The life cycle of Chloromyxum auratum (Myxozoa) from goldfish, Carassius auratus (L.), involves an antonactinomyxon actinospore

The myxozoan parasite Chloromyxum auratum Hallett, Atkinson, Holt, Banner & Bartholomew, 2006 , was shown experimentally to have a two‐host life cycle which involved a previously undescribed antonactinomyxon actinospore stage. Myxospores obtained from gall bladders of naturally infected feral goldfish, Carassius auratus (L.), were used to infect samples of mixed species of oligochaete worms obtained from the same locality as the fish: Fern Ridge Dam, Oregon, USA. After some 110 days post‐exposure, actinospores were detected from the water above the oligochaetes. The 18S rDNA sequence of these actinospores was identical to the original myxospores. Spore release was sporadic, of low intensity and short duration, which confounded efforts to identify the host oligochaete species and infect naïve fish. This is the first life cycle that incorporates an actinospore of the collective group Antonactinomyxon, and the first life cycle demonstrated in the laboratory for a species of Chloromyxum.     

Molecular and light microscopy evidence for the transfer of Myxobolus honghuensis from Carassius auratus gibelio broodfish to progeny

Myxozoans usually have a complex life cycle involving indirect transmission between vertebrate and invertebrate hosts. The vertical transmission of these parasites in vertebrate hosts has not been documented so far. Here, we assessed whether the myxozoan parasite Myxobolus honghuensis is vertically transmitted in naturally infected allogynogenetic gibel carp Carassius auratus gibelio (Bloch). M. honghuensis infection of broodfish, fertilized eggs and laboratory-cultured progeny was monitored in 2018 and 2019. The presporogonic stage was microscopically observed in the pharynx of broodfish and their progeny. In situ hybridization confirmed the presence of M. honghuensis presporogonic stage in the pharynx of broodfish and progeny. Nested PCR results showed that M. honghuensis was present in tissues and eggs of broodfish, fertilized eggs and their corresponding progeny. The sequences obtained from broodfish and progeny showed 98.0−99.8% similarity with ITS-5.8S rDNA of M. honghuensis. This study provides molecular and light microscopy evidence for the transfer of M. honghuensis from broodfish to progeny via the eggs, but it is insufficient to assert that M. honghuensis can transmit vertically in naturally infected allogynogenetic gibel carp. This is the first record about vertical transfer of myxozoan in the vertebrate host.     

Life cycle inference and phylogeny of Ortholinea labracis n. sp. (Myxosporea: Ortholineidae), a parasite of the European seabass Dicentrarchus labrax (Teleostei: Moronidae), in a Portuguese fish farm

Ortholinea labracis n. sp. is described and its life cycle is inferred from a Southern Portuguese fish farm, with basis on microscopic and molecular procedures. This myxosporean parasite infects the urinary bladder of the European seabass Dicentrarchus labrax and the intestinal epithelium of a marine oligochaete of the genus Tectidrilus. Myxospores subspherical in valvular view and ellipsoidal in sutural view measuring 7.6 ± 0.3 (6.8–8.7) μm in length, 7.2 ± 0.2 (6.7–7.7) μm in width and 6.5 ± 0.4 (5.8–7.7) μm in thickness. Two polar capsules, 3.0 ± 0.2 (2.6–3.4) μm long and 2.4 ± 0.1 (2.0–2.9) μm wide, located at the same level, but with divergent orientation and opening to opposite sides of the suture line. Sequencing of the SSU rRNA gene revealed a similarity of 100% between the analysed myxospores and triactinomyxon actinospores. The phylogenetic setting of O. labracis n. sp. shows subgrouping in correlation with tissue tropism, but identifies this parasite as another exception to the main division of Myxosporea into the main freshwater and marine lineages.     

A Minchinia mercenariae‐like parasite infects cockles Cerastoderma edule in Galicia (NW Spain)

The cockle Cerastoderma edule fishery has traditionally been the most important shellfish species in terms of biomass in Galicia (NW Spain). In the course of a survey of the histopathological conditions affecting this species in the Ria of Arousa, a haplosporidan parasite that had not been observed in Galicia was detected in one of the most productive cockle beds of Galicia. Uni‐ and binucleate cells and multinucleate plasmodia were observed in the connective tissue mainly in the digestive area, gills and gonad. The parasite showed low prevalence, and it was not associated with abnormal cockle mortality. Molecular identification showed that this parasite was closely related to the haplosporidan Minchinia mercenariae that had been reported infecting hard clams Mercenaria mercenaria from the Atlantic coast of the United States. The molecular characterization of its SSU rDNA region allowed obtaining a fragment of 1,796 bp showing 98% homology with M. mercenariae parasite. Phylogenetic analysis supported this identification as this parasite was clustered in the same clade as M. mercenariae from the United States and other M. mercenariae‐like sequences from the UK, with bootstrap value of 99%. The occurrence of M. mercenariae‐like parasites infecting molluscs outside the United States is confirmed.     

