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International Journal of Zoology and Animal Biology Research Article 30 min read

Morphological and Molecular Evidence of a Trematode Parasite Infected the Liver of Garfish Xenentodon cancila (Beloniformes: Belonidae) in India

Chaudhary A*, Singh K, Sharma B and Singh HS
* Corresponding author
ISSN: 2639-216X  10.23880/izab-16000437  Received: December 26, 2022  Published: January 30, 2023
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Keywords
Trematode Posthodiplostomum 18S ITS Cluster 28S Meerut India
Abstract

Morphological traits to describe trematode parasites are sometimes difficult to identify and validate a species, especially in cases where many species are described from a single host. The current work uses molecular data to describe diplostomoid metacercariae supplemented with morphology found in freshwater garfish, Xenentodon cancila (Hamilton) collected from the River Ganga in district Bijnour, Meerut (Uttar Pradesh), India. The metacercariae were identified as Posthodiplostomum species, collected from the liver showed a high mass of cysts embedded in and surrounded by partially damaged liver tissue. Partial DNA sequences of the 18S, internal transcribed spacers (ITS1–5.8S–ITS2), and 28S of nuclear ribosomal DNA were generated and compared with available sequences of other congeners on the Genbank database. The phylogenetic analysis of 18S, the ITS cluster (ITS1–5.8S–ITS2), and 28S rDNA of Posthodiplostomum sp. from India fell within the superfamily Diplostomoidea, along with other members of Posthodiplostomum, which confirms its distinct status and places it close to other Indian species. In the Indian region, with morphology alone, many species are described as Neascus-type metacercariae that are awaiting their validation to be supplemented with molecular data. Furthermore, the validity of a few species of the genus Posthodiplostomum is also discussed in the present study.

Introduction

Transmission of infectious parasites by the increasing trade of fish poses a risk to human health [1]. One of the important trades of species introduction globally is aquaculture, which causes the transmission of infectious diseases like bacterial and parasitic diseases to wild and aquaculture-raised fish [2, 3, 4]. Piscivorous migratory birds as definitive hosts are also a major factor related to parasite transmission between the continents [5]. Posthodiplostomum Dubois, 1936, is a trematode genus rich in diversity and comprises parasites that are widespread worldwide [6, 7, 8, 9, 10, 11, 12]. Initially, for the identification of strigeid larva types, they were categorized into different types, i.e., Tetracotyle Filippi, 1854; Codonocephalus Diesing, 1850; Neascus Hughes, 1927; and Diplostomulum Brandes, 1892. Hughes (1927) proposed a group of larval trematodes, i.e., Neascus, to contain metacercariae, and Dubois (1936) described the genus Posthodiplostomum, which is in the family Diplostomidae. Parasites of this genus have a three-host life cycle, with snails as first intermediate hosts, fish acting as second intermediate hosts, and the fish-eating birds as definitive hosts [11, 13]. According to research, during the initial phase of infection, metacercaria penetration and migration cause mechanical damage, hemorrhage, and other secondary bacterial infections in fishes [15]. Infection caused by these trematodes is called black-spot disease and may have its origins in fish muscles due to the deposition of melanocytes around metacercariae [14]. A few studies have mentioned Posthodiplostomum cuticola (Nordmann, 1832) as the causative agent of black-spot disease, and it is one of the common diseases that affect many fish species [15, 16, 17, 18, 19, 20]. Fish infected with black-spot diseases have had reduced growth along with other deformities and mortalities [12, 19, 21, 22]. Sometimes, the presence of metacercaria in the liver of infected fish can be associated with changes in hepatic tissues like parenchymal atrophy, necrosis, and fibrosis that lead to dysfunction in digestion and malnutrition [23]. Additionally, Posthodiplostomum species infection in fishes may cause weight loss, slow developmental processes, and mortality, making them less able to avoid predation by definitive hosts, i.e., fish-eating birds [15, 24, 25, 26].

Many species of Posthodiplostomum have been described worldwide, while more than twenty-five species are reported from India, described as Neascus-type metacercariae and as Posthodiplostomum species [27]. Their life cycle is complex, and it is very difficult to study or link different stages in the laboratory. In India, most of the Posthodiplostomum species are characterised based on morphology alone, which cannot be considered reliable due to a lack of descriptions or deposited specimens. Difficulties in identification can be overcome by the generation of molecular data to link different developmental stages, as suggested in various studies [28, 29, 30]. Molecular identification would eventually improve our understanding of the correct diversity of Posthodiplostomum species that are scarce and limited, especially in India and help in linking the adult and metacercariae stages.

As part of an ongoing survey of diplostomid metacercaria (Neascus type) from freshwater fishes in India, Posthodiplostomum species was collected in the liver of the garfish Xenentodon cancila with severe liver infection. A combined morphological, histological, and molecular analysis using the 18S, ITS cluster (ITS1–5.8S–ITS2), and 28S rDNA gene sequences was provided to evaluate the phylogenetic position of our specimens in relation to other closely related species.

