Transcriptomic evidence of a fourth mininucleovirus (Mininucleoviridae): A rapidly growing family among the Nucleo-Cytoplasmic Large DNA viruses (NCLDVs).

The Mininucleoviridae are crustacean-infecting viruses thought to drive mortality across aquatic biomes. Three have been characterised from Carcinus maenas, Panulirus argus, and Dikerogammarus haemobaphes. We screened 202 SRA datasets (NCBI) for novel mininucleoviruses from 44 amphipod species. Three metatranscriptome datasets from Gammarus lacustris contained sequences with similarity to Dikerogammarus haemobaphes mininucleovirus. Assembly resulted in 19 transcripts, 16 were putatively polycistronic. The putative Gammarus lacustris mininucleovirus shares 46 homologues with other mininucleoviruses (similarity range: 24.07 - 78.2 %). The transcripts from this putative virus highlight its likely association with the Mininucleoviridae.

Symbiotic survey of the bay scallop (Argopecten irradians) from the Gulf coast of Florida, USA.

The bay scallop Argopecten irradians supported a commercial fishery in Florida but their population declined and the fishery closed in 1994. A recreational fishery remains open along the west coast of Florida despite continued threats from overfishing and a changing environment. Disease is among those threats, as it is for bivalve fisheries globally. We examined the relationship between bay scallop population density, its symbiotic microbiome, and geographic location. We focused on three sites within the range of Florida's recreational scallop fishery: St. Joseph Bay (northern extent), offshore of the Steinhatchee River (central), and offshore of Hernando County (southern extent). The study was conducted prior to the seasonal opening of the fishery to minimize the impact of fishing on our results. We also sampled caged scallops that are used for restocking in St. Joseph Bay to assess the effect of artificially high density and confinement on the scallop pathobiome. Using a combination of traditional histological methods, molecular diagnostics, and metagenomics, a suite of 15 symbionts were identified. Among them, RNA-seq data revealed four novel + ssRNA viral genomes: three picorna-like viruses and one hepe-like virus. The DNA-seq library revealed a novel Mycoplasma species. Histological evaluation revealed that protozoan, helminth and crustacean infections were common in A. irradians. These potential pathogens add to those already known for A. irradians and underscores the risk they pose to the fishery.

A new member of the Nudiviridae from the Florida stone crab (Menippe mercenaria).

Menippe mercenaria, the Florida stone crab, supports an unconventional fishery across the southern USA and Caribbean that involves claw-removal and the return of de-clawed animals to the sea. We provide pathological, ultrastructural, and genomic detail for a novel hepatopancreatic, nucleus-specific virus - Menippe mercenaria nudivirus (MmNV) - isolated from M. mercenaria, captured during fisheries-independent monitoring. The virus has a genome of 99,336 bp and encodes 84 predicted protein coding genes and shows greatest similarity to Aratus pisonii nudivirus (ApNV) (<60% protein similarity and 31 shared genes of greatest similarity), collected from the Florida Keys, USA. MmNV is a member of the Gammanudivirus genus (Naldaviricetes: Lefavirales: Nudiviridae). Comparisons of virus genome size, preferred host environment, and gene number revealed no clear associations between the viral traits and phylogenetic position. Evolution of the virus alongside the diversification of host taxa, with the potential for host-switching, remain more likely evolutionary pathways.

Histopathological screening of Pontogammarus robustoides (Amphipoda), an invader on route to the United Kingdom.

