Morphological and Molecular Characterization of Three Myxosporean Species of the Genera Myxobolus, Henneguya, and Myxidium (Cnidaria: Myxozoa) Infecting Freshwater Fish, Isolated for the First Time in Japan.

The majority of myxosporean species (Cnidaria: Myxozoa) of the genera Myxobolus (35 species), Henneguya (8 species), and Myxidium (9 species) from freshwater or brackish fish in Japan were recorded more than 30 years ago (accumulatively 81.1% [43/53]). The re-discovery and molecular-genetic characterization of these species is a current research priority. During our myxosporean survey in Japanese freshwater fish, we detected three species that had never been recorded in Japan, but in the Russian Far East (Sakhalin Island, and Maritime Province): Myxobolus tribolodonus sp. n., forming cysts in the gills of Tribolodon sachalinensis (syn. M. marinus sensu Aseeva, 2000; M. marinus sensu Sokolov et Frolova, 2015, recorded from the gills of Pseudaspius (syn. Tribolodon) spp.); Henneguya pungitii Achmerov, 1953, forming cysts in the subcutis of external skin and buccal submucosa of Pungitius sinensis; and Myxidium salvelini Konovalov et Shulman, 1966, in the urinary bladder of Oncorhynchus masou ishikawae. These new isolates were characterized by integrated taxonomic approaches, i.e., myxospore morphology and molecular-genetic characterization of the small subunit ribosomal RNA gene (SSU rDNA). These new isolates were phylogenetically differentiated from any species whose SSU rDNA sequences were deposited in the DNA databases, and concurrently compared with recorded species based on classical morphological criteria. All three species were differentiated from myxosporeans previously recorded in Japan, indicating new distribution records out of the Russian Far East. For reliable species identification, accumulation of at least SSU rDNA sequences of known species worldwide is critically important.

Massive branchial henneguyosis of catfish: A distinct, myxozoan-induced gill disease caused by severe interlamellar Henneguya exilis infection in catfish aquaculture.

Proliferative gill disease (PGD), caused by the myxozoan Henneguya ictaluri, has been the most notorious parasitic gill disease in the US catfish aquaculture industry. In 2019, an unusual gill disease caused by massive burdens of another myxozoan, Henneguya exilis, was described in channel (Ictalurus punctatus) × blue (Ictalurus furcatus) hybrid catfish. Targeted metagenomic sequencing and in situ hybridization (ISH) were used to differentiate these conditions by comparing myxozoan communities involved in lesion development and disease pathogenesis between massive H. exilis infections and PGD cases. Thirty ethanol-fixed gill holobranchs from 7 cases of massive H. exilis infection in hybrid catfish were subjected to targeted amplicon sequencing of the 18S rRNA gene and compared to a targeted metagenomic data set previously generated from clinical PGD case submissions. Furthermore, serial sections of 14 formalin-fixed gill holobranchs (2 per case) were analyzed by RNAscope duplex chromogenic ISH assays targeting 8 different myxozoan species. Targeted metagenomic and ISH data were concordant, indicating myxozoan community compositions significantly differ between PGD and massive branchial henneguyosis. Although PGD cases often consist of mixed species infections, massive branchial henneguyosis consisted of nearly pure H. exilis infections. Still, H. ictaluri was identified by ISH in association with infrequent PGD lesions, suggesting coinfections occur, and some cases of massive branchial henneguyosis may contain concurrent PGD lesions contributing to morbidity. These findings establish a case definition for a putative emerging, myxozoan-induced gill disease of farm-raised catfish with a proposed condition name of massive branchial henneguyosis of catfish (MBHC).

Species-specific in situ hybridization confirms arrested development of Henneguya ictaluri in hybrid catfish (Channel Catfish × Blue Catfish) under experimental conditions, with notes on mixed-species infections in clinical cases of proliferative gill disease from Mississippi catfish aquaculture.

