Five Species of Wild Freshwater Sport Fish in Wisconsin, USA, Reveal Highly Diverse Viromes.

Studies of marine fish have revealed distant relatives of viruses important to global fish and animal health, but few such studies exist for freshwater fish. To investigate whether freshwater fish also host such viruses, we characterized the viromes of five wild species of freshwater fish in Wisconsin, USA: bluegill (Lepomis macrochirus), brown trout (Salmo trutta), lake sturgeon (Acipenser fulvescens), northern pike (Esox lucius), and walleye (Sander vitreus). We analyzed 103 blood serum samples collected during a state-wide survey from 2016 to 2020 and used a metagenomic approach for virus detection to identify known and previously uncharacterized virus sequences. We then characterized viruses phylogenetically and quantified prevalence, richness, and relative abundance for each virus. Within these viromes, we identified 19 viruses from 11 viral families: Amnoonviridae, Circoviridae, Coronaviridae, Hepadnaviridae, Peribunyaviridae, Picobirnaviridae, Picornaviridae, Matonaviridae, Narnaviridae, Nudnaviridae, and Spinareoviridae, 17 of which were previously undescribed. Among these viruses was the first fish-associated coronavirus from the Gammacoronavirus genus, which was present in 11/15 (73%) of S. vitreus. These results demonstrate that, similar to marine fish, freshwater fish also harbor diverse relatives of viruses important to the health of fish and other animals, although it currently remains unknown what effect, if any, the viruses we identified may have on fish health.

A NEW SPECIES OF THELOHANELLUS (CNIDARIA: MYXOSPOREA: MYXOBOLIDAE) FROM THE GILL OF QUILLBACK, CARPIODES CYPRINUS (CYPRINIFORMES: CATOSTOMIDAE), FROM THE ARKANSAS RIVER DRAINAGE OF OKLAHOMA.

During May 2022 and again in March 2023, 5 quillbacks, Carpiodes cyprinus, were collected from the Verdigris River, Wagoner County, Oklahoma (n = 1), and the Black River, Lawrence County, Arkansas (n = 4), and their gill, gallbladder, fins, integument, musculature, and other major organs were macroscopically examined for myxozoans. Gill lamellae from the single quillback from the Verdigris River was infected with a new myxozoan, Thelohanellus oklahomaensis n. sp. Qualitative and quantitative morphological data were obtained from fresh and formalin-fixed preserved myxospores, and molecular data consisted of a 1,767 base pair sequence of the partial small subunit (SSU) ribosomal RNA gene. Phylogenetic analysis grouped T. oklahomaensis n. sp. with myxozoans known to infect North American catostomids and Eurasian cyprinids. Histological examination localized plasmodia to an intralamellar developmental site and revealed a possible vestige of a second polar capsule. Although plasmodia markedly expanded lamellae, there were no associated epithelial or inflammatory changes. Thelohanellus oklahomaensis n. sp. is the only member of the genus known to infect the gills of C. cyprinus.

Viruses of Freshwater Mussels during Mass Mortality Events in Oregon and Washington, USA.

Freshwater mussels (Unionida) are globally imperiled, in part due to largely unexplained mass mortality events (MMEs). While recent studies have begun to investigate the possibility that mussel MMEs in the Eastern USA may be caused by infectious diseases, mussels in the Western USA have received relatively little attention in this regard. We conducted a two-year epidemiologic investigation of the role of viruses in ongoing MMEs of the Western pearlshell (Margaritifera falcata) and the Western ridged mussel (Gonidea angulata) in the Chehalis River and Columbia River watersheds in the Western USA. We characterized viromes of mussel hemolymph from 5 locations in 2018 and 2020 using metagenomic methods and identified 557 viruses based on assembled contiguous sequences, most of which are novel. We also characterized the distribution and diversity of a previously identified mussel Gammarhabdovirus related to pathogenic finfish viruses. Overall, we found few consistent associations between viruses and mussel health status. Variation in mussel viromes was most strongly driven by location, with little influence from date, species, or health status, though these variables together only explained ~1/3 of variation in virome composition. Our results demonstrate that Western freshwater mussels host remarkably diverse viromes, but no single virus or combination of viruses appears to be associated with morbidity or mortality during MMEs. Our findings have implications for the conservation of imperiled freshwater mussels, including efforts to enhance natural populations through captive propagation.

