Novel insights into immune stress markers associated with myxosporeans gill infection in Nile tilapia (molecular and immunohistochemical studies).

Nile tilapia (Oreochromis niloticus) is valued in aquaculture because of its quick development and ability to thrive in various environments. Myxosporeans are among the fish parasites that affect fish productivity, as they impact fish growth and reproduction, resulting in large fish deaths in farms and hatcheries. This study has been focused on morpho-molecular identification for the myxosporean parasites infecting Nile tilapia from three governorates in Egypt and assessment of gene expression of different cytokines (Interleukin-1ßeta (IL-1ß), major histocompatibility complex class II (MHC-II), and clusters of differentiation 4 (CD-4) and 8 (CD-8)) in tissues. Additionally, this work aimed to correlate the developed histopathological alterations and inflammatory reactions in gills with immunohistochemical expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α). Finally, the infected fish's cortisol levels and blood glucose were assessed. Results of BLAST sequence analysis of the 18S rRNA for the collected protozoans confirmed Myxobolus agolus, M. brachysporus, M. tilapiae, and Henneguya species. The molecular characterization of the immunological status of gills revealed marked upregulation of different inflammatory cytokines in the gills of infected fish. There was a significantly increased serum cortisol and glucose level in infected fish compared with control, non-infected ones. Severe histopathological alterations were observed in the infected fish gills, associated with increased expression of iNOS and TNF-α and related to myxosporean infection. The present study provides new insights into oxidative stress biomarkers in Nile tilapia infected with Myxosporeans and elucidates the gill's immune status changes as a portal of entry for protozoa that contribute to tissue damage.

The myxozoans Myxobolus cerebralis and Tetracapsuloides bryosalmonae modulate rainbow trout immune responses: quantitative shotgun proteomics at the portals of entry after single and co-infections.

Introduction: Little is known about the proteomic changes at the portals of entry in rainbow trout after infection with the myxozoan parasites, Myxobolus cerebralis, and Tetracapsuloides bryosalmonae. Whirling disease (WD) is a severe disease of salmonids, caused by the myxosporean M. cerebralis, while, proliferative kidney disease (PKD) is caused by T. bryosalmonae, which instead belongs to the class Malacosporea. Climate change is providing more suitable conditions for myxozoan parasites lifecycle, posing a high risk to salmonid aquaculture and contributing to the decline of wild trout populations in North America and Europe. Therefore, the aim of this study was to provide the first proteomic profiles of the host in the search for evasion strategies during single and coinfection with M. cerebralis and T. bryosalmonae. Methods: One group of fish was initially infected with M. cerebralis and another group with T. bryosalmonae. After 30 days, half of the fish in each group were co-infected with the other parasite. Using a quantitative proteomic approach, we investigated proteomic changes in the caudal fins and gills of rainbow trout before and after co-infection. Results: In the caudal fins, 16 proteins were differentially regulated post exposure to M. cerebralis, whereas 27 proteins were differentially modulated in the gills of the infected rainbow trout post exposure to T. bryosalmonae. After co-infection, 4 proteins involved in parasite recognition and the regulation of host immune responses were differentially modulated between the groups in the caudal fin. In the gills, 11 proteins involved in parasite recognition and host immunity, including 4 myxozoan proteins predicted to be virulence factors, were differentially modulated. Discussion: The results of this study increase our knowledge on rainbow trout co-infections by myxozoan parasites and rainbow trout immune responses against myxozoans at the portals of entry, supporting a better understanding of these host-parasite interactions.

Synopsis of the species of Ortholinea Shulman, 1962 (Cnidaria: Myxosporea: Ortholineidae).

A synopsis of Ortholinea Shulman, 1962 (Cnidaria: Myxosporea: Ortholineidae) is presented and identifies 26 nominal species presently allocated within this genus. Species morphological and morphometric features, tissue tropism, type-host, and type-locality are provided from original descriptions. Data from subsequent redescriptions and reports is also given. Accession numbers to sequences deposited in GenBank are indicated when available, and the myxospores were redrawn based on original descriptions. The information gathered shows that Ortholinea infect a wide taxonomic variety of freshwater and marine fish. Nonetheless, the broad host specificity reported for several species is not fully supported by morphological descriptions and requires molecular corroboration. The members of this genus are coelozoic and mainly parasitize the urinary system, with few species occurring in the gallbladder. Ortholinea visakhapatnamensis is the only exception, being histozoic in the visceral peritoneum. Molecular data of the small subunit ribosomal RNA gene (SSU rDNA) is available for about one third of Ortholinea species, with genetic interspecific variation ranging between 1.65% and 29.1%. Phylogenetic analyses reveal Ortholinea to be polyphyletic, with available SSU rDNA sequences clustering within the subclades of the highly heterogenous freshwater urinary clade of the oligochaete-infecting lineage. The life cycles of two Ortholinea species have been clarified based on molecular inferences and identify triactinomyxon actinospores as counterparts, and marine oligochaetes of the family Naididae as permissive hosts to this genus.

