Mitochondrial Genomes of Two Thaparocleidus Species (Platyhelminthes: Monogenea) Reveal the First rRNA Gene Rearrangement among the Neodermata.

Phylogenetic framework for the closely related Ancylodiscoidinae and Ancyrocephalinae subfamilies remains contentious. As this issue was never studied using a large molecular marker, we sequenced the first two Ancylodiscoidinae mitogenomes: Thaparocleidus asoti and Thaparocleidus varicus. Both mitogenomes had two non-coding regions (NCRs) that contained a number of repetitive hairpin-forming elements (RHE). Due to these, the mitogenome of T. asoti (16,074 bp) is the longest among the Monogenea; especially large is its major NCR, with 3500 bp, approximately 1500 bp of which could not be sequenced (thus, the total mitogenome size is ≈ 17,600 bp). Although RHEs have been identified in other monopisthocotyleans, they appear to be independently derived in different taxa. The presence of RHEs may have contributed to the high gene order rearrangement rate observed in the two mitogenomes, including the first report of a transposition of rRNA genes within the Neodermata. Phylogenetic analyses using mitogenomic dataset produced Dactylogyrinae embedded within the Ancyrocephalinae (paraphyly), whereas Ancylodiscoidinae formed a sister-group with them. This was also supported by the gene order analysis. 28S rDNA dataset produced polyphyletic Dactylogyridae and Ancyrocephalinae. The phylogeny of the two subfamilies shall have to be further evaluated with more data.

Identification of Gangesia oligonchis Roitman & Freze, 1964 (Cestoda: Onchoproteocephalidea) from Tachysurus fulvidraco Richardson in central China: implications for the validity of Gangesia pseudobagrae Chen, 1962.

Owing to the brief and incomplete original description of Gangesia pseudobagrae Chen, 1962 (Cestoda: Onchoproteocephalidea) and high morphological similarity to Gangesia oligonchis Roitman & Freze, 1964 parasitising the same host Tachysurus fulvidraco Richardson, the taxonomic validity of G. pseudobagrae in China remains questionable. Therefore, we sampled and identified specimens of Gangesia Woodland, 1924 from the intestine of T. fulvidraco from three lakes in central China. Morphologically, the sampled specimens almost perfectly corresponded both to G. oligonchis and the limited available description of G. pseudobagrae: rostellum-like organ armed with a single complete circle of hooks (24-31 in number); four uniloculate suckers covered with minute hooklets; genital pore irregularly alternated; testes medullary, spherical to oval; ovary medullary, bi-lobed, follicular; cirrus-sac thick-walled and long; uterus medullary. 28S rDNA sequence also exhibited the highest similarity to G. oligonchis (99.4-99.7%). Phylogenetic analysis showed that 11 individuals of Gangesia from the three lakes in China clustered with G. oligonchis from Russia (no sequence of G. pseudobagrae available on GenBank). Based upon the high similarity of morphology and high similarity of 28S rDNA sequences, the specimens of Gangesia from T. fulvidraco in central China were identified as G. oligonchis. Our results indicate that there is only one species of Gangesia in T. fulvidraco from the Palaearctic region, and thereby support the proposed synonymisation of G. pseudobagrae and G. oligonchis.

Three new Diplozoidae mitogenomes expose unusual compositional biases within the Monogenea class: implications for phylogenetic studies.

BACKGROUND: As the topologies produced by previous molecular and morphological studies were contradictory and unstable (polytomy), evolutionary relationships within the Diplozoidae family and the Monogenea class (controversial relationships among the Discocotylinea, Microcotylinea and Gastrocotylinea suborders) remain unresolved. Complete mitogenomes carry a relatively large amount of information, sufficient to provide a much higher phylogenetic resolution than traditionally used morphological traits and/or single molecular markers. However, their implementation is hampered by the scarcity of available monogenean mitogenomes. Therefore, we sequenced and characterized mitogenomes belonging to three Diplozoidae family species, and conducted comparative genomic and phylogenomic analyses for the entire Monogenea class. RESULTS: Taxonomic identification was inconclusive, so two of the species were identified merely to the genus level. The complete mitogenomes of Sindiplozoon sp. and Eudiplozoon sp. are 14,334 bp and 15,239 bp in size, respectively. Paradiplozoon opsariichthydis (15,385 bp) is incomplete: an approximately 2000 bp-long gap within a non-coding region could not be sequenced. Each genome contains the standard 36 genes (atp8 is missing). G + T content and the degree of GC- and AT-skews of these three mitogenome (and their individual elements) were higher than in other monogeneans. nad2, atp6 and nad6 were the most variable PCGs, whereas cox1, nad1 and cytb were the most conserved. Mitochondrial phylogenomics analysis, conducted using concatenated amino acid sequences of all PCGs, indicates that evolutionary relationships of the three genera are: (Eudiplozoon, (Paradiplozoon, Sindiplozoon)); and of the three suborders: (Discocotylinea, (Microcotylinea, Gastrocotylinea)). These intergeneric relationships were also supported by the skewness and principal component analyses. CONCLUSIONS: Our results show that nad2, atp6 and nad6 (fast-evolving) would be better candidates than cox1 (slow-evolving) for species identification and population genetics studies in Diplozoidae. Nucleotide bias and codon and amino acid usage patterns of the three diplozoid mitogenomes are more similar to cestodes and trematodes than to other monogenean flatworms. This unusual mutational bias was reflected in disproportionately long branches in the phylogram. Our study offsets the scarcity of molecular data for the subclass Polyopisthocotylea to some extent, and might provide important new insights into the evolutionary history of the three genera and three suborders.