OBJECTIVE: Proliferative gill disease (PGD) in Channel Catfish Ictalurus punctatus and hybrid catfish (Channel Catfish × Blue Catfish I. furcatus) is attributed to the myxozoan Henneguya ictaluri. Despite evidence of decreased H. ictaluri transmission and impaired parasite development in hybrid catfish, PGD still occurs in hybrid production systems. Previous metagenomic assessments of clinical PGD cases revealed numerous myxozoans within affected gill tissues in addition to H. ictaluri. The objective of this study was to investigate the development and pathologic contributions of H. ictaluri and other myxozoans in naturally and experimentally induced PGD. METHODS: Henneguya species-specific in situ hybridization (ISH) assays were developed using RNAscope technology. Natural infections were sourced from diagnostic case submissions in 2019. Experimental challenges involved Channel Catfish and hybrid catfish exposed to pond water from an active PGD outbreak, and the fish were sampled at 1, 7, 10, 12, 14, 16, 18, and 20 weeks postchallenge. RESULT: Nine unique ISH probes were designed, targeting a diagnostic variable region of the 18S ribosomal RNA gene of select myxozoan taxa identified in clinical PGD cases. Partial validation from pure H. ictaluri, H. adiposa, H. postexilis, and H. exilis infections illustrated species-specific labeling and no cross-reactivity between different myxozoan species or the catfish hosts. After experimental challenge, mature plasmodia of H. ictaluri and H. postexilis formed in Channel Catfish but were not observed in hybrids, suggesting impaired or delayed sporogenesis in the hybridized host. These investigations also confirmed the presence of mixed infections in clinical PGD cases. CONCLUSION: Although H. ictaluri appears to be the primary cause of PGD, presporogonic stages of other myxozoans were also present, which may contribute to disease pathology and exacerbate respiratory compromise by further altering normal gill morphology. This work provides molecular confirmation and more resolute developmental timelines of H. ictaluri and H. postexilis in Channel Catfish and supports previous research indicating impaired or precluded H. ictaluri sporogony in hybrid catfish.

Characterizing Two New Henneguya Species in the Respiratory Organs of African Sharptooth Catfish.

Henneguya species are myxozoans, a suborder of Cnidaria, which can affect the gills and extrarespiratory organs of the African sharptooth catfish, Clarias gariepinus. This research describes natural infection-induced histological alterations caused by the Henneguya species present. The Henneguya species were also identified molecularly using DNA sequenced from infected tissue cysts, and phylogenetically analyzed. Clinical investigations revealed cyst-like nodules on the fish gill filaments and extrarespiratory organs. Within a milky fluid inside the cysts were several Henneguya-like spores. Henneguya sp. infested 27.5% of the fish, with the highest prevalence in the gills compared to the extrarespiratory organs. The Henneguya species parasitized the gill and the dendritic tissues, resulting in histopathological characteristics. The plasmodia's developmental stages resulted in destructive damage which manifested as marked necrosis, which was replaced by a focal aggregation of inflammatory cells. Amplification of the 18S ribosomal DNA from the fish parasites was followed by sequencing, which confirmed their identities as new species Henneguya qenabranchiae n. sp. and Henneguya qenasuprabranchiae n. sp. with 99.53 and 99.64% identities, respectively, to Henneguya sp. 1 HS-2015. The two C. gariepinus myxozoans shared some characteristics based on morphologic and phylogenetic analysis as previously published, where it was proposed that they were a sister lineage to Henneguya species in Egypt, and it is now proposed that they are new species.

Co-infection of Candidatus Piscichlamydia Trichopodus (Order Chlamydiales) and Henneguya sp. (Myxosporea, Myxobolidae) in Snakeskin Gourami Trichopodus pectoralis (Regan 1910).