Ichthyophonus‐infected walleye pollock Theragra chalcogramma (Pallas) in the eastern Bering Sea: a potential reservoir of infections in the North Pacific

In 2003, the Alaska walleye pollock industry reported product quality issues attributed to an unspecified parasite in fish muscle. Using molecular and histological methods, we identified the parasite in Bering Sea pollock as Ichthyophonus. Infected pollock were identified throughout the study area, and prevalence was greater in adults than in juveniles. This study not only provides the first documented report of Ichthyophonus in any fish species captured in the Bering Sea, but also reveals that the parasite has been present in this region for nearly 20 years and is not a recent introduction. Sequence analysis of 18S rDNA from Ichthyophonus in pollock revealed that consensus sequences were identical to published parasite sequences from Pacific herring and Yukon River Chinook salmon. Results from this study suggest potential for Ichthyophonus exposures from infected pollock via two trophic pathways; feeding on whole fish as prey and scavenging on industry‐discharged offal. Considering the notable Ichthyophonus levels in pollock, the low host specificity of the parasite and the role of this host as a central prey item in the Bering Sea, pollock likely serve as a key Ichthyophonus reservoir for other susceptible hosts in the North Pacific.     

The placement of Gyrodactylus salmonis (Yin & Sproston) in the molecular phylogeny of studied members of the Gyrodactylus wageneri‐group parasitizing salmonids

The molecular phylogeny of Gyrodactylus salmonis from brook charr, Salvelinus fontinalis, rainbow trout, Oncorhynchus mykiss, cutthroat trout, O. clarkii, and Atlantic salmon, Salmo salar, in Canada is presented using sequences from ITS‐rDNA and the mitochondrial COX 1 gene. Sequence variation among G. salmonis specimens from the different North American hosts was consistent with within‐species variation reported for other Gyrodactylus. Sequence data are compared to those from other members of the wageneri group parasitizing salmoniform fishes in northern Europe (G. derjavini, G. derjavinoides, G. lavareti, G. salaris, G. salvelini, G. teuchis and G. truttae) and Asia (G. brachymystacis). Sequence divergence between G. salmonis and the recently described G. salvelini on Arctic charr, Salvelinus alpinus, in Finland was consistent with within‐species levels of variation in Gyrodactylus; however, phylogenetic analyses and morphological comparisons provided evidence of their distinctiveness such that they appear to be sister species. They grouped with G. lavareti (a parasite of a coregonid) to form a clade separate from European and Asian species of the wageneri lineage known from salmonid fish. Further study of gyrodactylids from across salmonid, coregonid and thymallid fish in the northern hemisphere would shed more light on the phylogeography of these parasites and serves as an important backdrop to understanding the evolution of their emergent virulence.     

Morphological and molecular biological studies on intramuscular Myxobolus spp. of cyprinid fish

The validity of Myxobolus species infecting the skeletal muscles of six cyprinid fish species was studied by morphological and molecular biological methods. Intracellularly developing Myxobolus spores identified as M. cyprini from the common carp, M. musculi from the barbel, and M. pseudodispar from the roach, rudd, common bream and white bream were very similar in their shape and size. Nonetheless, in species identified as M. pseudodispar, the occurrence of spores with an asymmetrical shape was higher than in M. cyprini, while asymmetrical spores were only occasionally found in M. musculi. The DNA sequence analysis of the polymerase chain reaction (PCR)‐amplified 18S rRNA gene of Myxobolus spores from these fish showed a similar phylogeny to that of their host species. As morphological studies and DNA sequence analysis demonstrated slight but real differences in the spores infecting muscles of the six cyprinid species, it is suggested that M. musculi, M. pseudodispar and M. cyprini are valid species.     

