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.

CERCARIAL LONGEVITY AND INFECTIVITY OF BOLBOPHORUS DAMNIFICUS, WITH NOTES ON METACERCARIAL PERSISTENCE AND SITE SPECIFICITY IN CHANNEL AND HYBRID CATFISH.

Advances in hybridization practices in U.S. catfish aquaculture have led to increased production of channel (Ictalurus punctatus) × blue catfish (Ictalurus furcatus) hybrids to capitalize on their more favorable production characteristics. However, the effects of typical channel catfish pathogens on hybrids are not well understood, including the digenean Bolbophorus damnificus, which has caused significant losses in channel catfish production. Three experiments were conducted to assess the longevity and site specificity of 2 life stages of B. damnificus impacting catfish production. The first experiment investigated the cercarial longevity and infectivity of B. damnificus over time. Channel catfish were individually challenged with 100 cercariae/fish with cercariae aged in 12-hr time intervals over 5 days (n = 5 fish/time point), with metacercarial cysts excised and enumerated 14 days postchallenge. There was a decrease in cercaria viability and encysted metacercariae over the first 36 hr, with the 12-hr time point having both the greatest cercaria survival and the highest number of metacercariae in exposed fish. The second experiment investigated the longevity of metacercariae within both channel and hybrid catfish. Fish (n = 30) were exposed to 2 treatments (75 or 150 cercariae/fish), and 2 fish from each treatment were sampled every 3 mo for 13 mo. Live metacercariae, based on motility observed after excystment, were found in both species up to 13 mo postchallenge, indicating the metacercariae of B. damnificus can persist throughout an entire growing season in both channel and hybrid catfish. The third experiment investigated the site specificity of metacercariae within both channel and hybrid catfish. Fish (n = 60/species) were challenged with 300 cercariae/fish and 9 fish/species were sampled after 90 days. Metacercariae were excised and enumerated from the anterior midsection (head and body), posterior midsection (trunk/caudal peduncle), ventral (belly), and caudal fin (tail) sections of each fish. Overall, the trunk/caudal peduncle had a 2-fold increase in the number of metacercariae excised, and although not significantly higher, results indicate this region should be the focal point of pondside assessment for the presence of B. damnificus because of ease of detection of encysted metacercariae.

Ithyoclinostomum yamagutii n. sp. (Digenea: Clinostomidae) in the great blue heron Ardea herodias L. (Aves: Ardeidae) from Mississippi, USA.

With only six recognised genera, the family Clinostomidae Lühe, 1901 remains a global research interest of parasitologists and ecologists. Recent efforts have focused on providing molecular data to investigate species diversity, elucidate life-cycles, and make inferences on the group's evolutionary history. Of the clinostomid genera, the monotypic Ithyoclinostomum Witenberg, 1926 has remained more enigmatic compared to the commonly encountered Clinostomum Leidy, 1856. Recent morphological and molecular evidence from metacercariae suggests a second Ithyoclinostomum species may exist in freshwater cichlids in Central America and Mexico. In a recent survey of great blue herons Ardea herodias L. from commercial catfish production farms in Mississippi, USA, two specimens of an abnormally large (> 20 mm) clinostomid were encountered in the oesophagus of a single bird. These specimens were identified as an Ithyoclinostomum sp. morphologically distinct from the only nominal species Ithyoclinostomum dimorphum (Diesing, 1850). Using morphological and molecular data these adult specimens were confirmed as conspecific with the larval metacercariae previously described from Central America and Mexico and represent the novel species, Ithyoclinostomum yamagutii n. sp.