Expression analysis of immune genes in goldfish (Carassius auratus) infected with the monogenean parasite Gyrodactylus kobayashii.

Heavy infection with the ectoparasite Gyrodactylus kobayashii commonly leads to high mortality of ornamental goldfish Carassius auratus. To understand the underlying mechanisms of goldfish against infection of gyrodactylids, transcription responses of immune-related genes including IL-1ß2, TNFα1, TNFα2, IFN-γ, TGFß, MHC II ß, TCRß1 and complement factor C3 were studied by real-time quantitative PCR analysis. Significant increases of expression of inflammatory genes such as IL-1ß2, TNFα1, TNFα2 and TGFß were detected at days 7 and 14 post-infection (pi). No significant differences of G. kobayashii load were observed in response to primary infection and re-infection. In addition, the transcript levels of genes involved in adaptive immunity such as MHC II ß and TCRß1 remained unchanged. Curiously, fish moderately infected with gyrodactylid showed elevated expression IL-1ß2, TNFα1 and TNFα2. In all the gyrodactylids-infected fish, expression of complement factor C3 was consistently inhibited. The results extend current knowledge to the understanding of gyrodactylid infection in fish and support the previous findings that innate immunity is indispensable for controlling parasite infection.

Effects of goldfish (Carassius auratus) population size and body condition on the transmission of Gyrodactylus kobayashii (Monogenea).

Field surveys indicate that host population size, rather than density, is the most important determinant of monogenean infection dynamics. To verify this prediction, epidemic parameters were monitored for 70 days at five host population sizes held at constant density using a goldfish - Gyrodactylus kobayashii laboratory model. During the first 20 days, the rate of increase of prevalence and mean abundance was faster in small host populations. Total mean prevalence and total mean abundance throughout the experiment were not significantly affected by host population sizes. Higher transmission rates were detected in larger host populations. However, there were no significant differences in effective contact rates among the five host populations on each sampling day during the first 20 days, implying that contact rates may be saturated at a sufficiently high host density. These results demonstrate that the epidemic occurs more quickly in smaller host populations at the beginning of the experiment. However, the epidemic is independent of the host population size due to the similar effective contact rates in the five population sizes. Significant negative influence of the initial body condition (Kn) of uninfected goldfish on total mean abundance of parasites suggests that susceptibility of hosts is also a determinant of parasite transmission.

Effects of Schyzocotyle acheilognathi (Yamaguti, 1934) infection on the intestinal microbiota, growth and immune reactions of grass carp (Ctenopharyngodon idella).

Our understanding of interactions among intestinal helminths, gut microbiota and host is still in its infancy in fish. In this study, the effects of Schyzocotyle acheilognathi infection on the intestinal microbiota, growth and immune reactions of grass carp were explored under laboratory conditions. 16S rDNA amplification sequencing results showed that S. acheilognathi infection altered the composition of intestinal microbiota only at the genus level, with a significant increase in the relative abundance of Turicibacter and Ruminococcus (P < 0.05) and a significant decrease in the relative abundance of Gordonia, Mycobacterium and Pseudocanthomonas (P < 0.05). Schyzocotyle acheilognathi infection had no significant effect (P > 0.05) on the alpha diversity indices (including Chao1, ACE, Shannon, Simpson index) of intestinal microbiota in grass carp, but PERMANOVA analysis showed that microbial structure significantly (P < 0.01) differed between hindgut and foregut. PICRUST prediction showed that some metabolism-related pathways were significantly changed after S. acheilognathi infection. The relative abundance of Turicibacter was positively correlated with the fresh weight of tapeworm (foregut: r = 0.48, P = 0.044; hindgut: r = 0.63, P = 0.005). There was no significant difference in the body condition of grass carp between the S. acheilognathi infected group and the uninfected group (P > 0.05). Intestinal tissue section with HE staining showed that S. acheilognathi infection severely damaged the intestinal villi, causing serious degeneration, necrosis and shedding of intestinal epithelial cells. The real-time fluorescent quantitative PCR results showed that S. acheilognathi infection upregulated the mRNA expression of the immune-related genes: Gal1-L2, TGF-ß1 and IgM.

