Molecular characterization and phylogeny based on ITS2 and 28S regions of rDNA of Microphallus sp. (Digenea: Microphallidae) parasitic in freshwater crabs of Manipur, India

doi:10.21203/rs.3.rs-4064777/v1

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Molecular characterization and phylogeny based on ITS2 and 28S regions of rDNA of Microphallus sp. (Digenea: Microphallidae) parasitic in freshwater crabs of Manipur, India

https://doi.org/10.21203/rs.3.rs-4064777/v1

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Freshwater crabs (Potamiscus manipuriensis), commonly consumed as local delicacies by the native people in the state of Manipur, were found to harbour metacercariae of Microphallus sp. (Family Microphyllidae), which were morphologically different from metacercariae of Microphallus indicus reported earlier from a different host (Barytelphusa lugubris mansoniana) in Meghalaya, another state in Northeast India. So, PCR-based molecular characterization of this metacercaria was done utilizing rDNA marker regions: larger subunit (LSU) or 28S and inter-transcribed spacer 2 (ITS2). Sequence and phylogenetic analyses confirmed that the taxon under study belonged to family Microphyllidae. The ITS2 secondary structure data analyses also confirmed the primary sequence analysis. The analysis also revealed sequence differences in one hundred and nineteen bases (with 38 transitions, 35 transversions and 46 indels) with regard to 28S, though ITS2 showed sequence differences in 25 bases (10 transitions, 7 transversions and 8 indels) between the present microphallid and M. indicus.

crabs

Potamiscus manipuriensis

metacercaria

Microphallidae

rDNA

The trematode flukes of the family Microphallidae Travassos, 1920, occur as intestinal parasites in all groups of vertebrates, mainly the birds and mammals (Martorelli et al. 2004; Deblock 2008). In their complex life cycle, crustaceans serve as second intermediate host harbouring encysted metacercarial larval form which is the infective form (Yamaguti 1975; Heard and Overstreet 1983; Pung et al. 2002; Diaz et al. 2004). Significant contributions have been made to study the life cycles of microphallid and also for their identification using traditional methods (Overstreet et al. 1992; Kostadinova et al. 2006; Guk et al. 2008; Chung et al. 2010; Diaz and Cremonte 2010; Lee et al. 2010; Goswami et al. 2013).

With the advent of molecular tools and technique in the recent years that utilize various regions of trematode rDNA, molecularly characterization has been increasing utilized supplementing morphological studies (Hillis and Dixon 1991). Trematode has complex life cycle with different larval stages completing in different intermediate host. These techniques using DNA-based PCR-amplification are useful in characterization at all larval stages (Dzikowski et al. 2004). They have been successfully used for species characterization and interpreting phylogenetic inter-relationships between various microphallid taxa (Tkach et al. 2003; Hust et al. 2004; Al-Kandari and Al-Bustan 2010; Pina et al. 2011; Goswami et al. 2013).

The commonly edible freshwater crab species in mountainous ranges of Manipur, Northeast India, are reported to be naturally infected with metacercariae representing the genera Paragonimus and Microphallus (Singh and Singh 1997; Singh 2002, 2003; Singh et al. 2006; Athokpam and Tandon 2015). The objective of the present study was to molecularly characterize the Microphallus sp. occurring in freshwater crabs in the region using ITS2 and 28S marker sequences and to compare it molecularly with M. indicus Indian isolate earlier reported from the crab host, Barytelphusa lugubris mansoniana, in the northeastern region of India by Goswami et al. (2013). The ITS2 secondary structure data were also analysed, so as to corroborate the species characterization.

Sample collection and morphology study

Metacercariae of Microphallus sp. were recovered from the muscle tissue of the crab host, Potamiscus manipuriensis by the artificial digestion technique, collected from susceptible foci of Manipur, Northeast India (Tandon et al. 2007; Athokpam and Tandon 2015).

Morphological analysis was carried out following the standard procedure as described elsewhere (Athokpam and Tandon 2014).