The present study describes a simultaneous infection of a novel Chlamydia-like organism (CLO) with a Myxozoa parasite, Henneguya sp. in snakeskin gourami Trichopodus pectoralis in Thailand. A new CLO is proposed "Candidatus Piscichlamydia trichopodus" (CPT) based on 16S rRNA phylogenetic analysis. Systemic intracellular CPT infection was confirmed by histological examination, in situ hybridization, PCR assay, and sequencing of 16S rRNA. This novel pathogen belongs to the order Chlamydiales but differs in certain aspects from other species. The histopathological changes associated with CPT infection were different from the typical pathological lesions of epitheliocystis caused by previously known CLO. Unlike other CLO, CPT localized in the connective tissue rather than in the epithelial cells and formed smaller clumps of intracellular bacteria that stained dark blue with hematoxylin. On the other hand, typical myxospores of the genus Henneguya with tails were observed in the gill sections. Infection with Henneguya sp. resulted in extensive destruction of the gill filaments, most likely leading to respiratory distress. Due to the frequency of co-infections and the unavailability of culture methods for CLO and Henneguya sp., it was difficult to determine which pathogens were directly responsible for the associated mortality. However, co-infections may increase the negative impact on the host and the severity of the disease. Given the commercial importance of the snakeskin gourami and its significant aquaculture potential, the findings of this study are important for further studies on disease prevention.

MYXOZOAN COMMUNITY COMPOSITION AND DIVERSITY IN CLINICAL CASES OF PROLIFERATIVE GILL DISEASE IN MISSISSIPPI CATFISH AQUACULTURE.

An abundance of morphologically variable Henneguya species complicates the understanding of disease relationships between ictalurid catfish and myxozoan (Phylum: Cnidaria) parasites on North American aquaculture operations. Henneguya ictaluri, the cause of proliferative gill disease (PGD) in channel and hybrid catfish, is arguably the most important parasite of commercial catfish aquaculture in the southeastern United States. While research indicates arrested development and limited sporogenesis of H. ictaluri in channel (Ictalurus punctatus) × blue (Ictalurus furcatus) hybrid catfish, incidents of PGD persist in hybrid production systems. This work investigated the influence of fish host on myxozoan community composition and diversity within naturally infected gill tissues from diagnostic case submissions to the Aquatic Research and Diagnostic Laboratory in Stoneville, Mississippi, from 2017 to 2019. Gills collected from farm-raised catfish with clinical PGD were subjected to metagenomic amplicon sequencing of the myxozoan 18S SSU rDNA gene diagnostic variable region 3 (DVR3). Myxozoan community composition significantly differed between channel and hybrid catfish PGD cases, with channel catfish having more diverse community structures. Channel catfish gills had a greater relative abundance of H. ictaluri in 2017 and 2019, while no differences were observed in 2018. Importantly, H. ictaluri was present in all channel and hybrid catfish PGD cases across all years; however, H. ictaluri was not the most abundant myxozoan in almost half the cases examined, suggesting other myxozoan species may also contribute to PGD pathology. The detection of numerous known and unclassified myxozoan sequences in addition to H. ictaluri provides evidence PGD may involve mixed species infections. Furthermore, the presence of numerous unclassified myxozoan sequences in gill samples from clinical PGD cases indicates the number of described species from U.S. farm-raised catfish vastly underestimates the true myxozoan diversity present within the varied pond microcosms associated with catfish aquaculture.

Henneguya species infecting the gastrointestinal tract of Clarias gariepinus from the Nile River.

As part of a study on parasitic infection in the African sharptooth catfish Clarias gariepinus, we found cysts of varying sizes in the stomach and intestine that contained myxospores with morphological features resembling those of the genus Henneguya. The present investigation was carried out with data on spore morphology and histopathology. Additionally, the myxozoan was identified using a molecular-based approach with 18S small subunit rDNA sequences. Based on the morphological characterization and tissue specificity of Myxozoa, 2 species of Henneguya were identified in the catfish stomach and intestine. Several histopathological changes were observed in the intestine which may affect fish performance and survival. The phylogenetic position of nucleotide sequences of the Henneguya species identified here were clustered with other fish-infecting Henneguya species. These sequences were deposited in GenBank. It appears that they potentially represent 2 species, denominated Henneguya sp. 1 and Henneguya sp. 2 according to the samples originating from the stomach and intestine, respectively. Although future investigations are needed for detailed morphological and molecular descriptions, this study documents the likely occurrence of infection with Henneguya noted for the first time, to our knowledge, in the digestive system of C. gariepinus in Egypt.