Materials and Methods

Parasite Collection and Morphology

During a helminthological survey in Bijnor (29° 23′ N, 79° 11′ E), Uttar Pradesh, India, between November 2020 and May 2021, a total of 57 garfish, Xenentodon cancila (Hamilton, 1822) (Beloniformes: Belonidae), were collected. After that, fish internal organs were examined with the aid of a stereomicroscope (Motic SMZ-168 series, Xiamen, People’s Republic of China) to detect the infection of parasites. Metacercariae were found to infect the liver, removed from the cysts with the help of a fine needle, and placed in a 0.9% saline solution for approximately 10–15 minutes. The morphology of metacercariae was investigated using both live and preserved specimens. For a live study of morphology, metacercariae were placed on a slide using a pipette in physiological saline (0.9%), coverslipped, and photographed by a Nikon Eclipse Ts2  microscope. Some of them were fixed in 10% formalin for permanent slide preparation, and some were immediately fixed in 95% ethanol for molecular analysis. For permanent slide preparation, they were stained with aceto-alum carmine, dehydrated in ascending grades of ethanol, cleared in xylene, and mounted in Canada balsam. Measurements were taken with the help of the digital images and NIS Elements 5.10 image analysis software. All measurements are given in millimeters. Abbreviations of the measurements are provided in the Table 2. Voucher specimens were deposited at the museum collection of the Department of Zoology, Chaudhary Charan Singh University, Meerut (U.P.), India, and at the Museum d’Histoire Naturelle, Geneva, Switzerland.

The infected fish liver sections were preserved immediately in 4% formalin and processed for histopathology. The samples were washed in 80% ethanol numerous times, implanted in paraffin wax, cut into 5- to 8-m-thick sections, and stained with hematoxylin and eosin. The histological sections were photographed by a Nikon Eclipse T_s_2 microscope equipped with NIS Elements 5.10 image analysis software.

DNA Isolation and Amplification

DNA extraction from ethanol-fixed metacercariae (n= 02) was performed using the QIAGEN DNeasyTM tissue kit (Qiagen, Hilden, Germany) as per manufacturer’s instructions. Sequences of the 18S, ITS cluster (ITS1–5.8S– ITS2), and 28S rDNA were obtained by PCR amplification according the primers suggested by Shinad et al. [31]. For all primers, PCR conditions were also performed according to Shinad et al. [31]. PCR products were then purified using Purelink™ Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Löhne, Germany) according to the manufacturer’s instructions. Sequencing reactions were performed in both directions using the ABI Big Dye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, California, USA) from the above mentioned primers and using the primers BD1 and BD2 [32] for the ITS cluster (ITS1-5.8S-ITS2) to acquire a long stretch of sequences.

Phylogenetic Analysis

The generated sequences were visualized, assembled, and edited using BioEdit software [33]. Sequences of other diplostomids available at GenBank were retrieved after a BLASTn search. The sequences used in the phylogenetic analyses are presented in Table 1. The obtained sequences were aligned separately using ClustalW, which was implemented in MEGA vr. 11 [48]. Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI) algorithms. The best nucleotide substitution models for 18S, ITS cluster (ITS1-5.8S-ITS2), and 28S rDNA data sets were invariant sites and gamma-distributed among site variation (GTR + I + G). Pairwise comparisons for each region were estimated using MEGA v. 11. The ML trees were computed in MEGA vr. 11 and nodal support values were estimated from 1000 bootstrap pseudo-replicates. The BI analyses were carried out in TOPALi [49] using Markov chain Monte Carlo (MCMC) searches with two concurrent runs of four chains for 1,000,000 generations, with every 100th tree saved. The first 25% of the sampled trees were discarded as “burn-in.” Tylodelphys species was selected as an out group for the 18S, ITS1–5.8S–ITS2 region, and 28S rDNA gene analyses.

Results

During the study, a total of 57 garfish specimens were collected, of which 54 were found infected with metacercariae. Metacercariae were found in the liver tissue of the infected fish in the form of numerous small cysts; the cyst wall was thin and delicate (Figure 1). Each fish was parasitized by 50–190 metacercariae. The excysted specimens of the present study clearly showed the morphological features of the genus Posthodiplosomum Dubois, 1936 and of species Posthodiplostomum pandei (=Neascus pandei) Rai and Pande, 1964 [50].

Morphological Description of Posthodiplostomum pandei (=Neascus pandei) Rai and Pande, 1964 (Figures 1-10)

Figure 1
Click to enlarge
Figure 1

Figures 1-10: Posthodiplostomum pandei (1,2) Encysted metacercariae embedded in liver tissue; (3) Histology of the liver shows encysted metacercaria within the liver tissue layer (arrow head); (4-6) Encysted metacercaria collected from X. cancila’s liver tissue; (7) Metacercaria exit from the cyst; (8, 9) Excysted metacercaria (10) Line drawing. Scale bars (1, 2) 100 µm; (3-6) 50 µm; (7-10) 100 µm.

Metacercariae (n=  14). Encysted metacercariae with a thin, single layer of cystic wall. Spatulate, divided into two parts, or bipartite body of excysted metacercaria. Leaf-shaped forebody, larger than the hindbody. Hindbody are typically bulb-shaped. Pseudosuckers absent. At the anterior end, sub- terminal, there is a spherical-shaped oral sucker. The ventral sucker is spherical, larger than the oral sucker, and situated in the body’s center, anterior to the holdfast organ. The holdfast organ is located near the forebody’s posterior margin. The distinction between genital primordials is poor. The testes are divided into two parts: the anterior and posterior testes. Ovary intra-testicular. The excretory bladder is oval and terminates with a genital pore.