Molecular study

DNA extraction, PCR amplification and Sequencing

Genomic DNA extraction was done from the metacercariae separated from a single crab host using QIAamp DNA Mini Kit (50) followings manufacturer’s instructions. The rDNA ITS2 and 28S regions were PCR amplified using the primers – for ITS2: [3S (forward): 5’- GGTACCGGTGGATCACTCGGCTCGTG-3’ and A28 (reverse): 5’- GGGATCCTGGTTAGTTTCTTTTCCTCCGC-3’] (Bowles et al., 1995) and for 28S: [Dig12 (forward): 5’-AAGCATATCACTAAGCGG-3’ and 1500R (reverse): 3’-GCTATCCTGAGGGAAACTTC-5’] (Tkach et al. 2000). PCR amplification was carried out following the standard protocol of White (1993) with minor modifications. The amplified products were stained with ethidium bromide and examined on 1.6% agarose gel in TAE buffer. They were purified with Genei Pure Quick PCR Puriﬁcation Kit (Bangalore, Karnataka, India) and sequenced in both directions using mentioned PCR primers by DNA sequencing services provided by Macrogen, Seoul, Korea.

Sequence analysis

Similarity search for the sequences generated during the study was done using Basic Local Alignment Search Tool (BLAST) available at http://www.ncbi.nlm.nih.go v/blast. The multiple sequence alignment of the sequences generated from the studied metacercaria, with related sequences from family Microphallidae retrieved from GenBank (Table 1), was done using ClustalW of Bioedit software (http://www. ebi.ac.uk/clustalw). Also, the sequence identity analysis was carried out using Bioedit version 7.0.9.0 (Hall 1999).

Phylogenetic tree construction

The rDNA ITS2 sequences of various microphallids were annotated using the hidden Markov models (HMM) (Keller et al. 2009) available at http://its2.bioapps.biozentrum.uni-wuerzburg.de/.Phylogenetic analyses were done based on the distance-based Neighbour Joining (NJ) method and character-based Maximum likelihood (ML) of MEGA11 and also profile neighbour-joining (PNJ) implemented in ProfDistS (Wolf et al. 2008; Tamura et al. 2021). In addition to the sequence data set used in the analysis, the Clinostomum sequence was included as the outgroup taxon. Bootstrapping analysis was done for all the phylogenetic trees constructed.

ITS2 secondary structure analysis

The secondary structures of these annotated ITS2 sequences were predicted using mfold webserver that utilizes minimum free energy folding algorithms, and the most stable structures with highest negative free energy were selected (Zuker 2003). The predicted secondary structures were aligned using 4SALE (Seibel et al. 2006), and the output file of sequence-structure alignment was save in .xfasta format. Then, the file was imported into ProfDistS 0.9.9 and allowed to run using PNJ implemented in ProfDistS, with GTR as selected correction number, 1,000 numbers of bootstraps and Q_ITS2 selected for Ratematrix Q. So, the data of ITS2 predicted secondary structures was also used for phylogenetic analysis to corroborate the tree construction with the primary data.

Morphology and morphometry

Based on the morphological analysis, the metacercaria was identified as belonging to genus Microphallus (Family: Microphallidae Ward, 1901) and is describe as follows.

Description (based on 5 specimens, Fig. 1a, b): Encysted form rounded in shape, thin-walled, 0.55–0.83 mm in diameter. Most of the adult organs developed in metacercarial stage; excysted metacercaria having globular body; tegument spinous, density of spines decreasing from anterior to posterior end; oral sucker subterminal; ventral sucker single, post equatorial; prepharynx present; pharynx well developed, muscular; oesophagus long, medium sized; intestinal caeca short, divergent just anterior to ventral sucker; testes two, postovarian, symmetrical, on each lateral side of body; seminal vesicle ovoid, intercaecal; ovary dextral to ventral sucker; vitellaria in two groups, each having cluster of 6–7 on lateral side of body, posterior or lateral to each testis; excretory pore terminal.

Morphometric measurements of various body parts of the mounted metacercaria are given in Table 2.

Molecular study

Molecular characterization and analysis

The selected marker regions were amplified and the generated sequences were deposited in GenBank [Accession numbers: KF738448, KF738451]. The amplicon size of ITS2 and 28S was 395 bp and 1002 bp in length, respectively. They were then compared with various Microphallidae taxa (a total of 28 accession numbers) retrieved from GenBank for analysis (Table 1). The metacercaria under the present study stands close to Microphallus spp, with high sequence identities of 92.8% (ITS2) with M. indicus and 91.6% (28S) with M. calidris (Tables 3, 4). The query sequences, when compared with M. indicus Indian isolate, revealed 25 nucleotide differences (with 10 transitions, 7 transversions and 8 indels) and 119 nucleotide differences (with 38 transitions, 35 transversions and 46 indels) for ITS2 and 28S, respectively (Table 5).