Biological invasions may act as conduits for pathogen introduction. To determine which invasive non-native species pose the biggest threat, we must first determine the symbionts (pathogens, parasites, commensals, mutualists) they carry, via pathological surveys that can be conducted in multiple ways (i.e., molecular, pathological, and histological). Whole animal histopathology allows for the observation of pathogenic agents (virus to Metazoa), based on their pathological effect upon host tissue. Where the technique cannot accurately predict pathogen taxonomy, it does highlight pathogen groups of importance. This study provides a histopathological survey of Pontogammarus robustoides (invasive amphipod in Europe) as a baseline for symbiont groups that may translocate to other areas/hosts in future invasions. Pontogammarus robustoides (n = 1,141) collected throughout Poland (seven sites), were noted to include a total of 13 symbiotic groups: a putative gut epithelia virus (overall prevalence = 0.6%), a putative hepatopancreatic cytoplasmic virus (1.4%), a hepatopancreatic bacilliform virus (15.7%), systemic bacteria (0.7%), fouling ciliates (62.0%), gut gregarines (39.5%), hepatopancreatic gregarines (0.4%), haplosporidians (0.4%), muscle infecting microsporidians (6.4%), digeneans (3.5%), external rotifers (3.0%), an endoparasitic arthropod (putatively: Isopoda) (0.1%), and Gregarines with putative microsporidian infections (1.4%). Parasite assemblages partially differed across collection sites. Co-infection patterns revealed strong positive and negative associations between five parasites. Microsporidians were common across sites and could easily spread to other areas following the invasion of P. robustoides. By providing this initial histopathological survey, we hope to provide a concise list of symbiont groups for risk-assessment in the case of a novel invasion by this highly invasive amphipod.

Alternosema astaquatica n. sp. (Microsporidia: Enterocytozoonida), a systemic parasite of the crayfish Faxonius virilis.

Crayfish have strong ecological impacts in freshwater systems, yet our knowledge of their parasites is limited. This study describes the first systemic microsporidium (infects multiple tissue types) Alternosema astaquatica n. sp. (Enterocytozoonida) isolated from a crayfish host, Faxonius virilis, using histopathology, transmission electron microscopy, gene sequencing, and phylogenetics. The parasite develops in direct contact with the host cell cytoplasm producing mature spores that are monokaryotic and ellipsoid in shape. Spores have 9-10 coils of the polar filament and measure 3.07 ± 0.26 µm (SD) in length and 0.93 ± 0.08 µm (SD) in width. Our novel isolate has high genetic similarity to Alternosema bostrichidis isolated from terrestrial beetles; however, genetic data from this parasite is restricted to a small fragment (396 bp) of the SSU gene. Additional data related to spore morphology and development, host, environment, and ecology indicate that our novel isolate is distinct from A. bostrichidis, which supports a new species description. Alternosema astaquatica n. sp. represents a novel member of the Orthosomella-like group which appears to be a set of opportunists within the Enterocytozoonida. The presence of this microsporidium in F. virilis could be relevant for freshwater ecosystems across this crayfish's broad geographic range in North America and may affect interactions between F. virilis and invasive rusty crayfish Faxonius rusticus in the Midwest USA.

Cambaraspora faxoni n. sp. (Microsporidia: Glugeida) from native and invasive crayfish in the USA and a novel host of Cambaraspora floridanus.

Crayfishes are among the most widely introduced freshwater taxa and can have extensive ecological impacts. Knowledge of the parasites crayfish harbor is limited, yet co-invasion of parasites is a significant risk associated with invasions. In this study, we describe a novel microsporidium, Cambaraspora faxoni n. sp. (Glugeida: Tuzetiidae), from two crayfish hosts in the Midwest USA, Faxonius virilis and Faxonius rusticus. We also expand the known host range of Cambaraspora floridanus to include Procambarus spiculifer. Cambaraspora faxoni infects muscle and heart tissue of F. rusticus and develops within a sporophorous vesicle. The mature spore measures 3.22 ± 0.14 µm in length and 1.45 ± 0.13 µm in width, with 8-9 turns of the polar filament. SSU sequencing indicates the isolates from F. virilis and F. rusticus were identical (100%) and 93.49% similar to C. floridanus, supporting the erection of a new species within the Cambaraspora genus. The novel parasite was discovered within the native range of F. rusticus (Ohio, USA) and within a native congeneric (F. virilis) in the invasive range of F. rusticus (Wisconsin, USA). Faxonius virilis is invasive in other regions. This new parasite could have been introduced to Wisconsin with F. rusticus or it may be a generalist species with a broad distribution. In either case, this parasite infects two crayfish species that have been widely introduced to new drainages throughout North America and could have future effects on invasion dynamics or impacts.