Hemagglutination in gill capillaries of sheepshead, Archosargus probatocephalus (Perciformes: Sparidae), infected by a myxosporidean / Hemaglutinação em capilares branquiais de sargo-de-dente, Archosargus probatocephalus (Perciformes: Sparidae) infectado por um mixosporídeo

Abstract During a survey Myxozoa, four specimens of the sheepshead (18 ± 1.5 cm and 59 ± 2.5 g) (Archosargus probatocephalus) were collected in the Ipioquinha river (Maceió/AL). Transmission electron microscopy observations revealed erythrocyte agglutinations in gill capillaries located near spherical cysts containing myxospores of the genus Henneguya. This hemagglutination partially or totally obstructed the gill capillaries. Erythrocytes occurred in close adherence to each other, with a closed intercellular space. A few lysed erythrocytes were observed among agglutinated cells. The reduced lumen of the capillaries was partially filled with amorphous dense homogenous material adhering to the erythrocytes. In addition, heterogeneous masses of irregular lower electron density were observed in the reduced channel of the capillary. The agglutinated erythrocytes appeared dense and homogenous, lacking cytoplasmic organelles. The nuclei had the appearance of normal condensed chromatin masses, generally without visible nucleoli. This occurrence of hemagglutination only in the capillaries located in close proximity to the developing myxozoan cysts suggests that parasite development may be a factor triggering erythrocyte agglutination. This is supported by previous experimental studies that showed a probable correlation between parasitic infections and hemagglutination. Nonetheless, further studies are necessary in order to better understand the physicochemical processes involved in this phenomenon.

Resumo Durante pesquisa de mixozoários foram coletados quatro espécimes do peixes sargo-de-dente (18 ± 1.5 cm e 59 ± 2.5 g) (Archosargus probatocephalus), no rio Ipioquinha (Maceió/AL). Observações por microscopia eletrônica de transmissão revelaram aglutinação de eritrócitos em capilares branquiais localizados próximos a cistos esféricos, contendo mixosporos do gênero Henneguya. Essa hemaglutinação obstruiu parcial ou totalmente os capilares branquiais. Os eritrócitos apareceram em forte aderência entre si, com espaço intercelular fechado. Foram observados poucos eritrócitos lisados entre as células aglutinadas. O lúmen reduzido dos capilares foi parcialmente preenchido com material homogêneo denso amorfo aderido aos eritrócitos, além de massas livres heterogêneas de densidade eletrônica baixa e irregular observadas no canal reduzido dos capilares. Os eritrócitos aglutinados pareciam densos e homogêneos, sem organelas citoplasmáticas. Os núcleos apareceram como massas normais de cromatina condensada, geralmente sem nucléolos visíveis. A ocorrência de hemaglutinação apenas nos capilares, localizados nas proximidades dos cistos mixozoários, sugere que o desenvolvimento parasitário pode ser um fator desencadeante da aglutinação eritrocitária. Isso é corroborado por estudos experimentais anteriores que mostraram uma provável correlação entre infecções parasitárias e hemaglutinação. No entanto, novos estudos são necessários para melhor compreender os processos físico-químicos envolvidos neste fenômeno.

Phylogeny and ultrastructure of Henneguya fusiformis Kostoïngué, Fall, Faye and Toguebaye 1999, a newly recorded species infecting the ovaries of Clarias gariepinus in Egypt.