Plasticity, Paralogy, and Pseudogenization: Rhabdoviruses of Freshwater Mussels Elucidate Mechanisms of Viral Genome Diversification and the Evolution of the Finfish-Infecting Rhabdoviral Genera.

Viruses in the family Rhabdoviridae display remarkable genomic variation and ecological diversity. This plasticity occurs despite the fact that, as negative sense RNA viruses, rhabdoviruses rarely if ever recombine. Here, we describe nonrecombinatorial evolutionary processes leading to genomic diversification in the Rhabdoviridae inferred from two novel rhabdoviruses of freshwater mussels (Mollusca: Bivalvia: Unionida). Killamcar virus 1 (KILLV-1) from a plain pocketbook (Lampsilis cardium) is closely related phylogenetically and transcriptionally to finfish-infecting viruses in the subfamily Alpharhabdovirinae. KILLV-1 offers a novel example of glycoprotein gene duplication, differing from previous examples in that the paralogs overlap. Evolutionary analyses reveal a clear pattern of relaxed selection due to subfunctionalization in rhabdoviral glycoprotein paralogs, which has not previously been described in RNA viruses. Chemarfal virus 1 (CHMFV-1) from a western pearlshell (Margaritifera falcata) is closely related phylogenetically and transcriptionally to viruses in the genus Novirhabdovirus, the sole recognized genus in the subfamily Gammarhabdovirinae, representing the first known gammarhabdovirus of a host other than finfish. The CHMFV-1 G-L noncoding region contains a nontranscribed remnant gene of precisely the same length as the NV gene of most novirhabdoviruses, offering a compelling example of pseudogenization. The unique reproductive strategy of freshwater mussels involves an obligate parasitic stage in which larvae encyst in the tissues of finfish, offering a plausible ecological mechanism for viral host-switching. IMPORTANCE Viruses in the family Rhabdoviridae infect a variety of hosts, including vertebrates, invertebrates, plants and fungi, with important consequences for health and agriculture. This study describes two newly discovered viruses of freshwater mussels from the United States. One virus from a plain pocketbook (Lampsilis cardium) is closely related to fish-infecting viruses in the subfamily Alpharhabdovirinae. The other virus from a western pearlshell (Margaritifera falcata) is closely related to viruses in the subfamily Gammarhabdovirinae, which until now were only known to infect finfish. Genome features of both viruses provide new evidence of how rhabdoviruses evolved their extraordinary variability. Freshwater mussel larvae attach to fish and feed on tissues and blood, which may explain how rhabdoviruses originally jumped between mussels and fish. The significance of this research is that it improves our understanding of rhabdovirus ecology and evolution, shedding new light on these important viruses and the diseases they cause.

A Bacteriological Comparison of the Hemolymph from Healthy and Moribund Unionid Mussel Populations in the Upper Midwestern U.S.A. Prompts the Development of Diagnostic Assays to Detect Yokenella regensburgei.

Recent bacteriological investigations of freshwater mussel mortality events in the southeastern United States have identified a variety of bacteria and differences in bacterial communities between sick and healthy mussels. In particular, Yokenella regensburgei and Aeromonas spp. have been shown to be associated with moribund mussels, although it remains unclear whether these bacteria are causes or consequences of disease. To further understand the role of bacteria in mussel epizootics, we investigated mortality events that occurred in the upper Midwest in the Embarrass River (Wisconsin) and the Huron River (Michigan). For comparison, we also studied mussels from an unaffected population in the St. Croix River (Wisconsin). Diverse bacterial genera were identified from these sites, including Y. regensburgei from moribund mussels in the Embarrass River (Wisconsin). This bacterium has also been consistently isolated during ongoing mortality events in the Clinch River (Virginia). Subsequently, we developed and validated molecular assays for the detection of Yokenella to use in future investigations of mussel mortality events and to identify environmental reservoirs of this bacterium.

Efficacy of hydrogen peroxide to reduce Gyrodactylus species infestation density on four fish species.

