The Molecular Profile of Paratrajectura Longcementglandatus Amin, Heckmann Et Ali, 2018 (Acanthocephala: Transvenidae) from Percid Fishes in the Marine Waters of Iran and Iraq.

Paratrajectura longcementglandatus Amin, Heckmann et Ali, 2018 (Transvenidae) was recently described from two species of percid fishes collected from the marine territorial waters of Iraq and Iran in the Persian Gulf. The genus Paratrajectura Amin, Heckmann et Ali, 2018 is a close relative to transvenid genera Trajectura Pichelin et Crib, 2001 and Transvena Pichelin et Crib, 2001. Morphologically, Paratrajectura is characterised by having apical proboscis cone, long, tubular cement glands, short lemnisci, prominent roots on all proboscis hooks, subterminal female gonopore, and males with long pre-equatorial testes. Molecular studies of P. longcementglandatus using 18S rDNA and cox1 genes compared with available data of members of other families of Echinorhynchida showed that P. longcementglandatus is grouped with species of the genus Transvena forming a clade within the family Transvenidae.

Introduction

Pichelin & Cribb (2001) described the family Transvenidae with two genera: monotypic Transvena with T. annulospinosa Pichelin et Cribb, 2001, and Trajectura with two species, T. ikedai (Machida, 1992) and T. perinsolens Pichelin et Cribb, 2001. Specimens of the two genera were recovered from wrasses (Labridae, Perciformes) in the Pacific off southern Australia and southern Japan. Lisitsyna et al. (2019) described two other species of the family Transvenidae, namely Transvena pichelinae Lisitsyna, Kudlai, Cribb et Smit, 2019, and Pararhadinorhynchus sodwanensis Lisitsyna, Kudlai, Cribb et Smit, 2019 from the marine fishes from the Sodwana Bay, South Africa. The other genus of this family, Paratrajectura, was established by Amin et al. (2018). It comprises one species Paratrajectura longcementglandatus Amin, Heckmann et Ali, 2018, which was described on the basis of worms from the Japanese threadfin bream Nemipterus japonicus Bloch (Nemipteridae) and the tigertooth croacker, Otolithes ruber Bloch et Schneider (Sciaenidae, Perciformes) caught in the marine territorial waters of Iraq and Iran, the Persian Gulf (Amin et al., 2018). The genus Paratrajectura is characterised by having apical proboscis cone, long, tubular cement glands, short lemnisci, prominent roots on all proboscis hooks, subterminal female gonopore, and males with long pre-equatorial testes.

While, several studies have been published about sequence data for acanthocephalans including two Transvena spp. (Westram et al., 2011; Garcia-Varela, et al., 2013; Pinacho-Pinacho et al., 2014; Lisitsyna et al., 2019), no sequence data has been published for P. longcementglandatus whose phylogenetic relationship with other acanthocephalans and related families was unknown. In this paper, we report the molecular profile of P. longcementglandatus, validate its generic affiliations, and explore its evolutionary relationships with related and other species and taxa based on partial 18S rDNA and cox1 genes.

Materials and Methods

DNA extraction and PCR amplification

For extraction of genomic DNA, five adult worms of P. longcementglandatus were washed with sterile distilled water several times to remove the ethanol residuals. Total DNA was extracted using Qiagen DNeasy Blood and Tissue kit (Qiagen Inc., Valencia, California, USA) according to manufacturer’s instructions and kept at −20 °C until use.

