Description of three new species of Kudoa Meglitsch, 1947 (Myxozoa: Multivalvulida) in commercial marine fishes from southern China, and new host records.

Multivalvulidan myxosporeans (Multivalvulida) of the genera Unicapsula Davis, 1924 and Kudoa Meglitsch, 1947 are mostly causative agents of latent and imperceptible infection in marine fishes. However, they are sometimes incriminated in causing post-mortem myoliquefaction or unsightly cyst formation in commercial fish. Despite the great commercial impacts of multivalvulidan infection, the biodiversity, host range and epidemiology of multivalvulidan species remain to be explored further, including infection of alternative annelid hosts. Therefore, this study aimed to identify multivalvulidan species and their host and/or distribution records in commercial fishes in China. Multivalvulidan infection was detected in ten commercial fish species of seven families from the South and East China Seas (Northwest Pacific Ocean) and the Eastern Central Atlantic Ocean (an imported Dagetichthys lusitanicus [de Brito Capello]). Based on morphological and molecular-genetic analyses of their small and large subunit of ribosomal RNA genes, five new host and/or geographical distribution records for five fish species are presented, and three new species in five fish species are described, namely Kudoa neoscomberomori sp. n. in Scomberomorus commerson (Lacépède); Kudoa pilosa sp. n. in Helicolenus hilgendorfi (Döderlein) (type host) and Sebastiscus tertius (Barsukov et Chen); and Kudoa tumidisporica sp. n. in Photopectoralis bindus (Valenciennes) (type host) and Nuchequula nuchalis (Temminck et Schlegel). This study provides new data on multivalvulidan diversity in the ocean ecosystem.

Detection and characterization of Kudoa thunni from uncooked yellowfin tuna (Thunnus albacares) in Southeast Asia.

Myxosporean parasites Kudoa spp. have been reported in several marine fish species worldwide. However, little is known about the contamination of these parasites in raw fish in Southeast Asia, where the consumption demand of uncooked fish is increasing. In 2019, the occurrence of several cases of raw yellowfin tuna (Thunnus albacares) obtained from retail shops with the presence of unknown white, nodular cysts within the musculature have raised public health concerns for the consumption of raw marine fish in Vietnam. Microscopic examination revealed numerous myxospores with the quadratic shape of the Kudoidae. Morphologically, stained spores detected in this study are suspected to Kudoa thunni. To confirm the suspected Kudoa species, further examination of the 18S small-subunit (SSU) was conducted and the results of nucleotide sequence analysis obtained from nodular cysts revealed 99.18-100% identity to that of Kudoa thunni sequences available in GenBank. Detection of K. thunni infection in tuna in Southeast Asia highlights the need for appropriate surveillance and control measures to ensure high quality standards and safety on raw fish production and consumption.

A new myxosporean species, Henneguya lata n. sp. (Myxozoa: Myxobolidae), from the gills of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae) in the Gulf of Tonkin, Vietnam.

During a survey of myxosporean parasites of marine fish in the coastal region of Vietnam, a species of the genus Henneguya (Myxosporea: Bivalvulida) was found in the gill of yellowfin seabream Acanthopagrus latus (Perciformes: Sparidae). White and oval cysts, measuring 145-220 µm in diameter, were detected in the gill lamellae of 4 of 15 fish examined (26.7%). Mature myxospores were elongate, with smooth valves, two similar polar capsules, and having the following dimensions: spore body length 9.9 ± 0.5 (8.9-12.5) µm, body width 6.7 ± 0.3 (6.1-7.6) µm, thickness 5.1 ± 0.2 (4.8-5.4) µm, caudal appendage length 10.0 ± 1 (8.3-11.6) µm, and total myxospore length 19.3 ± 1.4 (16.5-21.5) µm. The polar capsules were ovoid, measuring 3.2 ± 0.2 (2.8-3.9) µm long and 1.9 ± 0.2 (1.5-2.3) µm wide. Each polar capsule has a polar filament with 4-5 coils. Histological analysis revealed plasmodia in the connective tissues of the gill lamellae, but inflammation and deformation of the gills were not observed. In the phylogenetic tree reconstructed from the small subunit ribosomal DNA (SSU rDNA), sequences of the Henneguya specimens found in this study form a distinct branch. Morphological characteristics and molecular data identified a new species, namely Henneguya lata n. sp.

