Morphologic and molecular (28S rDNA) characterization of Dactylogyrus spp. in Cyprinus carpio and Ctenopharyngodon idella in Mashhad, Iran

Abstract

Dactylogyrids are a group of monogenean parasites that have a high species intensity on the gills of cyprinid fish. In this study, 89 common carp (Cyprinus carpio) and 25 grass carp (Ctenopharyngodon idella) were collected from Mashhad, in northeastern Iran. A total of 31 Dactylogyrus specimens, 20 and 11 specimens from the gills of common carp and grass carp, respectively, were collected and studied by morphologic analysis and molecular analysis based on 28S rDNA. Four lineages were revealed: D. anchoratus, D. extensus, D. lamellatus, and a new species. Phylogenetic analysis showed that 2 species, namely D. extensus and D. lamellatus, were identical to those previously reported. In addition, nucleotide sequencing showed the greatest homology (93.01%) of D. anchoratus to be with a species registered as D. inexpectatus in the Basic Local Alignment Search Tool database. The new Dactylogyrus species formed a distinct clade of its own in the phylogram. From the morphologic, molecular, and phylogenetic evidence, we propose that this isolate is a single new species within the genus Dactylogyrus. Further phylogenetic analysis, however, including the incorporation of additional molecular targets, is required to infer relationships among species in the Dactylogyrus genus.

Introduction

Dactylogyrus (Platyhelminthes: Monogenoidea) is a highly diverse genus with more than 900 described species that primarily parasitize the gills of cyprinid fish (1,2). As a group of ectoparasites with a direct life cycle, Dactylogyrus seems to be an appropriate model for studying parasite diversification, mainly because of its species richness and its morphologic and ecologic diversity (3). Moreover, Dactylogyrus is highly host- and niche-specific. Congeneric species could coexist on the same host owing to a low level of interspecies competition (4–7). To study the process of parasite diversification, several models of congeneric monogenean species have been investigated (8,9).

The taxonomy of dactylogyrids depends on accurate description of small differences in the size and shape of solid body parts, most notably the copulatory complex. Since the introduction of molecular tools into taxonomy, systematics, and phylogeny, many species descriptions have been re-evaluated. In classic morphologic analysis, speciation may lead to an underestimate of the number of species, whereas phenotypic plasticity may induce the reverse effect. Accurate identification of parasites is an essential first step in understanding their biologic and ecologic features, geographic distribution, habitat specificity, and transmission, as well as in designing effective control mechanisms (10).

Epidemiologically, more than 70 species belonging to the genus Dactylogyrus have been reported from wild and farmed freshwater fish of Iran (11,12). However, published data about genetic characterization and/or construction of a phylogenetic classification of these species are limited. Previous molecular phylogeny studies have shown that conserved ribosomal regions are suitable markers for evaluating different levels of taxonomic divergence and ideal in Platyhelminthes (13). Moreover, 28S rDNA has been successfully used for investigating the DNA sequence variations of Dactylogyrus isolates (14,15). Therefore, the objective of the present study was to compare morphologic identification with molecular (28S rDNA) characterization of Dactylogyrus species infecting common carp (Cyprinus carpio) and grass carp (Ctenopharyngodon idella) in northeastern Iran.

Materials and methods

Parasite sampling

This study was carried out in Mashhad County, which is the capital of Razavi Khorasan province in northeastern Iran. During April 2012 to June 2013, a total of 114 fresh cyprinids, including 89 C. carpio and 25 C. idella, with a fork length of 24 to 45 cm and a weight of approximately 1200 to 1800 g, were collected from various fish farms with a mean depth of 2.1 m in Mashhad.

Parasites were removed from the gills, placed on slides, covered by a coverslip, and identified with the use of a light microscope equipped with phase contrast, interference contrast, and digital image analysis (MicroImage 4.0 for Windows; Olympus Optical Company, Hamburg, Germany). The sclerotized parts of the parasite attachment organ (the opisthaptor, consisting of central hooks called anchors, 7 pairs of marginal hooks, and 1 or 2 connective bars) and reproductive organs (vaginal armaments and copulatory organs) were used for parasite identification according to Gusev (16,17). Some parasite specimens were fixed in a mixture of glycerin and ammonium picrate and deposited in the collection of the Department of Pathobiology, Ferdowsi University of Mashhad, Mashhad, Iran. Other parasites were preserved in 95% ethanol before DNA extraction.

