Uncovering the monogenean species diversity of cyprinoid fish in Iraq using an integrative approach.

The freshwaters of Iraq harbour a high diversity of endemic and phylogenetically unique species. One of the most diversified fish groups in this region is cyprinoids, and although their distribution is relatively well known, their monogenean parasites have only rarely been investigated. Herein, we applied an integrative approach, combining morphology with molecular data, to assess the diversity and phylogeny of cyprinoid-associated monogenean parasites. A total of 33 monogenean species were collected and identified from 13 endemic cyprinoid species. The highest species diversity was recorded for Dactylogyrus (Dactylogyridae, 16 species) and Gyrodactylus (Gyrodactylidae, 12 species). Four species of Dactylogyrus and 12 species of Gyrodactylus were identified as new to science and described. Two other genera, Dogielius (Dactylogyridae) and Paradiplozoon (Diplozoidae), were represented only by 4 and 1 species, respectively. Phylogenetic analyses of the Dactylogyrus and Gyrodactylus species revealed that the local congeners do not form a monophyletic group and are phylogenetically closely related to species from other regions (i.e. Europe, North Africa and Eastern Asia). These findings support the assumption that the Middle East served as an important historical crossroads for the interchange of fauna between these 3 geographic regions.

Anti-parasitic activity of garlic (Allium sativum) and onion (Allium cepa) extracts against Dactylogyrus spp. (Monogenean) in Nile tilapia (Oreochromis niloticus): Hematology, immune response, histopathological investigation, and inflammatory cytokine genes of gills.

BACKGROUND: Gills monogenean infestation causes significant mortalities in cultured fishes as a result of respiratory manifestation. Medicinal plants are currently being heavily emphasized in aquaculture due to their great nutritional, therapeutic, antimicrobial activities, and financial value. METHODS: The current study is designed to assess the effect of garlic (Allium sativum) and onion (Allium cepa) extracts as a water treatment on the hematological profile, innate immunity, and immune cytokines expression besides histopathological features of gills of Nile tilapia (Oreochromis niloticus L.) infected with gills monogenetic trematodes (Dactylogyrus sp.). Firstly, the 96-hour lethal concentration 50 (96 h-LC50) of garlic extract (GE) and onion extract (OE) were estimated to be 0.4 g/ L and 3.54 g/ L for GE and OE, respectively. Moreover, the in-vitro anti-parasitic potential for (GE) was found between 0.02 and 0.18 mg/mL and 0.4 to 1.8 mg/mL for OE. For the therapeutic trial, fish (n = 120; body weight: 40-60 g) were randomly distributed into four groups in triplicates (30 fish/group, 10 fish/replicate) for 3 days. Group1 (G1) was not infected or treated and served as control. G2 was infected with Dactylogyrus spp. and not exposed to any treatment. G3, G4 were infected with Dactylogyrus sp. and treated with 1/10 and 1/5 of 96 h LC50 of OE, respectively. G5, G6 were infected with Dactylogyrus sp. and treated with 1/10 and 1/5 of 96 h LC50 of GE, respectively. RESULTS: No apparent signs or behaviors were noted in the control group. Dactylogyrus spp. infected group suffered from clinical signs as Pale color and damaged tissue. Dactylogyrus spp. infection induced lowering of the hematological (HB, MCH, MCHC and WBCs), and immunological variables (lysozyme, nitric oxide, serum Anti- protease activities, and complement 3). the expression of cytokine genes IL-ß and TNF-α were modulated and improved by treatment with A. sativum and A. cepa extracts. The obtained histopathological alterations of the gills of fish infected with (Dactylogyrus spp.) were hyperplasia leading to fusion of the gill filament, lifting of epithelial tissue, aneurism and edema. The results indecated that G4 and G5 is more regenarated epithelium in compare with the control group. CONCLUSION: A. sativum and A. cepa extracts enhance the blood profile and nonspecific immune parameters, and down-regulated the expression level of (IL-1ß and TNF-α).

Monogeneans and chubs: Ancient host-parasite system under the looking glass.