Evolution of myxozoan mitochondrial genomes: insights from myxobolids.

BACKGROUND: Myxozoa is a class of cnidarian parasites that encompasses over 2,400 species. Phylogenetic relationships among myxozoans remain highly debated, owing to both a lack of informative morphological characters and a shortage of molecular markers. Mitochondrial (mt) genomes are a common marker in phylogeny and biogeography. However, only five complete myxozoan mt genomes have been sequenced: four belonging to two closely related genera, Enteromyxum and Kudoa, and one from the genus Myxobolus. Interestingly, while cytochrome oxidase genes could be identified in Enteromyxum and Kudoa, no such genes were found in Myxobolus squamalis, and another member of the Myxobolidae (Henneguya salminicola) was found to have lost its entire mt genome. To evaluate the utility of mt genomes to reconstruct myxozoan relationships and to understand if the loss of cytochrome oxidase genes is a characteristic of myxobolids, we sequenced the mt genome of five myxozoans (Myxobolus wulii, M. honghuensis, M. shantungensis, Thelohanellus kitauei and, Sphaeromyxa zaharoni) using Illumina and Oxford Nanopore platforms. RESULTS: Unlike Enteromyxum, which possesses a partitioned mt genome, the five mt genomes were encoded on single circular chromosomes. An mt plasmid was found in M. wulii, as described previously in Kudoa iwatai. In all new myxozoan genomes, five protein-coding genes (cob, cox1, cox2, nad1, and nad5) and two rRNAs (rnl and rns) were recognized, but no tRNA. We found that Myxobolus and Thelohanellus species shared unidentified reading frames, supporting the view that these mt open reading frames are functional. Our phylogenetic reconstructions based on the five conserved mt genes agree with previously published trees based on the 18S rRNA gene. CONCLUSIONS: Our results suggest that the loss of cytochrome oxidase genes is not a characteristic of all myxobolids, the ancestral myxozoan mt genome was likely encoded on a single circular chromosome, and mt plasmids exist in a few lineages. Our findings indicate that myxozoan mt sequences are poor markers for reconstructing myxozoan phylogenetic relationships because of their fast-evolutionary rates and the abundance of repeated elements, which complicates assembly.

Exotic myxozoan parasites establish complex life cycles in farm pond aquaculture.

Myxozoans are obligate parasites with complex life cycles, typically infecting fish and annelids. Here, we examined annelids from fish farm pond sediments in the Beit Shean Valley, in the Syrian-African Rift Valley, Israel, for myxozoan infections. We examined 1486 oligochaetes, and found 74 (5 %) were infected with actinospore stages. We used mitochondrial 16S sequencing to infer identity of 25 infected annelids as species of Potamothrix, Psammoryctides, Tubifex and Dero. We identified 7 myxozoan types from collective groups Neoactinomyxum and Sphaeractinomyxon, and characterized them by small subunit ribosomal DNA sequencing. The Neoactinomyxum type was genetically most similar (∼93 %) to cyprinid fish-infecting Myxobolus spp. The six Sphaeractinomyxon types were genetically similar (93-100 %) to Mugilid-infecting Myxobolus spp.; with one being the previously unknown actinospore stage of a myxospore that infects mullet from aquaculture from the Israeli coast of the Mediterranean Sea. As the farm pond system is artificial and geographically isolated from the Mediterranean, the presence of at least seven myxozoans in their annelid hosts demonstrates introduction and establishment of these parasites in a novel, brackish environment.

Henneguya (Cnidaria: Myxobolidae) species infecting Oligosarcus jenynsii (Characiformes: Characidae) in a Neotropical shallow lake from Argentina: morphological and molecular characterisation.