All the measurements of Posthodiplostomum species collected in the present study were compared in their morphology with those of other congeneric species in Table 2. Based on the morphology, the present species is recognised and differentiated as Neascus pandei Rai and Pande, 1964, and molecular biology confirms it as a Posthodiplostomum species. In histological sections, the cysts of P. pandei (=N. pandei) Rai and Pande, 1964 were found inside the liver and had a heavy infection (Figure 3).

  • Host: Garfish, Xenentodon cancila Hamilton, 1822 (Beloniformes: Belonidae).
  • Locality: Bijnor (29° 23′ N, 79° 11′ E), Uttar Pradesh, India.
  • Site of infection: Liver (cysts embedded in the liver tissue).
  • Prevalence in intermediate hosts: 95% (54 infected out of 57 hosts).
  • Specimens deposited: Voucher specimens were submitted to the collection of the Museum, Department of Zoology, Chaudhary Charan Singh University, Meerut (U.P.), India (HS-TR/2022/01) and to the Museum d’Histoire Naturelle, Geneva, Switzerland (MHNG- PLAT-0144332).
  • Representative DNA sequence: The newly generated sequences were deposited in GenBank under the following accession numbers: 18S rDNA gene: OP324624 (1068 bp), OP324628 (1079 bp); ITS1-5.8S-ITS2 region: OP324627 (1190 bp), OP324629 (1202 bp); 28S rDNA gene: OP324625 (1019 bp), OP324626 (1001 bp).

Remarks

Based on the shape of body parts and morphometric measurements, we identified our specimens as Posthodiplostomum pandei (=Neascus pandei) Rai and Pande, 1964. Many descriptions of Posthodiplostomum or Neascus-like metacercaria from X. cancila liver show inadequate data that results in taxonomic confusion in the identification of species, which leads to the addition of new species. Many previous studies from India were based solely on morphology and described as Neascus-type metacercaria species. In India, about twenty-six species of Neascus– type metacercariae and five of Posthodiplostomum were described (Table 3). Eleven of these have been reported solely from X. cancila as mentioned in Table 2, although they show minor variations in their morphometry. N. mesentriformis, N. baughi, N. nanaksagrensis, N. simhai, N. moghei, N. vedi, and N. khurramnagarensis have larger body size and width compared to our specimens, which clearly differentiate them from P. pandei (Table 2). There are no substantial morphometrical differences found in the body parts and morphology of N. xenentodoni, N. hepatica, and N. srivastavi with P. pandei, showing they are most closely related in nearly all metrics, which predicts they might be similar/closest species to P. pandei (Table 2). Although N. xenentodoni is close in morphology to P. pandei, as the molecular data of N. xenentodoni becomes available, it will be better to discuss it further. As we are not able to find out the deposited type specimens of N. hepatica and N. srivastavi for further clarification, it is too early to suggest them as synonyms with P. pandei. Thus, we consider P. pandei to be a valid species, and anyhow, these species (N. hepatica and N. srivastavi) should be taken into account in future studies in order to uncover their validity or synonymies with P. pandei.

Phylogenetic Analysis

The amplification and sequencing of the 18S gene of rDNA were successfully performed from two isolates of Posthodiplostomum pandei (=N. pandei) in the present study. The generated sequences were aligned with other diplostomids available in GenBank, as shown in the table (Table 1). The 18S sequences generated in this study show no intraspecific variations (Figure 11). The 18S sequence of P. pandei differed from other Posthodiplostomum species from different geographical regions by 1.5–3.9%. The phylogenetic tree obtained by ML and BI analyses of 18S sequences showed similar topologies. For comparison of the 18S gene with Indian species, only one sequence is available to date from C. punctata (KF738455), which is far away from our specimens. The analysis of 18s gene shows our specimens formed a well-supported clade found close to the Mexican isolate of P. minimum (PMU88074) and showing interspecific divergence of 1.5% (Figure 11).