PCR reactions were performed in 30 μL volumes containing 2 × red PCR premix (Ampliqon, Odense, Denmark), 20 pmol of each primer and 3 μL of extracted DNA. The partial 18S rRNA gene was amplified using the forward primer (5′-AGATTAAGCCATGCATG-CGTAAG-3′) and reverse primer (5′- ACCCACCGAATCAAGAAA-GAG-3′). Also, primers used for the amplification of the partial mitochondrial cytochrome oxidase subunit1 (cox1) gene were COI-F (5′-AGTTCTAATCATAARGATATYGG-3′) and COI-R (5′-TAAACT-TCAGGGTGACCAAAAAATCA-3′) (Folmer et al., 1994). PCR conditions for 18S rRNA gene amplification included of an initial denaturing step of 95 °C for 5 min and 35 cycles followed by denaturing step at 95 °C for 30 s, annealing step of 61 °C for 30 s, and 60 s of extension at 72 °C, and 72 °C for 7 min as a final extension. The thermal PCR profiles for cox1 gene consisted of initial denaturation at 95 °C for 6 minutes followed by 35 cycles of 95 °C for 30 s (denaturation), 55 °C for 30 s (annealing), and at 72 °C for 60 s (extension) with a final extension of 72 °C for 6 minutes. PCR products were analysed on 1.5 % agarose gel and visualized with UV transluminator. Next, the PCR products were sequenced in both directions using the same PCR primers with ABI 3130 sequencer.

The obtained sequence results were manually edited and trimmed using Chromas software v.2.01 (Technelysium Pty Ltd., Brisbane, Queensland, Australia). Next, generated sequences were compared with GenBank submitted sequences using the Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih gov/). Also, Clustal W method of Bioedit software v.7.0.9 was used for multiple sequence alignment (Larkin et al., 2007). The sequences of 18S rRNA and cox1 genes were submitted to GenBank database (Accession Numbers: MK770616 for 18S rRNA and MK770615 for cox1)

Phylogenetic analysis

The phylogenetic tree was constructed using Maximum-Likelihood model and Tamura-3-parameter model by Molecular and Evolution Genetic Analysis software v.6 (MEGA 6). The reliability of topology of the tree was supported with Bootstrap value based on 1000 replications. The whole scientific names of acanthocephalan species, names of host species, localities, and GenBank accession numbers used in the phylogenetic analysis are listed in Table 1.

Table 1

Acanthocephalan species represented in the phylogenetic analysis with their family, host species, GenBank accession numbers, locations, and references.