DNA extraction and amplification

Individual parasites were removed from ethanol and dried, then DNA was extracted by means of the DNA extraction MBST Kit (Molecular Biological System Technology, Tehran, Iran) according to the manufacturer’s instructions.

Total DNA obtained from individual parasites was suspended in 25 μL of distilled water. Partial 28S rDNA amplicons were amplified in 1 round with use of the forward primer F1 (5′-GCGAGTGAACGGAGATTAGC-3′) and the reverse primer R1 (5′-CCATTATTGACCGTGATGTATG-3′). These primers were designed with the software Premier 5.0 (Premier Biosoft International, USA) from conserved regions of the 28S rDNA sequence of Dactylogyrus species that were obtained from GenBank, National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA. Each amplification reaction was done in a final volume of 25 μL containing 3 μL of genomic DNA extract (50 ng/μL), 10× polymerase chain reaction (PCR) buffer, 0.4 mM of deoxynucleotide triphosphates, 10 pM of each primer pair, 1 U of Taq polymerase (Biotools, Jupiter, Florida, USA) and Milli-Q water in a Mastercycler Personal thermocycler (Eppendorf, Hamburg, Germany) under the following conditions: 5 min at 95°C (initial denaturation), followed by 35 cycles of 30 s at 94°C (denaturation), 45 s at 52.5°C (annealing), and 45 s at 72°C (extension), and then a final extension at 72°C for 10 min. The PCR products were examined on 1.5% agarose–TBE (Tris–borate and ethylene diamine tetraacetic acid) gels, stained with ethidium bromide, and visualized under ultraviolet light.

DNA sequencing

The PCR products were purified with the use of the AccuPrep PCR Purification Kit (Bioneer, Daejeon, Korea) and sequenced directly in a capillary DNA analyzer (ABI 3730; Applied Biosystems, Foster City, California, USA). Nucleotide sequence analysis was undertaken with the Basic Local Alignment Search Tool (BLAST) programs and databases of the NCBI. In addition, DNA sequences of closely related species were downloaded and used in the phylogenetic analysis.

Phylogenetic analysis of Dactylogyrus

Multiple sequence alignments were made with Clustal W in Molecular Evolutionary Genetics Analysis (MEGA) software, version 7.0, using default parameters, and later refined by eye using BioEdit (18). Molecular phylogenetic analysis was conducted in MEGA 7.0 software (19). The evolutionary history was inferred by using the maximum likelihood method based on the Tamura–Nei model (20). Support values for internal nodes were estimated by a bootstrap resampling procedure with 1000 replicates. Initial trees for the heuristic search were obtained automatically by applying neighbor-joining (NJ) and BioNJ algorithms to a matrix of pairwise distances estimated by the maximum composite likelihood approach and then selecting the topology with superior log likelihood values. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps, and missing data were eliminated. There were 440 positions in the final dataset. In addition, D. tripathii (JX993982) was used as an outgroup taxon on the basis of a previous analysis (21).

Results

Dactylogyrus specimens (n = 31) were collected from the gills of freshwater fish belonging to the family Cyprinidae from Mashhad in northeastern Iran, 20 from C. carpio and 11 from C. idella. The morphologic identities are listed along with the associated fish host in Table I. These samples are representative of the fauna in Mashhad. Nine PCR products, all 556 base pairs in length, were generated, and their nucleotide sequences were compared with those of other Dactylogyrus species in GenBank. Phylogenetic analysis grouped the Iranian isolates into 4 distinct clusters. The morphologically identified D. extensus and D. lamellatus were 100% homologous with D. extensus AJ969944 and AY553629 and with D. lamellatus EF100533, AJ969948, and AY307019, respectively, in GenBank. No published sequences were found for D. anchoratus in GenBank. However, an isolated D. anchoratus revealed a particularly close relationship (93.01% homology) with D. inexpectatus (AJ969945). The 2 isolates of Dactylogyrus that were not identified by morphologic techniques formed a group distinct from the other defined Dactylogyrus species. From morphologic and molecular phylogenetic evidence, it seems that these isolates may be regarded as representing a single new species within the Dactylogyrus genus. There was also strong bootstrap support (99%) for sister taxon relationships between this novel taxon and D. cryptomeres (AJ969947).