SpeciesHostLocationGenBank accession no.References
18SITS region28S
Ornithodiplostomum
scardinii		Scardinius
erythrophthalmus		Czech
Republic		KX931443-KX931427Stoyanov, et al.
[11]
O. scardiniiAmpullaceana
balthica		Denmark-MW001049,
MW001051		-Duan, et al. [8]
Ornithodiplostomum sp.HNACanada-KY951727-Blasco-costa, et
al. [29]
Mesoophorodiplostomum
pricei		Morone americanaCanadaHM064959HM064960-Locke, et al. [34]
Posthodiplostomum priceiL. delawarensisUSA--MZ710972Achatz, et al.
[35]
Posthodiplostomum
centrarchi		L. gibbosusSlovakiaKX931442--Stoyanov, et al.
[11]
P. centrarchiL. gibbosusBulgariaKX931441--Stoyanov, et al.
[11]
Posthodiplostomum
centrarchi		L. gibbosusUSA--OM638425Koxlien, et al.
2022
P. centrarchiL. gibbosusHungary-MN080277,
MN080281		-Cech, et al. [7]
P. centrarchiArdea herodiasCanada-MH521251-Locke, et al. [36]
Posthodiplostomum sp.L. gibbosusCanadaHM064958HM064955-Locke, et al. [34]
Posthodiplostomum sp.Micropterus
salmoides		CanadaHM064962--Locke, et al. [34]
Posthodiplostomum sp.L. gibbosusSlovakia-KX931442-Stoyanov, et al.
[11]
Posthodiplostomum
brevicaudatum		Gasterosteus
aculeatus		BulgariaKX931431,
KX931432		--Stoyanov, et al.
[11]
P. brevicaudatumPerca fluviatilisCzech
Republic		--KX931426Stoyanov et al.
2017 [11]
Posthodiplostomum
minimum		HNAMexicoU88074--Campos, et al.
[37]
Posthodiplostomum
pandei†		Xenentodon cancilaIndiaOP324624,
OP324628		OP324627,
OP324629		OP324625,
OP324626		Present study
Posthodiplostomum sp.L. gibbosusCanadaFJ469590--Moszczynska, et
al. [38]
Posthodiplostomum
cuticola		Ardea cinereaCzech
Republic		MK089352--Heneberg, et al.
[39]
P. cuticolaNycticorax
nycticorax		Ukraine--MZ710955Achatz, et al.
[35]
P. cuticolaAnisus vortexDenmark-MW001124,
MW001121		-Duan, et al. [8]
P. cuticolaAbramis bramaHungary-MN080266,
MN080287		-Cech, et al. [7]
Posthodiplostomum sp.L. gibbosusCanada-HM064949-Locke, et al. [34]
Posthodiplostomum sp.Tilapia sparrmaniiSouth
Africa		-MK604881-Hoogendoorn, et
al. [19]
Posthodiplostomum sp.Channa argusJapanAB693170AB693170-Nguyen et al.
2012 [40]
Posthodiplostomum sp.Channa punctataIndiaKF738455KF738447KF738450Athokpam, et al.
[41]
P. minimumLepomis
macrochirus		USAKY809062--Lovy, et al. [42]
P. minimumN. nycticoraxUSA--MZ710962Achatz, et al.
[35]
P. centrarchiA. herodiasCanadaMH521251--Locke, et al. [36]
P. minimumHNAUSAAY245767--Flowers, et al.
2003
Posthodiplostomum sp.Nannopterum
brasilianus		MexicoMF398354-MF398331Hernandez-
mena, et al. [43]
Posthodiplostomum
macrocotyle		Busarellus
nigricollis		Brazil--MZ710958Achatz, et al.
[35]
Posthodiplostomum
microsicya		Tigrisoma lineatumBrazil--MZ710960Achatz, et al.
[35]
Posthodiplostomum
erickgreenei		Pandion haliaetusUSA--MZ710956Achatz, et al.
[35]
Posthodiplostomum
eurypygae		Eurypyga heliasBrazil--MZ710957Achatz et al.
2021 [35]
Posthodiplostomum nanumArdea albaUSA--MZ710963Achatz, et al.
[35]
P. nanumGundlachia ticagaBrazil-MH358392-Lopez-
Hernandez, et al.
[23]
P. nanumPoecilia reticulataBrazil-MH358393-Lopez-
Hernandez,
2018 [23]
Posthodiplostomum sp.A. herodiasUSA--MZ710994Achatz, et al.
[35]
Posthodiplostomum
xenentodoni (=Neascus
xenentodoni)		X. cancilaIndia-KY234201KY234203Choudhary, et al.
[44]
Posthodiplostomum sp.Channa striataVietnam--MT394045Sokolov and
Gordeev, 2020
[45]
Posthodiplostomum sp.Trichopodus
trichopterus		Vietnam--MT394051Sokolov, et al.
[45]
Posthodiplostomum
hanumanthai (=Neascus
hanumanthai)		C. punctataIndia-KY234199KY042122Choudhary, et al.
[44]
Posthodiplostomum gussevi
(=Neascus gussevi)		Colisa fasciataIndia-KY234200KY234202Choudhary, et al.
[44]
Posthodiplostomum
pacificus		Larus californicusUSA--MZ710967Achatz, et al.
[35]
Posthodiplostomum cf.
anterovarium		L. gibbosusUSA--OM688205Koxlien, et al.
2022
P. cf. anterovariumL. gibbosusUSA--MZ710942Achatz, et al.
[35]
Posthodiplostomum cf.
podicipitis		Lophodytes
cucullatus		USA--MZ710969Achatz, et al.
[35]
Posthodiplostomum sp.Physa sp.USA--MZ710982Achatz, et al.
[35]
Posthodiplostomum
recurvirostrae		Recurvirostra
americana		USA--MZ710975Achatz, et al.
[35]
Posthodiplostomum
ptychocheilus		Mergus merganserUSA--MZ710974Achatz, et al.
[35]
Posthodiplostomum sp.Trichopodus
pectoralis		ThailandON614094--Nguyen, 2022
Posthodiplostomum sp.Skiffia lermaeMexico-OK315760,
OK315761		-Perez-Ponce de
Leon, et al. [46]
Posthodiplostomum sp.Allotoca dugesiiMexico-OK315771-Perez-Ponce de
Leon, et al. [46]
Posthodiplostomum sp.Gambusia sp.Mexico-OK315772-Perez-Ponce de
Leon, et al. [46]
Posthodiplostomum sp.Pimephales
promelas		Mexico-OK315775-Perez-Ponce de
Leon, et al. [46]
Outgroups
Tylodelphys immerGavia immerCanadaMH521252--Locke, et al. [36]
Tylodelphys aztecaePodilymbus
podiceps		Mexico--MF398337Hernandez-
mena, et al. [43]
Tylodelphys sp.Aechmophorus
occidentalis		Mexico-MK177831-Sereno-Uribe, et
al. [47]