Mitochondrial genomes and 28S rDNA contradict the proposed obsoletion of the order Tetraonchidea (Platyhelminthes: Monogenea).

Due to the incongruence of morphology-based hypotheses and scarcity of molecular data, validity of the order Tetraonchidea remains contentious. The only complete mitogenome currently available for the entire order is that of Paratetraonchoides inermis (Tetraonchoididae). To study the phylogeny of Tetraonchidea from mitogenomic perspective, we sequenced the first mitogenome for the family Tetraonchidae: Tetraonchus monenteron (Tetraonchidea). To get a nuclear-data perspective, we also sequenced nuclear 28S rDNA gene of both species. The mitogenome of T. monenteron does not have high A + T content, nor tRNA pseudo-genes, both of which were unique features reported in P. inermis. However, T. monenteron exhibits a unique gene order, with a large number of tRNA rearrangements in comparison to P. inermis and other monogeneans. Phylogenetic analyses conducted using Bayesian inference and maximum likelihood methods, complemented with partitioning, consistently support the sister-group relationship of T. monenteron (Tetraonchidae) and P. inermis (Tetraonchoididae). This is also partially supported by the 28S rDNA data and two morphologic apomorphies. This close relationship of Tetraonchidae and Tetraonchoididae challenges the latest major morphology-based classification, which proposed obsoletion of the Tetraonchidea order, and grouped Tetraonchoididae into the Gyrodactylidea clade. The validity of this order shall have to be further confirmed with more data.

Homoplasy or plesiomorphy? Reconstruction of the evolutionary history of mitochondrial gene order rearrangements in the subphylum Neodermata.

Recent mitogenomic studies have exposed a gene order (GO) shared by two classes, four orders and 31 species ('common GO') within the flatworm subphylum Neodermata. There are two possible hypotheses for this phenomenon: convergent evolution (homoplasy) or shared ancestry (plesiomorphy). To test those, we conducted a meta-analysis on all available mitogenomes to infer the evolutionary history of GO in Neodermata. To improve the resolution, we added a newly sequenced mitogenome that exhibited the common GO, Euryhaliotrema johni (Ancyrocephalinae), to the dataset. Phylogenetic analyses conducted on two datasets (nucleotides of all 36 genes and amino acid sequences of 12 protein coding genes) and four algorithms (MrBayes, RAxML, IQ-TREE and PhyloBayes) produced topology instability towards the tips, so ancestral GO reconstructions were conducted using TreeREx and MLGO programs using all eight obtained topologies, plus three unique topologies from previous studies. The results consistently supported the second hypothesis, resolving the common GO as a plesiomorphic ancestral GO for Neodermata, Cestoda, Monopisthocotylea, Cestoda + Trematoda and Cestoda + Trematoda + Monopisthocotylea. This allowed us to trace the evolutionary GO scenarios from each common ancestor to its descendants amongst the Monogenea and Cestoda classes, and propose that the common GO was most likely retained throughout all of the common ancestors, leading to the extant species possessing the common GO. Neodermatan phylogeny inferred from GOs was largely incongruent with all 11 topologies described above, but it did support the mitogenomic dataset in resolving Polyopisthocotylea as the earliest neodermatan branch. Although highly derived GOs might be of some use in resolving isolated taxonomic and phylogenetic uncertainties, we conclude that, due to the discontinuous nature of their evolution, they tend to produce artefactual phylogenetic relationships, which makes them unsuitable for phylogenetic reconstruction in Neodermata. Wider and denser sampling of neodermatan mitogenomic sequences will be needed to infer the evolutionary pathways leading to the observed diversity of GOs with confidence.

Morphology is not a reliable taxonomic tool for the genus Lernaea: molecular data and experimental infection reveal that L. cyprinacea and L. cruciata are conspecific.