Host-parasite coevolution is one of the fundamentals of evolutionary biology. Due to the intertwined evolutionary history of two interacting species and reciprocal coadaptation processes of hosts and parasites, we can expect that studying parasites will shed more light onto the evolutionary processes of their hosts. Monogenea (ectoparasitic Platyhelminthes) and their cyprinoid fish hosts represent one of the best models for studying host-parasite evolutionary relationships using a cophylogenetic approach. These parasites have developed remarkably high host specificity, where each host species often serves as a potential host for its own host-specific monogenean species. Here, the cophylogenetic relationships in the Dactylogyrus-Squalius system was investigated, as Squalius is one of several cyprinoid genera with puzzling phylogeography and inhabits all four major peri-Mediterranean peninsulas. Of 29 endemic Squalius species examined for the presence of Dactylogyrus parasites, a total of 13 Dactylogyrus species were collected from the gills of 20 Squalius species across a wide range of distribution. Phylogenetic reconstruction revealed a polyphyletic origin for Dactylogyrus species parasitizing congeneric Squalius, with four major clades being recognized. On the basis of the delimitation of host specificity, strict specialists parasitizing single host species, geographic specialists parasitizing congeners in a limited geographical region, and true generalists parasitizing congeners in various geographical regions were recognized in Dactylogyrus species parasitizing Squalius. The phylogenetic reconstruction of Squalius hosts revealed two major clades, the first encompassing only peri-Mediterranean species and the second including species from other Euro-Asian regions. Distance-based cophylogenetic methods did not reveal a statistically significant global cophylogenetic structure in the studied system; however, several host-parasite links among Iberian endemic species contributed significantly to the overall structure. The widest host range and associated genetic variability were recorded for D. folkmanovae, parasitizing nine Squalius species, and D. vistulae, parasitizing 13 Squalius species. Two different dispersion mechanisms and morphological adaptations to Squalius hosts were clearly reflected in the contrasting cophylogenetic patterns for these two species with different levels of host specificity. While host-parasite cospeciation plays an important role in diversification within D. folkmanovae, diversification within D. vistulae is driven mainly by host switching.

Synthesis and anti-parasites efficacy of coumarin derivatives against Dactylogyrus intermedius.

Dactylogyrus is one of the most common parasitic diseases in fish and causes huge losses to the aquaculture industry. With the advantages of safety, low toxicity and easy degradation, plant-derived drugs are ideal for the creation of green aquatic ingredients. The use of plant-derived drugs in aquaculture is limited by their low content and high processing costs, which is a challenge that can be solved by the chemical synthesis of plant-derived drugs. Eleven new coumarin derivatives were synthesized and assessed for their anthelmintic activity in this study. Among them, the derivative 7-((1-tosyl-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (N11) has good anthelmintic activity and its mean anthelmintic efficacy against D. intermedius at a concentration of 10 µM reached 99.84%, which is even better than the anthelmintic activity of the positive control mebendazole. Further studies showed that N11 had concentration values of 3.31 and 1.94 µM for 50% maximal effect (EC50 ) against D. intermedius at 24 and 48 h, respectively. Furthermore, scanning electron microscopy revealed that N11 caused damage to D. intermedius. What is more noteworthy is that a substantial reduction in the ATP content of the parasite was observed following in vitro and in vivo administration of N11. Moreover, it was also found that N11 was able to inhibit the horizontal transmission of D. intermedius. Furthermore, real-time quantitative PCR analysis was utilized to determine the expression profile of genes associated with anti-inflammatory cytokines (IL-10, TGF-ß and IL-4) in goldfish. In all examined organs, it was observed that the expression of anti-inflammatory cytokines increased subsequent to treatment with N11, according to the results. Thus, these results all suggest that N11 possesses good anthelmintic activity and is a potentially effective agent for the control of D. intermedius.

First report of Dactylogyrus formosus from India: a case of co-existence with Paradactylogyrus catlaius causing mass mortality in Indian major carp, Catla catla.

The family Dactylogyridae, known for its role as a frequent pathogen in Cyprinids, was identified in a case of mass mortality involving Catla catla fingerlings (measuring 6.5 ± 2.0 cm and weighing 40.5 ± 10 g) in West Bengal, India. Microscopic examination revealed the presence of two co-existing parasites, Dactylogyrus formosus and Paradactylogyrus catlaius, attached to different sections of the gill filament. Despite their coexistence, these parasites exhibited marked differences in their haptoral hard parts, genital organs, and preferred habitats. Molecular analysis of the Internal Transcribed Spacer 1 and 4 genes indicated more than 90% similarity between the detected parasite and D. formosus previously reported in China. Histopathological observations illustrated the parasites' specific attachment to the distal end of the primary gill lamellae, gradually causing destruction to a maximum number of secondary lamellae. Internally, infiltration of eosinophilic granular cells was observed in gill and kidney blood vessels, while the liver exhibited hepatocytes filled with hemosiderin. The infected fish were treated for 24 h with a safe dose of common salt (5.6 ppt) and albendazole (62 ppm). The survivability rate was significantly higher (p < 0.05) in both treated groups compared to the control. Interestingly, the group treated with common salt showed superior results compared to the albendazole-treated fish. This study presents the sympatric speciation of D. formosus in a new host (C. catla) and explores its host specificity, histopathology, and treatment methods. This case marks the first report of D. formosus causing substantial mortality in cultured Catla in India, alongside the coexistence with Paradactylogyrus catlaius.

