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International Journal of Zoology and Animal Biology Research Article 15 min read

Molecular Analysis of Pomphorhynchus kashmirensis based on 18S rDNA and ITS-rDNA

Sharifdini M and Amin OM*
* Corresponding author
ISSN: 2639-216X  10.23880/izab-16000372  Received: April 08, 2022  Published: April 22, 2022
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Keywords
Pomphorhynchus kashmirensis 18S rDNA ITS-rDNA Phylogeny
Abstract

Pomphorhynchus kashmirensis Kaw, 1941 was redescribed to correct inadequacies in the original description. We provide a molecular characterization to qualify its now completed morphological description and to compare with related taxa. Sequences of the 18S rDNA and ITS-rDNA datasets of specimens of P. kashmirensis were generated for assessment of gene diversity and the phylogenetic analyses. Comparative sequence analysis indicated interspecific variation between P. kashmirensis with different species of Pomphorhynchus was 0.2-1.4% and 2.4-36.4% based on 18S rDNA and ITS-rDNA datasets, respectively. The ITS-rDNA region is more variable than 18S rDNA within members of the family Pomphorhynchidae and it appears appropriate for assessment of their biodiversity. Our phylogenetic analyses showed that taxonomic position of the species P. kashmirensis is closely related with P. tereticollis and P. laevis. Also, the systematic status of Tenuiproboscis and Longicollum is uncertain within the family Pomphorhynchidae. Therefore, further molecular investigations will be needed for better understanding of the phylogenetic relationships in this family.

Introduction

Pomphorhynchus kashmirensis Kaw, 1941 is one of 9 species of Pomphorhynchus Monticelli, 1905 known from the Jammu-Kashmir regions of the Northern Indian Subcontinent. The original description from Triplophysa kashmirensis Hora, 1922 (Nemacheilidae) was inadequate as much of its morphological features could not be adequately visualized or confirmed in text or in illustrations [1]. Only one taxonomic treatment by Fotedar and Dhar was reported since its original description and before the redescription from specimens obtained from Schizothorax plagiostomus Heckel, 1838 by Amin et al. In the redescription, comparisons with the Kaw and Fotedar and Dhar accounts were made and 12 SEM images were added presenting new features not previously provided [2, 3]. This study complements the morphological aspects of P. kashmirensis as redescribed by providing its molecular profile for the first time based on of the 18S rDNA and ITS-rDNA datasets. Furthermore, its phylogenetic relationships with other species of the genus Pomphorhynchus and other members of Pomphorhynchidae are analyzed and discussed.

Materials and Methods

DNA Extraction and PCR Amplification

The P. kasmirensis material was collected from Schizothorax plagiostomus in the Jammu-Kashmir regions in the Northern Indian subcontinent. For molecular analysis, the genomic DNA was extracted from ethanol-preserved worms (n=3) using Qiagen DNeasy tissue kit (Qiagen Inc., Valencia, California, USA) according to manufacturer’s instructions. Finally, purified DNA was kept at − 20 °C until use.

The PCR mixture was performed in a final volume 30 μL containing 15 μL of 2X PCR premix with 1.5 mM MgCl2 (Ampliqon, Denmark), 20 ρM of each primer and 2 μL of the extracted DNA. The partial 18S region was amplified using the forward primer (5′-AGATTAAGCCATGCATGCGTAAG-3′) [4] and reverse primer (5′- ACCCACCGAATCAAGAAAGAG-3′) [5]. PCR amplification of the ITS1-5.8S-ITS2 ribosomal RNA gene was performed using the primers BD1 (5′ GTCGTAACAAGGTTTCCGTA-3′) and BD2 (5-TATGCTTAAATTCAGCGGGT-3′) [6].