The genus Henneguya is the second largest within the class Myxosporea, which infects marine and freshwater fish. One hundred Clarias gariepinus specimens were collected alive from a branch of the Nile River in Kafrelsheikh, Egypt. Microscopic and molecular procedures were used to describe how Henneguya fusiformis infects the ovaries of C. gariepinus. The infected fish showed no pathogenic changes except for macroscopic creamy whitish nodules in their ovaries with the highest prevalence during the spring season. The mature spores are spindle-shaped. The total spore length, spore body length and width are 53.4 ± 0.8 (52.5-54.3) µm, 29.8 ± 0.5 (29.2-30.4) µm and 6.5 ± 0.3 (6.1-6.9) µm, respectively. The spore anterior end consisted of two equal polar capsules, located in a tandem position, each one measuring 4.2 × 2.1 µm. The polar filament formed a coil with 6-8 turns. The measurement of the spore end with two extended processes was 24.3 ± 0.4 (23.9-24.8) µm. Phylogenetic analyses of the 18S ribosomal RNA gene sequence revealed that H. fusiformis are clustered together with other myxobolids that are histozoic in channel catfish, Ictalurus punctatus, and Asian Redtail catfish, Hemibagrus nemurus" (Clariidae) in the United States and Malaysia, respectively. To the best of our knowledge, this is the first record of H. fusiformis in Egypt. Additionally, our study is the first record of H. fusiformis in the ovaries of C. gariepinus.

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.

Morphological and molecular analysis of Henneguya tietensis n. sp. (Cnidaria: Myxosporea), parasitizing the gill filaments of Prochilodus lineatus (Valenciennes, 1837) from Brazil.

The present study describes Henneguya tietensis n. sp. parasitizing Prochilodus lineatus (Valenciennes, 1837) from Brazil, through morphological and molecular analysis. Myxospores were found in the gill tissue of P. lineatus with a prevalence of 10%. The myxospores consisted of two elongate, elliptical shell valves each with a long, tapering caudal appendage. The morphology was consistent with Henneguya and the myxospore measured as follows (mean ± SD): total length 55.5 ± 2.1 µm, body length 16.2 ± 1.1 µm, body width 5.5 ± 0.1 µm, caudal appendages length 39.0 ± 2.0 µm. The polar capsules were 7.3 ± 0.2 µm long, 1.7 ± 0.2 µm wide and contained a polar filament coiled 11 to 13 turns. Histological analysis showed the plasmodia developing in the middle region of each gill filament and caused a focally extensive distension by forming a space-occupying mass within the gill filament. The ultrastructural analysis allowed the observation of the presence of mature myxospores throughout the plasmodium. Phylogenetic analysis with the SSU rDNA gene showed Henneguya tietensis n. sp. as a sister species of the subclade formed by Henneguya piaractus and Henneguya brachypomus that infect fishes of the genus Piaractus. The genetically closest species was H. piaractus, which showed a similarity of 82.4%. Using molecular and morphological characterization, the myxozoan parasite described herein represents a new species of the genus Henneguya.

Description of myxosporeans (Cnidaria: Myxozoa) infecting the popular food fish Notopterus notopterus (Pisces: Notopteridae) in Malaysia and India.

This study was a co-operative investigation of myxosporean infections of Notopterus notopterus, the bronze featherback, which is a popular food fish in the South Asian region. We examined fish from Lake Kenyir, Malaysia and the River Ganga, Hastinapur, Uttar Pradesh, India, and observed infections with two myxosporeans: Myxidium cf. notopterum (Myxidiidae) and Henneguya ganapatiae (Myxobolidae), respectively. These species were identified by myxospore morphology, morphometry and host tissue affinity, and the original descriptions supplemented with small-subunit ribosomal DNA sequences and phylogenetic analysis. Free myxospores of M. cf. notopterum were found in the gallbladder, and measured 14.7 ±â€¯0.6 µm long and 6.3 ±â€¯0.6 µm wide; host, tissue and myxospore dimensions overlapped with the type, but differed in morphological details (spore shape, valve cell ridges) and locality (Malaysia versus India). Plasmodia and spores of H. ganapatiae were observed in gills, and myxospores had a spore body 9.7 ±â€¯0.4 µm long, 4.5 ±â€¯0.5 µm wide; sample locality, host, tissue, spore morphology and morphometry matched the original description. Small-subunit ribosomal DNA sequences were deposited in GenBank (M. cf. notopterum MT365527, H. ganapatiae MT365528) and both differed by >7% from congeneric species. Although the pathogenicity and clinical manifestation of myxozoan in humans are poorly understood, consumption of raw fish meat with myxozoan infection was reported to be associated with diarrhea. Identification of current parasite fauna from N. notopterus is an essential first step in assessing pathogen risks to stocks of this important food fish.