BACKGROUND: Species belonging to the genus Lernaea are cosmopolitan parasites that can infect many different freshwater fish hosts. Due to a high degree of morphological intraspecific variability and high levels of interspecific similarities, their classification is extremely difficult and controversial. Although the suitability of the shape of cephalic horns has been questioned decades ago by some experimental infection studies, this character still plays the central role in the identification of Lernaea spp. METHODS: We used the nominal species Lernaea cyprinacea and Lernaea cruciata to test the hypothesis that the shape of the anchor can exhibit host-induced morphological variability, and that the two taxa may be synonymous. RESULTS: We examined 517 wild or farmed specimens of five host fish species (four cyprinids and a mosquitofish), and found that all 16 parasite specimens collected from mosquitofish could be morphologically identified as L. cruciata, whereas the remaining 25 parasite specimens were all identified as L. cyprinacea. We experimentally infected goldfish and mosquitofish specimens with offspring (copepodids) of a single L. cyprinacea specimen: the adult parasites from goldfish were morphologically identified as L. cyprinacea, and those from mosquitofish as L. cruciata. We then used molecular data to corroborate that all these specimens are conspecific. CONCLUSIONS: Our results suggest that L. cyprinacea and L. cruciata may be synonyms, misidentified as different species as a result of host-induced morphological variation. Given the current shortage of molecular data for the genus Lernaea, in order to resolve the taxonomy of this genus (determine the exact number of species), future studies should aim to sequence as much molecular data as possible, and conduct further experimental infections.

Effect of intestinal tapeworms on the gut microbiota of the common carp, Cyprinus carpio.

BACKGROUND: Parasitic protozoans, helminths, alter the gut microbiota in mammals, yet little is known about the influence of intestinal cestodes on gut microbiota in fish. In the present study, the composition and diversity of the hindgut microbiota were determined in the intestine of common carp (Cyprinus carpio) infected with two tapeworm species, Khawia japonensis and Atractolytocestus tenuicollis. RESULTS: The intestine contained a core microbiota composed of Proteobacteria, Fusobacteria and Tenericutes. Infection with the two cestode species had no significant effect on the microbial diversity and richness, but it altered the microbial composition at the genus level. PCoA analysis indicated that microbial communities in the infected and uninfected common carp could not be distinguished from each other. However, a Mantel test indicated that the abundance of K. japonensis was significantly correlated with the microbial composition (P = 0.015), while the abundance of A. tenuicollis was not (P = 0.954). According to Pearson's correlation analysis, the abundance of K. japonensis exhibited an extremely significant (P < 0.001) positive correlation with the following gut microbiota taxa: Epulopiscium, U114, Bacteroides, Clostridium and Peptostreptococcaceae (0.8< r < 0.9); and a significant (P < 0.05) correlation with Enterobacteriaceae, Micrococcaceae, Rummeliibacillus, Lysinibacillus boronitolerans, Veillonellaceae, Oxalobacteraceae, Aeromonadaceae (negative), Marinibacillus and Chitinilyticum (0.4< r < 0.7). CONCLUSIONS: These results suggest that the composition of gut microbiota was somewhat affected by the K. japonensis infection. Additionally, increased ratios of pathogenic bacteria (Lawsonia and Plesiomonas) were also associated with the K. japonensis infection, which may therefore increase the likelihood of disease.

Evidence for Adaptive Selection in the Mitogenome of a Mesoparasitic Monogenean Flatworm Enterogyrus malmbergi.

Whereas a majority of monogenean flatworms are ectoparasitic, i.e., parasitize on external surfaces (mainly gills) of their fish hosts, Enterogyrus species (subfamily Ancyrocephalinae) are mesoparasitic, i.e., parasitize in the stomach of the host. As there are numerous drastic differences between these two environments (including lower oxygen availability), we hypothesized that this life-history innovation might have produced adaptive pressures on the energy metabolism, which is partially encoded by the mitochondrial genome (OXPHOS). To test this hypothesis, we sequenced mitochondrial genomes of two Ancyrocephalinae species: mesoparasitic E. malmbergi and ectoparasitic Ancyrocephalus mogurndae. The mitogenomic architecture of E. malmbergi is mostly standard for monogeneans, but that of A. mogurndae exhibits some unique features: missing trnL2 gene, very low AT content (60%), a non-canonical start codon of the nad2 gene, and exceptionally long tandem-repeats in the non-coding region (253 bp). Phylogenetic analyses produced paraphyletic Ancyrocephalinae (with embedded Dactylogyrinae), but with low support values. Selective pressure (PAML and HYPHY) and protein structure analyses all found evidence for adaptive evolution in cox2 and cox3 genes of the mesoparasitic E. malmbergi. These findings tentatively support our hypothesis of adaptive evolution driven by life-history innovations in the mitogenome of this species. However, as only one stomach-inhabiting mesoparasitic monogenean was available for this analysis, our findings should be corroborated on a larger number of mesoparasitic monogeneans and by physiological studies.