Two previously undescribed myxozoan species, Henneguya sardellae sp. n. and H. margaritae sp. n., found infecting connective tissues of the Neotropical characid fish Oligosarcus jenynsii (Günther) from Argentina are morphologically and molecularly characterised. Mature spores of H. sardellae sp. n. are ellipsoid, with two, straight and visibly fused caudal appendages cleaved at its blunt terminal end; measuring 33.5 ± 1.2 (30.9-35.5) µm in total length, spore body 17.5 ± 0.6 (16.3-18.6) µm, 7.8 ± 0.4 (7.0-8.8) µm wide and 6.9 ± 0.2 (6.6-7.2) µm thick, with two elongated, unequally-sized polar capsules situated at anterior end, and 11-13 turns of polar tubules. Mature spores of H. margaritae sp. n. are pyriform, with two caudal appendages visible fused together and much longer than spore body, with unequal endings; measuring 35.9 ± 2.8 (29.2-40.7) µm in total length, spore body 11.5 ± 0.9 (9.2-13.0) µm long, 5.8 ± 0.4 (5.1-6.7) µm wide and 5.5 ± 0.2 (5.1-5.8) µm thick, with two polar capsules similar in size, pyriform polar capsules containing polar tubules with 4-5 coils. Both species showed a membraneous sheath surrounding the spore body and caudal appendages; in H. sardellae sp. n. this feature can deploy laterally. Phylogenetic analyses based on SSU rDNA sequences showed that H. sardellae sp. n. and H. margaritae sp. n. clustered with other myxobolids parasitising Characiformes in Brazil, Cichliformes in Mexico and Cyprinodontiformes in Mexico and the United States. The description of these two new species of Henneguya as the first described species of the genus that parasitise freshwater fish in Argentina highlights the importance of further research on the diversity and distribution of myxozoans in this region.

New molecular evidence on the members of the genus Ortholinea (Cnidaria, Myxozoa) and the description of Ortholinea hamsiensis n. sp. infecting the urinary bladder of European anchovy Engraulis engrasicolus in the Black Sea.

Members of the genus Ortholinea are among the worldwide distributed myxozoan parasites that mainly infect marine fish. In this study, a new myxosporean species, Ortholinea hamsiensis n. sp., was isolated from the urinary bladder of European anchovy Engraulis engrasicolus collected from the Sinop coasts of the Black Sea. The prevalence and density values of infection were 1.4% and 1­5 individuals in the field of view (1 + ), respectively. Mature myxospores are subspherical with slight tapering down to the less pronounced tip in the frontal view and subspherical in the sutural view. Myxospores measured 9.1 ± 0.25 (8.8­9.9) µm in length, 9.2 ± 0.11 (8.9­9.4) µm in thickness, and 8.4 ± 0.33 (8.2-9.1) µm in width. Two polar capsules equal in size measured 3.1 ± 0.11 (3.0­3.3) µm in length and 2.7 ± 0.11 (2.6­2.9) µm in width. The polar tubule had 3­4 coils. Along with morphological peculiarities, the results of the 18S rDNA also revealed it to be a new species for science compared to the other species of the genus. In this study, another myxosporean species O. gobiusi was also detected in round goby Neogobius melanostomus with a prevalence of infection value of 4.8% and a density of 1­5 individuals in the field of view (1 + ). The present study also provided the first data of 18S rDNA of O. gobiusi from N. melanostomus and type species of the genus O. divergens from Gobius niger and the phylogenetic relationships of these species with other Ortholinea species have been revealed.

A NEW SPECIES OF THELOHANELLUS (CNIDARIA: MYXOSPOREA: MYXOBOLIDAE) FROM THE GILL OF QUILLBACK, CARPIODES CYPRINUS (CYPRINIFORMES: CATOSTOMIDAE), FROM THE ARKANSAS RIVER DRAINAGE OF OKLAHOMA.

During May 2022 and again in March 2023, 5 quillbacks, Carpiodes cyprinus, were collected from the Verdigris River, Wagoner County, Oklahoma (n = 1), and the Black River, Lawrence County, Arkansas (n = 4), and their gill, gallbladder, fins, integument, musculature, and other major organs were macroscopically examined for myxozoans. Gill lamellae from the single quillback from the Verdigris River was infected with a new myxozoan, Thelohanellus oklahomaensis n. sp. Qualitative and quantitative morphological data were obtained from fresh and formalin-fixed preserved myxospores, and molecular data consisted of a 1,767 base pair sequence of the partial small subunit (SSU) ribosomal RNA gene. Phylogenetic analysis grouped T. oklahomaensis n. sp. with myxozoans known to infect North American catostomids and Eurasian cyprinids. Histological examination localized plasmodia to an intralamellar developmental site and revealed a possible vestige of a second polar capsule. Although plasmodia markedly expanded lamellae, there were no associated epithelial or inflammatory changes. Thelohanellus oklahomaensis n. sp. is the only member of the genus known to infect the gills of C. cyprinus.

