Prospective enzymes for omega-3 PUFA biosynthesis found in endoparasitic classes within the phylum Platyhelminthes.

The free-living infectious stages of macroparasites, specifically, the cercariae of trematodes (flatworms), are likely to be significant (albeit underappreciated) vectors of nutritionally important polyunsaturated fatty acids (PUFA) to consumers within aquatic food webs, and other macroparasites could serve similar roles. In the context of de novo omega-3 (n-3) PUFA biosynthesis, it was thought that most animals lack the fatty acid (FA) desaturase enzymes that convert stearic acid (18:0) into ɑ-linolenic acid (ALA; 18:3n-3), the main FA precursor for n-3 long-chain PUFA. Recently, novel sequences of these enzymes were recovered from 80 species from six invertebrate phyla, with experimental confirmation of gene function in five phyla. Given this wide distribution, and the unusual attributes of flatworm genomes, we conducted an additional search for genes for de novo n-3 PUFA in the phylum Platyhelminthes. Searches with experimentally confirmed sequences from Rotifera recovered nine relevant FA desaturase sequences from eight species in four genera in the two exclusively endoparasite classes (Trematoda and Cestoda). These results could indicate adaptations of these particular parasite species, or may reflect the uneven taxonomic coverage of sequence databases. Although additional genomic data and, particularly, experimental study of gene functionality are important future validation steps, our results indicate endoparasitic platyhelminths may have enzymes for de novo n-3 PUFA biosynthesis, thereby contributing to global PUFA production, but also representing a potential target for clinical antihelmintic applications.

Alternative splicing of coq-2 controls the levels of rhodoquinone in animals.

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In normoxia, they use ubiquinone (UQ), but in anaerobic conditions inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. We previously showed the switch from UQ to RQ synthesis is driven by a change of substrates by the polyprenyltransferase COQ-2 (Del Borrello et al., 2019; Roberts Buceta et al., 2019); however, the mechanism of substrate selection is not known. Here, we show helminths synthesize two coq-2 splice forms, coq-2a and coq-2e, and the coq-2e-specific exon is only found in species that synthesize RQ. We show that in Caenorhabditis elegans COQ-2e is required for efficient RQ synthesis and survival in cyanide. Importantly, parasites switch from COQ-2a to COQ-2e as they transit into anaerobic environments. We conclude helminths switch from UQ to RQ synthesis principally via changes in the alternative splicing of coq-2.

The first mitochondrial genomes of endosymbiotic rhabdocoels illustrate evolutionary relaxation of atp8 and genome plasticity in flatworms.

The first three mitochondrial (mt) genomes of endosymbiotic turbellarian flatworms are characterised for the rhabdocoels Graffilla buccinicola, Syndesmis echinorum and S. kurakaikina. Interspecific comparison of the three newly obtained sequences and the only previously characterised rhabdocoel, the free-living species Bothromesostoma personatum, reveals high mt genomic variability, including numerous rearrangements. The first intrageneric comparison within rhabdocoels shows that gene order is not fully conserved even between congeneric species. Atp8, until recently assumed absent in flatworms, was putatively annotated in two sequences. Selection pressure was tested in a phylogenetic framework and is shown to be significantly relaxed in this and another protein-coding gene: cox1. If present, atp8 appears highly derived in platyhelminths and its functionality needs to be addressed in future research. Our findings for the first time allude to a large degree of undiscovered (mt) genomic plasticity in rhabdocoels. It merits further attention whether this variation is correlated with a symbiotic lifestyle. Our results illustrate that this phenomenon is widespread in flatworms as a whole and not exclusive to the better-studied neodermatans.

Molecular Phylogenetic Analysis of Acotylea (Platyhelminthes: Polycladida).