Phylogenetic analysis

Phylogenetic trees were constructed using the ITS2 and 28S data of microphallid taxa available in GenBank (Table 1). The ML and NJ phylogenetic trees constructed for marker genes depicted the same topology of taxa with minor differences. In the analysis of ITS2 sequences, the NJ tree is shown with sum of branch length = 0.97763611 (Fig. 2a), which was drawn on scale, branch lengths in the same units with those of evolutionary distances used to infer the phylogenetic tree. The ML tree with the highest log likelihood (-1571.3102) is shown (Fig. 2b), which was drawn to scale, with branch lengths measured in the number of substitutions per site. Similarly, for 28S sequence, the NJ tree with sum of branch length = 0.67117473 (Fig. 3a) and ML tree with the highest log likelihood (-4108.0370) (Fig. 3b) are shown. All the trees revealed that the present microphallid fluke clades with M. indicus, with significant bootstrap values of 98-100% (Figs.2a, b and 3a, b). In all trees, the outgroup, genus Clinostomum, formed a seperate clade.

ITS2 RNA secondary structures analysis

The ITS2 secondary structure of the presently studied Microphallus sp. shows the typical four-helix model with helix I and IV being short; helix III- the longest and showing UGG motifs and helix II showing U-U mismatch (Fig. 4). The Profile neighbour joining (ProfDistS) analysis of the ITS2 secondary sequences also showed the same topology as that shown by the primary sequence analysis (Fig. 5), the present metacercaria Microphallus sp. clading with M. indicus with significant bootstrap value (91%).

Ward (1901) created the genus Microphallus with Microphallus opacus (Ward 1894) as its type species from intestine of Amia calva. Many species of the genus have been reported from various definitive hosts such as birds, shrew and mammals – M. abortivus Deblock 1974; M. bittii Prevot 1973; M. breviatus Deblock and Maillard 1975; M. crociduri Mikhail and Fahmy 1968; M. forresteri Kinsella and Deblock 1997; M. fusiformis Reimer 1963; M. gracile Baer 1944; M. hoffmanni Rebecq 1964; M. kenyensis Canaris 1971; M. kinsellai Canaris and Beblock 2000; M. longicaecum Chen 1956; M. minus Ochi 1928; M. minutum Johnston 1948; M. montanus Beljakova and Kulkina 1998; M. oblonga Ching 1965; M. orientalis Yurakhno 1968; M. pearsoni Deblock and Canaris 1997 and M. oblongus Ching 1965 (see Yamaguti, 1971; Global Names Index, 2010).

Various Microphallus spp at their larval stages have been reported from crustaceans and molluscan hosts; these are M. ovatus Osborn 1919; M. progeneticus Sogandares-Bernal 1962; M. pachygrapsi Deblock and Cable 1966; M. scolectroma Deblock and Tran Van Ky 1966; M. papillornatus Deblock and Pearson 1969; M. pisodonphidis Reimer and Szuks 1973; M. tauricus Stenko 1973; M. helicicola Belopolskaya and Soboleva 1977; M. pseudopygmaeus Galaktionov 1980; M. triangulatus Galaktionov 1980; M. piriformes Galaktionov 1983; M. paragrapsi Smith 1983; M. fonti Overstreet, Heard and Lotz 1992; M. selector Kinsella and Deblock 1997 and M. sabanensis Diaz, Bashirullah and Hernandez 2004 (Anantaraman and Subramoniam 1976; Jayasree et al. 2001; Goswami et al. 2013). In India, so far, only few microphallid taxa have been identified, namely: Levinseniella indica Lal 1936; Basantisia ramai Pande 1938; Pseudospeloterma indicum Murhar 1960; Mehraformes jabalpurensis Bharadwaj 1963; Spelotrema narii Rao 1965; Microphallus indicus Mukherjee and Ghosh 1967; Megalatriotrema hispidum Rao 1969 and S. chauhani Gupta and Jahan 1975 (Yamaguti 1971; Global Names Index, 2010; Goswami et al. 2013).