Morphological and Molecular Characterization of Two New Species of Ceratomyxa Thélohan, 1892 (Cnidaria: Myxosporea) from the Marine Ornamental Fish Zanclus cornutus (Linnaeus, 1758) off Lakshadweep Islands, Arabian Sea.

PURPOSE: The present study provides the complete morphological and molecular description of two new species of myxosporeans, Ceratomyxa zancli n. sp. and Ceratomyxa cornuti n. sp. infecting the gallbladder of Zanclus cornutus from the Lakshadweep Islands, Arabian Sea. METHODS: Zanclus cornutus were screened for the presence of myxosporeans, and the recovered myxospores were morphologically characterized using Nomarski Differential Interference Contrast (DIC) optics. The sequences of SSU rDNA were employed for molecular and phylogenetic studies. RESULTS: Both the parasites exhibited a prevalence of 21% each. C. zancli n. sp. is characterized by broadly cresentic myxospores with convex anterior and slightly concave to straight posterior margins and rounded ends. Spore valves two, unequal, measured 9.6 ± 0.7 µm × 25.2 ± 1.3 µm. Polar capsules two, unequal, spherical, measured 4 ± 0.6 µm × 3.5 ± 0.6 µm. Polar filament exceptionally long and arranged irregularly. Myxospores of C. cornuti n. sp. are elongated with convex anterior and slightly concave to straight posterior margins. Spore valves two, unequal, measured 7.00 ± 0.4 µm × 26.56 ± 1.8 µm. Polar capsules spherical, unequal, measured 3.52 ± 0.2 × 3.36 ± 0.35. Molecular analysis of C. zancli n. sp. (ON818297) and C. cornuti n. sp. (ON818298) resulted in 1469 and 1491 bp long SSU rDNA sequences, respectively. Molecularly C. zancli n. sp. is close to C. diplodae and C. barnesi with 91.39% similarity, while C. cornuti n. sp. appears closer to C. robertsthomsoni with 97.46% similarity. In phylogenetic analyses, C. zancli n. sp. branched separately within the Ceratomyxa clade while C. cornuti n. sp. clustered with C. robertsthomsoni and C. thalassomae. CONCLUSION: Based on the differences in morphological, morphometric, molecular, and phylogenetic characteristics, as well as differences in the host and geographic location, the above two species of myxosporeans are considered novel. The study forms the first report of a species of Ceratomyxa from Z. cornutus.

RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari.

RNA interference (RNAi) is an effective approach to suppress gene expression and monitor gene regulation. Despite its wide application, its use is limited in certain taxonomic groups, including cnidarians. Myxozoans are a unique group of cnidarian parasites that diverged from their free-living ancestors about 600 million years ago, with several species causing acute disease in farmed and wild fish populations. In this pioneering study we successfully applied RNAi in blood stages of the myxozoan Sphaerospora molnari, combining a dsRNA soaking approach, real-time PCR, confocal microscopy, and Western blotting. For proof of concept, we knocked down two unusual actins, one of which is known to play a critical role in S. molnari cell motility. We observed intracellular uptake of dsRNA after 30 min and accumulation in all cells of the typical myxozoan cell-in-cell structure. We successfully knocked down actin in S. molnari in vitro, with transient inhibition for 48 h. We observed the disruption of the cytoskeletal network within the primary cell and loss of the characteristic rotational cell motility. This RNAi workflow could significantly advance functional research within the Myxozoa, offering new prospects for investigating therapeutic targets and facilitating drug discovery against economically important fish parasites.

Kudoa tanakai n. sp. (Myxozoa: Myxosporea: Multivalvulida), a new kudoid species with spheroid myxospores from the scalpel sawtail (Actinopterygii: Prionurus scalparum) from western Japan.