Salinity and temperature affect the symbiont profile and host condition of Florida USA blue crabs Callinectes sapidus.

Subtropical Florida blue crabs, Callinectes sapidus, exhibit differing life history traits compared to their temperate counterparts, likely influencing symbiont infection dynamics. Little information exists for Florida C. sapidus symbiont profiles, their distribution among various habitats, and influence on crab condition. Using histopathology, genomics, and transmission electron microscopy, we describe the first symbiont profiles for Florida C. sapidus occupying freshwater to marine habitats. Twelve symbiont groups were identified from 409 crabs including ciliophorans, digenean, microsporidian, Haplosporidia, Hematodinium sp., Nematoda, filamentous bacteria, gregarine, Callinectes sapidus nudivirus, Octolasmis sp., Cambarincola sp., and putative microcell. Overall, 78% of C. sapidus were documented with one or more symbiont groups demonstrating high infection rates in wild populations. Environmental variables water temperature and salinity explained 48% of the variation in symbiont groups among Florida habitats, and salinity was positively correlated with C. sapidus symbiont diversity. This suggests freshwater C. sapidus possess fewer symbionts and represent healthier individuals compared to saltwater populations. Crab condition was examined using the reflex action mortality predictor (RAMP) to determine if reflex impairment could be linked to symbiont prevalence. Symbionts were found positively correlated with crab condition, and impaired crabs were more likely to host symbionts, demonstrating symbiont inclusion may boost predictive ability of the RAMP application. The microsporidian symbiont group had a particularly strong effect on C. sapidus reflex response, and impairment was on average 1.57 times higher compared to all other symbiont groups. Our findings demonstrate the importance of considering full symbiont profiles and their associations with a spatially and temporally variable environment to fully assess C. sapidus population health.

Comparative genomics for Agmasoma sp. (Microsporidia) parasitising invasive Carcinus aestuarii and Carcinus maenas in Argentina.

Carcinus spp. are global aquatic invaders and carriers of several parasites, including a taxonomically unrecognised microsporidian recently observed from Argentina. We provide genome drafts for two parasite isolates, one from Carcinus maenas and one from Carcinus aestuarii, and use multi-gene phylogenetics and genome comparison methods to outline their similarities. Their SSU genes are 100 % similar and other genes have an average similarity of 99.31 %. We informally name the parasite Agmasoma carcini, terming the isolates Ac. var. aestuarii and Ac. var. maenas, following the wealth of genomic data available for each. This study follows on from Frizzera et al. (2021), where this parasite was first histologically identified.

Cross-Hemispheric Genetic Diversity and Spatial Genetic Structure of Callinectes sapidus Reovirus 1 (CsRV1).

The movement of viruses in aquatic systems is rarely studied over large geographic scales. Oceanic currents, host migration, latitude-based variation in climate, and resulting changes in host life history are all potential drivers of virus connectivity, adaptation, and genetic structure. To expand our understanding of the genetic diversity of Callinectes sapidus reovirus 1 (CsRV1) across a broad spatial and host life history range of its blue crab host (Callinectes sapidus), we obtained 22 complete and 96 partial genomic sequences for CsRV1 strains from the US Atlantic coast, Gulf of Mexico, Caribbean Sea, and the Atlantic coast of South America. Phylogenetic analyses of CsRV1 genomes revealed that virus genotypes were divided into four major genogroups consistent with their host geographic origins. However, some CsRV1 sequences from the US mid-Atlantic shared high genetic similarity with the Gulf of Mexico genotypes, suggesting potential human-mediated movement of CsRV1 between the US mid-Atlantic and Gulf coasts. This study advances our understanding of how climate, coastal geography, host life history, and human activity drive patterns of genetic structure and diversity of viruses in marine animals and contributes to the capacity to infer broadscale host population connectivity in marine ecosystems from virus population genetic data.

Pseudohepatospora borealis n. gen. n. sp. (Microsporidia: Enterocytozoonida): A microsporidian pathogen of the Jonah crab (Cancer borealis).

