A novel birnavirus identified as the causative agent of summer atrophy of pearl oyster (Pinctada fucata (Gould)).

The Akoya pearl oyster (Pinctada fucata (Gould)) is the most important species for pearl cultivation in Japan. Mass mortality of 0-year-old juvenile oysters and anomalies in adults, known as summer atrophy, have been observed in major pearl farming areas during the season when seawater temperatures exceed about 20 °C since 2019. In this study, we identified a novel birnavirus as the pathogen of summer atrophy and named it Pinctada birnavirus (PiBV). PiBV was first presumed to be the causative agent when it was detected specifically and frequently in the infected oysters in a comparative metatranscriptomics of experimentally infected and healthy pearl oysters. Subsequently, the symptoms of summer atrophy were reproduced by infection tests using purified PiBV. Infection of juvenile oysters with PiBV resulted in an increase in the PiBV genome followed by the atrophy of soft body and subsequent mortality. Immunostaining with a mouse antiserum against a recombinant PiBV protein showed that the virus antigen was localized mainly in the epithelial cells on the outer surface of the mantle. Although the phylogenetic analysis using maximum likelihood method placed PiBV at the root of the genus Entomobirnavirus, the identity of the bi-segmented, genomic RNA to that of known birnaviruses at the full-length amino acid level was low, suggesting that PiBV forms a new genus. The discovery of PiBV will be the basis for research to control this emerging disease.

Aureispira anguillae sp. nov., isolated from Japanese eel Anguilla japonica leptocephali.

A novel filamentous eel-leptocephalus pathogenic marine bacterium, designated strain EL160426T, was isolated from Japanese eel, Anguilla japonica, leptocephali reared at a laboratory in Mie, Japan. In experimental infection studies on eel larvae, the strain EL160426T caused massive larval mortality and was reisolated from moribund leptocephali. Characteristically, observations of infected larvae found that EL160426T forms columnar colonies on the cranial surface of larvae. The novel isolate exhibited growth at 15-30 °C, pH 7-9, and seawater concentrations of 60-150% (W/V). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EL160426T was most closely related to Aureispira maritima 59SAT with 97.7% sequence similarity. The whole genome sequence analysis of the strain EL160426T showed that the strain maintained a circular chromosome with a size of approximately 7.58 Mbp and the DNA G + C content was 36.2%. The major respiratory quinone was MK-7 and the predominant cellular fatty acids were 16:0, 20:4 w6c (arachidonic acid), 17:0 iso and 16:0 N alcohol. DNA relatedness between the closest phylogenetic neighbor strain EL160426T and A. maritima (JCM23207T) was less than 13%. On the basis of the polyphasic taxonomic data, the strain represents a novel species of the genus Aureispira, for which the name Aureispira anguillae sp. nov. is proposed. The type strain is EL160426T (= JCM 35024 T = TSD-286 T).

Pathogenicity, genomic analysis and structure of abalone asfa-like virus: evidence for classification in the family Asfarviridae.

This paper presents the rationale for classifying abalone asfa-like virus (AbALV) in the family Asfarviridae based on analyses of the host, whole genome and electron microscopic observations. AbALV caused >80 % cumulative mortality in an experimentally infected mollusc, Haliotis madaka. The AbALV genome was found to be linear, approximately 281 kb in length, with a G+C content of 31.32 %. Of the 309 predicted ORFs, 48 of the top hits with African swine fever virus (ASFV) genes in homology analysis were found to be in the central region of the genome. Synteny in the central region of the genome was conserved with ASFV. Similar to ASFV, paralogous genes were present at both ends of the genome. The pairwise average amino acid identity (AAI) between the AbALV and ASFV genomes was 33.97 %, within the range of intra-family AAI values for Nucleocytoviricota. Electron microscopy analysis of the gills revealed ~200 nm icosahedral virus particles in the cytoplasm of epithelial cells, and the size and morphology resembled ASFV. In addition to swine, ASFV also infects ticks, which are protostomes like abalone. The overall genome structure and virion morphology of AbALV and ASFV are similar, and both viruses infect protostomes, suggesting that AbALV is a new member of the family Asfarviridae.

