First molecular confirmation of the Dactylogyrus anchoratus and D . vastator ( Monogenea , Dactylogyridae ) from Carassius auratus in western India

Dactylogyrus anchoratus and Dactylogyrus vastator (Monogenea, Dactylogyridae) are distributed worldwide as the most frequent ectoparasites of goldfish (Carassius auratus). This is the first report of D. anchoratus and D. vastator from India. The monogeneans were identified using morphometric measurements of hard parts, the morphology of the haptoral parts and the shape of the male copulatory organ. Molecular characterization by phylogenetic analyses of 18S and 28S ribosomal RNA gene sequences supported the morphological identifications.

The Goldfish Carassius auratus (Linnaeus, 1758) is a freshwater fish of the family Cyprinidae (order Cypriniformes).C. auratus was introduced to India from Japan many years ago, but the year of introduction is unknown (http://www.fao.org/fishery/introsp/230/en).It has been widely distributed by the booming aquarium industry, which has made it the most popular ornamental fish in the world (Morgan and Beatty 2007;Gupta and Banerjee 2009).Many studies have suggested that C. auratus is one of the most common host parasitized by Dactylogyrus spp.(Šimková et al. 2004;Jalali and Barzegar 2005;Shamsi et al. 2009;Molnár 2009, Rasouli et al. 2012;Borisov 2013;Tu et al. 2015).In India, most studies of Dactylogyrus spp.have been based only on morphology.However, accurate identification of such a large group of species requires the use of molecular tools such as the ribosomal 18S and 28S genes that are used to identify and distinguish monogenean species (Chisholm et al. 2001;Šimková et al. 2004;Wu et al. 2007;Chaudhary andSingh 2012, 2013).

Parasite collection
Thirty six Gold fish obtained from the aquarium market in Meerut (29º01′N; 77º45′E), U.P., India were killed by a sharp blow on the top of the head, and their gill filaments were dissected out.For the collection of Dactylogyrus species, gill filaments were examined using a Motic SMZ-168 series stereomicroscope and parasites were removed.Parasites collected from the same individual fish were used for both morphological and sclerotized structure studies.For morphological examination permanent slides of whole individual parasites were prepared by staining with acetocarmine, dehydrating with ascending grades of alcohol and mounting in Canada balsam.For the study of sclerotized structures, whole parasites were cleared gradually in water and mounted in glycerin.Specimens were examined using light microscopy.Identification was via the morphology of haptoral hard parts and the copulatory complex.Line drawings were made with a camera lucida connected to the Motic digital microscope (DMB series).All measurements were made directly from the drawings and also confirmed with the inbuilt measuring software (Motic Image Plus 2.0 for Windows).Measurements are given in micrometers unless otherwise stated.

