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Pollution, biological invasions and climate change pose the greatest threats to biodiversity worldwide. Few studies explore the interlink between invasive species and local pollution sites. This study describes the type and cytochemical properties of haemocytes from invasive Carcinus maenas on the southwestern Atlantic coast (Argentina). Moreover, the morphology of haemocytes and the total haemocyte count (THC) as well as the differential haemocyte count (DHC) of crabs sampled at clean or unpolluted and polluted sites were compared, to determine whether environmental stresses were evident in their haemocytes. Our results revealed two groups or subpopulations of haemocytes: hyalinocytes, as well as granulocytes and semi-granulocytes. The type of haemocyte from crabs from both sites showed no differences in their morphology. For cytochemical assays, only neutral red dye showed a more intense staining of crab haemocytes at the polluted site. All haemocytes were positive to Periodic Acid-Schiff and Sudan Black. THC of crabs from the clean site were significantly higher, as well as having a significantly higher proportion of hyalinocytes and a lower proportion of granular cells. This study provides a baseline immunological assessment for C. maenas haemocytes, suggesting a differential haemocytic response to environmental stress.

Invasive Non-Native Species (INNS) can co-transport externally and internally other organisms including viruses, bacteria and other eukaryotes (including metazoan parasites), collectively referred to as the symbiome. These symbiotic organisms include pathogens, a small minority of which are subject to surveillance and regulatory control, but most of which are currently unscrutinised and/or unknown. The (putatively) pathogenic symbionts co-transported by an INNS host may be latent or associated with asymptomatic infection and unable to cause disease in the INNS, but may be opportunistic pathogens of other hosts, causing impact to one or more hosts in their new range. These pathogens potentially pose diverse risks to other species, with implications for increased epidemiological risk to agriculture and aquaculture, wildlife/ecosystems, and human health (zoonotic diseases). Aquatic INNS and their symbionts have many introduction pathways, including commodity and trade (releases, escapes, contaminant), transport (stowaway), and dispersal (corridor, unaided). The risks and impacts arising from co-transported pathogens, including other symbionts of unknown pathogenic virulence, remain largely unexplored, unlegislated, and difficult to identify and quantify. Here, we propose a workflow to determine any known and potential pathogens of aquatic INNS. This workflow acts as a prerequisite for assessing the nature and risk posed by co-transported symbionts of INNS. A better understanding of co-transported organisms, the risks they pose and their impact, is necessary to inform policy and INNS risk assessments. This leap in evidence will be instrumental to devise an appropriate set of statutory responsibilities with respect to these symbionts, and to underpin new and more effective legislative processes relating to the disease screening and risk assessment of INNS.

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.

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.

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.

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 Nudiviridae are a family of large double-stranded DNA viruses that infects the cells of the gut in invertebrates, including insects and crustaceans. The phylogenetic range of the family has recently been enhanced via the description of viruses infecting penaeid shrimp, crangonid shrimp, homarid lobsters and portunid crabs. Here we extend this by presenting the genome of another nudivirus infecting the amphipod Dikerogammarus haemobaphes. The virus, which infects cells of the host hepatopancreas, has a circular genome of 119,754 bp in length, and encodes a predicted 106 open reading frames. This novel virus encodes all the conserved nudiviral genes (sharing 57 gene homologues with other crustacean-infecting nudiviruses) but appears to lack the p6.9 gene. Phylogenetic analysis revealed that this virus branches before the other crustacean-infecting nudiviruses and shares low levels of gene/protein similarity to the Gammanudivirus genus. Comparison of gene synteny from known crustacean-infecting nudiviruses reveals conservation between Homarus gammarus nudivirus and Penaeus monodon nudivirus; however, three genomic rearrangements in this novel amphipod virus appear to break the gene synteny between this and the ones infecting lobsters and penaeid shrimp. We explore the evolutionary history and systematics of this novel virus, suggesting that it be included in the novel Epsilonnudivirus genus (Nudiviridae).