Growing diversity supports radiation of an Ellipsomyxa lineage into the Amazon freshwater: Description of two novel species parasitizing fish from Tapajós and Amazon rivers.

Species of the genus Ellipsomyxa Køie, 2003, parasitize mostly marine and brackish fish around the world. In the present study, we describe two novel species of Ellipsomyxa: Ellipsomyxa plagioscioni n. sp. parasitizing the gall bladder of Plagioscion squamosissimus (Sciaenidae), a freshwater fish but commonly found in brackish water in the Amazonian estuarine environment; and Ellipsomyxa paraensis n. sp. infecting Cichla monoculus (Cichlidae), a strictly freshwater fish. The host specimens were caught from the Amazon and Tapajós rivers, in the municipal region of Santarém, in the State of Pará, Brazil. The study was performed using a combination of morphological, biological, and SSU rDNA-based phylogeny, which suggested that marine transgressions of the Miocene epoch, in the central region of South America, were a pathway for the adaptation and radiation of these cnidarian parasites in the freshwater environment. Both disporic plasmodia and mature myxospores were found floating freely in the bile. Mature myxospores from both species were ellipsoidal in the valvular and sutural views, with thin smooth valves elongated in the direction perpendicular to the plane of the transverse sutural line. Ellipsomyxa plagioscioni n. sp. myxospores measured 11.1 (10.2-12.8) µm in length and 6.6 (5.6-7.6) µm in width. Two pyriform polar capsules discharging on opposite sides, some distance from both the sutural line and the spore ends, measured 3.8 (3.2-4.4) µm in length and 2.8 (2.3-3.3) µm in width, with 5-6 coil polar tubules. Ellipsomyxa paraensis n. sp. myxospores measured 11.5 (10.5-12.4) µm in length and 7.5 (6.6-8.6) µm in width. Two pyriform polar capsules which discharged on opposite sides some distance from both the sutural line and spore ends, measured 3.2 (2.1-3.9) µm in length and 2.6 (2.0-3.3) µm in width, with 2-3 coil polar tubules. Valvular protrusions were observed, associated with the tips of the polar capsules. Molecular analysis based on the SSU rDNA sequences indicated that the two novel Ellipsomyxa species were distinct from all other sequences deposited in the GenBank database. The phylogenetic trees clustered E. plagioscioni n. sp. as a basal species of a lineage of the marine/estuarine Ellipsomyxa, while E. paraensis n. sp. clustered together with other Amazonian species.

A New Species of ThelohanellusKudo, 1933 (Myxozoa: Bivalvulida) Infecting Skeletal Muscle of Blacktail Shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in the Chattahoochee River Basin, Georgia.

Thelohanellus magnacysta n. sp. (Bivalvulida: Myxobolidae) infects the skeletal muscle of blacktail shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in Bull Creek, Chattahoochee River Basin, eastern Georgia. Although numerous members of ThelohanellusKudo, 1933 have overlapping myxospore dimensions with the new species, it differs from all nominal congeners by polar filament coil number and polar capsule width as well as by lacking a mucous envelope, iodinophilic vacuole, and sutural markings. With the use of novel primers for Myxozoa, a phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) suggests that the new species shares a recent common ancestor with a clade of cyprinid-infecting species of Myxobolus Bütschli, 1882 (Bivalvulida: Myxobolidae) and Thelohanellus. Consistent with other published research concerning the systematics of Thelohanellus, this result suggested that Thelohanellus and Myxobolus are polyphyletic and need revision. Histological sections of infected blacktail shiners confirmed that myxospores were only found within a plasmodium and only infected skeletal muscle and that plasmodia were encapsulated by a granuloma comprising varying degrees of acute granulomatous inflammation. The new species is the fourth of Thelohanellus reported from North America and the first reported from Cyprinella, as well as the first myxozoan described from the blacktail shiner.