Molecular analysis
Prior to DNA analysis, worms were identified on the basis of morphology and then preserved in 95% ethanol at −20°C in separate vials.Genomic DNA was extracted from a pool of ten parasites for both the Dactylogyrus species collected using the Qiagen DNeasy ™ tissue kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions.The partial fragments of 18S and 28S ribosomal RNA genes were amplified with primers 18S (forward, 5'-CGGTTGCAATTTTTATGTGG-3' and reverse, 5'-GAGTGATCCACCACTTGCAG-3') (Chiary et al. 2014) and 28S (forward, 5'-TCTAGTAACGGCGA GTGAACG-3' and reverse, 5'-GGTGGAAGGTCT ACCTCAGC-3') (Chiary et al. 2014).Amplification reactions were performed as reported by Chiary et al. (2014).PCR cycles were carried out for 35 cycles as follows: 3 min at 94 °C (initial denaturation) followed by 30 sec at 94 °C for further denaturation, annealing for 45 sec at 55°C (18S primer pair) or 59°C (28S primer pair), 1 min at 72 °C followed by a final extension for 10 min at 72 °C.PCR products were examined on 1%agarose-TAE gels, stained with ethidium bromide and visualized under UV transillumination.PCR products were purified by Purelink ™ Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Löhne, Germany), according to the manufacturer's instructions, and sequenced using a Big Dye Terminator version 3.1 cycle sequencing kit in an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) using both sets of primers.
The 18S and 28S rDNA sequence data were subjected to BLASTn searches for related species of Dactylogyrus in GenBank (http://www.ncbi.nlm.nih.gov/genbank) for phylogenetic analysis.Genetic divergences were calculated using a p-distance model for each gene region using MEGA v. 6 (Tamura et al. 2013).For the phylogeny of Dactylogyrus spp., related sequences were retrieved and aligned using the Clustal W (Thompson et al. 1994) multiple alignment option with default parameters.Maximum likelihood (ML) and Bayesian inference (BI) analyses were calculated under the GTR+I+G model, selected by Model Test, using MEGA v. 6 (Tamura et al. 2013) and TOPALI 2.5 (Milne et al. 2009) respectively.The data were tested for the nucleotide substitution model of best fit using Akaike's Information Criterion (AIC) (Akaike 1973).Bootstrap values were generated based on 1,000 resampled datasets.Cichlidogyrus ergensi (GenBank accession number HE792788) and C. falcifer (HQ010024) were used as respective outgroups in the 18S and 28S phylogenetic trees generated.
Voucher  Based on the morphological study of hard parts, D. anchoratus showed morphological similarities with D. arcuatus Yamaguti, 1942 andD. formosus Kulwiec, 1927, which also parasitize C. auratus.D. anchoratus can easily be distinguished from D. arcuatus by the morphology of the male copulatory organ, which is slightly sinusoidal in D. anchoratus, but strongly curved in D. arcuatus.Additionally, D. anchoratus shows some similarities with D. formosus in the shape of the haptoral parts; however, all parts of the haptor of D. anchoratus are significantly larger than D. formosus.Based on the morphological study of the hard parts of haptor and male copulatory organ, D. vastator shows some morphological similarities with D. intermedius, another C. auratus parasite.However, D. vastator can be readily distinguished from D. intermedius on the basis of comparatively larger roots and hook length.
For D. anchoratus, the BLASTn search of the 510bp 18S fragment showed an overall 99% identity to the other two sequences of the same species isolated from Cyprinus carpio (AJ490161) and Carassius auratus (AJ564111) from the Czech Republic (Šimková et al. 2004).P-distance analysis revealed that the 18S rRNA gene sequence isolated from Indian D. anchoratus diverged from the C. auratus isolate by only 0.024% and from the C. carpio isolate by 0.026%; the former thus being genetically more similar to the present specimens.Besides this, the 18S sequence of D. anchoratus generated in this study also displayed 98 to 99% identity to D. formosus and D. arcuatus, and these share some morphological resemblance.In the 18S phylogenetic analysis, D. anchoratus (Indian isolate) grouped together with the same species isolates collected from the Czech Republic, along with the closely related sister species, D. formosus and D. arcuatus, using both ML and BI analyses (Figure 2).
The 790 bp 28S rRNA gene sequence of the Indian D. anchoratus lineage also shows 99% identity to two available sequences of the same species from Iran (JX524546 from Cyprinus carpio and JX524547 from Ctenopharyngodon idella).Further analysis of the 28S rRNA gene sequence showed that the Indian isolate differed only by 0.01%.Moreover, both ML and BI methods clustered D. anchoratus with the two Iranian isolateswith high bootstrap support (99/1.00), with this clade grouped along with another sister species, D. inexpectatus (supported by 100% bootstrap values) (Figure 3).
The BLASTn search of the 730bp 18S fragment for D. vastator showed 92-96% overall identity to other isolates of D. vastator from the Czech Republic (AJ564159) and China (KM487695, KJ854363 and KC876016), which were collected from Cyprinus carpio and Carassius auratus, respectively (Figure 2) (Simková et al. 2004;Tu et al. 2015;Ling et al. 2016).Pairwise distance analysis of the aligned 18S sequences revealed a low genetic difference, with an intraspecific variation of only 0.04-0.06%(Chinese isolates) and 0.05% (Czech Republic), in comparison with the Indian isolate, which shows maximum similarity with a Chinese isolate (0.04%; KC876016) collected from C. auratus.The Indian isolates of D. vastator cluster together with the four other isolates of the same species, along with another sister species, D. intermedius, in a clade with high bootstrap support (100/1.00) in agreement with Tu et al. (2015) (Figure 2).
In the analysis of 28S fragment (490bp), as no sequence of the same species is available on GenBank, D. vastator grouped with other isolates of Dactylogyrus with high bootstrap support (99/1.00) based on both ML and BI methods (Figure 3).