Anthelmintic effects of peppermint (Mentha piperita), lemon (Citrus limon), and tea tree (Melaleuca alternifolia) essential oils against Monogenean parasite (Dactylogyrus sp.) on carp (Cyprinus carpio).

Dactylogyrus sp. (Monogenea) is one of the most dangerous pathogens causing parasitic infections in carp (Cyprinus carpio) and other freshwater fish. Due to the adverse effects of conventional chemical treatments on the environment and fish, the use of herbal products in aquaculture against various diseases has increased. In the present study, anthelmintic effects of peppermint (Mentha piperita), lemon (Citrus limon), and tea tree (Melaleuca alternifolia) essential oils against Dactylogyrus sp. found on the gills of carp were studied using in vitro and in vivo experiments. In in vitro experiments, 1, 2.5, 5, and 10 µl/ml concentrations of these essential oils were tested on the parasites and cumulative mortalities were observed to reach 100 % after treatment with peppermint, lemon, and tea tree essential oils of 1 µl/ml concentration in approximately 15, 10 and 2 min, respectively. The in vitro experiments demonstrated that the cumulative mortality of the parasites increased with essential oil concentration and exposure duration. As the concentration of essential oil used increased, the observed mean time to death of parasites decreased. In in vivo experiments, median effective concentrations (EC50) as assessed by in vitro tests for peppermint and lemon essential oils in 5 min exposure and EC50 concentration for tea tree essential oil in 2 min exposure was applied on fish as a single bath and there was a significant decrease in the mean parasite intensities (p<0.05). Antiparasitic efficacies of peppermint, lemon, and tea tree essential oils were determined as 28.23 %, 30.95 %, and 35.31 %, respectively. The tea tree oil was the most effective and peppermint oil was the least effective in both in vitro and in vivo experiments. All three herbal essential oils tested in this study have weak antiparasitic potential against monogenean infections in fish.

Effects of different regimes of low temperature on egg hatching of Dactylogyrus vastator (Monogenea: Dactylogyridae).

The development of dactylogyrids is dependent on water temperature, and their eggs fail to hatch below 5 °C. In the field, however, mean abundance of Dactylogyrus species increases and reaches a high level in winter, which suggests that infective oncomiracidia hatch from eggs in winter. Therefore, the effect of low water temperature on in vitro egg hatching of D. vastator was determined in laboratory. D. vastator hatching success was 65.3%, 62.7%, 42.6% and 22.3% when eggs were firstly incubated for 0, 7, 14 and 21 days at 5 °C and then consecutively maintained for 15 days at 20 °C. When eggs were directly incubated at 5 °C, eggs failed to hatch within one month. However, hatching success was 69.8% and 66.7%, respectively, when maintained at 5 °C after 12 and 24 h incubation at 20 °C. The results suggested that egg incubation for more than 1 week at 5 °C had significant impacts on hatching success of D. vastator subsequently maintained at 20 °C. But low temperature (5 °C) had no effect on hatching success when eggs were firstly exposed to room temperature (20 °C) for one day.

Monogenean parasites infect ornamental fish imported to Australia.

The ornamental fish trade provides a pathway for the global translocation of aquatic parasites. We examined a total of 1020 fish imported from Singapore, Malaysia, Thailand, or Sri Lanka to Australia (including freshwater and marine fish species) for monogenean ectoparasites. Fish were received following veterinary certification that they showed no clinical signs of pests and diseases from the exporting country and visual inspection at Australian border control. Australian import conditions require mandatory treatment for goldfish with parasiticides (e.g. trichlorfon, formaldehyde, sodium chloride) for the presence of gill flukes (Dactylogyrus vastator Nybelin, 1924 and Dactylogyrus extensus Mueller and Van Cleave, 1932) prior to export. Over 950 individual parasites were detected in five imported fish species, representing 14 monogenean species. Seven Dactylogyrus spp. including D. vastator and three Gyrodactylus spp. infected goldfish, Carassius auratus Linnaeus, 1758, from Malaysia, Singapore, and Thailand. Dactylogyrus ostraviensis Rehulka, 1988, infected rosy barb, Pethia conchonius Hamilton, 1822, from Singapore, Sri Lanka, and Thailand while two Trianchoratus spp. infected three spot gourami, Trichopodus trichopterus Pallas, 1970 and pearl gourami Trichopodus leerii Bleeker, 1852, from Sri Lanka. Urocleidoides reticulatus Mizelle & Price, 1964, infected guppy, Poecilia reticulata Peters, 1859, from Sri Lanka. The discovery of D. vastator in goldfish, as well as 13 other monogenean species, shows that pre-export health requirements, which include chemical treatment of goldfish, and inspection of all ornamental fish species did not prevent infection by monogeneans. Inspection prior to exportation and at border control must account for the highly cryptic nature of monogenean parasites and consider alternatives to current pre-export conditions and visual inspection at border control.