Morphology and molecular data of two novel cnidarian myxosporean (Myxobolidae) infecting Piaractus brachypomus from the Amazon basin.

The objective of this study was reports, through morphological and small subunit ribosomal DNA (SSU rDNA) sequencing, two novel myxobolid myxosporeans infecting Piaractus brachypomus, an economicaly important Amazonian fish popularly known as "pirapitinga". Of a total of 25 specimens of P. brachypomus examined 68% had the gill filament parasitized by Henneguya tapariensis n. sp. and 16% had infection of Myxobolus arapiuns n. sp. in the pyloric cecum. The morphological analysis revealed H. tapariensis n. sp. myxospores with an ellipsoid shape and caudal process larger than the length of the body. The polar capsules of same size were elongated and occupied less than half the body. Sequencing of the SSU rDNA generated a partial sequence of 1946 bp. In M. arapiuns n. sp. the myxospores had oval-shaped body and polar capsules of the same size, occupying less than half the body. Sequencing of the SSU rDNA generated a partial sequence of 1950 bp. Phylogenetic analysis revealed a cluster according to the order/family of the host, where H. tapariensis n. sp. was grouped in a subclade with Henneguya brachypomus and Henneguya piaractus and M. arapiuns grouped in a subclade with Myxobolus colossomatis, Myxobolus matosi and Myxobolus pirapitingae.

Novel myxosporean species parasitizing an economically important fish from the Amazon basin.

This paper provides morphological and phylogenetic analyses of two new myxobolid species found infecting Piaractus brachypomus from the Amazon basin. The fish were caught in the Tapajós River, in the municipality of Santarém, in the state of Pará, Brazil. The plasmodial development of Henneguya brachypomus n. sp. occurred in the gill lamellae while Myxobolus pirapitingae n. sp. developed in the pyloric cecum. Morphological analyses did not identify inflammatory infiltrate for either species, but H. brachypomus n. sp. induced stretching of the epithelium, compression of the adjacent tissues, and displacement and deformation of the neighboring lamellae. The mature myxospores of H. brachypomus n. sp. were ellipsoid, with a length of 11.7-13.8 µm, a width of 4.0-4.6 µm, and a thickness of 3.5-4.3 µm. The polar capsules were elongated, with a length of 5.6-7.3 µm and a width of 1.3-2.0 µm, and each contained a polar filament with 8-9 coils. The caudal process was 40.5-48.1 µm long and the total length of the myxospore was 52.4-61.6 µm. Myxobolus pirapitingae n. sp. exhibited rounded mature myxospores measuring 10.0-11.1 µm in length, 7.0-7.6 µm in width, and 5.4-6.3 µm in thickness. The polar capsules were of equal size and occupied less than half the myxospore, measuring 3.5-4.0 µm in length and 2.0-2.6 µm in width, with each containing a polar filament with 6-7 coils. Phylogenetic analysis based on partial small subunit ribosomal DNA (ssrDNA) sequences showed that H. brachypomus n. sp. clustered as a sister species of Henneguya piaractus, while M. pirapitingae n. sp. was grouped in a sub-clade together with Myxobolus matosi and Myxobolus colossomatis.

Native parasite affecting an introduced host in aquaculture: cardiac henneguyosis in the red seabream Pagrus major Temminck & Schlegel (Perciformes: Sparidae) caused by Henneguya aegea n. sp. (Myxosporea: Myxobolidae).