Epitope mapping of the monoclonal antibody IP5B11 used for detection of viral haemorrhagic septicaemia virus facilitated by genome sequencing of carpione novirhabdovirus.

The monoclonal antibody (mAb) IP5B11, which is used worldwide for the diagnosis of viral haemorrhagic septicaemia (VHS) in fish, reacts with all genotypes of VHS virus (VHSV). The mAb exceptionally also reacts with the carpione rhabdovirus (CarRV). Following next generation genome sequencing of CarRV and N protein sequence alignment including five kinds of fish novirhabdoviruses, the epitope recognized by mAb IP5B11 was identified. Dot blot analysis confirmed the epitope of mAb IP5B11 to be associated with the region N219 to N233 of the N protein of VHSV. Phylogenetic analysis identified CarRV as a new member of the fish novirhabdoviruses.

Immunostimulation of shrimp through oral administration of silkworm pupae expressing VP15 against WSSV.

White spot syndrome virus (WSSV) is one of the most concerning pathogens in penaeid shrimp and can cause severe loss in shrimp aquaculture worldwide. Among the WSSV structural proteins, VP15, a DNA-binding protein located in the WSSV nucleocapsid, is an antiviral protein candidate to protect kuruma shrimp (Marsupenaeus japonicus) from WSSV infection. We identified that the truncated VP15, VP15(26-57), is responsible for the protective effect against the WSSV. This study attempts to develop an immunizing agent against WSSV using silkworm pupa as a delivery vector through oral administration. The VP15, VP15(26-57), and SR11 peptide derived from VP15(26-57) were expressed in silkworm pupae. Oral administration of feed mixed with the powdered pupae that expressed VP15-derived constructs enhanced the survivability of kuruma shrimp with an overall relative percent survival (RPS) higher than 70%. There is no death for the group receiving pupa/VP15(26-57), and the RPS is 100%. In addition, we also investigated the relative mRNA expression levels of immune-related genes by qPCR at different time points. Our results indicate that the oral administration of pupa/VP15-derived products could provide a high protective effect against WSSV and be a practical approach for controlling WSSV in aquaculture.

Concentration and reduction efficiency of vancomycin-resistant heterotrophic bacteria and vanA and vanB genes in wastewater treatment unit processes.

OBJECTIVES: This study elucidated the distribution and fate of vancomycin (VCM)-resistant heterotrophic bacteria (HTB) and resistance genes, vanA and vanB, during each treatment unit process of a wastewater treatment plant (WWTP). METHODS: Several bacterial counts as well as copy numbers of vanA and vanB genes were determined in each wastewater and sludge sample. In addition, HTB strains isolated from wastewater and sludge were analyzed for VCM susceptibility. Then, the fate and reduction ratios of each bacterial count, copy number of vanA and vanB genes, and the existence ratio of VCM-resistant HTB strains in the wastewater treatment unit process were evaluated. RESULTS: VCM-resistant HTB were detected in all wastewater and sludge samples, and their existence ratio decreased along the treatment process (92.9% in influent wastewater to 39.4% in chlorinated water). Notably, most of the HTB isolated from the influent wastewater were resistant to 8.0 µg/mL of VCM, strongly suggesting that a significant number of antibiotic-resistant bacteria are flowing into the WWTP from urban areas through the sewage system. The vanA and vanB genes were also detected in all wastewater and sludge, with high copy numbers (102-104 copies/mL) even in chlorinated water samples. CONCLUSIONS: Results revealed that residual VCM-resistant HTB, and resistance genes, which could not be completely removed, were ubiquitously released into the aquatic environment. Furthermore, a high existence ratio of VCM-resistant HTB and high copy numbers of resistance genes were also detected in the sludge, indicating that they are constantly circulating in the WWTP via the returned sludge.