Phylogenetic relationships of three Kudoa spp. with morphologically similar myxospores (K. iwatai, K. lutjanus, and K. bora), with the redescription of K. uncinata and K. petala and description of a new species (K. fujitai n. sp.) in fishes in the South China Sea.

Members of the myxozoan genus Kudoa (Myxosporea: Multivalvulida: Kudoidae) are characterized as having four or more shell valves in a myxospore, with a corresponding number of polar capsules. Certain Kudoa spp. are critical pathogens in fish, causing postmortem myoliquefaction, unmarketable fish musculature due to unsightly macroscopic cysts, and reduced aquaculture production due to the outbreaks of neurological symptoms or cardiac diseases. Molecular genetic techniques have enabled the differentiation of Kudoa spp. with morphologically similar myxospores. In the present study, we employed integrated taxonomic approaches on five Kudoa spp. forming cysts between the trunk muscle myofibers (K. bora from Osteomugil perusii and K. lutjanus from Acanthopagrus latus), or cysts in the gallbladder wall (K. petala from Sillago sihama), and pseudocysts in the trunk muscle myofibers (K. uncinata from Nuchequula nuchalis and K. fujitai n. sp. from O. perusii). These four host fishes, which originated in the South China Sea, were purchased in the wet markets in Zhanjiang City, Guangdong Province, China, between August 2016 and April 2018. We have redescribed the four Kudoa spp. (K. bora, K. lutjanus, K. petala, and K. uncinata) on which little data are available after their original descriptions. Particularly, genetic characterization of K. bora and K. lutjanus, which are known to have myxospores morphologically similar to those of K. iwatai, was performed based on the nuclear ribosomal RNA gene and partial mitochondrial DNA genes such as cytochrome c oxidase subunit I and small and large ribosomal genes, demonstrating the validity and independence of these three kudoid species. We also provide description of a new species-K. fujitai n. sp.-in the present study. Application of integrated taxonomic approaches to known species characterized solely based on morphological criteria, as well as unknown species (e.g., K. fujitai n. sp. in the present study), contributes to better understanding of the biodiversity of Kudoa and multivalvulid myxosporeans.

Pathologic Changes Associated With Respiratory Compromise And Morbidity Due To Massive Interlamellar Henneguya Exilis Infection In Channel × Blue Hybrid Catfish.

There are multiple Henneguya spp. (Myxozoa: Myxobolidae) endemic to North American catfish aquaculture that affect the gills of channel catfish and their hybrids. These parasites are morphologically similar, and confusion exists regarding the predilection sites and pathologic changes associated with different species. In the spring of 2018, channel (Ictalurus punctatus) female × blue (Ictalurus furcatus) male hybrid catfish from 2 separate commercial operations in northwest Mississippi were submitted for diagnostic assessment in response to observed morbidity and reduced feeding activity. Fish presented with unusually heavy infections of Henneguya spp. plasmodia in the gills. The majority of gill filaments contained widespread, pinpoint, raised, white nodules corresponding microscopically to myxospore-filled plasmodia that obliterated interlamellar spaces. The bipolar myxospores were consistent with Henneguya spp. described from North American ictalurids, possessing slender fusiform spore bodies and elongate bifurcate caudal processes. Associated microscopic lesions included lamellar fusion, epithelial hyperplasia, infrequent, localized, granulomatous branchitis, and rare cartilage lysis, suggesting impaired gill function. Mature plasmodia were excised by laser capture microdissection from ethanol-fixed, hematoxylin and eosin-stained histologic sections for molecular analysis. Fragments (700 bp) of a highly variable region of the 18S rRNA gene, diagnostic for the Myxobolidae, were 100% similar at the nucleotide level to Henneguya exilis. Although mortality was negligible, fish in the affected ponds exhibited signs of respiratory distress similar to proliferative gill disease (PGD) caused by Henneguya ictaluri in channel and hybrid catfish. However, gross and microscopic lesions differed markedly from PGD, known colloquially as "hamburger gill disease." While H. exilis has been reported from channel catfish, it is not typically associated with morbidity and mortality and has not previously been reported from channel × blue catfish hybrids. This work characterizes lesions and confirms the etiology of gill disease induced by the myxozoan H. exilis. In addition to PGD and other non-parasitic conditions, massive interlamellar H. exilis infection should be a differential consideration in pond-raised channel and hybrid catfish experiencing signs of respiratory distress.

