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Characterising myxozoan taxa parasitising fish hosts in catfish aquaculture ponds is crucial to understanding myxozoan community dynamics in these diverse and complex ecological systems. This work investigated the myxozoan fauna of the western mosquitofish, Gambusia affinis, a common, incidental species found in catfish aquaculture ponds in the southeastern United States. 598 fish were sampled in May of 2018 and 2019 from the pond facility of the Thad Cochran National Warmwater Aquaculture Center in Stoneville, Mississippi, USA. Fish were examined microscopically using wet mount preparations of fresh tissue and histology for myxozoans. 18S rRNA gene sequences were amplified from myxospores obtained at necropsy. Updated morphologic, histologic, and 18S rRNA gene sequence features are provided for Henneguya gambusi, Myxobolus pharyngeus, and Myxidium phyllium. Two potentially novel myxozoans were observed during this survey, an undocumented Myxobolus sp. associated with chondrolysis of bones throughout the body and a putative Myxobilatus sp. observed histologically in the renal tubules, ureters, and urinary bladder. However, inadequate samples were obtained for proper species descriptions. Lastly, the life cycle of M. pharyngeus, which is thought to utilize the oligochaete worm Dero digitata as their definitive host, was putatively confirmed by 18S rRNA sequence matching to actinospore stages from oligochaetes in catfish ponds in Mississippi. This work provides novel and expanded morphologic, histologic, molecular and biologic data of five myxozoan parasites of G. affinis, expanding our knowledge of myxozoan diversity in catfish aquaculture ponds.

Henneguya Thélohan, 1892 is the second most species rich genus of myxozoans, with reports from freshwater and marine fish worldwide. In the Great Lakes region of North America, muskellunge Esox masquinongy is an important game fish species that serves as an apex predator in the ecosystems of many inland lakes. The myxozoan fauna of esocid fish, especially muskellunge, remains largely understudied. During fish health assessments, muskellunge were examined for parasitic infections and myxozoan pseudocysts were observed on gill clip wet mounts. When ruptured under pressure, the intralamellar pseudocysts released thousands of myxospores consistent with those of the genus Henneguya. The myxospores were 67.3-96.6 (79.1 ± 5.9) µm in total length. The spore body was 18.6-22.6 (20.9 ± 1.0) µm × 5.4-6.9 (6.3 ± 0.4) µm in valvular view and 3.5-4.0 (3.8 ± 0.3) µm wide in sutural view. The two pyriform polar capsules positioned at the anterior of the spore body were 6.4-7.7 (7.0 ± 0.4) µm × 1.8-2.1 (2.0 ± 0.1) µm and each contained a tightly coiled polar filament with 9-10 turns. Two tapering caudal processes extended from the posterior of the spore body and were 47.3-75.6 (58.3 ± 5.8) µm in length. Histologically, large intralamellar polysporic plasmodia were surrounded by plump pillar cells and a distinct layer of plasma. Mild inflammation was present peripherally, with small numbers of necrotic germinative cells and intraplasmodial phagocytes internally. Ribosomal 18S rRNA gene sequence data were obtained from three gill pseudocysts. The three ~2000-bp sequences were identical, but shared no significant similarity with any publicly available sequence data. Phylogenetic analyses demonstrated sequence data from this Henneguya fell within a well-supported clade of Henneguya spp. reported from northern pike Esox lucius in Europe. Based on the distinct morphological, histological and molecular data, this species is designated as Henneguya michiganensis n. sp. from muskellunge in Michigan, USA.

The present work aims to morphologically and molecularly characterize an Eimeria sp. recovered from an adult, captive barred owl Strix varia in Mississippi, USA. While the owl was shedding oocysts, no clinical signs of coccidiosis were observed. Oocysts collected from a barred owl, S. varia were isolated from feces by fecal flotation and characterized by light microscopy. The oocysts were allowed to sporulate in K2Cr2O7 for 7 days and measured for comparison with previously described Eimeria species. Genomic DNA was extracted from measured oocysts and the cytochrome c oxidase subunit 1 mitochondrial gene was amplified and sequenced for comparison and phylogenetic analyses with publicly available sequence data. Oocysts collected from S. varia were morphologically and molecularly consistent with all previous accounts of Eimeria megabubonis from the great horned owl Bubo virginianus and morphologically consistent with the only account of Eimeria varia from S. varia. Phylogenetic analyses grouped the E. megabubonis sequence from the present study with previously reported sequences from E. megabubonis. Morphological and molecular data from oocysts collected in this study reveal S. varia is a host for E. megabubonis. Agreement between current data, morphological and molecular data of E. megabubonis, and morphological data from the original description of E. varia indicates E. varia is a junior synonym of E. megabubonis.