Application of Environmental DNA for Monitoring Red Sea Bream Iridovirus at a Fish Farm.

Red sea bream iridoviral disease (RSIVD) causes high economic damage in mariculture in Asian countries. However, there is little information on the source of infection and viral dynamics in fish farms. In the present study, the dynamics of RSIV in a fish farm that mainly reared juveniles and broodstocks of red sea bream (Pagrus major) were monitored over 3 years (2016 to 2018) by targeting environmental DNA (eDNA) of seawater. Our monitoring demonstrated that red sea bream iridovirus (RSIV) was detected from the eDNA at least 5 days before an RSIVD outbreak in the juveniles. The viral loads of eDNA during the outbreak were highly associated with the numbers for daily mortality, and they reached a peak of 106 copies/liter seawater in late July in 2017, when daily mortality exceeded 20,000 fish. In contrast, neither clinical signs nor mortality was observed in the broodstocks during the monitoring periods, whereas the broodstocks were confirmed to be virus carriers by an inspection in October 2017. Interestingly, the viral load of eDNA in the broodstock net pens (105 copies/liter seawater) was higher than that in the juvenile net pens (104 copies/liter seawater) just before the RSIVD outbreak in late June 2017. After elimination of all RSIV-infected surviving juveniles and 90% of broodstocks, few RSIV copies were detected in the eDNA in the fish farm from April 2018 onward (fewer than 102 copies/liter seawater). These results imply that the virus shed from the asymptomatically RSIV-infected broodstock was transmitted horizontally to the juveniles and caused further RSIVD outbreaks in the fish farm. IMPORTANCE Environmental DNA (eDNA) could be applied in monitoring waterborne viruses of aquatic animals. However, there are few data for practical application of eDNA in fish farms for the control of disease outbreaks. The results of our field research over 3 years targeting eDNA in a red sea bream (Pagrus major) fish farm implied that red sea bream iridoviral disease (RSIVD) outbreaks in juveniles originated from virus shedding from asymptomatically virus-infected broodstocks. Our work identifies an infection source of RSIVD in a fish farm via eDNA monitoring, and it could be applied as a tool for application in aquaculture to control fish diseases.

Mass mortality of pearl oyster (Pinctada fucata (Gould)) in Japan in 2019 and 2020 is caused by an unidentified infectious agent.

Mass mortality of 0-year-old pearl oysters, Pinctada fucata (Gould), and anomalies in adults were observed in Japan's major pearl farming areas in the summer of 2019 and 2020. Although adult oyster mortality was low, both adult and juvenile oysters underwent atrophy of the soft body, detachment of the mantle from nacre (the shiny inner surface of the valves), deposition of brownish material on the nacre, and loss of nacre luster. Infection trials were conducted to verify the involvement of pathogens in this phenomenon. Healthy adult pearl oysters were obtained from areas where this disease had not occurred to use as the recipients. The sources of infection were either affected adult oysters with atrophied soft bodies or batches of juveniles in which mortality had reached conspicuous levels. Transmission of the disease to the healthy oysters were tested either by cohabitation with affected oysters or by injections of the hemolymph of affected animals. The injection infection test examined the effects of filtration and chloroform exposure on the pathogen. Occurrence of the disease was confirmed by the appearance of brown deposits on the nacre and loss of nacre luster. The abnormalities of nacre were clearly reproduced in recipient shells in three out of four cohabitation trials with affected oysters. The disease was also reproduced in six out of six injection trails either with hemolymph filtered through 100 nm filter or with hemolymph treated with chloroform. In a serial passage with hemolymph injections, the disease was successfully transmitted through eight passages. These results suggest that the etiology of the disease is a non-enveloped virus with a diameter ≤100 nm.

Strategy for understanding the biological defense mechanism involved in immune priming in kuruma shrimp.