The temperature profile for the 18S rRNA gene included an initial denaturation step at 95°C for 5 min followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 61°C for 30 s and extension at 72°C for 60 s, followed by a final extension step at 72°C for 7 min. The PCR conditions of ITS-rDNA region amplification consisted of initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 90 s, followed by a final extension at 72°C for 10 min. The PCR products were separated by electrophoresis on a 1.5% agarose gel and visualized with UV transluminator (Vilber Lourmat, Collégien, France). Finally, PCR products were sequenced by an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

The sequence results were edited and trimmed by Chromas software v.2.01 (Technelysium Pty Ltd., Brisbane, Queensland, Australia). Edited sequences were compared with GenBank submitted sequences using the Basic Local Alignment Search Tool (BLAST) [7]. The sequences of 18S rRNA and ITS-rDNA were deposited in GenBank (Accession Numbers: MZ381411-MZ381413 for 18S rRNA and MZ381446-MZ381448 for ITS-rDNA).

Phylogenetic Analysis

Phylogenetic trees were constructed using Maximum- likelihood algorithm and tamura-3- parameter model in MEGA6 software [8]. Also, genetic distances were calculated with p-distance model. The bootstrap value with 1000 replications were used for evaluation of reliability of the tree. The sequences used for the phylogenetic analysis are listed in Tables 1 & 2.

SpeciesHostLocationGenBank accession no.Reference
Pomphorhynchus kashmirensis (Kaw, 1941)Schizothorax plagiostomusIndiaMZ381411-MZ381413This study
Pomphorhynchus tereticollis (Rudolphi, 1809)Gammarus pulexFranceAY423347[9]
Pomphorhynchus laevis (Zoega in Müller, 1776)Squalius cephalusCroatiaKF559309[10]
Pomphorhynchus laevis (Zoega in Müller, 1776)Rutilus rutilusGermanyJX014223[11]
Pomphorhynchus zhoushanensis (Li, Chen, Amin & Yang, 2017)Oplegnathus fasciatus Temminck & SchlegelChinaKY490049-59[12]
Longicollum pagrosomi (Yamaguti, 1935)Oplegnathus fasciatus Temminck & SchlegelChinaKY490052[12]
Longicollum pagrosomi (Yamaguti, 1935)Red sea breamSouth KoreaKX641270[13]
Rhadinorhynchus lintoni (Cable & Linderoth, 1963)Selar crumenophthalmusUSAJX014224[11]
Rhadinorhynchus pristis (Rudolphi, 1802)Gempylus serpensIndonesiaJX014226[11]
Echinorhynchus borealis (Linstow, 1901)Perca fluviatilisFinlandMW172281[14]
Echinorhynchus truttae (Schrank, 1788)Thymallus thymallusNaAY830156[15]
Echinorhynchus gadi (Zoega in Müller, 1776)Macrourus berglaxGreenlandJX014222[11]
Echinorhynchus gymnocyprii (Liu, Wang & Yang, 1981)Ptychobarbus kaznakoviChinaMT162051Lei et al. (unpublished)
Echinorhynchus gymnocyprii (Liu, Wang & Yang, 1981)Gymnocypris przewalskiiChinaMT162047Lei et al. (unpublished)
Acanthocephaloides propinquus (Dujardin, 1845)Gobius bucchichiiNaAY830149[15]
Acanthocephalus lucii (Müller, 1776)Perca fluviatilisNaAY830152[15]
Acanthocephalus clavula (Dujardin, 1845)Anguilla anguillaIrelandMW172278[14]
Acanthocephalus rhinensis (Amin, Thielen, Münderl, Taraschewski & Sures, 2008)Anguilla anguillaGermanyMW172279[14]
Acanthocephalus dirus (Van Cleave, 1931)Asellus aquaticusNaAY830151[15]
Acanthocephalus anguillae (Müller, 1780)Asellus aquaticusSloveniaMN394415[16]
Serrasentis sagittifer (Linton, 1889)Lethrinus laticaudisAustraliaMF426939[17]
Gorgorhynchoides bullock (Cable & Mafarachisi, 1970)Eugerres plumiereNaAY830154[15]
Outgroup
Oligacanthorhynchus tortuosa (Leidy, 1850)Didelphis virginianaNaAF064817[18]