OBJECTIVE: The ability to effectively treat parasitic infestations of fish is of high importance for fish culture facilities. However, tools or approved therapies for treating infestations on fish are limited. This paper summarizes results from four separate clinical field studies that evaluated the efficacy of hydrogen peroxide (H2 O2 ; 35% PEROX-AID) for reducing Gyrodactylus spp. infestation density. METHODS: Three species of Gyrodactylus were studied (G. salmonis, hosts: Brook Trout Salvelinus fontinalis and Lake Trout S. namaycush; G. freemani, host: Yellow Perch Perca flavescens; G. hoffmani, host: Fathead Minnow Pimephales promelas) before and after the application of immersion H2 O2 therapy. RESULT: Parasite density was significantly reduced for each parasite × host combination to which H2 O2 therapy was applied. Two clinical field studies in salmonids were found to demonstrate substantial effectiveness that enabled 35% PEROX-AID approval. CONCLUSION: Further assessments of Gyrodactylus spp. could expand the use of H2 O2 for controlling these parasites in aquaculture. Specifically, H2 O2 was effective at all levels tested (50 or 75 mg H2 O2 /L for 60 min for the Yellow Perch and Fathead Minnow clinical field studies; 100 or 150 mg H2 O2 /L for 30 min regardless of salt pre-treatment for the Brook Trout study; and 100 mg H2 O2 /L for 30 min or 50 mg H2 O2 /L for 60 min for the Lake Trout study).

A new Myxobolus (Cnidaria: Myxosporea: Myxobolidae) from the gills of the southern striped shiner, Luxilus chrysocephalus isolepis (Cypriniformes: Leuciscidae), from southwestern Arkansas, USA.

The southern striped shiner, Luxilus chrysocephalus isolepis (Hubbs & Brown) is a relatively large minnow belonging to the true minnow family Leuciscidae Bonaparte. Between May 2020 and January 2022, 55 L. c. isolepis were collected from watersheds in Montgomery (n = 6), Polk (n = 17) and Sevier (n = 32) counties, Arkansas, USA, and their gills, gallbladders, urinary bladders, fins, integument, other major organs, and musculature were examined for myxozoans. Gills of 11 (34%) individual southern striped shiners from Sevier County were infected with a new myxozoan, Myxobolus carlhubbsi n. sp. A qualitative and quantitative morphological description was based on formalin-fixed preserved myxospores, and molecular data consisted of a 1,970 base pair sequence of the partial small subunit rRNA gene from ethanol-preserved specimens. Histologically, plasmodia filled and expanded interlamellar troughs. Hyperplastic epithelial and goblet cells filled interlamellar troughs adjacent to plasmodia, but inflammatory response was limited to scattered lymphocytes. Phylogenetic analysis revealed that M. carlhubbsi n. sp. is a member of a clade of species with pyriform myxospores parasitizing North American Pogonichthyinae Girard and North American and Eurasian Leuciscinae Bonaparte. This is the first report of a myxozoan from L. c. isolepis. This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as urn:lsid:zoobank.org:pub:D10D71C2-2C75-4A1C-80ED-B98FF36CB509.

Pseudomurraytrema fergusoni n. sp. (Monogenea: Dactylogyridae) from gills of Pealip Redhorse, Moxostoma pisolabrum (Catostomidae) from Arkansas, USA, with a description of gill pathology.

Pseudomurraytrema fergusoni n. sp. is described from the Pealip Redhorse, Moxostoma pisolabrum from the Black River (White River drainage), Lawrence County, Arkansas, USA. This represents the second monogenean described from M. pisolabrum as well as the second species of Pseudomurraytrema reported from an Arkansas catostomid. The description includes partial 18S rRNA and 28S rRNA gene sequences (732 bp and 851 bp, respectively), helping fill a void in sequence data from North American monogeneans, particularly those in the genus Pseudomurraytrema. In addition, histopathologic changes associated with the infection resulted in severe localized pathologic lesions in gills of the host, suggesting compromise of respiratory surfaces within affected areas adjacent to the worms.

Freshwater Mussels Show Elevated Viral Richness and Intensity during a Mortality Event.