The microsporidian diversity catalogued so far has resulted in the development of several taxonomic groups, one of which is the Enterocytozoonida - a group of generalist 'ultimate opportunists', which infect many fished and aquacultured animals, as well as a broad suite of host taxa, including humans. In this study, we provide phylogenetic, ultrastructural, developmental, and pathological evidence for the creation of a new genus and species to hold a microsporidian parasite of the Jonah crab, Cancer borealis. Cancer borealis represents a species of commercial interest and has become the target of a recently developed fishery on the USA and Canadian Atlantic coast. This species was found to harbour a microsporidian parasite that develops in the cytoplasm of alpha and beta cells of the hepatopancreas. We retrieved a 937 bp fragment of the parasite SSU region, alongside developmental and ultrastructural data that suggests this species is âˆ¼ 87 % similar to Parahepatospora carcini and develops in a similar manner in direct association with the host cell cytoplasm. The mature spores are ovoid in shape and measure 1.48 ± 0.15 µm (SD) in length and 1.00 ± 0.11 µm (SD) in width. Phylogenetically, the new parasite clades in the Enterocytozoonida on the same branch as P. carcini. We provide a new genus and species to hold the parasite: Pseudohepatospora borealis n. gen. n. sp. (Microsporidia: Enterocytozoonida) and explore the likelihood that this species may fit into the Hepatoporidae family.

Rediscovering "Baculovirus-A" (Johnson, 1976): The complete genome of 'Callinectes sapidus nudivirus'.

Callinectes sapidus, or the 'blue crab', supports an extensive east-coast USA fishery and was one of the first crustacean species in which viruses were observed. Pioneering research by Dr Phyllis Johnson led to these initial discoveries, one of which included the discovery of a virus termed "Baculovirus-A". This virus was considered a potential member of the Baculoviridae, Nimaviridae, or Nudiviridae, in which all viral members are rod-shaped dsDNA viruses found in the nucleus of their host cell. With the availability of genomic and bioinformatic tools, such as Illumina HiSeq and assembly programs, it is now possible to assemble the genomes of viruses and gain additional genomic insight, which can shed light on viral taxonomy. Using these tools, alongside electron micrographs and histology slides, we reveal that the hepatopancreas-infecting 'Baculovirus-A' from Callinectes sapidus is a member of the Nudiviridae, resembling genetic and protein similarity to other crab and lobster infecting nudiviruses from the Gammanudivirus genus. Histologically, the virus causes nuclear hypertrophy as observed for other gammanuriviruses. The genome of the virus is circular, 122,436 bp in length, and encodes a predicted 98 protein coding genes, including all of the nudivirus core genes. The prevalence of virus from across Florida, USA, is provided alongside a genomic comparison of the new viral genome against other Gammanudivirus species, revealing the average prevalence to be 2.2% and that Callinectes sapidus nudivirus is distantly similar to the recently described Carcinus maenas nudivirus from Canada.

Systematic identity and phylogenetic analysis of Knowlespora clinchi (Knowles et al. 2022) gen. et comb. nov. From pheasantshell mussels (Actinonaiais pectorosa).

A microsporidian parasite infecting the pheasantshell mussel, Actinonaiais pectorosa, was discovered in a freshwater system in the USA. The original description of this species placed it into the holding genus "Microsporidium"; however, the availability of ecological, environmental, histological, electron microscopy, and genetic data, suffice to provide a complete formal taxonomic account of this species. In this note, the genus Knowlespora n. gen. is erected and described to hold the type species: Knowlespora clinchi gen. et comb. nov., originally described as 'Microsporidium clinchi'. A discussion is presented to suggest that this novel species is unlikely to be closely related to other molluscan microsporidians (all of which lack genetic data, to date) and highlights the diversity of oocyte-infecting species across the Microsporidia.

Microsporidia: a new taxonomic, evolutionary, and ecological synthesis.