BACKGROUND: Henneguya Thélohan, 1892 (Myxobolidae) is one of the most species-rich genera of myxosporean parasites infecting fish. Although common in nature, there are few reports of these parasites causing important disease in aquaculture. In this paper, we describe a new species of Henneguya infecting Pagrus major (Temminck & Schlegel), a fish host introduced to the Mediterranean Sea from Japan in the late 1980s. RESULTS: Large plasmodia of the parasite were found in the bulbus arteriosus and in the ventricle of the infected fish. Spores were found mainly in the kidney and heart and were accompanied by melanized macrophages or vascular intimal proliferation mixed with a mild non-suppurative response, respectively. Comparisons of morphometric data for spore and polar capsule length and width, suggest a unique combination of features in the newly described species. Molecular analysis, based on 18S rDNA sequence of the parasite, followed by phylogenetic analysis, indicated that the parasite described here is a novel species of Henneguya, clustered with the marine congeneric species. CONCLUSIONS: Henneguya aegea n. sp. infects in aquaculture P. major, a host introduced as eggs to the Mediterranean from Japan. Despite the high host specificity of the myxobolid parasites, H. aegea n. sp. seems to be able to use P. major as a host and propagate successfully, causing morbidity and mortality. This could result in spillback of the new species from high density cultured non-native P. major to native fish hosts.

Henneguya (Cnidaria: Myxosporea: Myxobolidae) infections of cultured barramundi, Lates calcarifer (Perciformes: Latidae) in an estuarine wetlands system of Malaysia: description of Henneguya setiuensis n. sp., Henneguya voronini n. sp. and Henneguya calcarifer n. sp.

Examination of 35 barramundi (Lates calcarifer) from aquaculture cages in Setiu Wetland, Malaysia, revealed a single fish infected with three Henneguya spp. (Cnidaria: Myxosporea). Characterization of the infections using tissue tropism, myxospore morphology and morphometry and 18S rDNA sequencing supported description of three new species: Henneguya setiuensis n. sp., Henneguya voronini n. sp. and H. calcarifer n. sp. Myxospores of all three species had typical Henneguya morphology, with two polar capsules in the plane of the suture, an oval spore body, smooth valve cell surfaces, and two caudal appendages. Spores were morphometrically similar, and many dimensions overlapped, but H. voronini n. sp. had shorter caudal appendages compared with H. calcarifer n. sp. and H. setiuensis n. sp. Gross tissue tropism distinguished the muscle parasite H. calcarifer n. sp. from gill parasites H. setiuensis n. sp. and H. voronini n. sp.; and these latter two species were further separable by fine-scale location of developing plasmodia, which were intra-lamellar for H. setiuensis n. sp. and basal to the filaments for H. voronini n. sp. small subunit ribosomal DNA sequences distinguished all three species: the two gill species H. setiuensis n. sp. and H voronini n. sp. were only 88% similar (over 1708 bp), whereas the muscle species H. calcarifer n. sp. was most similar to H. voronini n. sp. (98% over 1696 bp). None of the three novel species was more than 90% similar to any known myxosporean sequence in GenBank. Low infection prevalence of these myxosporeans and lack of obvious tissue pathology from developing plasmodia suggested none of these parasites are currently a problem for barramundi culture in Setiu Wetland; however additional surveys of fish, particularly at different times of the year, would be informative for better risk assessment.

Two Novel Myxozoans from Pirate Perch Aphredoderus sayanus (Gilliams, 1824) in the Upper Mississippi River, Including the First North American Species of Hennegoides Lom, Tonguthai, & Dyková, 1991.