With only six recognised genera, the family Clinostomidae Luhe, 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.

A Henneguya sp., morphologically resembling Henneguya nyongensis Fomena & Bouix, 1996, was isolated from the gills of Peter’s elephantnose fish, Gnathonemus petersii Gunther, imported from Nigeria. Plasmodia were located between lamellae and within the gill epithelium, often leading to lamellar fusion. Although slightly smaller, the myxospores from these fish were morphologically consistent with H. nyongensis. In valvular view, spores are elongate, pyriform with a rounded posterior and tapering caudal processes. Myxospore bodies are 9.6–12.3 (mean 11.2) µm long and 4.0–4.7 (mean 4.3) µm wide. Polar capsules are pyriform, elongate, 4.5–5.2 (4.7) µm long and 1.3–1.6 (1.4) µm wide, with a characteristic neck-like structure at the apical end. Sequence generated for the 18S small subunit rRNA gene did not directly match any sequences available on GenBank, but demonstrated 91% nucleotide similarity to an unpublished Henneguya sp. infecting Mormyrus kannume Forsskal. Herein, the description of H. nyongensis is supplemented with new data on histopathology, molecular characterisation, and expanded host and geographical range.

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.

Neodiplostomum americanum Chandler and Rausch, 1947 has been reported from six species of owls in North America. At present, there are no molecular data for this species and gene sequence data from Neodiplostomum Railliet, 1919 are limited. A freshly deceased specimen of the Great Horned Owl Bubo virginianus Gmelin, 1788 and a freshly deceased specimen of the Eastern Screech Owl Megascops asio Linnaeus, 1758 were collected in Oktibbeha County, Mississippi in 2014 and 2016, respectively. Neodiplostomum americanum were recovered from both hosts. Herein, updated morphological descriptions are supplemented with gene sequence data from conserved (18S, ITS1-5.8S, ITS2, and 28S rRNA) and fast-evolving (cytochrome c oxidase subunit 1 mtDNA) regions. Preliminary phylogenetic analysis of the genus based on cytochrome c oxidase subunit 1 gene sequence data supports the placement of N. americanum within a discrete phylogroup of the family Diplostomidae. The life history of N. americanum is unknown and currently limited to the description of the adult stage in avian hosts. The molecular data generated in this study offer insight into the phylogenetic placement of N. americanum within the Diplostomatidae and will aid in identifying different life stages in putative intermediate hosts.

A novel species of Henneguya was isolated from flathead catfish (Pylodictis olivaris) captured in the upper Mississippi River near Lansing (Allamakee County), IA, and La Crosse (La Crosse County), WI. Designated Henneguya laseeae n. sp., this novel species is described using critical morphological features, histology, and 18S ribosomal RNA gene sequence. Ovoid cysts, ranging from 1200 to 1800 μm in width, tended to be at filament tips or in the distal third, often directly on the filament midline, but occasionally paramedian. Lanceolate-shaped myxospores were consistent with those of the genus Henneguya. The spore body was 16.2 ± 0.5 μm (mean ± standard deviation; range = 15.1-17.0 μm) in length, 6.0 ± 0.4 μm (5.1-6.6 μm) in width, and 4.7 ± 0.2 μm (4.4-4.9 μm) thick. The two polar capsules at the anterior of the spore body were 5.9 ± 0.3 μm (5.3-6.3 μm) in length and 1.8 ± 0.1 μm (1.6-2.1 μm) in width and contained six to seven turns in the polar filament. The caudal processes tapered to fine points and were 54.3 ± 2.9 μm (49.1-61.7 μm) in length. Total spore length was 70.4 ± 3.3 μm (64.5-79.4 μm). The spores and plasmodium of this species are of similar size and morphology to other species of Henneguya from ictalurid fishes. Additionally, the 18S rRNA gene sequences placed this isolate within a clade populated by Henneguya spp. from North American ictalurids. This is the first reported species of Henneguya from flathead catfish.

