eggs, genital pore sub-median. Excretory bladder tubular, opening by a median excretory aperture in the caudal notch. Eggs small, operculated. P. parorchium Jaiswal, 1957 (Fig. 1b) Description: Body aspinose, less crenulated margin, broad, oral sucker globular, subterminal or terminal, ventral sucker rounded, equatorial, larger than an oral sucker, pharynx absent, esophagus tubular, leading into two broad intestinal
In the phylogenetic trees built based on these regions (Fig. 3), metacercaria found in the present study consistently grouped with P. srivastava, with P. parorchium grouping closely but distinctly from them.

Discussion
The taxonomic status of two species of Phyllodistomum in India was investigated through a comprehensive analysis combining morphology and molecular techniques. The morphological characteristics of the specimens closely resembled the original description of P. srivastava, in terms of the ratio of suckers, diagonal arrangement of Fig. 2 Phyllodistomum spp. found in the present study: A Adult P. srivastava, B detail of male and female reproductive complex of adult P. srivastava, C adult P. parorchium, D detail of male and female reproductive complex of adult P. parorchium, E metacercaria of Phyllodistomum sp. Note hind body with more or less crenated margin, elongated narrow esophagus, lobed testes positioned asymmetrically, and ovary with smooth margin in P. srivastava versus hind body with broad less crenulated margin, tubular esophagus, deeply lobed testes positioned almost symmetrically and lobulated ovary in P. parorchium  testes, bipartite vesicula seminalis, and paired vitellaria (Table 4). However, some minor differences in body shape were observed, which could potentially be attributed to variations in fixation and mounting techniques used during the study. A consistent finding in our measurements was that the specimens appeared smaller compared to those documented by Rai (1964). Additionally, Rai (1964) reported presence of papillae in live specimen which gradually disappeared after fixation. This observation underscores the importance of considering the impact of fixation on morphological features, as it could lead to changes or loss of certain characteristics, potentially affecting the accuracy of taxonomic comparisons. Cribb (1987) described Pseudophyllodistomum johnstoni which included five previously described species of Phyllodistomum. These species were reclassified under the new genus as follows: P. macrobrachicola (Yamaguti 1934) comb. nov., P. lesteri (Wu, 1938 comb. nov., P. srivastava (Rai 1964) comb. nov., P. lucknowense (Pandey 1970) comb. nov., andP. mingense (Tang, 1985) comb. nov. However, there are clear distinguishing features between P. johnstoni and P. srivastava. P. johnstoni which can be readily distinguished from P. srivastava by shape and size of body, its nearly equal oral and ventral sucker, single chambered seminal vesicle, and saccular excretory bladder. Additionally, P. johnstoni occur as a parasite of the urinary bladder of the Australian freshwater fish Leiopotherapon unicolor, whereas the P. srivastava recovered from the Indian cat fish Heteropneustes fossilis (Bloch 1974).
In case of P. parorchium, the present specimens closely resembled the description given by Jaiswal (1957) in terms of the ratio of suckers, position of genital pore, shape of testes, ovary, and vitellaria (Table 4). However, there are significant distinguishing traits between P. srivastava and P. parorchium. These characteristics include shape and size of body, absence of a crenulated margin on the hind body, presence of a large ventral sucker, a single chambered seminal vesicle, and deeply lobed vitellarium. Pandey (1970) described metacercaria od P. lucknowense, which was found in the same host, Macrobrachium dayanum (Henderson). The metacercaria P. lucknowense differs from P. srivastava in several aspects, such as the presence of a crenulated body margin, a spinose body, a saccular vesicula seminalis, and shape of the testes. Additionally, the present specimens of P. srivastava also differ from P. lucknowense Pandey 1970 by spinose body, saccular vesicula seminalis, and size of eggs. So far, only two metacercariae of Phyllodistomum, namely, P. srivastava and P. lucknowense, have been reported in the Indian subcontinent from shrimp, and their adults were obtained experimentally by feeding on Heteropneustes fossilis (Rai 1964;Pandey 1970). The metacercaria and adult of P. srivastava were found in the same locality and recovered from naturally infected hosts. The development of adult P. srivastava in fish is attributed to carnivorous nature of the fish host, H. fossilis, and the availability of insects and crustacean preys in its vicinity.
The comparative morphology of the present metacercaria showed a close resemblance to the adult of P. srivastava Rai 1964, including the ratio of suckers, the arrangement of testes, bipartite vesicula seminalis, and paired vitelline masses, but they differ from P. parorchium which has symmetrical testes, less crenulated margin, and deeply lobed vitelline follicles.
The morphological studies were supported by molecular findings as well. Phyllodistomum over the last decade, particularly for 28S rRNA, ITS 1, and CoxI sequences. Over the last decade, the increasing availability of genetic resources has greatly enhanced our understanding of the diversity and phylogenetic relationships of gorgoderid trematodes (Razo-Mendivil et al. 2013;de León et al. 2015a;de León et al. 2015b;Pinacho-Pinacho et al. 2021).
Regarding the comparison of base pair (bp) differences for different regions, our results align with previous studies. Razo-Mendivil et al. (2013) reported no intra-specific variation in isolates of P. inecoli and P. brevicecum for 28S and ITS1, whereas inter-specific variation ranged from 3.2 to 4.4% for 28S r DNA and from 8.3 to 14.7% for ITS1. In contrast, Petkevičiūtė et al. (2015) observed 0.3% intra-specific variation for P. folium and 0.5% for P. macrocotyle in 28S sequences. European species of Phyllodistomum, on the other hand, exhibited high inter-specific variation (8.5% to 14%). In another study by Petkevičiūtė  (2015), no intra-specific variation was found in 28S sequences of different isolates of European species P. folium.
In general, the intra-specific variation within the ITS region and mt CoxI is generally low, but consistent inter-specific differences exist (Nolan and Cribb 2005;Pinacho-Pinacho et al. 2021). To obtain a clear understanding of exact number of Phyllodistomum species in India, further studies are necessary. These studies should encompass both morphological and molecular analyses of metacercariae and adults, which will significantly contribute to the phylogenetic revision of gorgoderids. The combination of these approaches will provide valuable insights into the taxonomy and evolutionary relationships of these trematode species. Certain parasites with indirect life cycles possess the capability to manipulate the behaviour of their hosts (Freire et al., 2022), thereby aiding in their transmission to definitive hosts. Given the discovery of Phyllodistomum metacercariae in shrimp and adults in fish during the current study, an interesting area for future research would involve exploring the potential impact of Phyllodistomum infection on shrimp behaviour. Certain digenean parasites, including Clinostomum and Paramphistomum, are recognized for their migration through gastrointestinal tissues before reaching adulthood in the host's stomach (Rolfe et al. 1994, Shamsi et al. 2013. This process leads to pathogenic effects in definitive hosts. Hence, an additional prospective area for future research lies in examining the health consequences and pathogenicity induced by these parasites in their fish hosts.