Among the many Callinectes spp. across the western Atlantic, the blue crab C. sapidus has the broadest latitudinal distribution, encompassing both tropical and temperate climates. Its life history varies latitudinally, from extended overwintering at high latitudes to year-round activity in tropical locations. Callinectes sapidus reovirus 1 (CsRV1) is a pathogenic virus first described in North Atlantic C. sapidus and has recently been detected in southern Brazil. Little information exists about CsRV1 prevalence at intervening latitudes or in overwintering blue crabs. Using a quantitative reverse transcription PCR (RT-qPCR) method, this study investigated CsRV1 prevalence in C. sapidus across latitudinal differences in temperature and crab life history, as well as in additional Callinectes spp. and within overwintering C. sapidus. CsRV1 prevalence in C. sapidus was significantly correlated with high water temperature and blue crab winter dormancy. Prevalence of CsRV1 in C. sapidus on the mid-Atlantic coast was significantly lower in winter than in summer. CsRV1 infections were not detected in other Callinectes spp. These findings revealed that CsRV1 is present in C. sapidus across their range, but not in other Callinectes species, with prevalence associated with temperature and host life history. Such information helps us to better understand the underlying mechanisms that drive marine virus dynamics under changing environmental conditions.

Crayfish are common hosts of microsporidian parasites, prominently from the genus

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.

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.

Multiple enveloped viruses with rod-shaped nucleocapsids have been described, infecting the epithelial cell nuclei within the hepatopancreas tubules of crustaceans. These bacilliform viruses share the ultrastructural characteristics of nudiviruses, a specific clade of viruses infecting arthropods. Using histology, electron microscopy and high throughput sequencing, we characterise two further bacilliform viruses from aquatic hosts, the brown shrimp (Crangon crangon) and the European shore crab (Carcinus maenas). We assembled the full double stranded, circular DNA genome sequences of these viruses (~113 and 132 kbp, respectively). Comparative genomics and phylogenetic analyses confirm that both belong within the family Nudiviridae but in separate clades representing nudiviruses found in freshwater and marine environments. We show that the three thymidine kinase (tk) genes present in all sequenced nudivirus genomes, thus far, were absent in the Crangon crangon nudivirus, suggesting there are twenty-eight core genes shared by all nudiviruses. Furthermore, the phylogenetic data no longer support the subdivision of the family Nudiviridae into four genera (Alphanudivirus to Deltanudivirus), as recently adopted by the International Committee on Taxonomy of Viruses (ICTV), but rather shows two main branches of the family that are further subdivided. Our data support a recent proposal to create two subfamilies within the family Nudiviridae, each subdivided into several genera.

Say’s mud crab, Dyspanopeus sayi (Brachyura: Panopeidae) is a native shallow subtidal and inter-tidal inhabitant of the Atlantic coastline of North America and an invasive species in the Mediterranean and Black Seas. Little is known about the microparasites of this host and the broader Panopeidae. We describe a novel microsporidian parasite infecting the musculature of D. sayi from Malagash, Nova Scotia (Canada), at a prevalence of 7%. Histopathology and molecular diagnostics were used to describe pathology and parasite phylogenetics, respectively. Based on SSU rDNA gene sequencing we propose that the microsporidian requires establishment of a new genus (Panopeispora n. gen.) and species (Panopeispora mellora n. sp.), due to significant differences to closest known taxa (e.g. Facilispora margolisi [81% similarity] and Thelohania butleri [80% similarity]), residing in Clade V of the Microsporidia. Archived, wax-embedded histological material was re-processed for transmission electron microscopy to obtain preliminary details of its intracellular development cycle and ultrastructure within the host musculature. The discovery of this pathogen is discussed with relevance to microsporidian taxonomy and the potential for achieving ultrastructural data from archived material.