Species	Host	GenBank Acc. no. 185rDNA	GenBank Acc. no. cox1	Location	Reference
Gymnorhadinorhynchidae
Gymnorhadinorhynchus sp.	Regalecus russelii	MK014866	MK012665	Japan	Steinauer et al. (2019)
Gymnorhadinorhynchus decapteri (Braicovich, Lanfranchi, Farber, Marvaldi, Luque et Timi, 2014)	Decapterus punctatus	KJ590123	KJ590125	Brazil	Braicovich et al. (2014)
Cavisomidae
Neorhadinorhynchus nudus (Harada, 1938)	Auxis thazard	-	MG757444	China	Li et al. (2018)
Filisoma bucerium (Van Cleave, 1940)	Kyphosus elegans	AF064814	DQ089722	Na*	García-Varela et al. (2000), García-Varela and Nadler (2006)
Filisoma rizalinum (Tubangui et Masilungan, 1946)	Scatophagus argus	JX014229	-	Indonesia	Verweyen et al. (2011)
Rhadinorhynchidae
Rhadinorhynchus laterospinosus (Amin, Heckmann et Van Ha, 2011)	Auxis rochei	MK457183	MK572744	Vietnam	Amin et al. (2019a)
Rhadinorhynchus sp.	Sciaenidae	AY062433	DQ089712	Na	García-Varela et al. (2002), García-Varela and Nadler (2006)
Serrasentis sagittifer (Linton, 1889)	Lutjanus sebae	-	MF134296	Australia	Barton et al. (2018)
Serrasentis sagittifer	Johnius coitor	JX014227	-	Indonesia	Verweyen et al. (2011)
Serrasentis nadakali (George et Nadakal, 1978)	Na	KC291715	KC291713	Na	Paul et al. (unpublished)
Gorgorhynchoides bullock (Cable et Mafarachisi, 1970)	Eugerres plumieri	AY830154	DQ089715	Na	Garcia-Varela and Nadler (2005, 2006)
Rhadinorhynchus lintoni (Cable et Linderoth, 1963)	Selar Crumrn- opht halmus	JX014224	-	USA	Verweyen et al. (2011)
Rhadinorhynchus pristis (Rudolphi, 1802)	Selar Crumrn- opht halmus	JX014226	-	USA	Verweyen et al. (2011)
Transvenidae
Paratrajectura longcementglandatus (Amin, Heckmann et Ali, 2018)	Percid fishes	MK770616	MK770615	Marine waters of Iraq and Iran	Present study
Transvena annulospinosa (Pichelin et Cribb, 2001)	Anampses neoguinaicus	AY830153	DQ089711	Na	Garcia-Varela and Nadler (2005, 2006)
Transvena pichelinae sp. n. (Lisitsyna, 2019)	Thalassoma purpureum	MN105736, MN105737	MN 104895, MN 104896	South Africa	Lisitsyna et al. (2019)
Pararhadinorhynchus sodwanensis sp. n. (Lisitsyna, 2019)	Pomadasys furcatus	MN 105738	-	South Africa	Lisitsyna et al. (2019)
Pararhadinorhynchus sp.	Siganus fuscescens	HM545903	-	China	Wang et al. (unpublished)
Echinorhynchidae
Pseudoacanthocephalus toshimai (Nakao, 2016)	Rana pirica	LC129278	LC100044	Japan	Nakao (2016)
Pseudoacanthocephalus lucidus (Van Cleave, 1925)	Rana ornativentris	LC129279	LC100057	Japan	Nakao (2016)
Acanthocephalus lucii (Müller, 1776)	Perca fluviatilis	AY830152		Na	Garcia-Varela and Nadler (2005), Benesh et al. (2006)
Acanthocephalus lucii	Perca fluviatilis		AM039837	England	Garcia-Varela and Nadler (2005), Benesh et al. (2006)
Acanthocephalus anguillae (Müller, 1780)	Perca fluviatilis		AM039865	Austria	Benesh et al. (2006)
Acanthocephalus dirus (Van Cleave, 1931)	Asellus aquaticus	AY830151	DQ089718	Na	Garcia-Varela and Nadler (2005, 2006)
Acanthocephalus clavula (Dujardin, 1845)	Perca fluviatilis		AM039866	Ireland	Benesh et al. (2006)
Acanthocephalus nanus (Van Cleave, 1925)	Cynops pyrrhogaster	LC129889		Japan	Nakao (2016)
Echinorhynchus salmonis (Müller, 1784)	Coregonus lavaretus		KP261017	Finland	Wayland et al. (2015)
