Species‐specific in situ hybridization confirms arrested development of Henneguya ictaluriin 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

Proliferative gill disease (PGD) in Channel Catfish Ictalurus punctatusand hybrid catfish (Channel Catfish × Blue Catfish I. furcatus) is attributed to the myxozoan Henneguya ictaluri. Despite evidence of decreased H. ictaluritransmission 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. ictaluriand other myxozoans in naturally and experimentally induced PGD. Henneguyaspecies‐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. 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. exilisinfections illustrated species‐specific labeling and no cross‐reactivity between different myxozoan species or the catfish hosts. After experimental challenge, mature plasmodia of H. ictaluriand H. postexilisformed 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. Although H. ictaluriappears 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. ictaluriand H. postexilisin Channel Catfish and supports previous research indicating impaired or precluded H. ictalurisporogony in hybrid catfish. Impact statementThis work implicates Henneguya ictalurias the primary cause of proliferative gill disease, a devastating parasitic disease of U.S. catfish aquaculture, but demonstrates other myxozoan parasites can contribute to disease severity. The study better defines the developmental timeline of H. ictaluriand H. postexilisin Channel Catfish and supports research indicating impaired maturation of H. ictaluriin hybrid catfish. This work implicates Henneguya ictalurias the primary cause of proliferative gill disease, a devastating parasitic disease of U.S. catfish aquaculture, but demonstrates other myxozoan parasites can contribute to disease severity. The study better defines the developmental timeline of H. ictaluriand H. postexilisin Channel Catfish and supports research indicating impaired maturation of H. ictaluriin hybrid catfish.