Kudoa ogawai (Myxosporea: Kudoidae) Infection in Cultured Olive Flounder Paralichthys olivaceus.

Since Kudoa septempuntata was identified as a causative agent of food poisoning associated with raw olive flounder Paralichthys olivaceus, interest and concern regarding the parasite have increased. However, there have been no investigations or reports of other Kudoa species infecting the fish (except for K. paralichthys, which infects the brain) in Korea. We found cysts filled with myxospores of Kudoa species in muscles of cultured olive flounder specimens and identified these to the species level. Mature spores were quadrate, measuring 8.7±0.5 µm in length, 9.2±0.4 µm in thickness, and 12.9±0.6 µm in width. The spores containing 4 polar capsules had a length of 2.1±0.2 µm and a width of 1.8±0.3 µm. The partial 18S and 28S rDNA of isolates showed 99-100% similarities with K. ogawai. Using these morphological and molecular analyses, the species was identified as K. ogawai. This study is the first report of K. ogawai infection in cultured olive flounder in Korea.

Comparison of infectious agents detected from hatchery and wild juvenile Coho salmon in British Columbia, 2008-2018.

Infectious diseases are potential contributors to decline in Coho salmon (Oncorhynchus kisutch) populations. Although pathogens are theoretically considered to pose higher risk in high-density rearing environments like hatcheries, there is no direct evidence that hatchery-origin Coho salmon increase the transmission of infectious agents to sympatric wild populations. This study was undertaken to compare prevalence, burden, and diversity of infectious agents between hatchery-reared and wild juvenile Coho salmon in British Columbia (BC), Canada. In total, 2,655 juvenile Coho salmon were collected between 2008 and 2018 from four regions of freshwater and saltwater in BC. High-throughput microfluidics qPCR was employed for simultaneous detection of 36 infectious agents from mixed-tissue samples (gill, brain, heart, liver, and kidney). Thirty-one agents were detected at least once, including ten with prevalence >5%. Candidatus Brachiomonas cysticola, Paraneuclospora theridion, and Parvicapsula pseudobranchiocola were the most prevalent agents. Diversity and burden of infectious agents were substantially higher in marine environment than in freshwater. In Mainland BC, infectious burden and diversity were significantly lower in hatchery smolts than in wild counterparts, whereas in other regions, there were no significant differences. Observed differences in freshwater were predominantly driven by three parasites, Loma salmonae, Myxobolus arcticus, and Parvicapsula kabatai. In saltwater, there were no consistent differences in agent prevalence between hatchery and wild fish shared among the west and east coasts of Vancouver Island. Although some agents showed differential infectious patterns between regions, annual variations likely contributed to this signal. Our findings do not support the hypothesis that hatchery smolts carry higher burdens of infectious agents than conspecific wild fish, reducing the potential risk of transfer to wild smolts at this life stage. Moreover, we provide a baseline of infectious agents in juvenile Coho salmon that will be used in future research and modeling potential correlations between infectious profiles and marine survival.

A new species Myxodavisia jejuensis n. sp. (Myxosporea: Sinuolineidae) isolated from cultured olive flounder Paralichthys olivaceus in South Korea.