Table 1: Trematode species are included in the phylogenetic analysis with information on the host, locality and GenBank accession

Body
parts		P. pandei
(=N.
pandei)		P. pandei
(=N.
pandei)		N. mesentri
formis		N. hepaticaP. xenen
todoni
(=N. xenen
todoni)		N. baughiN. nanaks
agrensis		N.
simhai		N.
moghei		N. vediN. sriva
stavi		N. khur
ram
naga
rensis
Refe
rence		Present
study		Rai and
Pande,
1964 [50]		Rai and
Pande, 1964
[50]		Chakrabarti,
1970 [51]		Pandey,
1971 [52]		Baugh and
Chakrabarti,
1977 [53]		Baugh and
Chakra
barti, 1977
[53]		Agrawal
and
Khan,
1982
[54]		Agrawal
and
Khan,
1982
[24]		Pandey
and
Tiwari,
1986
[55]		Pandey
and
Pandey,
2000
[56]		Gupta,
et al.
[57]
Infected
Organ		Cysts in
Liver		LiverMesentryCysts in
Liver		Liver and
cranium		Body cavityCysts in
liver and
gonads		Body
muscles		LiverBody
muscles		LiverLiver
CSTL0.61
(0.49-0.76)		0.51-0.71.4-1.80.72-0.870.52-0.671.32-1.501.05-1.031.17-
1.19		0.99-
1.00		1.13-
1.33		0.58-
0.69		1.13 -
1.33
CSTW0.37
(0.28-0.46)		0.31-0.60.8-0.90.44-0.52-0.65-0.760.50-0.680.76-
0.78		0.62-
0.64		0.90-
1.20		0.41-
0.43-		0.90 -
1.20
WBL0.89
(0.51-1.2)		0.78-0.850.64-0.72---------
WBW0.30
(0.11z-0.42)		0.25-0.34----------
FBL0.62
(0.38-0.86)		--0.46-0.750.67-1.051.28-1.650.78-1.051.07-
1.20		0.97-
0.98		0.47-
1.03		0.54-
0.64		0.47 -
1.03
FBW0.30
(0.11-0.42)		--0.23-0.390.24-0.330.90-1.080.61-0.760.54-
0.60		0.37-
0.38		0.19-
0.21		0.28-
0.44		0.19 -
0.21
HBL0.23
(0.12-0.33)		--0.18-0.260.18-0.300.62-0.820.41-0.650.37-
0.39		0.26-
0.27		0.19-
0.22		0.20-
0.25		0.19 -
0.22
HBW0.16
(0.07-0.20)		--0.15-0.240.14-0.270.56-0.710.53-0.700.27-
0.22		0.28-
0.29		0.13-
0.19		0.18-
0.27		0.13 -
0.19
OSL0.02
(0.01-0.03)		0.024-0.040.04-0.050.04-0.07-0.05-0.080.04-0.060.05-
0.06		0.07-
0.75		0.03-
0.04		0.02-
0.04		0.03 -
0.04
OSW0.02
(0.01-0.02)		0.024-0.040.04-0.050.03-0.05-0.03-0.05-0.03-
0.04		-0.02-
0.03		0.02-
0.03		0.02 -
0.03
VSL0.04
(0.01-0.05)		0.04-0.060.04-0.050.05-0.090.04-0.070.06-0.090.06-0.090.09-
0.10		-0.03-
0.04		0.04-
0.06		0.03 -
0.04
VSW0.04
(0.02-0.05)		0.04-0.050.05-0.09-0.06-0.09-0.07-
0.08		-0.02-
0.03		0.05-
0.08		0.02
-0.03
HOL0.12
(0.06-0.18)		0.12-0.140.14-0.16----0.14-
0.16		--0.12 -
0.17
HOW0.14(0.06-
0.19)		0.12-0.14--------0.10 -
0.16
ATL0.05
(0.02-0.07)		0.06-0.07-0.10-0.15-0.20-0.260.17-0.200.10-
0.12		0.13-
0.14		0.02-
0.07		0.03-
0.05		0.02-
0.07
ATW0.07
(0.03-0.10)		0.02-0.03-0.06-0.10-0.25-0.320.16-0.230.13-
0.14		0.04-
0.045		0.10-
0.16		0.06-
0.09		0.06 -
0.12
PTL0.06
(0.03-0.07)		0.08-0.1-0.16-0.22-0.41-0.540.35-0.400.23-
0.25		0.13-
0.14		0.01-
0.03		0.03-
0.05		0.01 -
0.03
PTW0.11
(0.04-0.15)		0.03-0.05-0.02-0.07-0.07-0.130.07-0.100.10-
0.13		0.04-
0.045		0.05
-0.09		0.06-
0.09		0.05 -
0.09
OL0.06
(0.02-0.09)		--0.08-0.12--0.05-0.070.13-
0.14		0.11-
0.12		0.01-
0.03		0.04-
0.05		-
OW0.08
(0.01-0.11)		--0.0-0.08--0.06-0.090.07-
0.08		0.01-
0.015		0.05-
0.09		0.05-
0.06		-
EBL0.07
(0.03-0.09)		-----------
EBW0.05
(0.02-0.07)		-----------
OSVS0.42
(0.24-0.63)		-----------
VSHO0.04
(0.02-0.07)		-----------