Table 1: Acanthocephalan species represented in the phylogenetic analysis with their host species, locations, GenBank accession n

SpeciesHostLocationGenBank accession no.Reference
Pomphorhynchus kashmirensis (Kaw, 1941)Schizothorax plagiostomusIndiaMZ381446-MZ381448This study
Pomphorhynchus tereticollis (Rudolphi, 1809)Platichthys flesusGermanyJF706705[19]
Pomphorhynchus tereticollis (Rudolphi, 1809)SalmonidsGermanyMT216144[20]
Pomphorhynchus laevis (Zoega in Müller, 1776)Phoxinus phoxinusSlovakiaAY135413[21]
Pomphorhynchus bosniacus (Kiskároly & Čanković, 1967)Alburnus alburnusBosnia and HerzegovinaMK157039[22]
Pomphorhynchus bosniacus (Kiskároly & Čanković, 1967)Barbus barbusBosnia and HerzegovinaMH319901[22]
Tenuiproboscis keralensis (Kaur, Shamal, Chandran, Binesh, Gishnu, Asokan & Sanil, 2017)Scatophagus argusIndiaKU726600 and KU726601[23]
Pomphorhynchus lucyi (Williams & Rogers, 1984)Micropterus salmonoidesUSAAY135418[21]
Echinorhynchus gymnocyprii (Liu, Wang & Yang, 1981)Ptychobarbus kaznakoviChinaMT162085[24]
Echinorhynchus gadi (Zoega in Müller, 1776)Gadus morhua morhuaBarents SeaEF107648[25]
Echinorhynchus truttae (Schrank, 1788)NaGermanyMT216137[20]
Acanthocephalus nanus (Van Cleave, 1925)Cynops pyrrhogasterJapanLC100043[26]
Acanthocephalus anguillae (Müller, 1780)Proteus anguinusSloveniaMN394425[16]
Rhadinorhynchus dorsoventrospinosus (Amin, Heckmann & Nguyen Van Ha, 2011)Decapterus kurroidesVietnamMH384822[27]
Neoechinorhynchus bullocki (Doolin & Reyda, 2018)Catostomus commersoniiUSAMK017784[28]
Floridosentis mugilis (Machado Filho, 1951)Mugil sp.Gulf of MexicoKC004179[29]

Table 2: Acanthocephalan species represented in the phylogenetic analysis with their host species, locations, GenBank accession n

Results

An approximately 1225 bp fragment of 18S rDNA gene and a 805 bp fragment of ITS-rDNA region were successfully amplified from the specimens of P. kashmirensis. Based on both datasets, there was no intraspecific genetic divergence found among the obtained isolates of P. kashmirensis. The 18S rDNA dataset (1144 nt) included 20 sequences of species representing five families (Pomphorhynchidae, Rhadinorhynchidae, Echinorhynchidae and Arhythmacanthidae) within the Echinorhynchida and the novel sequences of P. kashmirensis. The ITS- rDNA dataset (747 nt) included 13 sequences of species of four families (Pomphorhynchidae, Rhadinorhynchidae and Echinorhynchidae) of Echinorhynchida and the new sequences of P. kashmirensis.