A new myxosporean parasite, Myxodavisia jejuensis n. sp. (Myxozoa; Bivalvulida) is described from the urinary bladder of olive flounder Paralichthys olivaceus cultured on Jeju Island, Korea. Two long lateral appendages with whip-like extensions were attached to mature spores of triangular to semi-circular shape. The spores were measured at 13.1 ± 1.1 µm in length, 17.2 ± 1.0 µm in thickness, and 13.1 ± 1.0 µm in width. Two spherical polar capsules, with a diameter of 5.0 ± 0.4 µm, were observed on opposite sides in the middle of the spore. The suture line was straight or slightly sinuous on the middle of spores. The 18S rDNA from M. jejuensis n. sp. was used in BLAST and molecular phylogenetic analysis. The results demonstrated that M. jejuensis n. sp. was closest to Sinuolinea capsularis and that the infection site tropism was correlated with the phylogeny of marine myxosporeans. In addition, we designed specific primers to detect the 18S rDNA gene of M. jejuensis n. sp.; the results showed specific amplification in M. jejuensis n. sp. among the myxosporeans isolated from the urinary bladder of the cultured olive flounder.

Kudoa sp. (Myxozoa, Multivalvulida): first report in five commercial fish species from the Canary Islands-FAO 34 (Macaronesia-Spain).

Kudoid myxozoans have been reported causing serious chronic problems in marine fisheries, by reducing the market value of infected fish through pathological damage to the host musculature. We report here the overall prevalence of a Kudoa species in 84/277 (30.3%) fishes from 20 different species of high commercial value captured between October 2011 and December 2013 from the United Nations Food and Agriculture Organization (FAO) 34 commercial fishing area, near the coast of the Canary Islands (Spain). Macroscopic examination showed myxozoan-like cysts in skeletal muscle from 5 of the 20 fish species examined, with the following prevalences: Pagellus acarne (86.7%), Pagellus erythrinus (46.5%), Serranus cabrilla (27.8%), Spondyliosoma cantharus (19.4%), and Sarpa salpa (28.6%). Infection intensity was determined based on spore counts following muscle tissue digestion. Morphometric studies to characterize the species and DNA sequence analysis results suggest that these infections are attributable to a Kudoa species closely related to Kudoa trachuri. This paper reports the first study on a multivalvulidan species to be identified from the Canary Islands. Furthermore, this is the first report of Kudoa parasites in all of the hosts mentioned above, with the exception of P. acarne.

New occurrence of Kudoa orbicularis parasitizing the freshwater catfish Trachelyopterus galeatus (Siluriformes: Auchenipteridae) in the Brazlian Amazon region / Nova ocorrência de Kudoa orbicularis parasitando peixe de água doce Trachelyopterus galeatus (Siluriformes: Auchenipteridae) na região Amazônica brasileira

Abstract The aim of this was describe an infection by Kudoa orbicularis in freshwater catfish Trachelyopterus galeatus. A sample of 80 specimens of T. galeatus was collected in the municipality of Cachoeira do Arari, Marajó Island, in the state of Pará, Brazil. Pseudocysts were found in the muscle fibers of the epaxial and hypaxial regions of 85.0% of the specimens analyzed, reflecting a high infection rate. The pseudocysts contained spores that were pseudo-square in shape, with a mean length of 4.65 µm (range: 4.04-5.54) and mean width of 1.53 µm (1.56-1.74). Analyses on the morphology of the spores and a partial 934-bp sequence of the SSU rDNA gene confirmed that the microparasite was Kudoa orbicularis. This is the second record of this microparasite in a siluriform host in the Brazilian Amazon region.

Resumo O objetivo deste estudo foi descrever a infecção por Kudoa orbicularis em Trachelyopterus galeatus. Foram analisados 80 espécimes de T. galeatus capturados no município de Cachoeira do Arari, ilha de Marajó, estado do Pará, Brasil. A presença de pseudocistos nas fibras musculares das regiões epiaxial e hipoaxial em 85,0% dos exemplares analisados, mostra alto grau de infecção. Os pseudocistos continham esporos de formato pseudoquadrado, medindo 4,65 (4,04-5,54) µm de comprimento e 5,25 (4,78-5,98) µm de largura, com quatro cápsulas polares de tamanho iguais medindo 2,22 (2,05-2,32) µm de comprimento e 1,53 (1,56-1,74) µm de largura. Através das análises morfológicas dos esporos e molecular de uma sequência parcial de 934bps do gene SSU rDNA, confirma que o microparasito é Kudoa orbicularis, sendo este o segundo registro desse microparasito em hospedeiro da ordem Siluriformes da Amazônia brasileira.