Table 2: Comparative measurements shown as mean (range) of _Neascus_-type and _Posthodiplostomum_ species, all infected the sa

CSTL= Cyst length, CSTW= Cyst width, WBL= Whole body length, WBW= Whole body width, FBL= Fore body length, FBW= Fore body width, HBL= Hind body length, HBW= Hind body width, OSL= Oral sucker length, OSW= Oral sucker width, VSL= Ventral sucker length, VSW= Ventral sucker width, HOL= Holdfast organ length, HOW= Holdfast organ width, ATL= Anterior testis length, ATW= Anterior testis width, PTL= Posterior testis length, PTW= Posterior testis width, OL= Ovary length, OW= Ovary width, EBL= Excretory bladder length, EBW= Excretory bladder width, OSVS= Distance between oral sucker and ventral sucker, VSHO= Distance between ventral sucker and holdfast organ. Table 2: Comparative measurements shown as mean (range) of Neascus-type and Posthodiplostomum species, all infected the same host (Xenentodon cancila) from India.

SpeciesHostSite of infectionSpecies status/
developmental
stage		Identification
evidence		References
P. botauriNAIntestineValid/AMorphologyVidyarthi [58]
N. vetastaiSchizothorax
esocinus, S.
micropogon, S.
niger		Cyst on skin and
viscera		Incertae sedis/MMorphologyKaw [59]
N. chelaiChela clupeoidesEncysted in the
integument and
muscles		Incertae sedis/MMorphologyKhera [60]
P. pandeiXenentodon
cancila		LiverValid/MMorphology/
Molecular*		Rai, et al. [50]
N. mesentriformisX. cancilaMesenteryIncertae sedis/MMorphologyRai, et al. [50]
P. milviNAIntestineValid/AMorphologyFotedar, et al. [61]
N. indicusNuria dursica,
Catla catla		Cyst in muscles
below scales		Incertae sedis/MMorphologyThapar [62]
N. cirrhinusCirrhinus
mrigala		Cyst in musclesIncertae sedis/MMorphologyThapar [62]
N. muscularisChanna punctataMusclesIncertae sedis/MMorphologyRai , et al. [50]
N. elongatusColisa fasciataMesenteries
inside body
cavity		Incertae sedis/MMorphologySingh [63]; Pandey 64]
N. hepaticaX. cancilaCyst in liverIncertae sedis/MMorphologyChakrabarti [51];
Vankara, et al. [65]
P. mehtaiMilvus migransIntestineValid/AMorphologyGupta, et al. 1974 [66]
N. channiC. punctataCraniumIncertae sedis/MMorphologyPandey [52]
P. xenentodoniX. cancilaLiver and
cranium		Valid/MMorphology/
Molecular		Pandey [52];
Choudhary, et al. [44]
N. komiyaiGlossgobius
giuris		Cyst attached to
stomach		Incertae sedis/MMorphologyPandey [67]
N. hoffmaniNandus nandusStomachIncertae sedis/MMorphologyPandey [67]
N. gusseviC. punctataCyst attached to
visceral organs		Valid/MMorphology/
Molecular		Chakrabarti [68];
Choudhary, et al. [44]
N. baughiX. cancilaBody cavityIncertae sedis/MMorphologyBaugh, et al [53]
N. nanaksagrensisX. cancilaCysts attached to
liver, gonads and
in liver		Incertae sedis/MMorphologyBaugh, et al. [53]
N. chauhaniHeteropneustus
fossilis		Free in body
muscles		Incertae sedis/MMorphologyAgrawal and Khan,
1982 [54]
N. hanumanthaiC. punctataCyst in musclesValid/MMorphology/
Molecular		Agrawal, et al. [54];
Choudhary, et al. [44]
N. simhaiX. cancilaCyst in body
muscles		Incertae sedis/MMorphologyAgrawal, et al. [54]
N. mogheiX. cancilaLiverIncertae sedis/MMorphologyAgrawal, et al. [54]
N. shahjahanpurensisClarius
batrachus		MusclesIncertae sedis/MMorphologyPandey, et al. [55]
N. ramalingamiLabeo rohitaBody musclesIncertae sedis/MMorphologyPandey and Tiwari,
1986 [55]
N. vediX. cancilaBody musclesIncertae sedis/MMorphologyPandey, et al. [55]
N. punctatusiC. punctataCranial cavityIncertae sedis/MMorphologyDhanukumari [69]
P. grayiiApocheilus
panchax		LiverValid/AMorphologyVerma, [70]; Madhavi,
et al. [71]
N. srivastaviX. cancilaLiverIncertae sedis/MMorphologyPandey, et al. [56]
Posthodiplostomum
sp.		C. punctataBody musclesValid/MMorphology/
Molecular		Athokpam, et al. [41]
N.
khurramnagarensis		X. cancilaLiverIncertae sedis/MMorphologyGupta, et al. [57]