The nucleotide divergence was noted between P. kashmirensis with P. laevis (KF559309 and JX014223), P. zhoushanensis (KY490049 and KY490050), P. tereticollis (AY423347), Longicollum pagrosomi (KY490052 and KX641270), Rhadinorhynchus lintoni (JX014224), R. pristis (JX014226), Acanthocephaloides propinquus (AY830149), Serrasentis sagittifer (MF426939), Gorgorhynchoides bullocki (AY830154), Echinorhynchus spp. and different species Acanthocephalus based on partial 18S rDNA sequence were 0.2-0.3%, 1.4%, 0.2%, 1.4%, 0.8%, 0.6%, 7.6%, 11.5%, 11.6%, 2.1-3.9% and 7.2-7.5%, respectively. Inter-generic differences between P. kashmirensis with P. tereticollis (JF706705 and MT216144), P. laevis (AY135413), P. bosniacus (MK157039 and MH319901), P. lucyi (AY135418), Tenuiproboscis keralensis (KU726600 and KU726601), Rhadinorhynchus dorsoventrospinosus (MH384822), Echinorhynchus spp. and Acanthocephalus spp. based on partial ITS-rDNA ITS-rDNA region were 2.5%, 2.4%, 5.5%, 36.4%, 15.5-15.6%, 49.2%, 36.3-38.2% and 49.6-50.1%, respectively.

The phylogenetic reconstruction based on the partial sequence spanning the 18S rDNA indicated that our sequences of P. kashmirensis (MZ381411-MZ381413) grouped with P. laevis (KF559309 and JX014223), P. tereticollis (AY423347), R. lintoni (JX014224) and R. pristis (JX014226). Additionally, P. zhoushanensis (KY490049 and KY490050) and L. pagrosomi (KY490052 and KX641270) appeared as sister group of mentioned clades (Figure 1).

Figure 1: Phylogenetic tree of isolates of the Pomphorhynchus kashmirensis obtained in this study (▲) and other members of the families Pomphorhynchidae, Echinorhynchidae, Arhythmacanthidae and Rhadinorhynchidae retrieved from GenBank based on the partial 18S rDNA gene. The tree was constructed based on the maximum likelihood method and the Tamura 3-parameter model in MEGA6. _Oligacanthorhynchus tortuosa_ sequence was used as the out group. Bootstrap values lower than 50 were omitted.
Click to enlarge
Figure 1: Phylogenetic tree of isolates of the Pomphorhynchus kashmirensis obtained in this study (▲) and other members of the families Pomphorhynchidae, Echinorhynchidae, Arhythmacanthidae and Rhadinorhynchidae retrieved from GenBank based on the partial 18S rDNA gene. The tree was constructed based on the maximum likelihood method and the Tamura 3-parameter model in MEGA6. Oligacanthorhynchus tortuosa sequence was used as the out group. Bootstrap values lower than 50 were omitted.

Figure 1: Phylogenetic tree of isolates of the Pomphorhynchus kashmirensis obtained in this study (▲) and other members of the families Pomphorhynchidae, Echinorhynchidae, Arhythmacanthidae and Rhadinorhynchidae retrieved from GenBank based on the partial 18S rDNA gene. The tree was constructed based on the maximum likelihood method and the Tamura 3-parameter model in MEGA6. Oligacanthorhynchus tortuosa sequence was used as the out group. Bootstrap values lower than 50 were omitted.

According to phylogenetic analyses based on the ITS- rDNA ITS-rDNA region, our sequences of P. kashmirensis (MZ381446-MZ381448) clustered with P. tereticollis (JF706705 and MT216144) and P. laevis (AY135413) but with marginal support. Also, P. bosniacus (MK157039 and MH319901) appeared as sister group of mentioned clades with higher bootstrap value. The sequences of T. keralensis (KU726600 and KU726601) as other genus of Pomphorhynchidae family were appeared to be a sister taxon of P. tereticollis, P. laevis and P. bosniacus with 100% of bootstrap support. However, the species of P. lucyi (AY135418) was located at the basal position to the members of the clade of Pomphorhynchidae (Figure 2).