Freshwater mussels (Unionida) are among the world's most imperiled taxa, but the relationship between freshwater mussel mortality events and infectious disease is largely unstudied. We surveyed viromes of a widespread and abundant species (mucket, Actinonaias ligamentina; syn: Ortmanniana ligamentina) experiencing a mortality event of unknown etiology in the Huron River, Michigan, in 2019-2020 and compared them to viromes from mucket in a healthy population in the St. Croix River, Wisconsin and a population from the Clinch River, Virginia and Tennessee, where a mortality event was affecting the congeneric pheasantshell (Actinonaias pectorosa; syn: Ortmanniana pectorosa) population. We identified 38 viruses, most of which were associated with mussels collected during the Huron River mortality event. Viral richness and cumulative viral read depths were significantly higher in moribund mussels from the Huron River than in healthy controls from each of the three populations. Our results demonstrate significant increases in the number and intensity of viral infections for freshwater mussels experiencing mortality events, whereas individuals from healthy populations have a substantially reduced virome comprising a limited number of species at low viral read depths.

A NEW SPECIES OF MYXOBOLUS (CNIDARIA: MYXOSPOREA: MYXOBOLIDAE) FROM THE GILLS OF CREEK CHUB, SEMOTILUS ATROMACULATUS (CYPRINIFORMES: LEUCISCIDAE: PLAGIOPTERINAE), FROM THE OUACHITA DRAINAGE OF ARKANSAS.

During October and November 2021, 33 creek chubs, Semotilus atromaculatus, were collected from 3 sites in Polk County, Arkansas (Ouachita River drainage), and their gills, gallbladder, fins, integument, musculature, and other major organs were examined for myxozoans. The gills of 9 (27%) were infected with a new myxozoan, Myxobolus fountainae n. sp. Qualitative and quantitative morphological data were from fresh and formalin-fixed preserved spores, while molecular data consisted of a 1918 base pair sequence of the partial small subunit ribosomal RNA gene. Phylogenetic analysis grouped M. fountainae n. sp. with the other leuciscid-infecting myxobolids from North America and within a larger clade of European myxozoans. In addition, histological information is provided on the infection. A previous record of Myxobolus muelleriBütschli, 1882, from the gills and ureters of S. atromaculatus is considered invalid and represents an unknown species. Myxobolus fountainae n. sp. is the only named myxozoan known to infect the gill filaments of S. atromaculatus, whereas Myxobolus pendula (Guilford, 1967) infects the gill arches.

A new species of Myxidium (Cnidaria: Myxosporea: Myxidiidae) from the gallbladder of pickerels, Esox spp. (Esociformes: Esocidae), from southwestern Arkansas, USA.

During April 2016 and again in November 2021, four Chain Pickerels, Esox niger were collected from Union (n = 3) and Nevada (n = 1) counties, Arkansas, USA, and 65 Grass Pickerels, Esox americanus vermiculatus were collected between January 2015 and December 2021 from four counties of Arkansas (n = 31) and McCurtain County, Oklahoma, USA (n = 34), and examined for myxozoans. Gallbladders of an individual E. niger from Nevada County, Arkansas, as well as a single individual of E. a. vermiculatus from Sevier County, Arkansas, were infected with a new myxozoan, Myxidium whippsi n. sp. Qualitative and quantitative morphological data were from formalin-fixed preserved myxospores while molecular data (SSU rRNA gene) consisted of 2031bp (host: E. niger) and 1723 bp (host: E. a. vermiculatus) partial sequences of the small subunit ribosomal RNA gene. Phylogenetic analysis placed M. whippsi n. sp. in a clade with two other myxozoans, Zschokkella nova and Myxidium truttae, previously reported from cyprinids and salmonids, respectively. We document the first report of a myxozoan from E. a. vermiculatus. This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as urn:lsid:zoobank.org:pub:A50FCEB3-68C3-428E-A04E-37A16790F1EB.

Mussel Mass Mortality and the Microbiome: Evidence for Shifts in the Bacterial Microbiome of a Declining Freshwater Bivalve.

Freshwater mussels (Unionida) are suffering mass mortality events worldwide, but the causes remain enigmatic. Here, we describe an analysis of bacterial loads, community structure, and inferred metabolic pathways in the hemolymph of pheasantshells (Actinonaias pectorosa) from the Clinch River, USA, during a multi-year mass mortality event. Bacterial loads were approximately 2 logs higher in moribund mussels (cases) than in apparently healthy mussels (controls). Bacterial communities also differed between cases and controls, with fewer sequence variants (SVs) and higher relative abundances of the proteobacteria Yokenella regensburgei and Aeromonas salmonicida in cases than in controls. Inferred bacterial metabolic pathways demonstrated a predominance of degradation, utilization, and assimilation pathways in cases and a predominance of biosynthesis pathways in controls. Only two SVs correlated with Clinch densovirus 1, a virus previously shown to be strongly associated with mortality in this system: Deinococcota and Actinobacteriota, which were associated with densovirus-positive and densovirus-negative mussels, respectively. Overall, our results suggest that bacterial invasion and shifts in the bacterial microbiome during unionid mass mortality events may result from primary insults such as viral infection or environmental stressors. If so, bacterial communities in mussel hemolymph may be sensitive, if generalized, indicators of declining mussel health.