In the southeastern USA, catfish aquaculture is burdened by predation from piscivorous birds and the digenetic trematodes they carry. In addition to cultured ictalurid fish, other forage or incidental fish species inhabit catfish production ponds. Of these, the inland silverside Menidia beryllina was recently found to harbor larval metacercariae of several trematode species. Three species of metacercariae were reported, two of which represent the first morphological descriptions of an Austrodiplostomum sp. and Bolbophorus sp. metacercaria, respectively. A total of 15 silversides were collected from a commercial catfish pond and examined for trematode infection. These fish were parasitized by metacercariae of an Austrodiplostomum sp. (100 % prevalence) in the eyes and brain, a Bolbophorus sp. (86.7 % prevalence) in the musculature, and Clinostomum marginatum (33.3 % prevalence) in the musculature and fins. All three trematode species were characterized morphologically and molecularly by sequencing of the cytochrome c oxidase subunit 1 gene (CO1). In addition, the internal transcribed spacer 1 region (ITS1), 5.8S rRNA gene, and ITS2 region were determined for the Bolbophorus sp., which linked this metacercaria to a Bolbophorus sp. cercaria from a planorbid snail Planorbella trivolvis and an unnamed Bolbophorus sp. adult from the American white pelican Pelecanus erythrorhynchos. Furthermore, Biomphalaria havanensis snails were collected from the same pond and found actively shedding cercariae morphologically and molecularly consistent with a diplostomid cercaria reported from Bi . havanensis in catfish ponds in Mississippi, USA. Sequence comparisons deemed these cercariae conspecific to the Austrodiplostomum sp. from inland silverside described here. Channel catfish fingerlings were exposed to these cercariae at doses of 50 and 100 cercariae per fish. The infectivity of this Austrodiplostomum sp. in channel catfish was assessed at 10 and 20 days post exposure (dpe). Metacercariae were observed in both the eyes and brain of infected channel catfish, supporting molecular data that suggests the cercaria and metacercaria are both stages of a previously unidentified Austrodiplostomum sp. life cycle.

Anemia is a widespread hematological disorder in vertebrates. In channel catfish (Ictalurus punctatus), anemia is a persistent problem in culture environments, however, its causes and physiological impacts are not well understood. To better understand the symptoms and characterize associated biomarkers of anemia, 1-D 1H and 2-D 1H J-resolved NMR were used to analyze metabolite changes in healthy and anemic channel catfish kidney and liver tissue. Additionally, standard physiological analytical techniques were used to analyze blood plasma. NMR analyses revealed energy sources such as glucose were depleted and many metabolites associated with anaerobic metabolism or alternative energy pathways such as lactate, creatine, alanine, acetate and myo-inositol had changed. Energy demanding processes were reduced, such as muscle function, as indicated by reductions in taurine and inosine, and protein synthesis. Stress and oxidative stress related metabolites changed, with increases in valine, leucine and isoleucine and decreases in glutathione. Inhibitory neurotransmitters such as 4-aminobutyric acid (GABA) increased, and excitatory neurotransmitters such as glutamine and glutamate generally decreased, although there were tissue-specific differences. Immune function also decreased in anemic fish. Blood analyses revealed decreased respiratory gas transport capabilities through reductions in erythrocytes and hemoglobin-markers. Taken together, these tissues and analytical techniques produced complementary results, and metabolite expression in anemic channel catfish was similar to that observed in other vertebrates exposed to anoxia, severe hypoxia or experiencing oxidative stress from deficiencies in iron homeostasis.

In the southeastern USA, the channel catfish Ictalurus punctatus is a host to at least eight different species of myxozoan parasites belonging to the genus Henneguya, four of which have been characterized molecularly using sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. However, only two of these have confirmed life cycles that involve the oligochaete Dero digitata as the definitive host. During a health screening of farm-raised channel catfish, several fish presented with deformed primary lamellae. Lamellae harbored large, nodular, white pseudocysts 1.25 mm in diameter, and upon rupturing, these pseudocysts released Henneguya myxospores, with a typical lanceolate-shaped spore body, measuring 17.1 ± 1.0 μm (mean ± SD; range = 15.0–19.3 μm) in length and 4.8 ± 0.4 μm (3.7–5.6 μm) in width. Pyriform-shaped polar capsules were 5.8 ± 0.3 μm in length (5.1–6.4 μm) and 1.7 ± 0.1 μm (1.4–1.9 μm) in width. The two caudal processes were 40.0 ± 5.1 μm in length (29.5–50.0 μm) with a spore length of 57.2 ± 4.7 (46.8–66.8 μm). The contiguous SSU rRNA gene sequence obtained from myxospores of five excised cysts did not match any Henneguya sp. in GenBank. The greatest sequence homology (91 % over 1,900 bp) was with Henneguya pellis, associated with blister-like lesions on the skin of blue catfish Ictalurus furcatus. Based on the unique combination of pseudocyst and myxospore morphology, tissue location, host, and SSU rRNA gene sequence data, we report this isolate to be a previously unreported species, Henneguya bulbosus sp. nov.