Acotylea is a suborder of Polycladida (Rhabditophora, Platyhelminthes) characterized by lack of a cotyl (sucker-like structure) on the ventral surface of the body. We newly determined partial sequences of two mitochondrial (16S ribosomal RNA and cytochrome c oxidase subunit I) and two nuclear (18S and 28S ribosomal RNA) genes from 24 acotylean species (12 families and 14 genera). Based on these sequences in addition to those available in public databases, we inferred the phylogeny of 16 families and 27 genera of Acotylea from molecular phylogenetic analyses (maximum likelihood and Bayesian inference) based on concatenated gene sequences. Our analyses supported three clades corresponding to Discoceloidea, Leptoplanoidea, and Stylochoidea. The phylogenetic position of Callioplanidae remains unclear. Among family- or genus-level taxa, Gnesiocerotidae, Stylochoplanidae, and Comoplana were not monophyletic. We discuss the validities of Notocomplanidae and Koinostylochus, and the family-level assignment of Mirostylochus.

Analysis of codon usage pattern of mitochondrial ND genes in Platyhelminthes.

Codon usage bias (CUB) is the nonrandom usage of synonymous codons in which some codons are more preferred to others.CUB can be determined by mutation pressure and selection. Various approaches have been used to understand the pattern of CUB in the mitochondrial ND (MT-ND or ND) genes involved in complex I of respiratory chain in five different classes of Platyhelminthes as no work was reported yet. The present study revealed that the CUB varies across MT-ND genes and the coding sequences showed the richness of A and T. Correspondence analysis implied the effect of mutational pressure and also the pattern of codon usage was different in different classes of platyhelminthes for MT-ND genes. Highly significant correlation was observed between overall nucleotide composition and its 3rd codon position in most of the homogeneous nucleotides such as A% and A3%, T% and T3%, G% and G3%, C% and C3%, GC% and GC3% and also some significant correlations observed among heterogeneous nucleotides in all the five classes for MT-ND genes suggested the role of mutational pressure as well as natural selection in affecting the CUB. Neutrality plot suggested that the contributions of natural selection and mutational pressure varied across different classes of platyhelminthes and also differed in different MT-ND genes.

A clustering package for nucleotide sequences using Laplacian Eigenmaps and Gaussian Mixture Model.

In this article, a new Python package for nucleotide sequences clustering is proposed. This package, freely available on-line, implements a Laplacian eigenmap embedding and a Gaussian Mixture Model for DNA clustering. It takes nucleotide sequences as input, and produces the optimal number of clusters along with a relevant visualization. Despite the fact that we did not optimise the computational speed, our method still performs reasonably well in practice. Our focus was mainly on data analytics and accuracy and as a result, our approach outperforms the state of the art, even in the case of divergent sequences. Furthermore, an a priori knowledge on the number of clusters is not required here. For the sake of illustration, this method is applied on a set of 100 DNA sequences taken from the mitochondrially encoded NADH dehydrogenase 3 (ND3) gene, extracted from a collection of Platyhelminthes and Nematoda species. The resulting clusters are tightly consistent with the phylogenetic tree computed using a maximum likelihood approach on gene alignment. They are coherent too with the NCBI taxonomy. Further test results based on synthesized data are then provided, showing that the proposed approach is better able to recover the clusters than the most widely used software, namely Cd-hit-est and BLASTClust.

Differential expression of disulfide reductase enzymes in a free-living platyhelminth (Dugesia dorotocephala).

A search of the disulfide reductase activities expressed in the adult stage of the free-living platyhelminth Dugesia dorotocephala was carried out. Using GSSG or DTNB as substrates, it was possible to obtain a purified fraction containing both GSSG and DTNB reductase activities. Through the purification procedure, both disulfide reductase activities were obtained in the same chromatographic peak. By mass spectrometry analysis of peptide fragments obtained after tryptic digestion of the purified fraction, the presence of glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), and a putative thioredoxin reductase (TrxR) was detected. Using the gold compound auranofin to selectively inhibit the GSSG reductase activity of TGR, it was found that barely 5% of the total GR activity in the D. dorotocephala extract can be assigned to GR. Such strategy did allow us to determine the kinetic parameters for both GR and TGR. Although It was not possible to discriminate DTNB reductase activity due to TrxR from that of TGR, a chromatofocusing experiment with a D. dorotocephala extract resulted in the obtention of a minor protein fraction enriched in TrxR, strongly suggesting its presence as a functional protein. Thus, unlike its parasitic counterparts, in the free-living platyhelminth lineage the three disulfide reductases are present as functional proteins, albeit TGR is still the major disulfide reductase involved in the reduction of both Trx and GSSG. This fact suggests the development of TGR in parasitic flatworms was not linked to a parasitic mode of life.