With over 10000 species of Amphipoda currently described, this order is one of the most diverse groups of freshwater and marine Crustacea. Members of this group are globally distributed, and many are keystone species and ecosystem engineers within their respective ecologies. As with most organisms, disease is a key factor that can alter population size, behaviour, survival, invasion potential and physiology of amphipod hosts. This review explores symbiont diversity and pathology in amphipods by coalescing a range of current and historical literature to provide the first full review of our understanding of amphipod disease. The review is broken into 2 parts. The first half explores amphipod microparasites, which include data pertaining to viruses, bacteria, fungi, oomycetes, microsporidians, dinoflagellates, myxozoans, ascetosporeans, mesomycetozoeans, apicomplexans and ciliophorans. The second half reports the metazoan macroparasites of Amphipoda, including rotifers, trematodes, acanthocephalans, nematodes, cestodes and parasitic Crustacea. In all cases we have endeavoured to provide a complete list of known species that cause disease in amphipods, while also exploring the effects of parasitism. Although our understanding of disease in amphipods requires greater research efforts to better define taxonomic diversity and host effects of amphipod symbionts, research to date has made huge progress in cataloguing and experimentally determining the effects of disease upon amphipods. For the future, we suggest a greater focus on developing model systems that use readily available amphipods and diseases, which can be comparable to the diseases in other Crustacea that are endangered, economically important or difficult to house.

This study provides morphological, ultrastructural and phylogenetic characterization of 2 novel species of Haplosporidia (Haplosporidium echinogammari n. sp. and H. orchestiae n. sp.) infecting amphipods of the genera Echinogammarus and Orchestia collected in southwestern England. Both parasites infect the connective tissues associated with the digestive gland and the tegument, and eventually infect other organs causing disruption of host tissues with associated motor impairment and fitness reduction. Prevalence of infection varied with host species, provenance and season, being as high as 75% for individuals of E. marinus infected with H. echinogammari in June (n = 50). Although no spores were found in any of the infected amphipods examined (n = 82), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the 2 new species. Phylogenetic analysis of the new species based on the small subunit (SSU) rDNA gene placed H. echinogammari close to H. diporeiae in haplosporidian lineage C, and H. orchestiae in a novel branch within Haplosporidium. Genetic diversity of the haplosporidians infecting these and other amphipod species was evaluated and compared to morphological and ultrastructural changes to host tissues. The phylogenetic relationship of haplosporidian infections in other crustacean hosts is discussed after inclusion into the analysis of 25 novel SSU rDNA sequences obtained from crabs, isopods and crayfish.

The amphipod Dikerogammarus haemobaphes is a high-risk carrier of parasites that impact wildlife in its non-native range. Studies using the mitochondrial genes, Cytochrome Oxidase Sub-Unit 1 (cox1) and small-subunit ribosomal RNA gene (16S), provide some nucleotide detail for understanding the evolution and phylogeography of this species. Despite this, the origins of the invasion remain unknown, as do the origins of its parasites. This study provides the full annotated mitochondrial genome (15,460 bp) of D. haemobaphes, consisting of 2 rRNAs, 24 tRNAs and 14 protein coding genes. Mitochondrial genes from the UK isolate are compared to existing data on NCBI and are used in a concatenated phylogenetic approach and identify D. haemobaphes as an early member of the Gammaridae (Amphipoda). Viral, bacterial, protistan and microsporidian parasites are present across the Gammaridae, including D. haemobaphes, suggesting the ancestor of the Gammaridae harboured related diseases, and that further screening of amphipods is likely to reveal further microparasite diversity. This correlation suggests that other gammarid invaders have the potential to harbour a range of microparasites. The mitochondrial genome of this species will act a resource to facilitate our understanding of geneflow, disease epidemiology and evolutionary history in this invasion-disease model.