We describe a new kudoid species, Kudoa tanakai n. sp., in the scalpel sawfish, Prionurus scalprum (Actinopterygii: Acanthuriformes: Acanthuridae), from the natural water around western Japan. The plasmodia were filamentous, localized in pseudocysts in the myofibers of the trunk muscles. The occurrence of plasmodia in the trunk muscle showed no site preference. Its myxospores were spheroid, measuring 6.6-7.6 (7.0) µm by 5.8-6.9 (6.3) µm in apical view (width) and 5.7-6.6 (6.2) in length (n = 30), with four shell valves and a corresponding number of spheroid polar capsules. Shell valves lacked apical protrusions, but scanning electron microscopy revealed that one of the four shell valves had two semi-lunar flaps at its apical terminus. Nucleotide sequencing of the small and large subunit ribosomal RNA genes of the present isolate showed phylogenetic affinities to kudoid species characterized by spheroid myxospores, such as K. musculoliquefaciens, K. hemiscylli, and K. carcharhini, but was molecularly and morphometrically distinct from these and other kudoid species. For direct comparison, Kudoa hemiscylli was collected from the Pacific spadenose shark, Scoliodon macrorhynchos (Elasmobranchii: Carcharhiniformes: Carcharhinidae), in the South China Sea off Guangdong Province, China, and the myxospore surface of the species was observed using scanning electron microscopy. Our study describes the new host and distribution record of this kudoid species originally described from a variety of elasmobranchs in the Australian Coral Sea.

First report of Kudoa species (Myxozoa, Multivalvulida) infection in purple-spotted Bigeye (Priacanthus tayenus) from the Saudi Arabian Gulf.

The purple-spotted bigeye, Priacanthus tayenus, is a marine benthic fish native to the Indian and Pacific Oceans, including the Arabian Gulf in Saudi Arabia. This study identified a myxozoan parasite infecting wild P. tayenus from the Saudi Arabian Gulf. These parasites produced spherical to ovoid-shaped, white plasmodia enclosed within pseudocysts in the fish musculature. The annual infection rate was 5.1%, with the highest prevalence in summer (7.6%), followed by spring (6%), and autumn (2.5%), while no infections were observed in winter. The number of plasmodia per fish ranged from 100 to 150 (135.1 ± 16.2). Their dimensions were 4-4.7 mm (4.3 ± 0.3 mm) in length and 4.5-7 mm (6 ± 1.1 mm) in width. Milky-colored exudates within the plasmodia contained mature spores measuring 8-9 µm (8.6 ± 0.4 µm) x 6-7.5 µm (6.9 ± 0.5 µm). The polar capsules of the spores exhibited dimensions of 2-5 µm (3.5 ± 0.5 µm) x 2.5-4.5 µm (3 ± 0.45 µm). Both morphological and genetic analyses confirmed these plasmodia as a novel Kudoa species. Histopathological examination revealed atrophy in the surrounding muscles without an inflammatory response. This study documents the first occurrence of a novel Kudoa sp. in P. tayenus at the Jubail landing site in Saudi Arabia, emphasizing the need for further surveillance and investigations to elucidate its pathogenesis and implications for wild fish stocks.

First report of Myxobolus neurofontinalis (Bivalvulida: Myxobolidae) infecting anadromous Brook Trout from Prince Edward Island, Canada.

OBJECTIVE: During routine histological examination of tissues from mortality events of anadromous Brook Trout Salvelinus fontinalis from Prince Edward Island (PEI), Canada, myxospores consistent with Myxobolus were observed infecting the central nervous system. The objective of this study was to identify the species of Myxobolus infecting the nervous system of anadromous Brook Trout from PEI, Canada. METHODS: Myxospore morphology, small subunit (SSU) ribosomal DNA (rDNA) sequence data, and histology were used to identify myxospores isolated from infected Brook Trout. RESULT: Myxospore measurements from the PEI samples matched those reported in the description of Myxobolus neurofontinalis from North Carolina. A 1057-bp fragment of the SSU rDNA from myxospores collected from Brook Trout in PEI was identical to an isolate of M. neurofontinalis (MN191598) collected previously from the type locality, New River basin, North Carolina. Histological sections confirmed infections were intercellular in the central nervous system. Minimal host response was observed, with only sparse mononuclear inflammatory infiltrates present at the periphery of and within dispersed myxospores, suggesting that infections are not pathogenic to Brook Trout. CONCLUSION: Myxospores were identified as M. neurofontinalis, which was previously described from the central nervous system of Brook Trout from the New River basin, North Carolina, USA. This constitutes the first time M. neurofontinalis has been documented outside of the New River basin in North Carolina.