Lineage-specific expansion and loss of tyrosinase genes across platyhelminths and their induction profiles in the carcinogenic oriental liver fluke, Clonorchis sinensis.

Tyrosinase provides an essential activity during egg production in diverse platyhelminths by mediating sclerotization of eggshells. In this study, we investigated the genomic and evolutionary features of tyrosinases in parasitic platyhelminths whose genomic information is available. A pair of paralogous tyrosinases was detected in most trematodes, whereas they were lost in cyclophyllidean cestodes. A pseudophyllidean cestode displaying egg biology similar to that of trematodes possessed an orthologous gene. Interestingly, one of the paralogous tyrosinases appeared to have been multiplied into three copies in Clonorchis sinensis and Opisthorchis viverrini. In addition, a fifth tyrosinase gene that was minimally transcribed through all developmental stages was further detected in these opisthorchiid genomes. Phylogenetic analyses demonstrated that the tyrosinase gene has undergone duplication at least three times in platyhelminths. The additional opisthorchiid gene arose from the first duplication. A paralogous copy generated from these gene duplications, except for the last one, seemed to be lost in the major neodermatans lineages. In C. sinensis, tyrosinase gene expressions were initiated following sexual maturation and the levels were significantly enhanced by the presence of O2 and bile. Taken together, our data suggest that tyrosinase has evolved lineage-specifically across platyhelminths related to its copy number and induction mechanism.

Pipeline for the identification and classification of ion channels in parasitic flatworms.

BACKGROUND: Ion channels are well characterised in model organisms, principally because of the availability of functional genomic tools and datasets for these species. This contrasts the situation, for example, for parasites of humans and animals, whose genomic and biological uniqueness means that many genes and their products cannot be annotated. As ion channels are recognised as important drug targets in mammals, the accurate identification and classification of parasite channels could provide major prospects for defining unique targets for designing novel and specific anti-parasite therapies. Here, we established a reliable bioinformatic pipeline for the identification and classification of ion channels encoded in the genome of the cancer-causing liver fluke Opisthorchis viverrini, and extended its application to related flatworms affecting humans. METHODS: We built an ion channel identification + classification pipeline (called MuSICC), employing an optimised support vector machine (SVM) model and using the Kyoto Encyclopaedia of Genes and Genomes (KEGG) classification system. Ion channel proteins were first identified and grouped according to amino acid sequence similarity to classified ion channels and the presence and number of ion channel-like conserved and transmembrane domains. Predicted ion channels were then classified to sub-family using a SVM model, trained using ion channel features. RESULTS: Following an evaluation of this pipeline (MuSICC), which demonstrated a classification sensitivity of 95.2 % and accuracy of 70.5 % for known ion channels, we applied it to effectively identify and classify ion channels in selected parasitic flatworms. CONCLUSIONS: MuSICC provides a practical and effective tool for the identification and classification of ion channels of parasitic flatworms, and should be applicable to a broad range of organisms that are evolutionarily distant from taxa whose ion channels are functionally characterised.

Major acid endopeptidases of the blood-feeding monogenean Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae).

In parasitic flatworms, acid endopeptidases are involved in crucial processes, including digestion, invasion, interactions with the host immune system, etc. In haematophagous monogeneans, however, no solid information has been available about the occurrence of these enzymes. Here we aimed to identify major cysteine and aspartic endopeptidase activities in Eudiplozoon nipponicum, an invasive haematophagous parasite of common carp. Employing biochemical, proteomic and molecular tools, we found that cysteine peptidase activities prevailed in soluble protein extracts and excretory/secretory products (ESP) of E. nipponicum; the major part was cathepsin L-like in nature supplemented with cathepsin B-like activity. Significant activity of the aspartic cathepsin D also occurred in soluble protein extracts. The degradation of haemoglobin in the presence of ESP and worm protein extracts was completely inhibited by a combination of cysteine and aspartic peptidase inhibitors, and diminished by particular cathepsin L, B and D inhibitors. Mass spectrometry revealed several tryptic peptides in ESP matching to two translated sequences of cathepsin L genes, which were amplified from cDNA of E. nipponicum and bioinformatically annotated. The dominance of cysteine peptidases of cathepsin L type in E. nipponicum resembles the situation in, e.g. fasciolid trematodes.