Discussion
Dactylogyrus is a group of monogenean parasites, having more than 900 described species (Gibson et al. 1996).Morphologically, monogeneans found in this study were identified as D. anchoratus and D. vastator.These parasites are native to Southeast Asia, i.e.China, but they have the potential to spread to other regions through introductions (Molnár 2009).Morphometric characters of D. anchoratus, such as total length and inner root, are larger than the most closely-related Dactylogyrus species, D. formosus.Differentiating D. vastator from its most closely related species, D. intermedius, is mostly via the dorsal anchor total length, and shaft and root, which are slightly larger in D. vastator than in D. intermedius.Both parasites were identified based on the shape of their haptoral armature and male copulatory complex, which are visible under the microscope (Figure 1).Besides morphology, molecular characterization permits validation and comparison with other congeners of Dactylogyrus ensuring correct identification.D. anchoratus and D. vastator are established on goldfishes worldwide as highly pathogenic gill parasites, causing hyper-trophy of gill tissue and ultimately death (Dove and Ernst 1998;Řehulková and Rehulka 1999;Di Cave et al. 2000;Řehulková and Gelnar 2005;Molnár 2009;Shinn andTun 2013, Fridman et al. 2014).Most species of Dactylogyrus are strictly host-specific or show specificity for phylogenetically related cyprinid species (Šimková et al. 2006).As Indian freshwaters contain a high cyprinid fish biodiversity (Johnson and Arunachalam 2009;Basu et al. 2012;Baliarsingh et al. 2013;Pawara et al. 2014).The introduction of D. anchoratus and D. vastator poses considerable risks to native cyprinids because these two species are categorized as generalist parasites, which have already been shown to be capable of parasitizing at least two unrelated host species (Jarkovský et al. 2004;Šimková et al. 2013).

Conclusions
Our study reports the occurrence of D. anchoratus and D. vastator from cultured goldfish in Meerut, U.P., India, using morphological and molecular methods.Further investigation is required to identify more species of Dactylogyrus molecularly for the diagnosis and prevention of the diseases caused by these parasites.

Figure 2 .
Figure 2.Phylogenetic tree generated by maximum likelihood analysis based on 18S rDNA sequences for selected species of Dactylogyrus with D. anchoratus (KT279400) and D. vastator (KT279398) from India.Accession numbers for all species is shown in tree next to the species name.Supported values at the branch are shown as ML/BI.Nodes unsupported by BI are marked with a hyphen.Species newly sequenced in this study are in bold.Cichlidogyrus ergensi was used as outgroup.

Figure 3 .
Figure 3. Maximum likelihood tree of selected Dactylogyrus species based on 28S sequences with Indian isolates (accession numbers, KT279401 (D. anchoratus) and KT279399 (D. vastator).Posterior probabilities for BI are given after the bootstrap values for ML.Nodes unsupported by BI are marked with a hyphen.Species newly sequenced in this study are in bold.Accession numbers are given next to the species name.Cichlidogyrus falcifer was used as outgroup.