Urocleidus sayani n. sp. (Monogenea: Dactylogyridae) from Gills of Pirate Perch (Aphredoderus sayanus).

Urocleidus sayani n. sp. is described from the gills of pirate perch (Aphredoderus sayanus) in the Wisconsin backwaters of the upper Mississippi River and was found in samples from the Southeastern United States. Urocleidus sayani n. sp. is the second monogenean described from the pirate perch and the first for this host within Dactylogyridae. The description includes a partial 18S rRNA gene sequence (623 bp), filling a void in sequence data from North American monogeneans.

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.

In vitro transcribed dsRNA limits viral hemorrhagic septicemia virus (VHSV)-IVb infection in a novel fathead minnow (Pimephales promelas) skin cell line.

The farming of baitfish, fish used by anglers to catch predatory species, is of economic and ecological importance in North America. Baitfish, including the fathead minnow (Pimephales promelas), are susceptible to infection from aquatic viruses, such as viral hemorrhagic septicemia virus (VHSV). VHSV infections can cause mass mortality events and have the potential to be spread to novel water bodies through baitfish as a vector. In this study, a novel skin cell line derived from fathead minnow (FHMskin) is described and its use as a tool to study innate antiviral immune responses and possible therapies is introduced. FHMskin grows optimally in 10% fetal bovine serum and at warmer temperatures, 25-30 °C. FHMskin is susceptible and permissive to VHSV-IVb infection, producing high viral titres of 7.35 × 107 TCID50/mL after only 2 days. FHMskin cells do not experience significant dsRNA-induced death after treatment with 50-500 ng/mL of in vitro transcribed dsRNA for 48 h and respond to dsRNA treatment by expressing high levels of three innate immune genes, viperin, ISG15, and Mx1. Pretreatment with dsRNA for 24 h significantly protected cells from VHSV-induced cell death, 500 ng/mL of dsRNA reduced cell death from 70% to less than 15% at a multiplicity of infection of 0.1. Thus, the novel cell line, FHMskin, represents a new method for producing high tires of VHSV-IVb in culture, and for studying dsRNA-induced innate antiviral responses, with future applications in dsRNA-based antiviral therapeutics.

Henneguya laseeae n. sp. from flathead catfish (Pylodictis olivaris) in the upper Mississippi River.

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.

Using silver and bighead carp cell lines for the identification of a unique metabolite fingerprint from thiram-specific chemical exposure.

Conservation biology often requires the control of invasive species. One method is the development and use of biocides. Identifying new chemicals as part of the biocide registration approval process can require screening millions of compounds. Traditionally, screening new chemicals has been done in vivo using test organisms. Using in vitro (e.g., cell lines) and in silico (e.g., computer models) methods decrease test organism requirements and increase screening speed and efficiency. These methods, however, would be greatly improved by better understanding how individual fish species metabolize selected compounds. We combined cell assays and metabolomics to create a powerful tool to facilitate the identification of new control chemicals. Specifically, we exposed cell lines established from bighead carp and silver carp larvae to thiram (7 concentrations) then completed metabolite profiling to assess the dose-response of the bighead carp and silver carp metabolome to thiram. Forty one of the 700 metabolomic markers identified in bighead carp exhibited a dose-response to thiram exposure compared to silver carp in which 205 of 1590 metabolomic markers exhibited a dose-response. Additionally, we identified 11 statistically significant metabolomic markers based upon volcano plot analysis common between both species. This smaller subset of metabolites formed a thiram-specific metabolomic fingerprint which allowed for the creation of a toxicant specific, rather than a species-specific, metabolomic fingerprint. Metabolomic fingerprints may be used in biocide development and improve our understanding of ecologically significant events, such as mass fish kills.