Mitochondrial genomes of two diplectanids (Platyhelminthes: Monogenea) expose paraphyly of the order Dactylogyridea and extensive tRNA gene rearrangements.

BACKGROUND: Recent mitochondrial phylogenomics studies have reported a sister-group relationship of the orders Capsalidea and Dactylogyridea, which is inconsistent with previous morphology- and molecular-based phylogenies. As Dactylogyridea mitochondrial genomes (mitogenomes) are currently represented by only one family, to improve the phylogenetic resolution, we sequenced and characterized two dactylogyridean parasites, Lamellodiscus spari and Lepidotrema longipenis, belonging to a non-represented family Diplectanidae. RESULTS: The L. longipenis mitogenome (15,433 bp) contains the standard 36 flatworm mitochondrial genes (atp8 is absent), whereas we failed to detect trnS1, trnC and trnG in L. spari (14,614 bp). Both mitogenomes exhibit unique gene orders (among the Monogenea), with a number of tRNA rearrangements. Both long non-coding regions contain a number of different (partially overlapping) repeat sequences. Intriguingly, these include putative tRNA pseudogenes in a tandem array (17 trnV pseudogenes in L. longipenis, 13 trnY pseudogenes in L. spari). Combined nucleotide diversity, non-synonymous/synonymous substitutions ratio and average sequence identity analyses consistently showed that nad2, nad5 and nad4 were the most variable PCGs, whereas cox1, cox2 and cytb were the most conserved. Phylogenomic analysis showed that the newly sequenced species of the family Diplectanidae formed a sister-group with the Dactylogyridae + Capsalidae clade. Thus Dactylogyridea (represented by the Diplectanidae and Dactylogyridae) was rendered paraphyletic (with high statistical support) by the nested Capsalidea (represented by the Capsalidae) clade. CONCLUSIONS: Our results show that nad2, nad5 and nad4 (fast-evolving) would be better candidates than cox1 (slow-evolving) for species identification and population genetics studies in the Diplectanidae. The unique gene order pattern further suggests discontinuous evolution of mitogenomic gene order arrangement in the Class Monogenea. This first report of paraphyly of the Dactylogyridea highlights the need to generate more molecular data for monogenean parasites, in order to be able to clarify their relationships using large datasets, as single-gene markers appear to provide a phylogenetic resolution which is too low for the task.

Composition and Diversity of Communities of Dactylogyrus spp. In Wild and Farmed Goldfish Carassius auratus.

Species composition and diversity of dactylogyrids were compared on gills of wild and cultured goldfish (silver crucian carp) Carassius auratus from 3 naturally populated lakes and 3 stocked aquaculture ponds in the Hubei province of China to examine the differences in the gill parasite community between these natural and farmed waters. Of the 7 Dactylogyrus spp. detected, all were found in lakes and 5 in ponds, with Dactylogyrus inexpectatus and Dactylogyrus anchoratus being absent from ponds. No significant correlation was found between the species richness and habitat area or host size, nor was there a significant difference in mean species richness between lakes (0.41-0.65) and ponds (0.30-0.76). Brillouin's diversity in lakes (0.049-0.067) was higher than that in ponds (0.024-0.046), but not significantly so. Although the diversity of parasite communities was higher in wild goldfish, higher mean abundance of some Dactylogyrus spp. was found in cultured goldfish. Based on Bray-Curtis similarity, it was difficult to differentiate parasite communities in lakes from those in ponds at the infracommunity level, whereas the 3 lakes and Guanqiao pond differed markedly from the remaining 2 ponds at the component community level. Although infracommunities differed among waterbodies, no effects of fish length or waterbody type were found on infracommunity or component community structure. Together, these results suggest that abundance and species richness of Dactylogyrus spp. on goldfish in lakes and farm ponds are influenced by habitat-specific environmental factors.

Basal position of two new complete mitochondrial genomes of parasitic Cymothoida (Crustacea: Isopoda) challenges the monophyly of the suborder and phylogeny of the entire order.