Since the 1970s, individuals that survive a specific infectious disease among crustaceans reportedly develop resistance to the given virulence factors. Quasi-immune response is a similar phenomenon of acquired resistance against white spot syndrome virus, also found in kuruma shrimp. This phenomenon, resembling immunological memory, is collectively called immune priming and recently attracts increasing attention. In this study, I review, along with recent findings, past attempts to immunize shrimp by administration of the pathogen itself or recombinant proteins of viral constituent factors. Moreover, I aimed at investigating the diversity of pattern recognition receptors in kuruma shrimp from the currently available information that allows for a better understanding of immune priming. This review would potentially help to elucidate the underlying mechanisms of immune priming in the future.

Identification of antigenic domains and peptides from VP15 of white spot syndrome virus and their antiviral effects in Marsupenaeus japonicus.

White spot syndrome virus (WSSV) is one of the most devastating pathogens in penaeid shrimp and can cause massive damage in shrimp aquaculture industries. Previously, the WSSV structural protein VP15 was identified as an antigenic reagent against WSSV infections. In this study, we truncated this protein into VP15(1-25), VP15(26-57), VP15(58-80), and VP15(1-25,58-80). The purified proteins from the E. coli expression system were assayed as potential protective agents in Kuruma shrimp (Marsupenaeus japonicus) using the prime-and-boost strategy. Among the four truncated constructs, VP15(26-57) provided a significant improvement in the shrimp survival rate after 20 days of viral infection. Subsequently, four peptides (KR11, SR11, SK10, and KK13) from VP15(26-57) were synthesized and applied in an in vivo assay. Our results showed that SR11 could significantly enhance the shrimp survival rate, as determined from the accumulated survival rate. Moreover, a multiligand binding protein with a role in the host immune response and a possible VP15-binding partner, MjgC1qR, from the host M. japonicus were employed to test its binding with the VP15 protein. GST pull-down assays revealed that MjgC1qR binds with VP15, VP15(26-57), and SR11. Taken together, we conclude that SR11 is a determinant antigenic peptide of VP15 conferring antiviral activity against WSSV.

Isolation and characterization of hirame aquareovirus (HAqRV): A new Aquareovirus isolated from diseased hirame Paralichthys olivaceus.

We isolated a novel Aquareovirus (hirame aquareovirus: HAqRV) from Japanese flounder Paralichthys olivaceus suffering from reovirus-like infection. In electron microscopy, the spherical virion (75 nm in diameter) was observed with multi-layered capsid structure. The viral genome consisted of 11 segments and regions encoding 7 virion structural proteins and 5 non-structural proteins were predicted. The deduced amino acid sequences of those proteins were highly similar to those of the aquareoviruses. However, the similarity of complete genome sequence between the HAqRV and other aquareoviruses was less than 60%. Phylogenetic analyses based on the deduced amino acid sequences suggested that the HAqRV is not classified into the known species of Aquareovirus. Pathogenicity of HAqRV was clearly demonstrated in accordance with Koch's postulates by experimental infection using Japanese flounder. The results suggest that the HAqRV is a new Aquareovirus species which is highly virulent for the Japanese flounder at early life stages.

Complete Genome Sequence of Carp Edema Virus Isolated from Koi Carp.

Carp edema virus disease (CEVD), or koi sleepy disease, is caused by CEV. Here, we report the complete genome sequence of CEV strain FTI2020, isolated from koi carp. This sequence information has great potential for improving our understanding of the genetic characteristics of this piscine poxvirus.

Immune toxicity of phenanthrene and its combined effects of white spot syndrome virus on the survival of kuruma shrimp (Penaeus Japonicus).