The pirate perch Aphredoderus sayanus is a relatively small fish species found in rivers throughout much of the eastern United States. Due to their cryptic nature, relatively little is known regarding their parasite fauna. A survey of pirate perch from the upper Mississippi River revealed 2 novel myxozoans. Hennegoides flockae n. sp. was observed in heavily infected gills where the lamellae featured irregular expansion by bulbous myxozoan polysporic plasmodia, typically affecting the middle to distal half of the filaments. When severe, infection of sequential filaments was such that the filaments were fused, forming what appeared as multicystic/lobular parasitic aggregates subdivided by fine epithelial cords. The total myxospore length of Hennegoides flockae was 35.4-46.4 (41.3 ± 3.3) and the spore body, asymmetrically ovoid in valvular view, was 15.4-18.7 (17.0 ± 0.7) × 7.1-8.7 (7.9 ± 0.4). Henneguya marcquenskiae n. sp. was observed in the liver with plasmodia present randomly and infrequently in the hepatocellular parenchyma. The total myxospore length for Henneguya marcquenskiae was 39.5-55.9 (48.4 ± 4.2), with the spore body being lanceolate, 13.9-16.5 (15.4 ± 0.7) × 7.1-9.0 (8.3 ± 0.5). Phylogenetic analysis of the SSU rRNA gene placed both Hennegoides flockae and Henneguya marcquenskiae as sisters to each other in a clade containing other Myxozoans known to infect the gills of esocids, percids, and centrarchids. These parasites represent the first reports of Henneguya and Hennegoides from pirate perch, with the latter being the first report of this genus outside of the Asian continent.

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.

Phylogeny of Myxobolidae (Myxozoa) and the evolution of myxospore appendages in the Myxobolus clade.

Genera Myxobolus Bütschli, 1882 and Henneguya Thélohan, 1892 (Myxobolidae) are specious myxozoan genera. They comprise nearly half of overall known myxozoan species diversity. A typical spore feature of Henneguya is the presence of two caudal appendages of the spore valves, which distinguishes them from species of the genus Myxobolus. Several Myxobolus spp., however, were reported to show aberrant spores with Henneguya-like caudal appendages. We found such aberrant spores in Myxobolus tsangwuensis and Myxobolus wulii. We studied the ultrastructure of M. wulii and Myxobolus oralis spores with caudal appendages by transmission electron microscopy (TEM). TEM of these aberrant spores revealed that their caudal appendages have the same ultrastructure as the appendages of Henneguya spp. Small caudal appendages of M. wulii spores observed only on TEM suggested that this character may be often overlooked and more Myxobolus species potentially have the ability to express the caudal appendages on the myxospore. In order to trace the evolution of this character, we performed broad phylogenetic analysis of all species of the family Myxobolidae which are available in GenBank including nearly 300 taxa. We found at least eight independent evolutionary origins of spores with two appendages, three origins of a single appendage and 12 apparent secondary losses of the spore projections. Therefore, genus Henneguya with typical two-tailed myxospores is polyphyletic, however a majority of its species has a common ancestor and groups in the second largest subclade of the Myxobolus clade. We also mapped the biological characteristics (host, site of infection and environment) of Myxobolidae species on the phylogenetic tree. We revealed an evident host-associated evolutionary pattern in all parts of the Myxobolus clade with a distinct and species-rich subclade containing almost exclusively species infecting species of the Order Cypriniformes.

Morphology and phylogeny of Henneguya oviperda infecting oocytes of Esox lucius, with description of parasite-induced histopathology.

Henneguya oviperda (Cohn, 1895) (Myxozoa: Myxobolidae) is a parasite infecting oocytes of the northern pike Esox lucius Linnaeus, 1758 (Actinopterygii: Esocidae). Infected oocytes are surrounded by all oocyte layers, some of them thinner and less visible than intact oocytes. A mature plasmodium of H. oviperda fills the entire internal space of the oocytes at the secondary growth phase, rendering the nucleus and organelles of the latter undetectable. Apart from the observed degradation of internal structures, alterations in the envelopes of the infected oocytes, and the deformation of the intact oocytes adjacent to them, no other developmental anomalies have been found in the reproductive products of female northern pike. Mature spores of H. oviperda have oval bodies with polar capsules of almost equal size and caudal projections that are on average equal to the spore body length. Phylogenetic analysis comparing 18S rDNA sequences placed H. oviperda into a clade of esocid-infecting species of the genus Henneguya and also supported H. psorospermica as a sister species.