Dactylogyrids (Platyhelminthes: Monogenea) from Sudanese Labeo Spp., with a Description of Dogielius Sennarensis n. Sp. and a Redescription of Dogielius Flosculus Guégan, Lambert & Euzet, 1989.

Dactylogyrid monogeneans of Labeo horie Heckel, 1847 and L. niloticus (Linnaeus, 1758) (Cyprinidae) were surveyed at two sites on the River Nile in Sudan. The present study reports the presence of ten species of Dactylogyrus Diesing, 1850 and three species of Dogielius Bychowsky, 1936 based on a morphometric evaluation of the sclerotised structures. The species found include: Dactylogyrus nathaliae Guégan, Lambert & Euzet, 1988; D. rastellus Guégan, Lambert & Euzet, 1988; D. retroversus Guégan, Lambert & Euzet, 1988; D. senegalensis Paperna, 1969, D. yassensis Musilová, Rehulková & Gelnar, 2009 and five other undescribed species of Dactylogyrus. The genus Dogielius was represented by Dogielius flosculus Guégan, Lambert & Euzet, 1989; the newly identified D. sennarensis n. sp., and one undescribed Dogielius species. While D. sennarensis n. sp. resembles D. intorquens, it differs from this species and other congeners by having a longer ventral bar and anchor points and nosclerotised vagina. In addition, this study redescribes D. flosculus based on the morphology of specimens collected from L. horie. All specimens studied had a single large vagina, in contrast to the original description which reported a vagina composed of two unconnected parts. All dactylogyrid species in this study represent new host and geographical records.

Parasites and a host's sense of smell: reduced chemosensory performance of fathead minnows (Pimephales promelas) infected with a monogenean parasite.

Parasites residing within the central nervous system of their hosts have the potential to reduce various components of host performance, but such effects are rarely evaluated. We assessed the olfactory acuity of fathead minnows (Pimephales promelas) infected experimentally with the monogenean Dactylogyrus olfactorius, the adults of which live within the host's olfactory chambers. Olfactory acuity was compared between infected and uninfected hosts by assessing electro-olfactography (EOG) neural responses to chemical stimuli that indicate the presence of food (L-alanine) or the presence of conspecifics (taurocholic acid). We also compared differences in gross morphology of the olfactory epithelium in infected and uninfected minnows. Differences in EOG responses between infected and uninfected minnows to both cue types were non-significant at 30 days post-exposure. By days 60 and 90, coincident with a two times increase in parasite intensity in the olfactory chambers, the EOG responses of infected minnows were 70-90% lower than controls. When infected fish were treated with a parasiticide (Prazipro), olfactory acuity returned to control levels by day 7 post-treatment. The observed reduction in olfactory acuity is best explained by the reduced density of cilia covering the olfactory chambers of infected fish, or by the concomitant increase in the density of mucous cells that cover the olfactory chambers. These morphological changes are likely due to the direct effects of attachment and feeding by individual worms or by indirect effects associated with host responses. Our results show that infection of a commonly occurring monogenean in fathead minnows reduces olfactory acuity. Parasite-induced interference with olfactory performance may reduce a fish's ability to detect, or respond to, chemical cues originating from food, predators, competitors or mates.

Synthesis and anthelmintic activity of arctigenin derivatives against Dactylogyrus intermedius in goldfish.

To control the parasitic disease of Dactylogyrus intermedius, a series of new arctigenin derivatives were designed, synthesized and tested in our study. The anthelmintic activity of most of the derivatives ranged from 1 to 10mg/L. Compared to traditional drug praziquantel (EC50=2.69mg/L), ether derivatives 2g and 2h exhibited slightly higher anti-parasitic activity, with the EC50 values of 2.48 and 1.52mg/L, respectively. Furthermore, the arctigenin-imidazole hybrids 4a and 4b also removed D. intermedius effectively, with the EC50 values of 2.13 and 2.07mg/L, respectively. The structure-activity relationship analysis indicated that four carbon atoms length of linker and imidazole substitute group could significantly increase the anthelmintic activity, and reduced the toxicity. Through the scanning electron microscope observation, compounds 4a and 4b caused the D. intermedius tegumental damage such as intensive wrinkles, holes and nodular structures. Overall, the structural optimization analysis of arctigenin suggested that 4a and 4b can be used for preventing and controlling Dactylogyrus infections and considered as promising lead compounds for the development of commercial drugs.