Table 3: Species of Neascus-type or Posthodiplostomum described from India on the basis of metacercariae specimens, infected host

Figure 2
Click to enlarge
Figure 2

Figure 11: Phylogenetic interrelationships among Posthodiplostomum species based on Maximum Likelihood (ML) and Bayesian Inference (BI) analyses of partial 18S rDNA gene sequences. The scale-bar indicates the number of substitutions per site. Species names are provided after GenBank accession numbers. The numbers represent bootstrap values greater than 70%. The present studied species, Posthodiplostomum pandei, is indicated by the blue line box, while the red line box shows the other Indian species.

In this study, the ITS (ITS1–5.8S–ITS2) sequences of two individuals of P. pandei were generated and aligned with other sequences of the diplostomids (Figure 12). The genetic divergence between the different species of Posthodiplostomum ranged from 0.92 to 4.1%. Moreover, ITS (ITS1–5.8S–ITS2) sequences were available for the following Indian species: N. hanumanthai (KY042122), N. gussevi (KY234202), N. xenentodoni (KY234203), and Posthodiplostomum sp. (KF738447), whose genetic divergence ranged from 0.92 to 2.6%. Maximum likelihood (ML) and Bayesian inference (BI) analyses produced a similar tree in topology that shows N. xenentodoni to be the closest to our specimens; both belong to the same host (Figure 12).

The 28S sequences in the current phylogenetic analysis clearly demonstrated the status of P. pandei within Posthodiplostomum (Figure 13). The Indian species N. hanumanthai (KY042122), N. gussevi (KY234202), N. xenentodoni (KY234203), and an unknown Posthodiplostomum sp. (KF738450) were positioned with Posthodiplostomum members, validated their status, and formed a well-supported clade. Choudhary, et al. [44] were not mentioned the status of N. xenentodoni regarding this is a species of Posthodiplostomum as the molecular data predicts_, the present study analysis confirmed that it is a species of _Posthodiplostomum with good bootstrap values (90% ML and 0.92 BI) and sister to the present specimens (Figure 13). In comparison, the genetic divergence among Posthodiplostomum and P. pandei ranged from 1.2 to 4.5%. P. pandei and other Indian Posthodiplostomum species had 1.2– 2.2% genetic variation. Analysis shows all the 28S sequences of Indian species and the data generated in this study are nested within a clade with strong bootstrap support and Bayesian posterior probability values of 83 and 0.9 (Figure 13).

Figure 3
Click to enlarge
Figure 3

Figure 12: Phylogenetic reconstruction using ITS region (ITS1-5.8S-ITS2) sequences of Posthodiplostomum species (shown in blue box). The numbers represent bootstrap values greater than 70%. Numbers above branches indicate nodal support as maximum likelihood (ML) and posterior probabilities from BI. The scale bar indicates the expected number of substitutions per site. Species names are provided after Gen Bank accession numbers. The blue line box represents the currently studied species P. pandei, while the red line box represents the other Indian species.

Figure 4
Click to enlarge
Figure 4

Figure 13: Phylogenetic relationship between Posthodiplostomum species inferred from sequences of the 28S rDNA gene based on maximum likelihood (ML) and Bayesian inference (BI) analyses. Nodal support is indicated as ML/BI. The numbers represent bootstrap values greater than 70%. The scale bar indicates the expected number of substitutions per site. Species names are provided after GenBank accession numbers. Posthodiplostomum pandei is indicated by the blue line box, while the red line box shows the other Indian species.

We also want to mention that in a molecular phylogenetic study by Achatz, et al. [35], the synonymy of Posthodiplostomum, Ornithodiplostomum Dubois, 1936, and Mesoophorodiplostomum Dubois, 1936, was suggested. To the best of our knowledge, the Genbank database only contains molecular data for the following Neascus-_type species from the Indian region to date: _N. hanumanthai, N. gussevi, N. xenentodoni, and one unknown Posthodiplostomum sp. The comparison of the genetic data of our specimens with the above-mentioned species molecular level and in variations of measurements of the body parts also easily differentiates them with P. pandei.