Shrimp inhabiting coasts that are frequented by humans are exposed to various pollutants. Additionally, viral infections that cause serious damage to shrimp populations have been observed in these environments. The present study sought to evaluate the immunotoxic effects of phenanthrene (Phe), a pollutant detected in coastal environments, on kuruma shrimp (Penaeus japonicus). We further examined the survival of shrimp following combined exposure to Phe (30 or 300 µg/L) and white spot syndrome virus (WSSV). Results show that exposure to Phe for seven days decreased immune system-related parameters, including total hemocyte count and phenoloxidase activity in hemolymph (p < 0.05). However, these effects were not detected after three days of exposure. Moreover, a combined exposure assay revealed that shrimp mortality increased following exposure to 300 µg/L Phe and infection with WSSV. The number of WSSV gene copies was also observed to increase in these co-exposed shrimp. Taken together, these results indicate that long-term Phe exposure impairs the immune system of P. japonicus, resulting in fatal proliferation of WSSV. Hence, considering that combined exposure to Phe and WSSV leads to increased mortality of shrimp, it is imperative that the detrimental effects elicited by multiple stresses be considered, and controlled, in areas inhabited by kuruma shrimp.

Molecular characterization and gene expression analysis of hypoxia-inducible factor and its inhibitory factors in kuruma shrimp Marsupenaeus japonicus.

In shrimp aquaculture, overcrowded farming causes fluctuations in dissolved oxygen concentrations. Low-oxygen conditions (hypoxia) affect shrimp growth. Hypoxia-inducible factor (HIF) is a transcriptional factor in the basic helix-loop-helix/PAS family and is activated in response to hypoxic stress. However, little is known about HIF and other inhibitors of the HIF pathway in crustaceans. In this study, we cloned MjHIF-1α, an inhibitory factor, MjFIH-1 (factor inhibiting HIF-1α), and MjVHL (Von Hippel-Lindau tumor suppressor) from kuruma shrimp (Marsupenaeus japonicus). MjVHL is the first crustacean VHL ortholog to be cloned. MjHIF-1α, MjFIH-1, and MjVHL exhibit significant sequence similarity and share key functional domains with previously described vertebrate and invertebrate genes. As a result of gene expression analysis in various tissues, MjHIF-1α and MjVHL were more highly expressed in the intestine than in any other organ tissues. In hypoxia experiments, HIF-induced expression levels of MjHIF-1α in the hypoxic group increased significantly for 24 h after initiating hypoxia stimulation and expression of MjVHL decreased significantly for 6 h after hypoxia stimulation (P < 0.05).

Purification and characterization of bioactive peptides RYamide and CCHamide in the kuruma shrimp Marsupenaeus japonicus.

To understand the regulation systems of appetite, bioactive peptides from the kuruma shrimp Marsupenaeus japonicus (Mj) were isolated and purified by reverse pharmacological assays using CHO cells expressing the Drosophila melanogaster G-protein-coupled receptors (GPCRs) CG5811 (a RYamide receptor) or CG14593 (a CCHamide-2 receptor). Four peptides having binding activity to GPCRs were obtained and named Mj RYamide-1, Mj RYamide-2, Mj RYamide-3, and Mj CCHamide. Genes encoding the prepropeptides of these peptides were identified using kuruma shrimp transcriptome databases. The Mj prepro-RYamide gene encodes a 130-amino acid polypeptide containing Mj RYamide-1, Mj RYamide-2, and Mj RYamide-3, whereas the Mj prepro-CCHamide gene encodes a 119-amino acid polypeptide containing a single Mj CCHamide peptide. The expression of these genes was confirmed in various neuronal organs including the brain and ventral nerve cord. In addition, prepro-RYamide gene expression is significantly reduced in the brain after starvation. RYamides may thus be associated with regulation of feeding or digestion. Changes in kayak (the c-fos ortholog in invertebrates) gene expression after administration of synthetic peptides were also investigated. Mj kayak expression levels are upregulated in hepatopancreas after treatment with Mj RYamide-3 or CCHamide. Thus, the peptides isolated in this study may have some regulatory effect on cellular metabolism in aquacultured invertebrates.