Echinorhynchus gadi (Müller, 1776)	Na	AY218123	AY218095	Na	Giribet et al. (2004)
Echinorhynchus bothniensis (Zdzitowiecki et Valtonen, 1987)	Osmerus eperlanus		KP261018	Finland	Wayland et al. (2015)
Echinorhynchus truttae (Schrank, 1788)	Thymallus thymallus	AY830156	DQ089710	Na	Garcia-Varela and Nadler (2005, 2006)
Echinorhynchus brayi (Wayland, Sommerville et Gibson, 1999)	Pachycara crassiceps		KP261015	Atlantic Ocean: Porcupine Seabight	Wayland et al. (2015)
Echinorhynchus cinctulus (Porta, 1905)	Lota lota		KP261014	Finland	Wayland et al. (2015)
Pomphorhynchidae
Longicollum pagrosomi (Yamaguti, 1935)	Pagrus major	LC195887	-	Japan	Mekata et al. (unpublished)
Longicollum pagrosomi	Oplegnathus fasciatus	-	KY490048	China	Li et al. (2017)
Pomphorhynchus zhoushanensis (Li, Chen, Amin et Yang, 2017)	Oplegnathus fasciatus	-	KY490045	China	Li et al. (2017)
Pomphorhynchus bulbocolli (Linkins, 1919)	Lepomis macrochirus	-	DQ089709	Na	Garcia-Varela and Nadler (2006)
Pomphorhynchus purhepechus (García-Varela, Mendoza-Garfias, Choudhury et Pérez-Ponce de León, 2017)	Moxostoma austrinum	-	KY911281	Na	Garcia-Varela et al. (2017)
Tenuiproboscis sp.	Epinephelus malabaricus	-	JF694273	Na	Vijayan et al. (unpublished)
Pomphorhynchus laevis (Zoega in Müller, 1776)	Gammarus pulex	AY423346	AY423348	France	Perrot-Minnot (2004)
Pomphorhynchus tereticollis (Rudolphi, 1809)	Gammarus pulex	AY423347	AY423351	France	Perrot-Minnot (2004)
Arhythmacanthidae
Acanthocephaloides propinquus (Dujardin, 1845)	Gobius bucchichii	AY830149	DQ089713	Na	Garcia-Varela and Nadler (2005, 2006)
Diplosentidae
Sharpilosentis peruviensis (Lisitsyna, Scholz et Kuchta, 2015)	Duopalatinus cf. peruanus	-	KP967562	Peru	Lisitsyna et al. (2015)
Illiosentidae
Dollfusentis chandleri (Golvan, 1969)	Na	-	DQ320484	Na	Baker and Sotka(unpublished)
Dentitruncus truttae (Sinzar, 1955)	Salmo trutta	JX460865	JX460877	Croatia	Vardić Smrzlić et al. (2013)
Illiosentis sp.	Na	AY830158	DQ089705	Na	Garcia-Varela and Nadler (2005, 2006)
Leptorhynchoides thecatus (Linton, 1891)	Lepomis cyanellus	AF001840	DQ089706	Na	Near et al. (1998), Garcia-Varela and Nadler (2006)
Pseudoleptorhynchoides lamothei (Salgado-Maldonado, 1976)	Ariopsis guatemalenis	EU090950	EU090949	Na	Near et al. (1998), Garcia-Varela and Nadler (2006)
Koronacantha pectinaria (Van Cleave, 1940)	Microlepidotus brevipinnis	AF092433	DQ089707	Na	García-Varela and Nadler (2005, 2006)
Koronacantha Mexicana (Monks et Pérez-Ponce de León, 1996)	Haemulopsis leuciscus	AY830157	DQ089708	Na	García-Varela and Nadler (2005, 2006)
Neoechinorhynchidae (Outgroup)
Neoechinorhynchus brentnickoli (Monks, Pulido-Flores and Violante- González, 2011)	Dormitator latifrons	-	JN830849	Na	Pinacho-Pinacho et al. (2012)
Neoechinorhynchus saginata (Van Cleave & Bangham, 1949)	Na	-	DQ089704	Na	García-Varela and Nadler (2006)
Floridosentis mugilis (Machado Filho, 1951)	Na	AF064811	DQ089723	Na	García-Varela and Nadler (2006), García-Varela et al. (2000)
Neoechinorhynchus crassus (Van Cleave, 1919)	Na	KU363969	-	Iran	Dadar and Adel (unpublished)
Neoechinorhynchus pseudemydis (Cable and Hopp, 1954)	Capoeta aculeata	KU363973	-	Iran	Dadar and Adel (unpublished)