A variety of molecular techniques have increasingly been utilized for various taxonomic issues related to describing new species or strains, supplementing traditional methods for characterizing based on their morphological studies (Thompson et al. 2004; Olson and Tkach 2005; Otachi et al. 2015). These techniques can be used even from the larval stages in addition to the adult stage of a taxon (Jousson et al. 1998; Rathinam et al. 2012; Chomchoei et al. 2022). In the present study, the microphallid metacercarial form harbouring the crab host Potamiscus manipuriensis was identified based on both morphometric and molecular analysis.

Microphallid species show a high degree of similarity in morphology between their larval and adult stages due to the precocious development of reproductive organs. Based on morphological study, the present metacercarial form was found to belong to the family Microphallidae Ward, 1901, possessing the typical morphological features of the genus Microphallus (Yamaguti 1971; Deblock 2008). In India, about eight species from the family have been reported as infecting various mammals, reptiles, amphibians and birds, with their infective metacercaria stage in crustacean host (Yamaguti 1971; Patterson et al. 2010; Goswami et al. 2013). For all these eight microphallids species reported so far, identification is based on morphological study alone, except for M. indicus, for which molecular characterization supplemented the morphology-based criteria (Goswami et al. 2013). Athokpam and Tandon (2015) had reported the microphallid species recovered from crabs in Manipur region to be morphologically differ with smaller morphological parameters from M. indicus earlier reported from Meghalaya, another state in Northeast India. In the present study, the sequence comparison revealed that the Microphallus sp. differs from M. indicus Indian isolate, with 25 nucleotide differences for ITS2, and 119 nucleotide differences for 28S regions, showing transition, transversion and indels.

Tkach et al. (2003) studied phylogenetic interrelationships of Microphalloidea taxa (representing the families Lecithodendriidae, Microphallidae, Pleurogenidae and Prosthogonimidae) using partial 28S sequences. In the public domain, data on molecular characterization using rDNA tools with phylogenetic analysis for various microphallid species are available (Hust et al. 2004; Al-Kandari and Al-Bustan 2010; Pina et al. 2011). In phylogenetic analysis based on rDNA ITS2 and 28S regions, our query sequence claded close to M. indicus and within the Microphallidae taxa, thus supplementing the morphology study, which were also supported by sequence identity analysis. The high bootstrap values thus confirmed the placing of Microphallus sp. under study within Microphallidae family; a bootstrap value greater than 70% gives reliable clading in a phylogenetic tree (Hillis and Bull 1993).

The ITS2 secondary structures include additional morphological information that are not found in the primary sequence and thus help in reconstructing the complete tree of life (Caetano-Anollés 2002; Grajales et al. 2007). A four-domain secondary structure model for ITS2 proposed by Michot et al (1993) boosts its value as a marker for megasystematics (Schultz et al. 2005). So, ITS2 region has high nucleotide mutation but their conserve nature of secondary structure has made them highly useful in various evolutionary studies (Coleman 2003). It is, therefore, necessary to consider both sequence and structure when calculating their alignments and in phylogenetic analysis (Keller et al. 2010). Hence, the ITS2 rDNA primary sequence and secondary structures have been utilized in many taxonomic studies including those on digenean taxa (Coleman 2007; Shylla et al. 2011; Ghatani et al. 2012; Athokpam and Tandon 2014). In the present study, Profile neighbour joining analysis of the secondary structure and sequence information of studied Microphallus sp. revealed the same topology as NJ and ML phylogenetic trees, clading with M. indicus with significant bootstrap value (91%), thus confirming the results of primary sequence analysis.

On the basis of morphological study, supplemented with molecular characterization, the presently studied microphallid metacercarial parasite occurring in Potamiscus crabs in Manipur region is identified as belonging to the genus Microphallus Ward, 1901. Limited data on the infection status of genus Microphallus are available in the public domain. So, more extensive works need to carry out to focus on finding possible intermediate hosts, completing their life cycle and for systematic analysis.

Acknowledgments

VDA acknowledges University Grants Commission (UGC), Government of India for awarding her ‘Research Fellowship in Science for Meritorious Students’ and Council of Scientific & Industrial Research (CSIR), Government of India, for Senior Research Fellowship.

Funding

This work was supported by Department of Information Technology, Government of India under the “North-East Parasite Information and Analysis Centre (NEPIAC)” sanctioned to VT et al. [Sanction no.: DIT/R&D/BIO/ 15(13)/2008 dated Sep. 29, 2008]. VDA was awarded University Grants Commission (UGC) ‘Research Fellowship in Science for Meritorious Students’ and Council of Scientific & Industrial Research (CSIR) ‘Senior Research Fellowship’.