Microsporidian diversity is vast. There is a renewed drive to understand how microsporidian pathological, genomic, and ecological traits relate to their phylogeny. We comprehensively sample and phylogenetically analyse 125 microsporidian genera for which sequence data are available. Comparing these results with existing phylogenomic analyses, we suggest an updated taxonomic framework to replace the inconsistent clade numbering system, using informal taxonomic names: Glugeida (previously clades 5/3), Nosematida (4a), Enterocytozoonida (4b), Amblyosporida (3/5), Neopereziida (1), and Ovavesiculida (2). Cellular, parasitological, and ecological traits for 281 well-defined species are compared with identify clade-specific patterns across long-branch Microsporidia. We suggest that future taxonomic circumscriptions of Microsporidia should involve additional markers (SSU/ITS/LSU), and that a comprehensive suite of phenotypic and ecological traits help to predict broad microsporidian functional and lineage diversity.

Microsporidian Pathogens of Aquatic Animals.

Around 57.1% of microsporidia occupy aquatic environments, excluding a further 25.7% that utilise both terrestrial and aquatic systems. The aquatic microsporidia therefore compose the most diverse elements of the Microsporidia phylum, boasting unique structural features, variable transmission pathways, and significant ecological influence. From deep oceans to tropical rivers, these parasites are present in most aquatic environments and have been shown to infect hosts from across the Protozoa and Animalia. The consequences of infection range from mortality to intricate behavioural change, and their presence in aquatic communities often alters the overall functioning of the ecosystem.In this chapter, we explore aquatic microsporidian diversity from the perspective of aquatic animal health. Examples of microsporidian parasitism of importance to an aquacultural ('One Health') context and ecosystem context are focussed upon. These include infection of commercially important penaeid shrimp by Enterocytozoon hepatopenaei and interesting hyperparasitic microsporidians of wild host groups.Out of ~1500 suggested microsporidian species, 202 have been adequately taxonomically described using a combination of ultrastructural and genetic techniques from aquatic and semi-aquatic hosts. These species are our primary focus, and we suggest that the remaining diversity have additional genetic or morphological data collected to formalise their underlying systematics.

Histopathological survey for parasite groups in Gammarus varsoviensis (Amphipoda).

Invasive non-native amphipods (Crustacea) are becoming a model system in which to explore the impact and diversity of invasive parasites-parasites that are carried along an invasion route with their hosts. Gammarus varsoviensis is a freshwater amphipod species that has a recently explored invasion history. We provide a histopathological survey for a putatively invasive non-native population of this amphipod, identifying 8 symbiotic groups: Acanthocephala, Rotifera, Digenea, ciliated protozoa, Haplosporidia, Microsporidia, 'Candidatus Aquirickettsiella', and a putative nudivirus, at various prevalence. Our survey indicates that the parasites have no sex bias and that each has the potential to be carried in either sex along an invasion route. We discuss the pathology and prevalence of the above symbiotic groups and whether those that are parasitic may pose a risk if G. varsoviensis were to carry them to novel locations.

The plot thickens: Ovipleistophora diplostomuri infects two additional species of Florida crayfish.

Ovipleistophora (Microsporidia) is a globally distributed genus of obligate parasites that infect fish, Crustacea, and trematodes. We report on two additional crayfish hosts, Procambarus pictus (Simms Creek, Florida) and Procambarus fallax (Santa Fe River, Florida), that exhibited signs of high-intensity microsporidian infection in the musculature. Sequence data (SSU) for the isolates were 99.79% and 99.97% similar to Ovipleistophora diplostomuri parasitizing Procambarus bivittatus. Additional screening of regional fish species (Lepomis macrochirus) revealed additional microsporidian diversity (Potaspora) but did not include Ovipleistophora. Detecting Clade V isolates in multiple host groups provides further evidence for trophic transmission among Clade V microsporidia.

Revising the Freshwater Thelohania to Astathelohania gen. et comb. nov., and Description of Two New Species.