Molecular characterization and confocal laser scanning microscopic study of Pygidiopsis macrostomum (Trematoda: Heterophyidae) parasites of guppies Poecilia vivipara

Pygidiopsis macrostomum and Ascocotyle (Phagicola) pindoramensis (Digenea: Heterophyidae) parasitize guppies as intermediate hosts and, respectively, fish‐eating mammals or birds as definitive hosts. Heterophyids have zoonotic potential, and molecular studies associated with morphological and ecological aspects have helped to clarify their taxonomy and phylogeny. Poecilia vivipara naturally parasitized by metacercariae of both species (100% prevalence) exhibit no external signs of parasitism. In this work, four new sequences of P. macrostomum (18S rDNA, 28S rDNA and ITS2 rDNA) and one new sequence of A. (P.) pindoramensis (mtDNA cox‐1) are presented. Phylogeny reconstructions linked P. macrostomum to other heterophyids, but the separation of the Heterophyidae and Opisthorchiidae remains unclear. Additionally, we used indirect immunocytochemistry and the phalloidin‐fluorescence techniques allied with confocal laser scanning microscopy to describe muscular and neuronal structures of P. macrostomum. A complex arrangement of muscular fibres is associated with the tegument, suckers, gut and reproductive system. Radial fibres around the ventral sucker are thick, branched and extend to the body wall. High‐resolution confocal imaging revealed a typical digenean muscular arrangement and important heterophyid morphological traits. These data will support future control measures to reduce the parasitism in guppies reared in fish farming systems, especially for aquarium and experimental purposes.     

Endolimax piscium sp. nov. (Amoebozoa), causative agent of systemic granulomatous disease of cultured sole,Solea senegalensis Kaup

A new amoeba species pathogenic for Senegalese sole is described based on ultrastructural analysis and SSU rDNA phylogenetic inference. The parasite presents round to ovoid trophozoites (<5 μm) with a high degree of intracellular simplification. No mitochondria were observed, but mitosome‐like organelles were present. No cysts could be detected. Phylogenetic analysis confirmed the Senegalese sole parasite as an amitochondriate Archamoeba related to Endolimax nana and Iodamoeba spp., and we tentatively describe it as a new species in the genus Endolimax, Endolimax piscium. However, the genetic distance with E. nana is quite large, with only 60% pairwise identity between both SSU rDNA genotypes. Although the overall topology of the Archamoebae cladograms containing E. piscium was consistent, the support for the branching of Endolimax spp. relative to its closest neighbours was variable, being higher with distance or parsimony‐based inference methods than with ML or Bayesian trees. The use of stringent alignment sampling masks also caused instability and reduced support for some branches, including the monophyly of Endolimax spp. in the most conservative data sets. The characterization of other Archamoebae parasitizing fish could help to clarify the status of E. piscium and to interpret the large genetic distance observed between Endolimax species.     

The Three‐spined Stickleback,Gasterosteus aculeatus Linnaeus 1758, plays a minor role as a host of Lepeophtheirus salmonis (Krøyer 1837) in the Gulf of Maine

The sea louse, Lepeophtheirus salmonis (Krøyer 1837), is a significant parasite of farmed salmon throughout the Northern Hemisphere. Management of on‐farm louse populations can be improved by understanding the role that wild fish play in sustaining and providing refuge for the local population of sea lice. In this study, 1,064 sticklebacks were captured. Of these animals, 176 individuals were carrying a total of 238 sea lice, yielding a prevalence and intensity of 16.5% and 1.4 lice per fish, respectively. Detailed examination of the sea lice on the three‐spined sticklebacks captured in Cobscook Bay found two L. salmonis individuals using three‐spined sticklebacks as hosts. A 2012 survey of wild fish in Cobscook Bay, Maine, found multiple wild hosts for Caligus elongatus (von Nordmann 1832), including three‐spined sticklebacks (Gasterosteus aculeatus L.), but no L. salmonis were found in this earlier study.     

Protein expression profiling in haemocytes and plasma of the Manila clam Ruditapes philippinarum in response to infection with Perkinsus olseni

The protein expression profiling in clam haemocytes and plasma in response to Perkinsus olseni was addressed. Adult Manila clams from a P. olseni‐free bed were experimentally challenged with parasite zoospores to analyse immune response. In another experiment, the effects of longer term infection were assessed in adult clams collected from a P. olseni‐affected bed, by comparing moderate to very heavily infected clams with non‐infected ones. Haemocyte and plasma proteins were separated by two‐dimensional electrophoresis; spot patterns were qualitatively compared between treatments within each experiment and the spots indicating differential protein expression associated with P. olseni challenge or with field infection were processed for protein identification. Fifteen clam proteins (four in haemocytes and eleven in plasma) of which expression was markedly affected by P. olseni were identified. Some of the identified proteins have a well‐known role in clam immune response against the parasite, such as lysozyme and lectins. Rho GTPase‐activating protein 6 could be a marker of resistance against P. olseni, which should be further studied.     