The authors declare that no funds or other support were received during the preparation of this manuscript.

Conflict of Interest

There is no conflict of interest.

Author’s Contributions

Veena Tandon prepare the study framework, critically analysed the manuscript; Voleentina Devi Athokpam carried out specimen preparation, analysis and drafting of the manuscript; Lalit Mohan Goswami help during analysis and drafting. All authors read and approved the final manuscript.

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Table 1. Microphallidae taxa sequences of rDNA used in the analysis with their respective GenBank accession numbers

Sl. No.	Parasite name	Locality	Genes
Sl. No.	Parasite name	Locality	ITS2	28S
1.	Microphallus indicus	India: Meghalaya	FJ966111.1	FJ966109.1
2.	Microphallus abortivus	UK	HM584174.1	AY220626.1
3.	Microphallus calidris	Russia	HM584183.1	HM584125.1
4.	Microphallus pseudopygmaeus	Russia	HM584198.1	HM584126.1
5.	Microphallus pygmaeus	Iceland	HM584190.1	HM584133.1
6.	Microphallus similis	Russia	HM584178.1	HM584138.1
7.	Microphallus piriformes	Iceland	HM584181.1	HM584122.1
8.	Microphallus primas	UK	-	AY220627.1
9.	Microphallus triangulates	Russia	HM584196.1	HM584139.1
10.	Microphallus fusiformis	UK	-	-
11.	Microphallus turgidus	USA	-	-
12.	Microphallus sp.	Russia	HM584188.1	HM584140.1
13.	Microphallus sp.	Russia	HM584175.1	-
14.	Maritrema subdolum	Russia	HM584172.1	AF151926.1
15.	Maritrema oocysta	UK	HM584170.1	AY220630.1
16.	Maritrema eroliae	Kuwait	HQ650132.1	JF826247.1
17.	Maritrema neomi	Ukraine	-	AF151927.1
18.	Maritrema arenaria	UK	-	AY220629.1

Table 2. Morphometric measurements of the excysted metacercaria of Microphallus sp.

Characters		Range (mm)	Mean	±SD
Body length		0.423 – 0.522	0.461	± 0.042
Body width (maximum)		0.369 – 0.495	0.434	± 0.052
Oral sucker (diameter)		0.048 – 0.054	0.050	± 0.005
Prepharynx length		0.003 – 0.018	0.009	± 0.008
Pharynx:	Length	0.018 – 0.021	0.020	± 0.010
Pharynx:	Width	0.021 – 0.024	0.022	± 0.011
Oesophagus		0.069 – 0.090	0.083	± 0.043
Ventral sucker (diameter)		0.027 – 0.048	0.038	± 0.009
Ovary:	Length	0.048 – 0.087	0.070	± 0.019
Ovary:	Width	0.036 – 0.063	0.052	± 0.012
Left testis:	Length	0.060 – 0.105	0.080	± 0.019
Left testis:	Width	0.063 – 0.108	0.083	± 0.019
Right testis:	Length	0.051 – 0.087	0.070	± 0.018
Right testis:	Width	0.060 – 0.099	0.072	± 0.018

Table 5. Difference in nucleotide positions between Microphallus sp. under study and M. indicus, Indian isolate. A) ITS2 B) 28S

(Ts –Transition; Tv – Transversion; Indel – Insertion/Deletion; * - Query sequence)

(a) ITS2

Sl. No.	Base position	Microphallus sp. *	M. indicus	Differences	Sr. No.	Base position	Microphallus sp.*	M. indicus	Differences
1.	8	T	A	Tv	13.	315	T	G	Tv
2.	28	T	C	Ts	14.	316	-	T	Indel
3.	100	A	T	Tv	15.	317	-	A	Indel
4.	142	A	G	Ts	16.	318	-	A	Indel
5.	153	-	C	Indel	17.	319	-	T	Indel
6.	154	-	C	Indel	18.	320	-	G	Indel
7.	237	A	G	Ts	19.	333	A	T	Tv
8.	248	T	G	Tv	20.	334	A	C	Tv
9.	251	T	G	Tv	21.	357	G	A	Ts
10.	265	T	C	Ts	22.	400	A	G	Ts
11.	295	-	T	Indel	23.	402	A	G	Ts
12.	297	G	A	Ts	24.	410	A	G	Ts
					25.	412	A	G	Ts