Carcinus maenas is in the top 100 globally invasive species and harbours a wide diversity of pathogens, including viruses. We provide a detailed description for a novel bunyavirus (Carcinus maenas Portunibunyavirus 1) infecting C. maenas from its native range in the Faroe Islands. The virus genome is tripartite, including large (L) (6766 bp), medium (M) (3244 bp) and small (S) (1608 bp) negative sense, single-stranded RNA segments. Individual genomic segments are flanked by 4 bp regions of similarity (CCUG). The segments encode an RNA-dependent RNA-polymerase, glycoprotein, non-structural protein with a Zinc-Finger domain and a nucleoprotein. Most show highest identity to the ‘Wenling Crustacean Virus 9’ from an unidentified crustacean host. Phylogenomics of crustacean-infecting bunyaviruses place them across multiple bunyavirus families. We discuss the diversity of crustacean bunyaviruses and provide an overview of how these viruses may affect the health and survival of crustacean hosts, including those inhabiting niches outside of their native range.

Crustaceans suffer from diseases that can alter their survival and ecology with additional economic consequences for fisheries and aquaculture. Many parasites have been described from crustaceans and with the advent of novel technologies such as next generation sequencing, the discovery of novel parasites has become increasingly efficient. Molecular techniques are beginning to surpass more conventional tools for parasite discovery, but they typically do not provide information on pathology. Histopathology remains one of the least expensive methods for parasite discovery and allows for both detection of parasites and descriptions of the pathology they cause. When used in concert with modern molecular and electron microscopy techniques, the approach is powerful; however, there are few informational tools for the interpretation of histological slides from crustaceans. Those available do not provide comprehensive images of all organs and early works were limited to lower resolution than currently available. More recent texts provide in-depth details of infection in histological section, but few provide images of healthy material or describe a baseline from which to compare. Here, we provide a series of image plates derived from histologically processed tissues from three palinurid lobsters: Panulirus argus, Palinurus elephas and Panulirus guttatus. Histology from these lobsters shows high visual similarity in all tissue types. We provide a histological atlas of healthy tissue that can be used as a baseline resource for pathobiologists working on these common species (and related crustaceans) and we discuss how disease may result in visual aberrations to these tissues.

The Caribbean spiny lobster Panulirus argus is susceptible to infection by Panulirus argus Virus 1 (PaV1), the only virus known to naturally infect any lobster species. However, P. argus is able to mitigate PaV1 transmission risk by avoiding infected individuals. White Spot Syndrome Virus (WSSV) has a particularly wide host range. WSSV has not been documented in wild populations of spiny lobsters, but has been experimentally transmitted to six other lobster species from the genus Panulirus spp. While WSSV has been detected intermittently in wild populations of shrimp in the Caribbean region, the risk to P. argus has not been evaluated. Potential emergence of the disease could result in fisheries losses and ecological disruption. To assess the risk to P. argus, we tested its susceptibility to WSSV via injection and waterborne transmission. We also tested whether healthy lobsters can detect and avoid conspecifics with qPCR-quantifiable WSSV infections. We found P. argus to be highly susceptible to WSSV via intramuscular injection, with mortality reaching 88% four weeks post inoculation. Panulirus argus was also susceptible to WSSV via waterborne transmission, but WSSV burden was low after four weeks via qPCR. Behavioral assays indicated that P. argus can detect and avoid conspecifics infected with WSSV and the avoidance response was strongest for the most heavily infected individuals – a response comparable to PaV1-infected conspecifics. Panulirus argus is the first spiny lobster found to be susceptible to WSSV in the Americas, but it is possible that a generalized avoidance response by healthy lobsters against infected conspecifics provides a behavioral defense and may reduce WSSV infection potential and prevalence. Such avoidance may extend to other directly transmitted pathogens in spiny lobster populations preventing them from becoming common in their population.Author SummaryErica P. Ross is a PhD candidate at the University of Florida, studying the disease ecology of the Caribbean spiny lobster, with a focus on chemosensory ecology. Donald C. Behringer is an associate professor at the University of Florida and his research focuses on disease ecology, epidemiology, and fishery ecology, with a focus on crustaceans and other marine invertebrates. Jamie Bojko received his PhD from the University of Leeds and is currently a post-doctorate associate at the University of Florida studying experimental and systemic crustacean pathology.