Combined effects of Chinese medicine feed and ginger extract bath on co-infection of Ichthyophthirius multifiliis and Dactylogyrus ctenopharyngodonid in grass carp.

Dactylogyrus ctenopharyngodonid and Ichthyophthirius multifiliis are two important ectoparasites of freshwater fish. Co-infection by the two parasites leads to high fish mortality and results in heavy economic losses. This study aimed to evaluate the efficacy of medicated feed and a ginger extract bath against D. ctenopharyngodonid and I. multifiliis on grass carp and investigate the hematological response of grass carp co-infected by the two parasites. These results demonstrated that red blood cell (RBC) and thrombocyte percentage among leucocytes significantly decreased after grass carp were co-infected by D. ctenopharyngodonid and I. multifiliis. The monocyte and neutrophil percentages significantly increased with the increment of parasite mean intensities, while the lymphocyte percentage decreased. The activities of serum acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and superoxide dismutase (SOD) significantly increased after co-infection. When grass carp treated with medicated feed containing 4% of Astragalus membranaceus, Allium sativum, Morus alba, and Glycyrrhiza uralensis, the activities of ACP, AKP, LZM, and SOD were significantly enhanced, and the mean intensities of D. ctenopharyngodonid and I. multifiliis were significantly decreased. When grass carp was treated with medicated feed and a 4-mg/L ginger extract bath, all parasites were eliminated during 28 days. The bath of ginger extract at a concentration of 4 mg/L kept a low mean intensity of I. multifiliis and D. ctenopharyngodonid, then the two parasites were eliminated by oral administration of the medicated feed with an immunostimulant (Chinese medicine compound).

Synthesis and anthelmintic activity of coumarin-imidazole hybrid derivatives against Dactylogyrus intermedius in goldfish.

With an intention to find more potent anti-parasite agents, four bromoalkane substituted coumarin derivatives (1-4) and twenty coumarin-imidazole hybrid derivatives were synthesized and screened for their anthelmintic activity and the acute toxicity. Anti-parasites results confirmed that most coumarin derivatives retained their anthelmintic activity against Dactylogyrus intermedius at the dose range from 1 to 10mg/L. Among the candidates, compound 23 showed the best anthelmintic activity than other compounds against D. intermedius infestation with EC50 value of 0.85mg/L. The structure-activity relationship analysis confirmed that the anthelmintic activities of derivatives were determined by the length of 'linker' (R1substitute position) and the substitute group in R2 position. The active data confirmed that six carbon atoms length of 'linker' and benzimidazole substitute group can increased the anthelmintic activity of compound, significantly. On the basis of these results, compound 23 can be used as a potential lead compound for the development of commercial drug against D. intermedius.

Morphometric and molecular characterization of Dactylogyrus vastator and D. intermedius in goldfish (Carassius auratus).

Goldfish is known to be parasitized by at least seven species of Dactylogyrus and considered as one of the most common hosts. Dactylogyrus vastator and Dactylogyrus intermedius are the dominant species living on the gills of goldfish. However, little information on morphometric characterization is available, which easily causes misidentification. The purpose of this study is to provide comprehensive morphometric and molecular characterization of D. vastator and D. intermedius collected form a fish farm in Henan, China. The characterization was presented based on the high-resolution images and standard molecular markers (18S ribosomal DNA subunit and the internal transcribed spacer region), as well as a total of 10 point-to-point morphometrics characters. In addition, a detailed comparison of morphometric and phylogenetic characterization in D. vastator versus D. intermedius was performed. The results demonstrated that all parameters measured differed significantly between these two species of Dactylogyrus, whereas molecular comparison showed subtle differences between them in nucleotide divergence and genetic distances. These findings suggested that these two species of Dactylogyrus can be distinguished more easily based on morphometric measurements than molecular data for 18S ribosomal DNA + internal transcribed spacer region (ITS-1).

Fighting fish parasites with photodynamically active chlorophyllin.