Post-mortem 'soft flesh' in three commercial fish species from off Atlantic Morocco associated with the myxosporean parasites Kudoa thyrsites and K. encrasicoli (Myxozoa).

Small pelagic fishes represent one of the most important food resources off the Northwest coast of Africa. Despite their economic significance, little is known about the infections with flesh invading myxosporean parasites of genus Kudoa (Cnidaria, Myxozoa). Heavy infections in the flesh may be associated with post-mortem myoliquefaction, commonly known as 'soft flesh'. This condition may reduce the quality and marketability of the fish fillet, resulting in both economic losses to the fishing industry and loss of consumer confidence. In this study, we investigated Kudoa-induced 'soft flesh' occurrence in European anchovy Engraulis encrasicolus, European pilchard Sardina pilchardus, and Atlantic chub mackerel Scomber colias caught in 2019 off the Moroccan Atlantic coast. Five hundred specimens of each fish species were examined for 'soft flesh' by texture testing and visual inspection 48 h post-catch. 'Soft flesh' occurred in 0.2 % of the European anchovies, 1.4 % of the European pilchard, and in 4.4 % of the Atlantic chub mackerel. Microscopic examination of muscle samples revealed that 'soft flesh'-affected fish were infected with myxospores of K. thyrsites-like morphotype. Analysis of the kudoid SSU rDNA sequence obtained from European pilchard and the Atlantic chub mackerel identified these as K. thyrsites (100 % identity), whereas analysis of the sequence from European anchovy identified the presence of K. encrasicoli (100 % identity). Even if there are no known human health consequences associated with the ingestion of these Kudoa species, the unsightly appearance of some infected fillets is a food quality issue, that can eventually lead to reduced marketability and value.

Comparison of specificity and sensitivity of diagnostic methods for Enteromyxum leei and Enteromyxum fugu detected from cultured tiger puffer, Takifugu rubripes in Korea.

Enteromyxum leei and Enteromyxum fugu, which are myxosporean parasites, were first found in cultured tiger puffer Takifugu rubripes in Korea. We collected four tiger puffers that showed severe emaciation signs for our experiments. DNA sequencing was confirmed that the tiger puffers were coinfected with E. leei and E. fugu. Furthermore, similar amounts of E. leei and E. fugu were confirmed using real-time PCR in the intestine. To the best of our knowledge, there have been no reports of E. fugu infection in the olive flounder Paralichthys olivaceus. However, the diagnosis of inflowing water, discharged water and olive flounder samples using highly sensitive diagnostic methods confirmed the presence of E. fugu in water and fish samples from olive flounder farms near the tiger puffer farm. Therefore, the present study aimed to develop highly sensitive diagnostic methods such as real-time and two-step PCR for early diagnosis and follow-up of the emaciation disease and multiplex PCR for rapid diagnosis. The multiplex PCR method exhibited the same sensitivity as the one-step PCR method developed in this study, demonstrating its efficacy for rapid diagnosis. Therefore, the suggested methods can be utilized for the early diagnosis and rapid diagnosis of emaciation diseases and reduction of economic losses through rapid disease control.

Morphology and phylogeny of Coccomyxa bragantinensis n. sp. (Cnidaria: Myxozoa) found parasitising the Coco Sea catfish, Bagre bagre (Siluriformes: Ariidae), captured off the coast of Northern Brazil.

The present study describes Coccomyxa bragantinensis n. sp., which was found parasitising the gallbladder of the Coco Sea catfish, Bagre bagre, captured off Ajuruteua beach, in the region of Bragança in Pará state, northern Brazil. Most (77.5%) of the 40 fish specimens examined (31/40) had myxospores floating in the bile liquid. These spores are partially ellipsoid, with a tapering anterior extremity and a rounded, elongated posterior extremity with a single piriform polar capsule containing a helicoidal polar filament, with 5-6 coils. A partial sequence of 957 bp of the SSU rDNA gene was obtained from the specimens and deposited in GenBank (xxx). The new species described here - Coccomyxa bragantinensis n. sp. - is phylogenetically similar to Coccomyxa morovi, although it differs from all the other Coccomyxa species and is the first species of this genus to be described from Brazil on the basis of molecular evidence.