Invasive crayfish and the introduction of non-native diseases pose a significant risk for the conservation of endangered, white-clawed crayfish (Austropotamobius pallipes). Continued pollution of waterways is also of concern for native species and may be linked with crayfish disease dynamics. We explore whether crayfish species or environmental quality are predictors of infection presence and prevalence in native A. pallipes and invasive signal crayfish (Pacifastacus leniusculus). We use a seven-year dataset of histology records, and a field survey comparing the presence and prevalence of infectious agents in three isolated A. pallipes populations; three isolated P. leniusculus populations, and three populations where the two species had overlapped in the past. We note a lower diversity of parasites (Simpson’s Index) in P. leniusculus (‘Pacifastacus leniusculus Bacilliform Virus’ - PlBV) (n = 1 parasite) relative to native A. pallipes (n = 4 parasites), which host Thelohania contejeani, ‘Austropotamobius pallipes bacilliform virus’ (ApBV), Psorospermium haeckeli and Branchiobdella astaci, at the sites studied. The infectious group present in both species was an intranuclear bacilliform virus of the hepatopancreas. The prevalence of A. astaci in A. pallipes populations was higher in more polluted water bodies, which may reflect an effect of water quality, or may be due to increased chance of transmission from nearby P. leniusculus, a species commonly found in poor quality habitats.

Jennings, Lucas A., Bojko, Jamie, Rotjan, Randi D., Behringer, Donald C. (2021): Cirolana westbyi, (Isopoda: Cirolanidae) a new species in the 'Cirolana parva-group' from the Turneffe Atoll, Belize. Journal of Natural History 54 (31-32): 2053-2069, DOI: 10.1080/00222933.2020.1837273, URL: http://dx.doi.org/10.1080/00222933.2020.1837273

Abstract This study provides a broad phylogenetic analysis for the Eubrachyura, with the inclusion of three new Panopeidae mitochondrial genomes: Eurypanopeus depressus (flatback mud crab) (15,854bp), Panopeus herbstii (Atlantic mud crab) (15,812bp) and Rhithropanopeus harrisii (Harris, or ‘white-fingered’ mud crab) (15,892bp). These new mitogenomes were analyzed alongside all available brachyuran mitochondrial genomes (n = 113), comprising 80 genera from 29 families, to provide an updated phylogenetic analysis of the infra-order Brachyura (“true crabs”). Our analyses support the subsection Potamoida within the Eubrachyura as the sister group to Thoracotremata. The family Panopeidae aligns with the family Xanthidae to form the Xanthoidea branch, which is supported by current morphological and genetic taxonomy. A unique gene arrangement termed ‘XanGO’ was identified for the panopeids and varies relative to other members of the subsection Heterotremata (within the Eubrachyura) via a transposition of the trnV gene. This gene arrangement is novel and is shared between several Xanthoidea species, including Etisus anaglyptus (hairy spooner crab), Atergatis floridus (brown egg crab), and Atergatis integerrimus (red egg crab), suggesting that it is a conserved gene arrangement within the Xanthoidea superfamily. Our study further reveals a need for taxonomic revision of some brachyuran groups, particularly the Sesarmidae. The inclusion of panopeid mitogenomes into the greater brachyuran phylogeny increases our understanding of crab evolution and higher level Eubrachyuran systematics.