BACKGROUND: Isopoda is a highly diverse order of crustaceans with more than 10,300 species, many of which are parasitic. Taxonomy and phylogeny within the order, especially those of the suborder Cymothoida Wägele, 1989, are still debated. Mitochondrial (mt) genomes are a useful tool for phylogenetic studies, but their availability for isopods is very limited. To explore these phylogenetic controversies on the mt genomic level and study the mt genome evolution in Isopoda, we sequenced mt genomes of two parasitic isopods, Tachaea chinensis Thielemann, 1910 and Ichthyoxenos japonensis Richardson, 1913, belonging to the suborder Cymothoida, and conducted comparative and phylogenetic mt genomic analyses across Isopoda. RESULTS: The complete mt genomes of T. chinensis and I. japonensis were 14,616 bp and 15,440 bp in size, respectively, with the A+T content higher than in other isopods (72.7 and 72.8%, respectively). Both genomes code for 13 protein-coding genes, 21 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and possess a control region (CR). Both are missing a gene from the complete tRNA set: T. chinensis lacks trnS1 and I. japonensis lacks trnI. Both possess unique gene orders among isopods. Within the CR of I. japonensis (284 bp), we identified a repetitive region with four tandem repeats. Phylogenetic analysis based on concatenated nucleotide sequences of 13 protein-coding genes showed that the two parasitic cymothoids clustered together and formed a basal clade within Isopoda. However, another parasitic cymothoid, Gyge ovalis Shiino, 1939, formed a sister group with the suborder Limnoriidea Brandt & Poore in Poore, 2002, whereas two free-living cymothoid species were located in the derived part of the phylogram: Bathynomus sp. formed a sister group with the suborder Sphaeromatidea Wägele, 1989, and Eurydice pulchra Leach, 1815 with a clade including Bathynomus sp., Sphaeromatidea and Valvifera G. O. Sars, 1883. CONCLUSIONS: Our results did not recover the suborders Cymothoida and Oniscidea Latreille, 1802 as monophyletic, with parasitic and free-living cymothoidans forming separate clades. Furthermore, two parasitic cymothoidans formed the sister-clade to all other isopods, separated from Epicaridea Latreille, 1825, which challenges currently prevalent isopod phylogeny. Additional mt genomes of parasitic and free-living isopods might confer a sufficient phylogenetic resolution to enable us to resolve their relationships, and ultimately allow us to better understand the evolutionary history of the entire isopod order.

Comparative mitogenomics supports synonymy of the genera Ligula and Digramma (Cestoda: Diphyllobothriidae).

BACKGROUND: After observing differences in the number of reproductive complexes per proglottid within the genus Ligula, the genus Digramma was erected. However, the validity of Digramma has been previously questioned due to a low variability in the cox1, nad1 and ITS rDNA sequences between the two genera. We undertook a study to greatly increase the amount of sequence data available for resolution of this question by sequencing and characterizing the complete mitogenomes of Digramma interrupta and Ligula intestinalis. RESULTS: The circular mtDNA molecules of Digramma interrupta and Ligula intestinalis are 13,685 bp and 13,621 bp in size, respectively, both comprising 12 PCGs, 22 tRNA genes, two rRNA genes, and two mNCRs. Both mitogenomes exhibit the same gene order and share 92.7% nucleotide identity, compared with 85.8-86.5% to the most closely related genus Dibothriocephalus. Each gene from D. interrupta and L. intestinalis is almost of the same size, and the sequence identity ranges from 87.5% (trnD) to 100% (trnH, trnQ and trnV). NCR2 sequences of D. interrupta and L. intestinalis are 249 bp and 183 bp in length, respectively, which contributes to the main difference in length between their complete mitogenomes. A sliding window analysis of the 12 PCGs and two rRNAs indicated nucleotide diversity to be higher in nad5, nad6, nad2, nad4 and cox3, whereas the most conserved genes were rrnL and rrnS. Lower sequence identity was also found in nad2, nad4, nad5, nad6 and cox3 genes between the two diphyllobothriids. Within the Diphyllobothriidae, phylogenetic analysis indicated Ligula and Digramma to be most closely related to one another, forming a sister group with Dibothriocephalus. CONCLUSIONS: Owing to higher nucleotide diversity, the genes nad2, nad4, nad5, nad6 and cox3 should be considered optimal candidates to use as molecular markers for population genetics and species identification between the two closely related species. The phylogenetic results in combination with the comparative analysis of the two mitogenomes, consistently support the congeneric status of L. intestinalis and D. interrupta.

The complete mitochondrial DNA of three monozoic tapeworms in the Caryophyllidea: a mitogenomic perspective on the phylogeny of eucestodes.