Molecular confirmation of Henneguya adiposa (Cnidaria: Myxozoa) and associated histologic changes in adipose fins of channel catfish, Ictalurus punctatus (Teleost).

Henneguya adiposa is one of ten known, closely related myxozoan species that parasitize a variety of tissue sites in the channel catfish, Ictalurus punctatus. Reported to specifically target the adipose fin, H. adiposa is not associated with morbidity or mortality, although detailed descriptions of its associated histologic pathology are lacking. The objective of this work was to confirm the presence of H. adiposa within fin lesions of affected channel catfish using DNA sequenced from histologic sections obtained by laser capture microdissection, as well as to describe pathologic changes induced by infection. The parasite formed large, white, elongate, nodular plasmodia that caused localized tissue damage and incited a granulomatous inflammatory response within a deep connective tissue layer at the base of the adipose fin. Myxospores released from ruptured plasmodia into adjacent tissue were observed to migrate superficially in tracts through the skin, indicating a portal of exit for environmental dispersal. Defects in the connective tissue layer created by ruptured plasmodia were infiltrated by granulomatous inflammation and fibroplasia, suggesting lesion resolution by scar formation over time. Sequencing of the 18S rRNA gene amplified from excised myxospores confirmed the myxozoan's identity as H. adiposa, with 100% similarity to the reference sequence from previous published work.

Skin nodules associated with parasitism with Henneguya sp. (Cnidaria: Myxosporea) in the neotropical fish Cyphocharax modestus.

A new myxozoan species, Henneguya sp., is described based on material from skin of Cyphocharax modestus. Mature myxospores are were elongate and ellipsoidal, measuring 21.4 ±â€¯1.2 (19.4-23.2) µm in total length, 5.1 ±â€¯0.3 (4.5-5.8) µm in width, 11.9 ±â€¯0.5 (10.9-12.7) µm in body length and 9.6 ±â€¯0.7 (8.4-10.5) µm in length of the caudal process. The polar capsules were elongated and had unequal sizes, with length of 5.1 ±â€¯0.4 (4.5-6.0) µm and 5.6 ±â€¯0.4 (4.9-6.3) µm for smaller and larger respectively and width of 1.8 ±â€¯0.2 (1.4-2.0) µm. The larger polar capsule had 8 turns in polar filament while the smaller polar capsule had 5 turns in polar filament. The macroscopic analysis revealed the presence of large nodules, which were located before and after the dorsal fin of the hosts. The histopathological analysis showed the development of nodules filled with plasmodia, surrounded by loose connective tissue, developed in the dermis of the skin. Many cysts containing countless spores, as well as free spores, were located in the dermis and hypodermis of the hosts, causing the disorganization of the connective tissue that is responsible for the support. This is the first record of a Henneguya species in C. modestus.

Simultaneous occurrence of two new myxosporean species infecting the central nervous system of Hypopygus lepturus from Brazil.

This paper describes 2 new myxosporean species, Henneguya lepturus sp. nov. and Thelohanellus lepturus sp. nov., simultaneously infecting the brain and spinal cord of Hypopygus lepturus Hoedeman, 1962 (Teleostei, Hypopomidae) from the Brazilian Amazon (Roraima State). Several spherical cysts of varying dimensions (up to 135 µm) were microscopically observed. The myxospores of H. lepturus sp. nov. measured 25.8 µm in total length, having an ellipsoidal body (12.4 × 6.4 × 2.2 µm) and 2 equal tapering tails (13.4 µm in length). Each of the 2 pyriform polar capsules measured 4.4 × 1.6 µm and possessed a polar filament coiled in 8-9 turns. The myxospores of T. lepturus sp. nov. were pyriform, formed by 2 equal valves (17.7 × 9.1 × 4.3 µm) surrounding a single polar capsule (10.9 × 3.5 µm) that had a coiled polar filament with 13-16 turns and a binucleated sporoplasm that contained several circular sporoplasmosomes. Molecular analysis of the small subunit (SSU) rRNA gene sequences of these 2 species were in agreement with the taxonomic classification derived from the ultrastructure of the myxospores. Histopathology of the host tissue showed degradation of the myelinated axons surrounding the cysts of both species, with the hosts displaying behavioural changes and erratic movements when observed in an aquarium.