Characterization of novel aurantiactinomyxon types (Cnidaria, Myxosporea) from the oligochaete Ilyodrilus templetoni (Southern, 1909), with a comprehensive phylogeny of the collective group.

Three new aurantiactinomyxon types are described from the oligochaete Ilyodrilus templetoni (Southern, 1909) (Naididae) collected from a northern Portuguese estuary, based on light microscopy and sequencing of the 18S rDNA. The addition of I. templetoni to the group of freshwater annelids known to be permissive for aurantiactinomyxon development reinforces the crucial role of naidids in the evolution and settlement of myxozoans in estuarine environments. Maximum likelihood and Bayesian inference analyses of a comprehensive 18S rDNA dataset placed the novel types within the Paramyxidium clade. This positioning suggests them as probable life cycle counterparts to Paramyxidium spp. that most likely infect the European eel Anguilla anguilla, as the sole representative of Elopomorpha in Portuguese rivers. Although distance estimation revealed a genetic difference of only 0.4 % between Aurantiactinomyxon types 1 and 3, this value was determined to be representative of interspecific variability based on the consistent matching of both genotypes with distinct actinospore morphologies, and potential richness of closely related species of Paramyxidium infecting the European eel in Portuguese waters. The clustering of aurantiactinomyxon types within distinct myxosporean lineages, representative of the suborders Variisporina and Platysporina, demonstrates that the aurantiactinomyxon morphotype is highly functional in promoting myxozoan infections in estuarine environments.

Strategies for describing myxozoan pathogens, dreadful fish diseases in aquaculture.

Myxozoans are obligate endoparasites, cosmopolitan in distribution with both vertebrate and invertebrate hosts. Their myxospores consist of shell valves, polar capsules with coiled polar tubules that are extrudible, and infective amoeboid germs. Myxozoan parasites are most abundant, and due to their increasing number in recent years, they can pose an emerging threat to the fish industry worldwide. Hence, the immediate need is to devise a strategy to understand and detect parasites and parasitism. They may proliferate to different organs with the advancement of infection. This all warrants the development/devising of strategies and results of integrative studies in order to identify these dreadful parasites and resolve taxonomic issues. Different methods whether classical methods including gross morphology or advanced methods such as electron microscopy (SEM, TEM, STEM), Confocal laser scanning microscopy (CLSM), histopathological studies, site preference, host and tissue specificity, a molecular approach using new markers can be clubbed for identification because these parasites are hidden and are difficult to recognize. This group was earlier classified only on the basis of myxospores morphology, but due to the high structural variability of this group advanced methods and approaches have to be implied which can minimize the problems in assigning new species.

First report of a Kudoa lutjanus outbreak in farmed Chicken Grunts Parapristipoma trilineatum.

OBJECTIVE: As part of the National Disease Surveillance Program for Taiwanese Aquaculture, we investigated the causative agent of disease outbreaks in farmed Chicken Grunts Parapristipoma trilineatum. METHODS: In this study, outbreak cases on two separate farms were noticed in coastal Pingtung County, Taiwan. In total, 50 juvenile fish showing clinical signs (such as emaciation and erratic swimming behavior) and broodstock (two females and two males) from both farms were collected to perform gross lesion assessment, histopathological examination, and molecular identification of the pathogen. RESULT: Clinical symptoms were infected fish exhibited erratic swimming behavior, such as whirling and floating on the surface of the water. In the following months, cumulative mortality had reached 19% and 24%, respectively. The gross lesions in the infected fish included white oval cysts in the muscle, serosa of the internal organs, sclera of the eyes, and cerebral meninges. After conducting a wet mount examination of cysts using a light microscope, we observed a significant quantity of spores with morphological characteristics, suggesting their affiliation with the Myxosporea group. The spores were semiquadrate, with four tiny suture notches at the periphery; the mean spore length was 7.3 µm (SD = 0.5), and the mean spore width was 8.2 µm (SD = 0.6). The mean length and width of the pyriform polar capsules (nematocysts) were 3.6 µm (SD = 0.5) and 2.2 µm (SD = 0.5), respectively. The 18S and 28S ribosomal RNA sequences of these specimens were identical to those of Kudoa lutjanus. CONCLUSION: As this was the first time an outbreak of K. lutjanus in Chicken Grunts was confirmed, its reappearance with substantial mortality should serve as a warning to the aquaculture industry.