Comparative phylogeography of two monogenean species (Mazocraeidae) on the host of chub mackerel, Scomber japonicus, along the coast of China.

In the present paper, the phylogeographies of two monogenean species, Pseudokuhnia minor and Kuhnia scombri, on the same species of host, Scomber japonicus, were studied. Fragments of the mitochondrial cytochrome c oxidase subunit 1 gene were sequenced for 264 individuals of P. minor and 224 individuals of K. scombri collected from 10 localities along the coast of China. Genetic diversity of K. scombri was higher than that of P. minor, which may imply that P. minor has a lower evolution rate and/or is a younger species. The neighbour-joining (NJ) trees of both parasites were comprised of two clades without association to sample sites, which is the signature of remixing populations following past division. Analyses of molecular variance and pairwise fixation index revealed different genetic structures for the populations of these two closely related species along the coast of China: P. minor without significant genetic structure, while K. scombri has some genetic differentiation. Both neutrality tests and mismatch distribution suggested that the populations of these two species of parasites experienced population expansion in the late Pleistocene era due to the glacial-interglacial cycles induced by climatic oscillations.

Genetic population structure of Gyrodactylus thymalli (Monogenea) in a large Norwegian river system.

The extent of geographic genetic variation is the result of several processes such as mutation, gene flow, selection and drift. Processes that structure the populations of parasite species are often directly linked to the processes that influence the host. Here, we investigate the genetic population structure of the ectoparasite Gyrodactylus thymalli Zitnan, 1960 (Monogenea) collected from grayling (Thymallus thymallus L.) throughout the river Glomma, the largest watercourse in Norway. Parts of the mitochondrial dehydrogenase subunit 5 (NADH 5) and cytochrome oxidase I (COI) genes from 309 G. thymalli were analysed to study the genetic variation and investigated the geographical distribution of parasite haplotypes. Three main clusters of haplotypes dominated the three distinct geographic parts of the river system; one cluster dominated in the western main stem of the river, one in the eastern and one in the lower part. There was a positive correlation between pairwise genetic distance and hydrographic distance. The results indicate restricted gene flow between sub-populations of G. thymalli, most likely due to barriers that limit upstream migration of infected grayling. More than 80% of the populations had private haplotypes, also indicating long-time isolation of sub-populations. According to a molecular clock calibration, much of the haplotype diversity of G. thymalli in the river Glomma has developed after the last glaciation.

Genetic diversity and population structure of Synthesium pontoporiae (Digenea, Brachycladiidae) linked to its definitive host stocks, the endangered Franciscana dolphin, Pontoporia blainvillei (Pontoporiidae) off the coast of Brazil and Argentina.

Pontoporia blainvillei (Gervais and d'Orbigny, 1844) is an endangered small cetacean endemic to South America with four Franciscana Management Areas (FMA) recognized as different population stocks. The role of the intestinal parasite Synthesium pontoporiae (Digenea: Brachycladiidae) as a possible biological marker to differentiate P. blainvillei stocks was evaluated using nuclear and mitochondrial DNA markers. Internal transcribed sequence 1 and 2 (ITS1 and ITS2) regions of S. pontoporiae did not show intraspecific variability. The mitochondrial NADH dehydrogenase subunit 3 (ND3) and cytochrome oxidase subunit I (COI) gene sequences suggested lack of population structure in S. pontoporiae and population expansion. The apparent panmixia of S. pontoporiae may be due to the high mobility of one or more of its intermediary hosts. Alternatively, it may be due to the small sample size. This result is incongruent with the previously proposed FMA.

Comparative genomic analysis of aspartic proteases in eight parasitic platyhelminths: insights into functions and evolution.