Water-soluble chlorophyll (chlorophyllin) was used in a phototoxic reaction against a number of fish ectoparasites such as Ichtyobodo, Dactylogyrus, Trichodina, and Argulus. Chlorophyllin is applied to the water at concentrations of several micrograms per milliliter for a predefined incubation time, and afterwards, the parasites are exposed to simulated solar radiation. Application in the dark caused only little damage to the parasites; likewise, light exposure without the addition of the photosensitizer was ineffective. In Ichthyobodo, 2 µg/mL proved sufficient with subsequent simulated solar radiation to almost quantitatively kill the parasites, while in Dactylogyrus, a concentration of about 6 µg/mL was necessary. The LD50 value for this parasite was 1.02 µg/mL. Trichodina could be almost completely eliminated at 2 µg/mL. Only in the parasitic crustacean Argulus, no killing could be achieved by a photodynamic reaction using chlorophyllin. Chlorophyllin is non-toxic, biodegradable, and can be produced at low cost. Therefore, we propose that chlorophyllin (or other photodynamic substances) are a possible effective countermeasure against several ectoparasites in ponds and aquaculture since chemical remedies are either forbidden and/or ineffective.

In vitro immunocompetence of two compounds isolated from Polygala tenuifolia and development of resistance against grass carp reovirus (GCRV) and Dactylogyrus intermedius in respective host.

The present study was undertaken to isolate some compounds from methanol extract of Polygala tenuifolia and evaluate their immunostimulatory properties and antiviral activity using grass carp Ctenopharyngodon idella kidney (CIK) cells and GCRV. By applying insecticidal bioassay-guided, chromatography techniques and successive recrystallization, two purified compounds were obtained. The changes of expression of selected immune genes (Mx1, IL-1ß, TNFα, MyD88 and IgM) in C. idella kidney cell lines were evaluated after exposure to these isolated compounds. The results showed that compound 1 and 2 up-regulated to varying degrees of Mx1, IL-1ß, TNFα, and MyD88 in C. idella kidney cells. WST-8 kit assay verified the two compounds has no toxic effects on CIK cell, and furthermore, have in vitro antivirus activity. Especially, that there is keeping 79% cell viability when exposure to compound 2 (100 mg L(-1)). According to in vivo insecticidal assays against Dactylogyrus intermedius, compound 2 exhibited higher efficacy than compound 1, which was found to be 87.2% effective at the concentrations of 5 mg L(-1) and safe to goldfish (Carassius auratus). Besides, the purified compounds were identified by spectral data as: (1) 1,5-Anhydro-D-glucitol and (2) 3,4,5-trimethoxy cinnamic acid. Overall, the results indicate that bath administration of these compounds modulates the immune related genes in C. idella kidney cells and to some extent, eliminate the virus and parasitic infections.

Complete mitochondrial genomes of Dactylogyrus crucifer and Dactylogyrus zandti reveal distinct patterns of codon usage within Dactylogyrus.

Monogeneans of the genus Dactylogyrus Diesing, 1850, the largest genus in the family Dactylogyridae, mostly parasitize the gills of cyprinoid hosts; however, only 3 Dactylogyrus' mitochondrial genomes (mitogenomes) are studied so far. The aim of this research is to extend our understanding of the mitogenomes of Dactylogyrus. We sequenced the mitogenomes of D. crucifer and D. zandti isolated from Rutilus rutilus and Abramis brama orientalis in northwest China, and then we compared these mitogenomes with other monogeneans. We used Illumina NovaSeq to sequence the entire mitochondrial genomes of D. crucifer and D. zandti and characterized the mitogenomes to understand the gene structure, gene identity, the secondary structures of the 22 tRNA genes, and relative synonymous codon usage. We used the analytic Bayesian Information and Maximum Likelihood methods to determine their associated phylogenetic trees. The mitogenomes of D. crucifer and D. zandti were 14,403 and 18,584 bp, respectively. Organization and positioning of these genes were in accordance with Dactylogyrus lamellatus and Dactylogyrus tuba. The nucleotide composition of Dactylogyridae was different from other families of Monogenea, and the A+T count of genus Dactylogyrus (54 - 58.4 %) was lower than other genus species of the family Dactylogyridea (63.9 - 78.4 %) in protein-coding genes. Dactylogyrus members displayed a codon usage bias. The relative synonymous codon used by Dactylogyrus was not conserved and was lower than other monogeneans. The codon use patterns of closely-related species isolated from closely-related hosts were identical. Phylogenetic analyses using mitogenomic dataset produced Dactylogyrus isolated from host subfamily Leuciscinae formed a sister-group. Our results contributed significantly to an increased database of mitogenomes, more than 50 %, for Dactylogyrus that may help future studies of mitochondrial genes and codon uses for the analysis of monogenean phylogenetics.

Mitochondrial phylogenomics provides conclusive evidence that the family Ancyrocephalidae is deeply paraphyletic.