Are brown trout Salmo trutta fario and rainbow trout Oncorhynchus mykiss two of a kind? A comparative study of salmonids to temperature‐influenced Tetracapsuloides bryosalmonae infection

Proliferative kidney disease (PKD) of salmonids caused by Tetracapsuloides bryosalmonae causes high mortalities of wild brown trout (Salmo trutta fario) and farmed rainbow trout (Oncorhynchus mykiss) at elevated water temperatures. Here the aim was to compare the temperature‐dependent modulation of T. bryosalmonae in the two salmonid host species, which display different temperature optima. We used a novel experimental set‐up in which we exposed brown trout and rainbow trout to an identical quantified low concentration of T. bryosalmonae for a short time period (1 hr). We followed the development of the parasite in the fish hosts for 70 days. PKD prevalence and parasite kinetics were assessed using qPCR. Exposures were performed at temperatures (12°C and 15°C) that reflect an environmental scenario that may occur in the natural habitat of salmonids. T. bryosalmonae infection was confirmed earliest in brown trout kept at 15°C (day 7 post‐exposure) while, in all other groups, T. bryosalmonae was not confirmed until day 15 post‐exposure. Moreover, significantly greater infection prevalence and a faster increase of parasite intensity were observed in brown trout kept at 15°C than in all other groups. These results indicate that PKD is differentially modulated by water temperature in related host species.     

Differences in uptake and killing of pathogenic and non‐pathogenic bacteria by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei, (Boone)

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non‐pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi‐granulocytes) have the main function in phagocytosis of both pathogenic and non‐pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non‐virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post‐inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non‐virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.     

Myxobolus cerebralis (Hofer) infection risk in native cutthroat trout Oncorhynchus clarkii (Richardson) and its relationships to tributary environments in the Yellowstone Lake Basin

Conservation of native species is challenged by the introduction of non‐native pathogens and diseases into aquatic and terrestrial environments worldwide. In the Yellowstone Lake basin, Yellowstone National Park, the invasive parasite causing salmonid whirling disease Myxobolus cerebralis (Hofer) has been identified as one factor contributing to population declines of native Yellowstone cutthroat trout Oncorhynchus clarkii bouvieri (Jordan & Gilbert). In 2002 and 2003, we examined relationships between the stream environment and severity of M. cerebralis infection in native trout. Coefficients of variation of environmental features were calculated to examine variability. Ten years later, we reassessed infection levels at 22 tributaries broadly across the system. Results of principal component analysis (PCA) of physical features (2003) were negatively correlated with infection severity, mostly in lower jaw cartilage of cutthroat trout, and PCA of chemical features (and temperature) correlated with infection severity in cranial cartilage. Pelican Creek, where M. cerebralis prevalence and severity was high 2002–2003, remained high in 2012. We did not find evidence that the parasite had dispersed further within the system. Variable environmental features (physiological stress) across short spatiotemporal scales within a stream or season may possibly predispose salmonids to infection in the wild and facilitate parasite establishment.     

Establishment of medium for laboratory cultivation and maintenance of Fredericella sultana for in vivo experiments with Tetracapsuloides bryosalmonae (Myxozoa)

The freshwater bryozoan Fredericella sultana (Blumenbach) is the most common invertebrate host of the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish. Culture media play an important role in hatching of statoblasts and maintaining clean bryozoan colonies for Malacosporea research. We developed a novel culture medium, Bryozoan Medium C (BMC), for the cultivation and maintenance of F. sultana under laboratory conditions. Statoblasts of F. sultana were successfully hatched to produce transparent‐walled, specific pathogen‐free (SPF) colonies that were maintained >12 months in BMC at pH 6.65. Tetracapsuloides bryosalmonae was successfully transmitted from infected brown trout, Salmo trutta L., to newly hatched F. sultana colonies in BMC, then from the infected bryozoan to SPF brown trout. This study demonstrated the utility of BMC (pH 6.65) for hatching statoblasts, long‐term cultivation of clean and transparent bryozoan colonies and maintenance of the Tetracapsuloides bryosalmonae life cycle in the laboratory for molecular genetic research and other studies such as host–parasiteinteraction.     