Crayfish are common hosts of microsporidian parasites, prominently from the genus Thelohania. Thelohania is a polyphyletic genus, with multiple genetically distinct lineages found from freshwater and marine environments. Researchers have been calling for a revision of this group for over a decade. We provide evidence that crayfish-infecting freshwater Thelohania are genetically and phylogenetically distinct from the marine Thelohania (Clade V/Glugeida), whilst also describing two new species that give further support to the taxonomic revision. We propose that the freshwater Thelohania should be transferred to their own genus, Astathelohania gen. et comb. nov., in a new family (Astathelohaniidae n. fam.). This results in the revision of Thelohania contejeani (Astathelohania contejeani), Thelohania montirivulorum (Astathelohania montirivulorum), and Thelohania parastaci (Astathelohania parastaci). We also describe two novel muscle-infecting Astathelohania species, A. virili n. sp. and A. rusti n. sp., from North American crayfishes (Faxonius sp.). We used histological, molecular, and ultrastructural data to formally describe the novel isolates. Our data suggest that the Astathelohania are genetically distinct from other known microsporidian genera, outside any described family, and that their SSU rRNA gene sequence diversity follows their host species and native geographic location. The range of this genus currently includes North America, Europe, and Australia.

'Candidatus Mellornella promiscua' n. gen. n. sp. (Alphaproteobacteria: Rickettsiales: Anaplasmataceae): An intracytoplasmic, hepatopancreatic, pathogen of the flatback mud crab, Eurypanopeus depressus.

Bacterial pathogens are a long-standing threat to the longevity and survival of crustacean hosts. Their presence and continuing emergence require close monitoring to understand their impact on fished, cultured, and wild crustacean populations. We describe a new bacterial pathogen belonging to the Anaplasmataceae family (Alphaproteobacteria: Rickettsiales), providing pathological, ultrastructural, phylogenetic, and genomic evidence to determine a candidate genus and species ('Candidatus Mellornella promiscua'). This bacterium was found to infect the mud crab, Eurypanopeus depressus, on the North Carolina coastline (USA) at a prevalence of 10.8%. 'Candidatus Mellornella promiscua' was often observed in co-infection with the rhizocephalan barnacle, Loxothylacus panopaei. The bacterium was only found in the hepatopancreas of the mud crab host, causing cytoplasmic hypertrophy, tubule necrosis, large plaques within the cytoplasm of the host cell, and an abundance of sex-pili. The circular genome of the bacterium is 1,013,119 bp and encodes 939 genes in total. Phylogenetically, the new bacterium branches within the Anaplasmataceae. The genome is dissimilar from other described bacteria, with 16S gene similarity observed at a maximum of 85.3% to a Wolbachia endosymbiont. We explore this novel bacterial pathogen using genomic, phylogenetic, ultrastructural, and pathological methods, discussing these results in light of current bacterial taxonomy, similarity to other bacterial pathogens, and the potential impact upon the surrounding disease ecology of the host and benthic ecosystem.

Mitochondrial Genomes, Phylogenetic Associations, and SNP Recovery for the Key Invasive Ponto-Caspian Amphipods in Europe.

The Ponto-Caspian region is the main donor of invasive amphipods to freshwater ecosystems, with at least 13 species successfully established in European inland waters. Dikerogammarus spp. and Pontogammarus robustoides are among the most successful, due to their strong invasive impact on local biota. However, genomic knowledge about these invaders is scarce, while phylogeography and population genetics have been based on short fragments of mitochondrial markers or nuclear microsatellites. In this study, we provide: (i) a reconstruction of six mitogenomes for four invasive gammarids (D. villosus, D. haemobaphes, D. bispinosus, and P. robustoides); (ii) a comparison between the structure of the newly obtained mitogenomes and those from the literature; (iii) SNP calling rates for individual D. villosus and D. haemobaphes from different invasion sites across Europe; and (iv) the first time-calibrated full mitogenome phylogeny reconstruction of several Ponto-Caspian taxa. We found that, in comparison to other gammarids, the mitogenomes of Ponto-Caspian species show a translocation between the tRNA-E and tRNA-R positions. Phylogenetic reconstruction using the mitogenomes identified that Ponto-Caspian gammarids form a well-supported group that originated in the Miocene. Our study supports paraphyly in the family Gammaridae. These provided mitogenomes will serve as vital genetic resources for the development of new markers for PCR-based identification methods and demographic studies.