BACKGROUND: Unresolved taxonomic classification and paraphyly pervade the flatworm class Monogenea: the class itself may be paraphyletic and split into Polyopisthocotylea and Monopisthocotylea; there are some indications that the monopisthocotylean order Dactylogyridea may also be paraphyletic; single-gene markers and some morphological traits indicate that the family Ancyrocephalidae is paraphyletic and intertwined with the family Dactylogyridae. METHODS: To attempt to study the relationships of Ancyrocephalidae and Monopisthocotylea using a phylogenetic marker with high resolution, we sequenced mitochondrial genomes of two fish ectoparasites from the family Dactylogyridae: Dactylogyrus simplex and Dactylogyrus tuba. We conducted phylogenetic analyses using three datasets and three methods. Datasets were ITS1 (nuclear) and nucleotide and amino acid sequences of almost complete mitogenomes of almost all available Monopisthocotylea mitogenomes. Methods were maximum likelihood (IQ-TREE), Bayesian inference (MrBayes) and CAT-GTR (PhyloBayes). RESULTS: Both mitogenomes exhibited the ancestral gene order for Neodermata, and both were compact, with few and small intergenic regions and many and large overlaps. Gene sequences were remarkably divergent for nominally congeneric species, with only trnI exhibiting an identity value > 80%. Both mitogenomes had exceptionally low A + T base content and AT skews. We found evidence of pervasive compositional heterogeneity in the dataset and indications that base composition biases cause phylogenetic artefacts. All six mitogenomic analyses produced unique topologies, but all nine analyses produced topologies that rendered Ancyrocephalidae deeply paraphyletic. Mitogenomic data consistently resolved the order Capsalidea as nested within the Dactylogyridea. CONCLUSIONS: The analyses indicate that taxonomic revisions are needed for multiple Polyopisthocotylea lineages, from genera to orders. In combination with previous findings, these results offer conclusive evidence that Ancyrocephalidae is a paraphyletic taxon. The most parsimonious solution to resolve this is to create a catch-all Dactylogyridae sensu lato clade comprising the current Ancyrocephalidae, Ancylodiscoididae, Pseudodactylogyridae and Dactylogyridae families, but the revision needs to be confirmed by another marker with a sufficient resolution.

Nearctic Dactylogyrus species (Platyhelminthes, Monogenea) parasitizing cypriniform fishes in the context of morphology and phylogeny, with descriptions of seven new species.

Ribosomal DNA sequences are currently available for 32 morphologically recognized species of Dactylogyrus parasitizing Nearctic cypriniforms, but only 16 of them are assigned to nominal species. Herein, morphological data on 28 of the 32 species are provided, together with comments on their phylogenetic relationships in the context of the morphology of taxonomically important structures. Seven new species of Dactylogyrus are described from five species of leuciscids and one species of catostomids, as follows: D. aduncus n. sp. from Campostoma spadiceum, D. cloutmani n. sp. from Luxilus chrysocephalus isolepis, D. cornifrons n. sp. from Cyprinella venusta, D. fimbratus n. sp. from Rhinichthys cataractae, D. mcallisteri n. sp. from Semotilus atromaculatus, and D. chieni n. sp. and D. haneki n. sp. from Hypentelium nigricans. Four previously described species of Dactylogyrus, D. atromaculatus from S. atromaculatus, D. eos from C. neogaeus, D. parvicirrus from Notemigonus crysoleucas and D. perlus from L. c. isolepis, are redescribed and/or figured. As for the remaining 17 species, only the male copulatory organs (MCOs) taken from the respective hologenophores are illustrated. On the basis of phylogenetic analyses, two main clades of Nearctic Dactylogyrus were recognized and supported by the different morphology of the MCO. The first one included 22 strictly Nearctic species sharing the same MCO type with Dactylogyrus spp. parasitizing cyprinids likely of Asian origin. The second clade encompassed Dactylogyrus spp. with diverse MCO morphology and was placed in the sister position to Dactylogyrus spp. parasitizing European leuciscids and North-West African cyprinids.