Molecular diagnosis of Myxobolus spirosulcatus associated with encephalomyelitis of cultured yellowtail, Seriola quinqueradiata Temminck & Schlegel

Mass mortality of cultured yellowtail, Seriola quinqueradiata, has recently been reported from fish farms in western Japan. Previous studies revealed that diseased fish were characterized by encephalomyelitis and presporogonic stages of a myxosporean‐like parasite in the spinal cord. However, the parasite has remained unidentified because of the lack of mature stages being present. Thus, in the present study, analysis of the small subunit ribosomal DNA (18S rDNA) of the parasite as well as in situ hybridization (ISH) studies using histological sections of the infected tissue was conducted. The 18S rDNA of the myxosporean had higher sequence similarities with those of bile‐duct‐infecting myxosporeans rather than those infecting nervous tissues and was identified as Myxobolus spirosulcatus. The ISH using specific probes demonstrated that the DNA amplified was derived from the multinuclear organisms found in histological sections. A highly sensitive and specific PCR‐based assay for M. spirosulcatus was developed, which revealed a high prevalence of infection in cultured yellowtail that exhibited the clinical signs of encephalomyelitis.     

Fish hosts of the freshwater mussel Unio foucauldianus Pallary, 1936

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Immunohistochemical characterization of polyclonal antibodies against Enteromyxum leei and Enteromyxum scophthalmi (Myxozoa: Myxosporea), intestinal parasites of fish

The enteric myxozoan parasites Enteromyxum leei (Diamant, Lom et Dyková) and Enteromyxum scophthalmi Palenzuela, Redondo et Álvarez‐Pellitero are responsible for high weight loss in infected fish, which leads to subchronic disease and low mortality rates in gilthead sea bream (GSB), Sparus aurata L., and to high mortality rates in turbot, Psetta maxima (L.). The detection of initial parasite stages in histological sections is particularly difficult, but can be simplified by means of specific antibodies. Rabbit polyclonal antibodies (pAbs) were raised against E. scophthalmi and E. leei, and direct enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry were used to characterize their sensitivity and specificity. Both pAbs were adsorbed (apAb) with non‐infected intestines to avoid non‐specific labelling of fish tissues and to improve their specificity. The highest titre obtained in ELISA was 1: 32 000 for apAb‐Eleei and 1:16 000 for apAb‐Escoph. Working dilutions in immunohistochemistry were 1:1000 for apAb‐Eleei and 1:8000 for apAb‐Escoph. Both apAbs labelled proliferative and sporogonic stages with high specificity. apAb‐Escoph was very specific, whereas apAb‐Eleei cross‐reacted with Sphaerospora dicentrarchi Sitjà‐Bobadilla et Álvarez‐Pellitero and Sphaerospora testicularis Sitjà‐Bobadilla et Álvarez‐Pellitero, suggesting the presence of shared antigens. These pAbs stand as new tools for antigenic characterization and the diagnosis of both Enteromyxum species.     

Isolation and screening of probiotic candidates from marron,Cherax cainii (Austin, 2002) gastrointestinal tract (GIT) and commercial probiotic products for the use in marron culture

Six strains of bacteria including Bacillus mycoides (A10) and Shewanella species (A12) isolated from healthy marron intestine, Bacillus species (PM1), Bacillus subtilis (PM3), Bacillus sp. (PM4) and Bacillus sp. (AQ) from commercial probiotic products were investigated for probiotic potential in marron culture. Antibiotic susceptibility tests indicated PM3 and PM4 were susceptible to all nine antibiotics evaluated. A10, A12 and AQ were resistant to class penicillins, whereas PM1 was resistant to class penicillin and macrolides. All strains were non‐pathogenic for marron. Strong inhibition against Vibrio mimicus and Vibrio cholerae non‐01 was exhibited by PM4 and PM3. A10 inhibited V. mimicus at 72 h of growth, but not V. cholerae non‐01, whereas A12 inhibited V. cholerae non‐01 but not V. mimicus, and AQ showed no inhibition activity. A wide range of enzymes were produced by A10 and AQ using the API ZYM test. Protease enzymes were produced by PM3, PM4, AQ and PM1. In order of effectiveness, the following bacteria have probiotic potential: B. subtilis (PM3), Bacillus sp. (PM4) and B. mycoides (A10). Further study is required to determine the bacterium or any combination that gives a multibeneficial effect on marron.