*Na = not available

Ethical Approval and/or Informed Consent

The authors declare compliance with all relevant ethical standards.

Results

The specimens of P. longcementglandatus successfully presented amplifications of about 1234 bp for the 18S rDNA gene and 664 bp for the cox1 gene. Comparisons of the18S rDNA and cox1 sequences from this parasite with other available acanthocephalan sequences in GenBank, using multiple sequence alignment, showed that it had the highest similarity with T. annulospinosa based on18S rDNA (98 %) and cox1 (77 %) genes. The 18S rDNA dataset (1129 nt) included 26 sequences for species of seven families within the Echinorhynchida and the novel sequence of P. longcementglandatus. The cox1 dataset (538 nt) included 39 sequences for species of nine families of Echinorhynchida and the sequence of P. longcementglandatus.

The phylogenetic reconstruction based on the partial sequence spanning the 18S rDNA showed that our sequence of P. longcementglandatus is clustered with Transvena annulospinosa (AY830153), T. pichelinae (MN105736 and MN105737), P. sodwanensis (MN105738) and an unidentified species of Pararhadinorhynchus (HM545903) with strong support forming a clade of the family Transvenidae. Also, the species of Gymnorhadinorhynchus sp. (MK014866) (Gymnorhadinorhynchidae) and Rhadinorhynchus laterospinosus (MK457183) (Rhadinorhynchidae) are very closely related with the family Transvenidae in the tree with 100 % of bootstrap support. The sequence of Gymnorhadinorhynchus decapteri (KJ590123) (Gymnorhadinorhynchidae) is located at the basal position to the members of the clade. Other families of the order Echinorhynchida including Rhadinorhynchidae, Pomphorhynchidae, Cavisomidae, Arhythmacanthidae and Echinorhynchidae located in a major sister clade (Fig. 1). Inter-generic differences are noted between P. longcementglandatus and T. annulospinosa, T. pichelinae, P. sodwanensis and Pararhadinorhynchus sp. from Transvenidae based on partial 18S rDNA sequence were 2.4 % (18 nt), 2.8 % (21 nt), 2.9 % (22 nt) and 2.9 % (22 nt), respectively. According to phylogenetic analyses based on the cox1 gene, our sequence of P. longcementglandatus (MK770615) is grouped with T. annulospinosa (DQ089711) and T. pichelinae (MN104895 and MN104896) with strong support in a clade of the family Transvenidae. The species of G. decapteri (KJ590125) and Gymnorhadinorhynchus sp. (MK012665) (Gymnorhadinorhynchidae), Neorhadinorhynchus nudus (MG757444) (Cavisomidae), R. laterospinosus (MK572744) and Rhadinorhynchus sp. (DQ089712) (Rhadinorhynchidae), appear as a sister group of the family Transvenidae (Fig. 2). The interspecific divergence between P. longcementglandatus and T. annulospinosa, T. pichelinae based on partial cox1 gene was 23.4 % (141 nt), 27.3 % (144 nt), respectively.

Fig. 1

Phylogenetic tree based on the Maximum likelihood analysis using 18S rDNA sequence of Paratrajectura longcementglandatus of current study and sequences of the closest-related members of the order Echinorhynchida deposited in the GenBank. Outgroup: Floridosentis mugilis, Neoechinorhynchus pseudemydis and N. crassus. Bootstrap values lower than 70 are omitted.

Fig. 2

Phylogenetic reconstruction based on the Maximum likelihood analysis using partial region of the cox1 sequence of Paratrajectura longcementglandatus of current study and sequences of the closest-related members of order Echinorhynchida deposited in the GenBank. Outgroup: Floridosentis mugilis, Neoechinorhynchus saginata and N. brentnickoli. Bootstrap values lower than 70 are omitted.

Discussion

Recently, molecular methods are applied for species identification, classification and phylogenetic analysis of acanthocephalan species (García-Varela et al., 2002). To date, molecular profile has been provided for few species of the family Transvenidae including T. annulospinosa, T. pichelinae, P. sodwanensis and Pararhadinorhynchus sp. (Pichelin & Cribb, 2001; Lisitsyna et al., 2019). In the current study, phylogenetic relationships of P. longcementglandatus as another genus of this family is described based on partial 18S rDNA and cox1 genes determining relationships with other acanthocephalan families.