A new species of Myxosporea, Henneguya quelen, from silver catfish Rhamdia quelen (Siluriforme: Pimelodidae) in the Amazonian region.

Henneguya quelen n. sp. (Myxosporea) is described from the kidney of the silver catfish, Rhamdia quelen (Quoy & Gaimard, 1824), from Marajó Island. In all, 40% of the 57 silver catfish collected from the Paracauari River in the municipality of Salvaterra presented cysts in the kidney. The cysts were whitish, spherical, and 0.45 mm in diameter. The histological analyses revealed tubular renal compression and cellular degeneration in the area adjacent to the xenomas. The spores are ellipsoid, with well-marked valves, caudal projection, and two elongated polar capsules of equal size. The mature spores measure approximately 15.6 µm in length, with the caudal appendage split posteriorly; 24.3 µm in length, for a total spore length of 40.0 µm; and a width of 4.1 µm. The polar capsules are pyriform, 5.5 µm long and 1.7 µm wide. A phylogenetic analysis based on Bayesian inference confirmed that the specimens represented a new species, which was denominated Henneguya quelen n. sp.

Development of a Real-Time PCR Assay for Detection of Kudoa iwatai (Myxosporea: Multivalvulida) in Japanese Seabass ( Lateolabrax japonicus).

Kudoa iwatai, a myxosporean parasite, has low host fish specificity, and consumers encounter commercial marine fish or marketed marine fish infected with this parasite in Japan. Although the presence of this parasite infection in fish samples is traditionally determined by the microscopic morphological examination of extracted spores, this method lacks sensitivity and specificity. In this study, we developed a real-time PCR assay for the detection of K. iwatai 18S rDNA to achieve the rapid and specific identification of K. iwatai in foreign substance inspection. We also evaluated the usefulness of real-time PCR for Japanese seabass ( Lateolabrax japonicus) with or without K. iwatai cysts. Our real-time PCR assay was able to reliably detect the target plasmid DNA over a 7-log range (from 4.0 × 101 to 4.0 × 107 copies per reaction) and displayed a linear relationship, with a correlation of determination value of 0.9993 and slope of -3.3651. Moreover, the mean value of the intra-assay coefficient of variation was 0.89% in triplicate assays, and the detection limit of this method was 2.5 copies of K. iwatai 18S rDNA per reaction. The sensitivity of the real-time PCR was the same or higher than that of an established conventional PCR when DNA extracts from eight Japanese seabass with or without K. iwatai were used as templates. The specificity of the real-time PCR was comparable with that of conventional PCR by using DNA extracts from fish samples infected with nine Kudoa species. Together, these results indicate that our real-time PCR assay is highly sensitive, reproducible, and specific for detecting K. iwatai 18S rDNA in foreign substance inspection. We believe that this highly sensitive real-time PCR may also be useful for understanding the gastrointestinal diseases associated with K. iwatai and for studying the yet unknown life cycle of K. iwatai.

First molecular evidence of Thelohanellus wallagoi Sarkar, 1985 (Myxozoa) from economically important food fish, freshwater shark Wallago attu (Siluridae) in India.