Quantification of vancomycin-resistant enterococci and corresponding resistance genes in a sewage treatment plant.

This study aimed to analyze vancomycin-resistant enterococci (VRE) and their resistance genes, vanA and vanB, to examine their presence in sewage treatment systems. Water samples were collected from primary sedimentation tank inlet, aeration tank, final sedimentation tank overflow outlet, and disinfection tank. Enterococcal strains were determined their vancomycin susceptibility by the minimum inhibitory concentration (MIC) test. Vancomycin-resistance genes (vanA and vanB) were quantified by real-time PCR. The sewage treatment process indeed decreased the number of most enterococci contained in the entering sewage, with a removal rate of ≥ 5 log. The MIC test showed that two enterococcal strains resistant to a high concentration of vancomycin (>128 µg mL(-1)). However, most of the enterococcal strains exhibited sensitivity to vancomycin, indicating that VRE were virtually absent in the sewage treatment systems. On the other hand, vancomycin-resistance genes were detected in all the sewage samples, including those collected from the chlorination disinfection tank. The highest copy numbers of vanA (1.5 × 10(3) copies mL(-1)) and vanB (1.0 × 10(3) copies mL(-1)) were detected from the water sample of effluent water and chlorinated water, respectively. Therefore, antibiotic resistance genes remain in the sewage treatment plant and might discharged into water environments such as rivers and coastal areas.

Deciphering of the Dual oxidase (Nox family) gene from kuruma shrimp, Marsupenaeus japonicus: full-length cDNA cloning and characterization.

In many physiological processes, including the innate immune system, free radicals such as nitric oxide (NO) and reactive oxygen species (ROS) play significant roles. In humans, 2 homologs of Dual oxidases (Duox) generate hydrogen peroxide (H(2)O(2)), which is a type of ROS. Here, we report the identification and characterization of a Duox from kuruma shrimp, Marsupenaeus japonicus. The full-length cDNA sequence of the M. japonicus Dual oxidase (MjDuox) gene contains 4695 bp and was generated using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). The open reading frame of MjDuox encodes a protein of 1498 amino acids with an estimated mass of 173 kDa. In a homology analysis using amino acid sequences, MjDuox exhibited 69.3% sequence homology with the Duox of the red flour beetle, Tribolium castaneum. A transcriptional analysis revealed that the MjDuox mRNA is highly expressed in the gills of healthy kuruma shrimp. In the gills, MjDuox expression reached its peak 60 h after injection with WSSV and decreased to its normal level at 72 h. In gene knockdown experiments of free radical-generating enzymes, the survival rates decreased during the early stages of a white spot syndrome virus (WSSV) infection following the knockdown of the NADPH oxidase (MjNox) or MjDuox genes. In the present study, the identification, cloning and gene knockdown of the kuruma shrimp MjDuox are reported. Duoxes have been identified in vertebrates and some insects; however, few reports have investigated Duoxes in crustaceans. This study is the first to identify and clone a Dual oxidase from a crustacean species.

Molecular cloning and characterization of the NADPH oxidase from the kuruma shrimp, Marsupenaeus japonicus: early gene up-regulation after Vibrio penaeicida and poly(I:C) stimulations in vitro.

Free radicals such as nitric oxide (NO) and reactive oxygen species (ROS) are involved in many physiological processes. In humans, there are 5 homologs of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxes) that generate superoxide (O(2)(-)), which can dismute to produce ROS, and play significant roles in innate immunity and cell proliferation. Though Noxes have been identified in vertebrates (humans and fishes) and some insects, there are very few reports investigating Noxes in crustaceans. In the present study, we describe the entire cDNA sequence (4216 bp) of Marsupenaeus japonicus (kuruma shrimp) Nox (MjNox) generated using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). The open reading frame of MjNox encodes a protein of 1280 amino acids with an estimated mass of 146 kDa that has 46.8% sequence homology with the Nox gene of the fruit fly, Drosophila melanogaster. Highly conserved amino acid sequences were observed in the NADPH binding domain. Transcriptional analysis revealed that MjNox mRNA is highly expressed in the lymphoid organ, hepatopancreas and hemocytes of the healthy kuruma shrimp. In the hemocytes, MjNox expression reached its peak 4 h after stimulation with either Vibrio penaeicida or poly(I:C) and decreased to its normal level after 12 h.This study is the first to identify and clone a Nox family member (MjNox) from a crustacean species.