(b) 28S

Sl. No.	Base position	Microphallus sp.*	M. indicus	Differences	Sr. No.	Base position	Microphallus sp.*	M. indicus	Differences
1.	5	G	C	Tv	61.	225	-	C	Indel
2.	10	A	-	Indel	62.	235	-	G	Indel
3.	11	C	-	Indel	63.	242	-	T	Indel
4.	12	C	-	Indel	64.	249	-	C	Indel
5.	18	G	T	Tv	65.	256	-	A	Indel
6.	22	G	T	Tv	66.	257	-	G	Indel
7.	25	T	-	Indel	67.	258	A	G	Ts
8.	26	G	-	Indel	68.	259	G	C	Tv
9.	27	T	-	Indel	69.	261	C	T	Ts
10.	28	T	-	Indel	70.	267	T	G	Tv
11.	29	T	-	Indel	71.	268	C	T	Ts
12.	30	G	-	Indel	72.	270	G	C	Tv
13.	43	A	T	Tv	73.	274	A	G	Ts
14.	45	A	T	Tv	74.	275	A	G	Ts
15.	47	T	G	Tv	75.	276	C	T	Ts
16.	52	G	C	Tv	76.	278	G	A	Ts
17.	53	G	-	Indel	77.	280	-	C	Indel
18.	54	T	-	Indel	78.	281	-	A	Indel
19.	55	A	-	Indel	79.	282	-	A	Indel
20.	56	A	-	Indel	80.	283	-	C	Indel
21.	64	C	T	Ts	81.	284	-	C	Indel
22.	67	A	-	Indel	82.	291	C	T	Ts
23.	68	G	-	Indel	83.	292	-	C	Indel
24.	70	C	T	Tv	84.	294	G	A	Ts
25.	76	A	-	Indel	85.	297	C	T	Ts
26.	82	A	C	Tv	86.	300	C	T	Ts
27.	83	C	T	Ts	87.	301	G	C	Tv
28.	92	T	A	Tv	88.	302	G	A	Ts
29.	101	A	C	Tv	89.	304	G	A	Ts
30.	102	G	A	Ts	90.	305	T	G	Tv
31.	103	T	G	Tv	91.	306	G	A	Ts
32.	104	A	T	Tv	92.	307	G	T	Tv
33.	105	C	T	Ts	93.	308	G	A	Ts
34.	106	C	A	Tv	94.	309	G	A	Ts
35.	107	G	C	Tv	95.	312	-	T	Indel
36.	114	A	C	Tv	96.	317	G	C	Tv
37.	119	T	G	Tv	97.	320	-	T	Indel
38.	124	A	G	Ts	98.	321	-	G	Indel
39.	131	T	G	Tv	99.	322	-	C	Indel
40.	134	-	G	Indel	100.	342	-	C	Indel
41.	139	A	G	Ts	101.	347	T	C	Ts
42.	140	G	A	Ts	102.	348	-	T	Indel
43.	147	C	A	Tv	103.	370	-	C	Indel
44.	148	A	C	Tv	104.	389	-	C	Indel
45.	149	G	A	Ts	105.	406	-	C	Indel
46.	154	-	G	Indel	106.	411	C	T	Ts
47.	157	A	C	Tv	107.	441	T	C	Ts
48.	169	G	A	Ts	108.	442	T	C	Ts
49.	171	-	G	Indel	109.	443	C	T	Ts
50.	172	-	T	Indel	110.	444	-	C	Indel
51.	176	A	C	Tv	111.	454	T	C	Ts
52.	186	T	G	Tv	112.	494	-	G	Indel
53.	188	G	T	Tv	113.	515	A	G	Ts
54.	189	A	G	Ts	114.	822	C	G	Tv
55.	194	-	G	Indel	115.	825	A	G	Ts
56.	198	A	C	Tv	116.	877	C	T	Ts
57.	215	-	T	Indel	117.	888	T	C	Ts
58.	222	C	G	Tv	118.	947	T	C	Ts
59.	223	G	C	Tv	119.	949	T	C	Ts
60.	224	-	G	Indel

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Molecular characterization and phylogeny based on ITS2 and 28S regions of rDNA of Microphallus sp. (Digenea: Microphallidae) parasitic in freshwater crabs of Manipur, India

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