The freshwater shark Wallago attu (Bl. and Schn.) is a frequent silurid in the River Ganga and one of the most commercially exploit fish in India. In a survey on its infection with myxosporeans, spore type belongs to Thelohanellus species was found in the gills, kidney and intestine respectively. Through morphological and molecular investigations, we identified the spore as Thelohanellus wallagoi Sarkar, 1985. They were pyriform in valvular view and slim in sutural view, and had one pyriform polar capsule with four to five turns. The spores measured 8.0 × 4.0 × 2.3 µm. T. wallagoi developed in small cysts in the gill lamellae, whereas cyst and scattered spores of T. wallagoi were also found in the kidney and intestine respectively. The 18S rDNA sequence of T. wallagoi isolates recovered from gills, kidney and intestine were found similar to each other and differed from any other Thelohanellus species available in GenBank and validated its status after 32 years of original description. Phylogenetic analysis signified that T. wallagoi was placed sister to Myxobolus species in the clade that indicated the polyphyletic nature of the genus Thelohanellus.

Identification of a new myxosporean parasite Thelohanellus indiana n. sp. (Myxosporea: Myxobolidae) isolated from three major organs of goldfish, Carassius auratus L. highlighted with its morphological and SSU rDNA sequence based molecular description.

Fish mortality and poor growth in surviving fish contribute substantial losses to the ornamental fish farms of India and revealed an infection of a new myxosporidian parasite Thelohanellus indiana n. sp. which has become one of the most important limiting factors for successful aquaculture management. The parasite infects Carassius auratus, an Indian goldfish, described on the basis of myxospores morphology and amplification of a part of 18 S rDNA gene. Three major attaching site of fish body have been explored for showing the location of attachment for the parasites. The whitish cysts of the parasites are about 2.5-3.5 mm contains large amount of lemon shaped mature myxospores measuring 12.1-15.2 (13.8) × 7.5-8.8 (8) µm. A single round or elliptical polar capsule located only at the anterior pole of the spore having 6.2-7.2 (6.8) × 3.3-4.7 (4.0) µm in diameter. The morphological characters have been assessed by both the light and scanning electron microscope. The most differentiating feature from closely related species was carried out by morpho-taxonomic affinities with previously described species which are tremendously supported by molecular taxonomy by partial sequencing of the 18 S rDNA gene resulted in a total of 2101 bp fragment of newly obtained SSU rRNA gene sequence of the new species which exhibit 79-91% homogeneity with other closely related species available in GenBank. The BLAST search of Thelohanellus sp. did not matches with any available sequences in GenBank and the phylogenetic analysis revealed that the novel species were sister to T. habibpuri and T. caudatus, in the Thelohanellus clade and form a closest neighboring branch as a subclade in phylogenetic tree from which the new Thelohanellus parasite is being placed. Both the branches are originating from monophyletic clade that are strongly supported by bootstrap values which indicate clearly about independent position of T. indiana n. sp.

Ceratomyxa gracillima n. sp. (Cnidaria: Myxosporea) provides evidence of panmixia and ceratomyxid radiation in the Amazon basin.

We describe a new freshwater myxosporean species Ceratomyxa gracillima n. sp. from the gall bladder of the Amazonian catfish Brachyplatystoma rousseauxii; the first myxozoan recorded in this host. The new Ceratomyxa was described on the basis of its host, myxospore morphometry, ssrDNA and internal transcribed spacer region (ITS-1) sequences. Infected fish were sampled from geographically distant localities: the Tapajós River, Pará State, the Amazon River, Amapá State and the Solimões River, Amazonas State. Immature and mature plasmodia were slender, tapered at both ends, and exhibited vermiform motility. The ribosomal sequences from parasite isolates from the three localities were identical, and distinct from all other Ceratomyxa sequences. No population-level genetic variation was observed, even in the typically more variable ITS-1 region. This absence of genetic variation in widely separated parasite samples suggests high gene flow as a result of panmixia in the parasite populations. Maximum likelihood and maximum parsimony analyses placed C. gracillima n. sp. sister to Ceratomyxa vermiformis in a subclade together with Ceratomyxa brasiliensis and Ceratomyxa amazonensis, all of which have Amazonian hosts. This subclade, together with other Ceratomyxa from freshwater hosts, formed an apparently early diverging lineage. The Amazonian freshwater Ceratomyxa species may represent a radiation that originated during marine incursions into the Amazon basin that introduced an ancestral lineage in the late Oligocene or early Miocene.