We performed genome-wide identifications and comparative genomic analyses of the predicted aspartic proteases (APs) from eight parasitic flatworms, focusing on their evolution, potentials as drug targets and expression patterns. The results revealed that: i) More members of family A01 were identified from the schistosomes than from the cestodes; some evidence implied gene loss events along the class Cestoda, which may be related to the different ways to ingest host nutrition; ii) members in family A22 were evolutionarily highly conserved among all the parasites; iii) one retroviral-like AP in family A28 shared a highly similar predicted 3D structure with the HIV protease, implying its potential to be inhibited by HIV inhibitor-like molecules; and iiii) retrotransposon-associated APs were extensively expanded among these parasites. These results implied that the evolutionary histories of some APs in these parasites might relate to adaptations to their parasitism and some APs might have potential serving as intervention targets.

Expression profile of the Schistosoma japonicum degradome reveals differential protease expression patterns and potential anti-schistosomal intervention targets.

Blood fluke proteases play pivotal roles in the processes of invasion, nutrition acquisition, immune evasion, and other host-parasite interactions. Hundreds of genes encoding putative proteases have been identified in the recently published schistosome genomes. However, the expression profiles of these proteases in Schistosoma species have not yet been systematically analyzed. We retrieved and culled the redundant protease sequences of Schistosoma japonicum, Schistosoma mansoni, Echinococcus multilocularis, and Clonorchis sinensis from public databases utilizing bioinformatic approaches. The degradomes of the four parasitic organisms and Homo sapiens were then comparatively analyzed. A total of 262 S. japonicum protease sequences were obtained and the expression profiles generated using whole-genome microarray. Four main clusters of protease genes with different expression patterns were identified: proteases up-regulated in hepatic schistosomula and adult worms, egg-specific or predominantly expressed proteases, cercaria-specific or predominantly expressed proteases, and constantly expressed proteases. A subset of protease genes with different expression patterns were further validated using real-time quantitative PCR. The present study represents the most comprehensive analysis of a degradome in Schistosoma species to date. These results provide a firm foundation for future research on the specific function(s) of individual proteases and may help to refine anti-proteolytic strategies in blood flukes.

Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes.

BACKGROUND: The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives. RESULTS: Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and 'Turbellaria') contain methylated cytosines within their genome compartments. CONCLUSIONS: Collectively, these findings provide the first direct evidence for a functionally conserved and enzymatically active DNA methylation system throughout the Platyhelminthes. Defining how this epigenetic feature shapes phenotypic diversity and development within the phylum represents an exciting new area of metazoan biology.

Thioredoxin glutathione reductase-dependent redox networks in platyhelminth parasites.

SIGNIFICANCE: Platyhelminth parasites cause chronic infections that are a major cause of disability, mortality, and economic losses in developing countries. Maintaining redox homeostasis is a major adaptive problem faced by parasites and its disruption can shift the biochemical balance toward the host. Platyhelminth parasites possess a streamlined thiol-based redox system in which a single enzyme, thioredoxin glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase (TR) domains, supplies electrons to oxidized glutathione (GSSG) and thioredoxin (Trx). TGR has been validated as a drug target for schistosomiasis. RECENT ADVANCES: In addition to glutathione (GSH) and Trx reduction, TGR supports GSH-independent deglutathionylation conferring an additional advantage to the TGR redox array. Biochemical and structural studies have shown that the TR activity does not require the Grx domain, while the glutathione reductase and deglutathionylase activities depend on the Grx domain, which receives electrons from the TR domains. The search for TGR inhibitors has identified promising drug leads, notably oxadiazole N-oxides. CRITICAL ISSUES: A conspicuous feature of platyhelminth TGRs is that their Grx-dependent activities are temporarily inhibited at high GSSG concentrations. The mechanism underlying the phenomenon and its biological relevance are not completely understood. FUTURE DIRECTIONS: The functional diversity of Trxs and Grxs encoded in platyhelminth genomes remains to be further assessed to thoroughly understand the TGR-dependent redox network. Optimization of TGR inhibitors and identification of compounds targeting other parasite redox enzymes are good options to clinically develop relevant drugs for these neglected, but important diseases.

Histochemical demonstration of five enzymes' activities in Macrogyrodactylus clarii (Monogenea: Gyrodactylidae) from the catfish Clarias gariepinus.