Figure 2: Phylogenetic tree of isolates of the _Pomphorhynchus kashmirensis_ obtained in this study (▲) and other members of the families _Pomphorhynchidae, Echinorhynchidae_ and _Rhadinorhynchidae_ retrieved from GenBank based on the ITS-rDNA region. The tree was constructed based on the maximum likelihood method and the Tamura 3-parameter model in MEGA6. Neoechinorhynchus bullocki and _Floridosentis mugilis_ sequences were used as the out group. Bootstrap values lower than 50 were omitted.
Click to enlarge
Figure 2: Phylogenetic tree of isolates of the Pomphorhynchus kashmirensis obtained in this study (▲) and other members of the families Pomphorhynchidae, Echinorhynchidae and Rhadinorhynchidae retrieved from GenBank based on the ITS-rDNA region. The tree was constructed based on the maximum likelihood method and the Tamura 3-parameter model in MEGA6. Neoechinorhynchus bullocki and Floridosentis mugilis sequences were used as the out group. Bootstrap values lower than 50 were omitted.

Figure 2: Phylogenetic tree of isolates of the Pomphorhynchus kashmirensis obtained in this study (▲) and other members of the families Pomphorhynchidae, Echinorhynchidae and Rhadinorhynchidae retrieved from GenBank based on the ITS-rDNA region. The tree was constructed based on the maximum likelihood method and the Tamura 3-parameter model in MEGA6. Neoechinorhynchus bullocki and Floridosentis mugilis sequences were used as the out group. Bootstrap values lower than 50 were omitted.

Discussion

The genus Pomphorhynchus presently contains twenty- nine species which are ordinary parasitic worms within the intestinal tract of freshwater fishes, and sometimes marine fishes and amphibians [30]. Until now, only few species of this genus have been characterized using molecular analysis [12, 19, 21, 31, 32, 33]. The validity of P. kashmirensis as redescribed by Amin, et al. is confirmed by the presented molecular analysis. Herein, molecular profile of P. kashmirensis is obtained based on partial 18S rDNA and ITS- rDNA genes for understanding its taxonomic relationships within the family Pomphorhynchidae and with other families of Echinorhynchida.

The level of interspecific nucleotide variation between P. kashmirensis and other species of Pomphorhynchus registered in Gen Bank was 0.2-1.4% and 2.4-36.4% based on 18S rDNA and ITS-rDNA sequences, respectively. These results indicated that ITS-rDNA is more variable between the species than 18S rDNA. Based on the partial 18S rDNA, intergeneric variation between P. kashmirensis and L. pagrosomi another genus of the family Pomphorhynchidae, was 1.4%. Also, based on partial ITS sequences, inter-generic variations between P. kashmirensis and T. keralensis sequences another genus of Pomphorhynchidae family was 15.5-15.6%. Similar to our study, other researchers claimed ITS rDNA sequences has high level of interspecific nucleotide variation in different species of Pomphorhynchus [21, 12]. In addition, some molecular studies on acanthocephalans illustrated that ITS rDNA region is a relatively variable region and has been used as a suitable molecular marker for inferring phylogenetic relationships in some acanthocephalan genera such as Acanthogyrus [34], Corynosoma [35], Neoechinorhynchus [36] and Leptorhynchoides [37].

The small subunit 18S rRNA gene is one of the most frequently used molecular markers to determine genetic diversity and phylogenetic relationships among acanthocephalans. This gene is fairly conserved with a slow evolutionary rate and is usually utilized to infer phylogenetic analysis among the major classes of Acanthocephala [38]. In this study, relatively small genetic variation was found between members of the genus Pomphorhynchus based on18S rRNA gene. Also, there were small genetic differences between species of Pomphorhynchus and Rhadinorhynchus based on this gene. This issue has been investigated and it has been concluded that these two sequences are not really Rhadinorhynchus, but actually Pomphorhynchids [39].