BACKGROUND: External segmentation and internal proglottization are important evolutionary characters of the Eucestoda. The monozoic caryophyllideans are considered the earliest diverging eucestodes based on partial mitochondrial genes and nuclear rDNA sequences, yet, there are currently no complete mitogenomes available. We have therefore sequenced the complete mitogenomes of three caryophyllideans, as well as the polyzoic Schyzocotyle acheilognathi, explored the phylogenetic relationships of eucestodes and compared the gene arrangements between unsegmented and segmented cestodes. RESULTS: The circular mitogenome of Atractolytocestus huronensis was 15,130 bp, the longest sequence of all the available cestodes, 14,620 bp for Khawia sinensis, 14,011 bp for Breviscolex orientalis and 14,046 bp for Schyzocotyle acheilognathi. The A-T content of the three caryophyllideans was found to be lower than any other published mitogenome. Highly repetitive regions were detected among the non-coding regions (NCRs) of the four cestode species. The evolutionary relationship determined between the five orders (Caryophyllidea, Diphyllobothriidea, Bothriocephalidea, Proteocephalidea and Cyclophyllidea) is consistent with that expected from morphology and the large fragments of mtDNA when reconstructed using all 36 genes. Examination of the 54 mitogenomes from these five orders, revealed a unique arrangement for each order except for the Cyclophyllidea which had two types that were identical to that of the Diphyllobothriidea and the Proteocephalidea. When comparing gene order between the unsegmented and segmented cestodes, the segmented cestodes were found to have the lower similarities due to a long distance transposition event. All rearrangement events between the four arrangement categories took place at the junction of rrnS-tRNA Arg (P1) where NCRs are common. CONCLUSIONS: Highly repetitive regions are detected among NCRs of the four cestode species. A long distance transposition event is inferred between the unsegmented and segmented cestodes. Gene arrangements of Taeniidae and the rest of the families in the Cyclophyllidea are found be identical to those of the sister order Proteocephalidea and the relatively basal order Diphyllobothriidea, respectively.

Sequencing of the complete mitochondrial genome of a fish-parasitic flatworm Paratetraonchoides inermis (Platyhelminthes: Monogenea): tRNA gene arrangement reshuffling and implications for phylogeny.

BACKGROUND: Paratetraonchoides inermis (Monogenea: Tetraonchoididae) is a flatworm parasitising the gills of uranoscopid fishes. Its morphological characteristics are ambiguous, and molecular data have never been used to study its phylogenetic relationships, which makes its taxonomic classification controversial. Also, several decades of unsuccessful attempts to resolve the relationships within the Monogenea present a strong indication that morphological datasets may not be robust enough to be used to infer evolutionary histories. As the use of molecular data is currently severely limited by their scarcity, we have sequenced and characterized the complete mitochondrial (mt) genome of P. inermis. To investigate its phylogenetic position, we performed phylogenetic analyses using Bayesian inference and maximum likelihood approaches using concatenated amino acid sequences of all 12 protein-coding genes on a dataset containing all available monogenean mt genomes. RESULTS: The circular mt genome of P. inermis (14,654 bp) contains the standard 36 genes: 22 tRNAs, two rRNAs, 12 protein-encoding genes (PCGs; Atp8 is missing) and a major non-coding region (mNCR). All genes are transcribed from the same strand. The A + T content of the whole genome (82.6%), as well as its elements, is the highest reported among the monogeneans thus far. Three tRNA-like cloverleaf structures were found in mNCR. Several results of the phylogenomic analysis are in disagreement with previously proposed relationships: instead of being closely related to the Gyrodactylidea, Tetraonchidea exhibit a phylogenetic affinity with the Dactylogyridea + Capsalidea clade; and the order Capsalidea is neither basal within the subclass Monopisthocotylea, nor groups with the Gyrodactylidea, but instead forms a sister clade with the Dactylogyridea. The mt genome of P. inermis exhibits a unique gene order, with an extensive reorganization of tRNAs. Monogenea exhibit exceptional gene order plasticity within the Neodermata. CONCLUSIONS: This study shows that gene order within monopisthocotylid mt genomes is evolving at uneven rates, which creates misleading evolutionary signals. Furthermore, our results indicate that all previous attempts to resolve the evolutionary history of the Monogenea may have produced at least partially erroneous relationships. This further corroborates the necessity to generate more molecular data for this group of parasitic animals.

Seasonal Dynamics and Spatial Distribution of the Dactylogyrus Species on the Gills of Grass Carp (Ctenopharyngodon idellus) from a Fish Pond in Wuhan, China.