Histochemical techniques were applied to whole mounts, to study the distribution of the enzymes alkaline phosphatase, acid phosphatase, adenosine triphosphatase, 5'-nucleotidase and glucose-6-phosphatase in the organs and tissues of a viviparous monogenean, Macrogyrodactylus clarii Gussev, 1961, from the gills of the North African catfish Clarias gariepinus (Burchell) in Egypt. The following organs and tissues were studied: head region, anterior adhesive glands, mouth region, pharynx, intestine, testis, vesicula seminalis, male accessory gland, male accessory reservoir, copulatory organ, receptaculum seminis, egg-cell forming region, embryonic cells, excretory system, nerve cells, haptor, muscle fibres and subtegumental cell bodies (cytons). The enzymes showed marked differences in their activities among the studied organs and tissues. Alkaline phosphatase and acid phosphatase activities were detected in many organs and tissues, while the activities of adenosine triphosphatase, 5'-nucleotidase and glucose-6-phosphatase were restricted to a few organs. Although no positive reaction for any enzyme was observed in the anterior adhesive gland cells, a positive reaction for acid phosphatase was detected in the anterior adhesive areas. All enzymes showed marked activity in the digestive and excretory systems. The distribution of the enzymes in the tissues and organs of M clarii is compared with those of other monogeneans, including other gyrodactylids parasitizing the same host fish. Some possible functions of the enzymes are discussed.

The adaptive evolution divergence of triosephosphate isomerases between parasitic and free-living flatworms and the discovery of a potential universal target against flatworm parasites.

Triosephosphate isomerase (TIM) is an important drug target or vaccine candidate for pathogenetic organisms such as schistosomes. Parasitic and free-living flatworms shared their last common ancestor but diverged from each other for adapting to parasitic and free-living lives afterwards, respectively. Therefore, adaptive evolution divergence must have occurred between them. Here, for the first time, TIMs were identified from three free-living planarian flatworms, namely Dugesia japonica, Dugesia ryukyuensis, and Schmidtea mediterranea. When these were compared with parasitic flatworms and other organisms, the following results were obtained: (1) planarian TIM genes each contain only one intron, while parasitic flatworm genes each contain other four introns, which are usually present in common metazoans, suggesting planarian-specific intron loss must have occurred; (2) planarian TIM protein sequences are more similar to those of vertebrates rather than to their parasitic relatives or other invertebrates. This implies that relatively rapid evolution occurred in parasitic flatworm TIMs; (3) All the investigated parasitic flatworm TIMs contain a unique tripeptide insert (SXD/E), which may imply its insertion importance to the adaptation of parasitic life. Moreover, our homology modeling results showed the insert region was largely surface-exposed and predicted to be of a B cell epitope location. Finally, the insert is located within one of the three regions previously suggested to be promising immunogenic epitopes in Schistosoma mansoni TIM. Therefore, this unique insert might be significant to developing new effective vaccines or specific drugs against all parasitic flatworm diseases such as schistosomiasis and taeniosis/cysticercosis.

Evidence of new species of Haematoloechus (Platyhelminthes: Digenea) using partial cox1 sequences.

BACKGROUND AND AIMS: Haematoloechus, digeneans parasites of amphibians, is a species-rich genus with more than 50 species around the globe. Establishing an accurate taxonomy for this group has been difficult due to high intraspecific variability. Nuclear DNA sequences have given independent information about species validity and phylogeny of the group. MATERIALS AND METHODS: In this paper, I test the performance of partial sequences of the mitochondrial cytochrome c oxidase subunit I (cox1) gene in the differentiation of recognized species of the genus and in the detection of potential new taxa. Samples from 13 nominal species were sequenced, plus four samples that could not be assigned to any described species based on morphology. RESULTS: Parsimony analysis of the amplified 360 bp fragment resulted in six most parsimonious trees showing the same grouping of samples, differing in the samples' arrangement within those groups. All 13 species were recovered on the trees, and five potential new species are shown. CONCLUSION: Additional sampling and sequencing is necessary to support this hypothesis, but with this preliminary information the search for diagnostic characters that allow the description of the new taxa is less difficult.