This study showed that the interspecific variation between P. longcementglandatus and species of Transvena based on partial 18S rDNA was 2.4 % – 2.8 % (18 – 21 nt) and between it and species of Pararhadinorhynchus was 2.9 % (22 nt). Also based on cox1 gene, inter-generic variations between P. longcementglandatus and T. annulospinosa was 23.4 % (141 nt). These results illustrate that sequence differences between the genera of the family based on cox1 gene is higher than18S rDNA and it is appropriate to consider for taxonomic studies at the generic level.

The phylogenetic analysis of the 18S rDNA sequence (Fig. 1) showed that P. longcementglandatus is grouped in a highly supported clade with T. annulospinosa (AY830153), T. pichelinae (MN105736 and MN105737), P. sodwanensis (MN105738) and Pararhadinorhynchus sp. (HM545903) forming a clade of the family Transvenidae. In the clade, the family Transvenidae grouped close to R. laterospinosus (MK457183) and G. decapteri (KJ590123) (Gymnorhadinorhynchidae). Our phylogenetic tree for 18S rDNA is similar to those of García-Varela et al. (2002) and Lisitsyna et al. (2019) where the family Transvenidae grouped close to different species of Rhadinorhynchus (Rhadinorhynchidae) and Gymnorhadinorhynchus (Gymnorhadinorhynchidae).

Our phylogenetic analysis of the cox1 gene (Fig. 2) confirmed that P. longcementglandatus is grouped with T. annulospinosa (DQ089711) and T. pichelinae (MN104895 and MN104896) which made the clade of the family Transvenidae with good statistical support. Also, the families Rhadinorhynchidae, Gymnorhadinorhynchidae and Cavisomidae appear as a sister group with the clade of family Transvenidae. Other families of Echinorhynchida such as Pomphorhynchidae, Echinorhynchidae, Cavisomidae, Illiosentidae, Rhadinorhynchidae, Gymnorhadinorhynchidae, Diplosentidae and Arhythmacanthidae are well separated in the later clade. In the present study, the higher level of variation in cox1 gene compared to the 18S rDNA gene provides better resolution of the relationships within closely related taxa. While Amin et al. (2019a) presented relationships in their analysis of Rhadinorhynchus based on cox1 sequences, it was not clearer than 18S rDNA due to the lack of sufficient sequences of this gene in GenBank.

One of the most commonly used molecular markers for classification of acanthocephalans is the small subunit from RNA ribosomal gene or 18S rRNA. This gene displays a slow evolution rate and is highly conserved. It was used to infer phylogenetic relationships among the major classes of Acanthocephala (García-Varela & Pérez-Ponce de León, 2015). Most of phylogenetic studies of acanthocephalans similar to this research showed that 18S rDNA sequences appear to be suitable marker for phylogenies among acanthocephalans (García-Varela ; Near, 2002; Herlyn et al., 2003; Verweyen et al., 2011; Amin et al., 2019b). Also, cox1 gene is commonly used for phylogenetic studies and to recognize and establish species limits in acanthocephalans (Guillen-Hernández et al., 2008; Alcántar-Escalera et al., 2013; García-Varela, et al., 2013). The present study confirmed that this gene has high genetic diversity among genera of the family and other families of Echinorhynchida which would be more particularly useful for phylogenetic analysis.

Finally, the genetic data collected in the current study provide a better understanding of the taxonomic status of P. longcementglandatus. Sequence variations within the family Transvenidae and among other families of Echinorhynchida based on cox1 gene is higher than18S rDNA that can be useful for achieving a proper assessment of biodiversity. More sequence data from other geographical isolates using more gene targets will be useful for exploring the phylogenetic relationships among species. On the other hand, using of molecular tools for identification of acanthocephalan species is still scarce due to the lack of sequences of different genera of acanthocephalans in GenBank (Amin et al., 2013; Salgado-Maldonado, 2013; Weaver & Smales, 2013; Amin et al., 2014; Smales, 2014; Gomes et al., 2015; Steinauer & Nickol, 2015). More molecular studies are recommended in order to elucidate acanthocephalans classification.