The phylogenetic tree of the 18S rDNA gene (Figure 1) showed that the sequences of P. kashmirensis (MZ381411-MZ381413) obtained in the current study grouped with P. laevis (KF559309 and JX014223) from Croatia and Germany and P. tereticollis (AY423347) from France. However, R. lintoni (JX014224) and R. pristis (JX014226) were nested within this cluster but the genus Rhadinorhynchus was located well apart from the members of the family Pomphorhynchidae in the ITS-rDNA tree. Our 18S rDNA phylogenetic tree is similar to a previously published tree [39, 40], where the genus Rhadinorhynchus was found to be clustered with these species of Pomphorhynchus. In addition, P. zhoushanensis (KY490049 and KY490050) from China and L. pagrosomi (KY490052 and KX641270) from China and Korea were grouped together in one cluster and appeared as a sister group of the mentioned clade with high statistical support. Some authors have also observed a similar situation in L. pagrosomi in Pomphorhynchus clade [12, 40].

Similar to the 18S rDNA tree, the phylogenetic tree of ITS-rDNA (Figure 2) also illustrated that the P. kashmirensis has a close relationship with P. tereticollis (JF706705 and MT216144) from Germany and P. laevis (AY135413) from Slovakia. Additionally, P. bosniacus isolates (MK157039 and MH319901) from Bosnia and Herzegovina clustered as a sister group of the mentioned clade with high statistical support. Similar to our study, Li, et al. showed that the genus Tenuiproboscis was nested within the core of Pomphorhynchus in the phylogenetic trees [12]. Meanwhile, the species of P. lucyi (AY135418) isolated from USA was placed separately from other members of genus Pomphorhynchus. In the current study, the higher level of variation in ITS-rDNA region compared to the 18S rDNA gene improved resolution of the relationships within the family Pomphorhynchidae with high bootstrap support.

Similar to our findings, Li et al. proposed that genus Pomphorhynchus is a polyphyletic taxon based on three different genetic markers (18S rDNA, ITS rDNA and Cox1). It is because members of the Pomphorhynchus were mixed with the genera Longicollum and Tenuiproboscis [12]. The current phylogenetic position of Longicollum and Tenuiproboscis remains questionable and more investigations required to elucidate the phylogenetic relationship between these genera.

Conclusion

The present phylogenetic analyses based on both 18S rDNA and ITS-rDNA sequence data showed the taxonomic position of P. kashmirensis being closely related with P. tereticollis and P. laevis. The ITS-rDNA sequences of members of genus Pomphorhynchus have more variation than 18S rDNA which it can be useful marker for achieving a proper assessment of gene diversity and phylogenetic analyses. In addition, the systematic status of Tenuiproboscis and Longicollum is uncertain within the Pomphorhynchidae. Therefore, obtaining sequence data from other members of the family Pomphorhynchidae and providing additional genetic markers would be useful for better understanding of the phylogenetic relationships in this family.

Acknowledgement

The authors are grateful for the financial support of the Department of Biology at Brigham Young University, Provo, Utah and for the Institutional Grant provided by The Parasitology Center, Scottsdale, Arizona, USA.

Compliance with Ethical Standards

Conflict of Interest: The authors declare no conflict of interest and compliance with all relevant ethical standards.

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@article{sharifdini2022,
  title   = {Molecular Analysis of Pomphorhynchus kashmirensis based on
18S rDNA and ITS-rDNA},
  author  = {Sharifdini M and Amin OM},
  journal = {International Journal of Zoology and Animal Biology},
  year    = {2022},
  volume  = {5},
  number  = {2},
  doi     = {10.23880/izab-16000372}
}
Sharifdini M and Amin OM (2022). Molecular Analysis of Pomphorhynchus kashmirensis based on
18S rDNA and ITS-rDNA. International Journal of Zoology and Animal Biology, 5(2). https://doi.org/10.23880/izab-16000372
TY  - JOUR
TI  - Molecular Analysis of Pomphorhynchus kashmirensis based on
18S rDNA and ITS-rDNA
AU  - Sharifdini M and Amin OM
JO  - International Journal of Zoology and Animal Biology
PY  - 2022
VL  - 5
IS  - 2
DO  - 10.23880/izab-16000372
ER  -