Class B scavenger receptor, Croquemort from kuruma shrimp Marsupenaeus japonicus: Molecular cloning and characterization.

The scavenger receptor, Croquemort is a member of the CD36 superfamily comprising transmembrane proteins involved in the recognition of polyanionic ligands. Various researchers have proved that members of the CD36 superfamily are involved in immunity and developmental processes. In the present study, we report a cDNA encoding the kuruma shrimp, Marsupenaeus japonicus Croquemort scavenger receptor (MjSCRBQ) obtained from a cDNA library of lymphoid organ by RACE amplification. The full-length cDNA of 2098 bp consists an open reading frame of 1596 nucleotides that translates into a 532-amino acid putative protein, with a 5' untranslated region of 323 bp and 3' UTR of 153 bp. The MjSCRBQ is constitutively expressed in gills, heart, hemolymph, hepatopancreas, intestine, lymphoid organ, muscle, nerve, and stomach and at high levels in the brain. Expression analysis in lymphoid organs of shrimp infected with white spot syndrome virus (WSSV) revealed high levels of MjSCRBQ 72 and 120 h post-infection. The MjSCRBQ contains putative functional domains including transmembrane domains and a CD36 domain. Multiple alignments of MjSCRBQ amino acid sequences showed significant identity with Drosophila melanogaster SCRBQ (31%), Salmo salar SCRBQ (29%), Homo sapiens SCRBQ (28%) and Rattus norvegicus SCRBQ (30%). In a phylogenetic analysis, MjSCRBQ was identified in the invertebrate scavenger receptor cluster. This is the first report in crustaceans of the identification and characterization of a Croquemort scavenging receptor.

Myxobolus supamattayai n. sp. (Myxosporea: Myxobolidae) from Thailand parasitizing the scale pellicle of wild mullet (Valamugil seheli).

A new myxosporean species, Myxobolus supamattayai n. sp., was isolated from wild mullet (Valamugil seheli) from the Andaman Sea, Thailand and described based on its morphology and molecular data. The myxosporean produced black plasmodia-like unique clinical sign on the skin with sporogonic stages and mature spores. Polysporous plasmodia, up to 2.5 mm in diameter, were found in epithelium tissue in the scale pocket. The spores measured 6.6 (6.2-7.0) µm in length, 6.5 (6.2-6.7) µm in width, smooth, and round board to ellipsoidal in valvular view. Spores were enclosed with intracapsular process which represents 5-7 and 11-12 in amount revealed in light microscopy and ultrastructure, respectively. The polar capsules were pyriform and of equal size, measuring 3.5 (3.4-3.6) µm in length and 2.0 (1.9-2.2) µm in width, with four to five turns of polar filament arranged perpendicularly to longitudinal axis of the polar capsule. In conclusion, this new species is entirely different from those previously described; however, this finding was assured by the partial sequence of SSU rRNA gene (1,666 bp) analysis that differed from all known species of Myxobolus Bütschli, 1882. The phylogenetic tree of the sequence data sets including those of freshwater and marine of Myxobolus spp. and the sister group (Henneguya spp.) was constructed to establish the relationship of this new species in Myxobolus clade and to explore its relations between their sister groups. Phylogenetic analysis indicated that a monophyletic group with Myxobolus spp. which infected mullet represents the newly formed species. These results suggested the presumably nearby evolution prospecting of Myxobolus species that were found in the same host.