Spatial distribution and seasonal variation in mean intensity and prevalence of monogeneans on the gills of grass carp ( Ctenopharyngodon idellus ) were investigated from September 2012 to December 2014 in a fish pond in Wuhan, China. During this period, 2 species of Dactylogyrus were found, i.e., Dactylogyrus lamellatus and Dactylogyrus ctenopharyngodonis. Dactylogyrus lamellatus was present during the entire duration of the investigation, whereas D. ctenopharyngodonis was only detected after January 2014, when another batch of grass carp fingerlings was added to the pond. Prevalence of the 2 Dactylogyrus spp. was relatively high throughout the year, but significant seasonal changes were detected in the mean intensity of the 2 species (P < 0.05). Mean intensity of D. lamellatus peaked in late winter and spring, and then dropped to the lowest point in summer. Dactylogyrus ctenopharyngodonis exhibited a high mean intensity in summer and autumn, but low in winter and spring. Spatial distribution of the 2 species was similar: the highest mean intensity was found on medial and distal parts of the second gill arch. Moreover, no evident change was detected in niche preference of D. lamellatus after the infection of D. ctenopharyngodonis occurred. There was no significant positive correlation in abundance between the 2 species (P > 0.05). In addition, there were no significant differences in the percentage of mean intensity of the 2 species on each gill arch among low-, medium-, and high-intensity groups (P > 0.05). These results suggest unsaturated gill niches and the absence of interspecific competition between the 2 Dactylogyrus species.

Population genetic structure of the acanthocephalan Acanthosentis cheni in anadromous, freshwater, and landlocked stocks of its fish host, Coilia nasus.

The acanthocephalan Acanthosentis cheni was found in anadromous, freshwater, and landlocked stocks of its fish host, Coilia nasus. To examine the genetic variations of the acanthocephalan among the 3 populations with the adaptation of the host to the freshwater, the genetic structure of the helminth was investigated in anadromous (Zhoushan and Chongming islands, and Anqing), freshwater (Anqing, Ezhou, and Poyang Lake), and landlocked (Tian'ezhou Reserve) populations by sequencing intergenic transcribed spacers (ITS) of the ribosomal RNA coding genes. Low Fst values and high gene flow were found among the 7 populations (Fst = 0.0135, P = 0.2723; Nm = 36.48) and the 3 ecotypes of Acanthosentis cheni (Fst = 0.0178, P = 0.1044; Nm = 27.67). On the other hand, significant genetic differentiation of the C. nasus host populations was detected between the upstream and downstream areas of Xiaogu Mountain (Fst = 0.1961, P = 0.0030; Nm = 2.05), which is the farthest location of spawning migration for C. nasus . However, the migration break of the fish host appeared not to cause significant genetic differentiation of A. cheni populations between the upper and lower reaches of Xiaogu Mountain. Other factors might promote genetic exchange of A. cheni populations such as dispersal of the intermediate host by flooding or other fish species serving as the definitive or paratenic hosts. In Anqing, nucleotide diversity of the acanthocephalan was highest in the freshwater population (0.0038) and lower in the anadromous population (0.0026). This suggested that new mutations may have occurred in the freshwater A. cheni population in Anqing when adapting to a freshwater environment.

Molecular phylogeny and host specificity of the larval Eustrongylides (Nematoda: Dioctophmidae) from freshwater fish in China.

The nematodes Eustrongylides spp. collected from different fish species in China were examined for their intra- and interspecific evolutionary variations using the molecular markers mitochondrial cytochrome oxidase c subunit 1 (COI) gene and internal transcribed spacer (ITS) rDNA regions. The phylogenetic analysis indicated that Eustrongylides species are divided into 3 well-supported clades. The ITS divergence between the clades suggested that clades 2 and 3 might represent the same species, whereas clade 1 represent another cryptic species. The host specificity of these nematodes was analyzed according to prevalence data, host range, and phylogenetic information. Clade 1 was found in 4 fish species, i.e., Odontobutis obscurus, Silurus asotus, Culter mongolicus, and Acanthogobius flavimanus, but was predominant in the 2 perciform species, O. obscurus and A. flavimanus. Clade 2 was found in 3 fish species, Monopterus albus, Channa argus, and Channa asiatica, but was predominant in M. albus, reported to feed primarily on oligochaetes, the first intermediate host of Eustrongylides sp. Clade 3 was found in 9 species, but its low prevalence suggests accidental infection in all species. Although the larval nematode presented low host specificity, it exhibited some host preference.