Title: Espèce néarctique de Dactylogyrus (Platyhelminthes, Monogenea) parasitant des poissons cypriniformes dans un contexte de morphologie et phylogénie, avec description de sept nouvelles espèces. Abstract: Des séquences d'ADN ribosomal sont actuellement disponibles pour 32 espèces morphologiquement reconnues de Dactylogyrus parasitant les cypriniformes néarctiques, mais seulement 16 d'entre elles sont attribuées à des espèces nominales. Ici, des données morphologiques sur 28 des 32 espèces sont fournies avec des commentaires sur leurs relations phylogénétiques dans le contexte de la morphologie des structures taxonomiquement importantes. Sept nouvelles espèces de Dactylogyrus sont décrites à partir de cinq espèces de leuciscidés et d'une espèce de catostomidés, comme suit : D. aduncus n. sp. de Campostoma spadiceum, D. cloutmani n. sp. de Luxilus chrysocephalus isolepis, D. cornifrons n. sp. de Cyprinella venusta, D. fimbratus n. sp. de Rhinichthys cataractae, D. mcallisteri n. sp. de Semotilus atromaculatus et D. chieni n. sp. et D. haneki n. sp. de Hypentelium nigricans. Quatre espèces de Dactylogyrus précédemment décrites, D. atromaculatus de S. atromaculatus, D. eos de C. neogaeus, D. parvicirrus de Notemigonus crysoleucas et D. perlus de L. c. isolepis, sont redécrites et/ou figurées. Comme pour les 17 espèces restantes, seuls les organes copulateurs mâles (OCM) prélevés sur les hologénophores respectifs sont illustrés. Sur la base d'analyses phylogénétiques, deux clades principaux de Dactylogyrus néarctiques ont été reconnus et soutenus par des morphologies différentes de l'OCM. Le premier comprenait 22 espèces strictement néarctiques partageant le même type d'OCM que les Dactylogyrus spp. parasitant des cyprinidés probablement d'origine asiatique. Le deuxième clade comprenait des Dactylogyrus spp. avec diverses morphologies d'OCM et était placé en position sœur des Dactylogyrus spp. parasitant les leuciscidés européens et les cyprinidés d'Afrique du Nord-Ouest.

Dactylogyrus spp. (Dactylogyridae, Monogenea) from tinfoil barb, Barbonymus schwanenfeldii imported into South Africa: morphometric and molecular characterisation.

This study reports on three species of Dactylogyrus Diesing, 1850 (Dactylogyridae) collected from tinfoil barb, Barbonymus schwanenfeldii (Bleeker) which were imported into South Africa as ornamental fish from Sri Lanka and Thailand. Supplementary morphometric characterisation and molecular data (partial 18S and 28S rDNA, and ITS1 region sequences) are presented for Dactylogyrus lampam (Lim & Furtado, 1986), Dactylogyrus tapienensis Chinabut & Lim, 1993 and Dactylogyrus viticulus Chinabut & Lim, 1993. Prevalence of Dactylogyrus spp. infection was 87% and 80% for fish from Sri Lanka and Thailand, respectively. Composition of the parasites between the fish of each origin differed. All three species were found to infect fish from Thailand, but only D. lampam was present on the fish received from Sri Lanka. Phylogenetic analysis revealed the position of studied species, with D. lampam clustering within the lineages of varicorhini-type species, while D. tapienensis and D. viticulus form a sister lineage to Dactylogyrus spp. associated with Cyprinus carpio L. and Carassius spp., species parasitising central African large cyprinids (Labeo Cuvier), and species parasitising African and Middle Eastern Carasobarbus spp.

Title: Dactylogyrus spp. (Dactylogyridae, Monogenea) de Barbonymus schwanenfeldii importé en Afrique du Sud : caractérisation morphométrique et moléculaire. Abstract: Cette étude porte sur trois espèces de Dactylogyrus Diesing, 1850 (Dactylogyridae), prélevées sur des Barbonymus schwanenfeldii (Bleeker) qui ont été importés en Afrique du Sud comme poissons d'ornement depuis le Sri Lanka et la Thaïlande. Une caractérisation morphométrique et des données moléculaires supplémentaires (ADNr 18S et 28S partiels et séquences de la région ITS1) sont présentées pour Dactylogyrus lampam (Lim & Furtado, 1986), Dactylogyrus tapienensis Chinabut & Lim, 1993 et Dactylogyrus viticulus Chinabut & Lim, 1993. La prévalence de l'infection par les Dactylogyrus spp. était respectivement de 87 % et 80 % pour les poissons du Sri Lanka et de Thaïlande. La composition des parasites entre les poissons des deux origines différait. Les trois espèces infectaient les poissons de Thaïlande, mais seul D. lampam était présent sur les poissons du Sri Lanka. L'analyse phylogénétique a révélé la position des espèces étudiées, D. lampam se regroupant dans les lignées d'espèces de type varicorhini, tandis que D. tapienensis et D. viticulus forment une lignée sœur des Dactylogyrus spp. associés à Cyprinus carpio L. et Carassius spp., espèces parasitant les grands cyprinidés d'Afrique centrale (Labeo Cuvier), et espèces parasitant les Carasobarbus spp. d'Afrique et du Moyen-Orient.