Discussion

Previous studies show that it is difficult to evaluate the host specificity with regard to larval helminthes because of their poor morphological identification [72, 73]. But molecular methods are the best tools to deal with this problem and achieve accurate identifications of larval trematodes in fish hosts [72, 74, 75]. Larval trematodes often harbour multiple sites in their hosts, but most of their infection site preferences or specificities are likely mistaken [76, 77, 78, 79]. In a study by Locke, et al. [34], different Posthodiplostomum species were recorded from viscera and muscles. Therefore, thorough examinations of infection sites supplemented with molecular data are presently required to decide if infection sites are the same or differ between Posthodiplostomum species. In the present study, P. pandei was molecularly characterised for the first time. The placement of our specimens in the phylogenetic trees produced in this study supports their settlement within Diplostomidae and separates them from other representatives of different geographical regions and India too. Our species appeared as a sister taxon to the Posthodiplostomum species isolate that belongs to Mexico in the 18S rRNA tree, though it was far from the Indian species (KF738455), which is the only sequence available for the Channa punctata. However, in the ITS analysis, P. pandei was grouped of N. xenentodoni, both species infected the same host, X. cancila; and were fairly distinct from each other. In the distribution of species by 28S rRNA tree, it was congruent with the 18S and ITS rRNA trees in many respects, with Posthodiplostomum species of C. punctata (KF738450) appearing along with two other species, N. hanumanthai and N. gussevi from India. N. xenentodoni from the same host appeared as a sister taxon to P. pandei in the 28S rRNA tree. The phylogenetic trees generated with all molecular markers in the present study revealed that P. pandei belongs to a clade containing  Posthodiplostomum  species Further work is a prerequisite to establishing and understanding the Indian Posthodiplostomum species infections in fish and avian hosts that need to be molecularly characterised to determine their diversity, which might be different from what we know today.

About 26 species of Neascus-type metacercariae have been described in India, mostly based on morphology (Table 3). For many species, at this point, it is hard to establish how many species are under the genus Posthodiplostomum, which shows their unstable taxonomic history in India. The reason behind the unknown status of diplostomid parasites in India is that they have not been well studied, leaving a big gap with regard to their true diversity. However, in recent years, a few studies have pointed it out, contributed to the understanding of diplostomid parasites, and tried to fill the gap [80, 81, 82, 83, 84, 85]. Based on morphological variation between specimens, previous authors [27] recommended the description of eleven Neascus-type metacercariae species from X. cancila (Tables 1 & 3). In our study, nearly similar morphological measurements were found in the overall body measurements for P. pandei, N. hepatica, and N. srivastavi. In all eleven species reported from the host X. cancila, only P. xenentodoni (= N. xenentodoni) has molecular sequences. With the exception of P. xenentodoni, no genetic sequences for the other Indian species were available for comparison from X. cancila. Another reason that also hampers the taxonomic studies and diversity of this group of parasites in India is the restrictions of ethical guidelines that are related to definitive hosts (birds), which make it challenging to study the relationship of adults in birds along with metacercariae and the completion of their life cycles.

In the case of diplostomid parasites, García-Varela, et al. [86] recently proposed that all South American species described on the basis of metacercaria morphology be considered incertae sedis. We are agreeing with that because studies based only on the morphology of metacercariae will make it difficult to reveal the relationship with their adult forms in future studies. Therefore, we also propose that, from India, those metacercariae for which molecular data is available have been considered valid species, as their phylogenetic relationships can be easily evaluated with their adults in future studies (Table 3). Under the incertae sedis status, we believe that those Posthodiplostomum/Neascus- type metacercariae species for which only morphological data is available should be taken into account, leaving the possibility for future studies to perform morphological and molecular comparisons that help to uncover their life cycle and clarify their status.

Conclusion

Overall, our identification of P. pandei is supported by its morphology as well as at the molecular level. Furthermore, molecular data generated here will be useful in future studies to determine whether Neascus-type metacercariae species belong to Posthodiplostomum or another genus. Though we robustly acclaim that no new species can be further described without molecular data under the genus Posthodiplostomum, We also proposes that some previously published Neasus-type species that had been misidentified should be reassigned on the basis of morphological and molecular data in future studies.

Conflicts of Interest

The authors declare no competing interests.

Acknowledgements

We are grateful to the Head, Department of Zoology, Chaudhary Charan Singh University, Meerut (Uttar Pradesh), 250004, India, for providing laboratory facilities.

Funding

This research was supported by grants from the DST (Department of Science and Technology), Government of India, New Delhi, India, under the WOS-A Scheme (SR/ WOS-A/LS-382/2018) in Delhi to AC.

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@article{chaudhary2023,
  title   = {Morphological and Molecular Evidence of a Trematode Parasite
Infected the Liver of Garfish Xenentodon cancila (Beloniformes:
Belonidae) in India},
  author  = {Chaudhary A, Singh K, Sharma B and Singh HS},
  journal = {International Journal of Zoology and Animal Biology},
  year    = {2023},
  volume  = {6},
  number  = {1},
  doi     = {10.23880/izab-16000437}
}
Chaudhary A, Singh K, Sharma B and Singh HS (2023). Morphological and Molecular Evidence of a Trematode Parasite
Infected the Liver of Garfish Xenentodon cancila (Beloniformes:
Belonidae) in India. International Journal of Zoology and Animal Biology, 6(1). https://doi.org/10.23880/izab-16000437
TY  - JOUR
TI  - Morphological and Molecular Evidence of a Trematode Parasite
Infected the Liver of Garfish Xenentodon cancila (Beloniformes:
Belonidae) in India
AU  - Chaudhary A, Singh K, Sharma B and Singh HS
JO  - International Journal of Zoology and Animal Biology
PY  - 2023
VL  - 6
IS  - 1
DO  - 10.23880/izab-16000437
ER  -