Table I.

Species of Dactylogyrus parasites isolated from the gills of common and grass carp on fish farms in Mashhad, northeastern Iran, and studied by molecular and phylogenetic analysis.

Species	Host	Number	GenBank accession number
D. anchoratus	Cyprinus carpio	2	JX524546, JX524547
	Ctenopharyngodon idella	1	KY039152
D. extensus	C. carpio	2	JX524548, KY039153
D. lamellatus	C. idella	2	JX524549, KY039154
Dactylogyrus sp.	C. carpio	2	KY039150, KY039151

Discussion

Molecular analysis confirmed the presence of at least 4 species of Dactylogyrus in northeastern Iran: D. anchoratus, D. extensus, D. lamellatus, and a novel taxon. The presence of these species has been observed morphologically in cyprinids in northeastern Iran (12), but the molecular phylogeny had not yet been reported. From the molecular phylogenetic evidence, we suggest that the new taxon, which we named Dactylogyrus mashhad carpio, can be considered a novel species. In addition, sequencing results obtained from D. anchoratus showed that this species is the taxon most closely related to D. inexpectatus. Unfortunately, further analysis was not possible since there were no published 28S rDNA data for D. anchoratus. No intraspecies differences based on the 28S rDNA sequence were noted among the Dactylogyrus isolates. The relative conservation of this region has been demonstrated in previous studies (14,15).

In previous studies the 28S rDNA gene was found to be the most conserved part of the ribosomal region among Dactylogyrus species (14,15,22–24). However, to go deeper into the phylogeny of Dactylogyrus, more variable regions, such as ITS2, can be used. Although it is expected that a short and conserved sequence such as the 28S rDNA gene will yield little phylogenetic information, the coding regions of the rRNA transcription unit have been extensively used to investigate phylogenetic relationships from phylum to genus level.

The present study revealed D. anchoratus to be the only Dactylogyrus species isolated from both C. carpio and C. idella. Because Dactylogyrus species form groups of phylogenetically closely related species within a host, it could be hypothesized that their mode of speciation (i.e., intrahost) is closely related to the morphologic evolution of attachment organs and the reproductive system, as suggested by Rohde (25).

The diversity of the Dactylogyrus species observed in this study might be related to host-specific characteristics of these parasites and variations in environmental conditions. In this study D. anchoratus was found in both C. carpio and C. idella, whereas D. extensus and D. lamellatus were collected from only C. carpio and C. idella, respectively. Notably, D. anchoratus has low host specificity: it can infect crucian carp and wild goldfish and appears throughout the year among small and large carp. It has been found in all bodies of water with a broad range of water temperature (from 1°C to 30°C) and salinity (up to 1.3%) in all seasons of the year (26,27). However, the main host of D. extensus is C. carpio, which can live and reproduce at both low and high temperatures (28,29). Moreover, D. lamellatus appears mainly among C. idella, which can reproduce and grow at water temperatures of 14°C to 29.5°C (30).

In conclusion, we suggest further phylogenetic investigation of the genus Dactylogyrus based on the 28S rDNA region in different parts of Iran. The conserved 28S gene has proven to be phylogenetically informative and might even be used as an aid in detecting the position of difficult species. Whereas morphological variation, expressed in shape and size of the attachment apparatus, is very low, molecular variation, expressed by variation in 28S rDNA region, is very high. This can be attributed to the fast evolving nature of the 28S rDNA region, or to the fact that this genus is constituted of groups of a higher taxonomic level than previously recognized. However, exploring another genetic marker and including more species should shed more light on this intriguing issue.

Figure 1.

Phylogenetic tree based on 28S rDNA sequence data, constructed according to the maximum likelihood method. Black dots represent the Iranian Dactylogyrus isolates. Numerals above the branches indicate bootstrap values (%) from 1000 replicates. The scale bar indicates the proportion of sites changing along each branch.