Unfortunately, the inclusion of original names (in non-Latin script) of the following authors caused problems with author name indexing in PubMed. Therefore, these original names were removed from XML data to correct the PubMed record. Mengji Cao, Yuya Chiaki, Hideki Ebihara, Jingjing Fu, George Fú Gāo, Tong Han, Jiang Hong, Ni Hong, Seiji Hongo, Masayuki Horie, Dàohóng Jiāng, Fujio Kadono, Hideki Kondō, Kenji Kubota, Shaorong Li, Longhui Li, Jiànróng Lǐ, Huazhen Liu, Tomohide Natsuaki, Sergey V. Netesov, Anna Papa, Sofia Paraskevopoulou, Liying Qi, Takahide Sasaya, Mang Shi, Xiǎohóng Shí, Zhènglì Shí, Yoshifumi Shimomoto, Jin‑Won Song, Ayato Takada, Shigeharu Takeuchi, Yasuhiro Tomitaka, Keizō Tomonaga, Shinya Tsuda, Changchun Tu, Tomio Usugi, Nikos Vasilakis, Jiro Wada, Lin‑Fa Wang, Guoping Wang, Yanxiang Wang, Yaqin Wang, Tàiyún Wèi, Shaohua Wen, Jiangxiang Wu, Lei Xu, Hironobu Yanagisawa, Caixia Yang, Zuokun Yang, Lifeng Zhai, Yong‑Zhen Zhang, Song Zhang, Jinguo Zhang, Zhe Zhang, Xueping Zhou. In addition, the publication call-out in the supplementary material was updated from issue 11 to issue 12. The original article has been corrected.

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.

The blue crab Callinectes sapidus is one of the most widely studied marine crustaceans due to its high economic value and ecological significance. Despite extensive research on the blue crab in North America, many questions remain about the distribution and abundance of the species in the subtropics and tropics. In many places, C. sapidus is sympatric with morphologically similar Callinectes spp., which has implications for seafood mislabeling. To enable rapid identification of the species, we designed and tested two PCR-based assays targeting the 12S rRNA mitochondrial gene. The first assay discriminates C. sapidus from other Callinectes spp. via post-PCR restriction digestion (PCR-RFLP) and the second assay discriminates among multiple Callinectes spp. through High Resolution Melting (HRM) analysis and supervised machine learning analyses. A total of 58 DNA samples from five Callinectes spp. (validated via 12S gene sequencing) were used for assay testing. The PCR RFLP assay was 100% accurate identifying C. sapidus from other Callinectes spp. HRM analysis of amplicons showed good discrimination among species, with distinct clusters formed between species with higher sequence homology. Linear discriminant analysis (LDA) classification of HRM curves was quite successful given the small dataset available, producing ~90-91% mean accuracy in classification over all species with 100-fold cross validation. Much of the error came from misclassifications between C. similis and C. danae, which are ~99% similar in sequence for the amplicon; collapsing them into a single class increased overall classification success to 94%. Error also arose from C. bocourti classifications, which had a reference set containing only three samples. Classification accuracy of C. sapidus alone via HRM was 97.5%. Overall, these assays show great promise as rapid and inexpensive methods to identify Callinectes spp. and have application for both ecological research and seafood identification or labeling.

Invasive non-native species (INNS) pose a risk as vectors of parasitic organisms (Invasive Parasites). Introducing invasive parasites can result in ecological disturbances, leading to biodiversity loss and native species illness/mortality, but occasionally can control INNS limiting their impact. Risks to human health and the economy are also associated with INNS and invasive parasites; however, we understand little about the diversity of symbiotic organisms co-invading alongside INNS. This lack of clarity is an important aspect of the ‘One Health’ prerogative, which aims to bridge the gap between human, wildlife, and ecosystem health. To explore symbiont diversity associated with the invasive crustacean group (including: crab, lobster, crayfish, shrimp, amphipod, isopod, copepod, barnacle, other) (n = 323) derived from 1054 aquatic invertebrates classed as INNS across databases, we compile literature (year range 1800–2017) from the native and invasive range to provide a cumulative symbiont profile for each species. Our search indicated that 31.2% of INN crustaceans were known to hold at least one symbiont, whereby the remaining 68.8% had no documented symbionts. The symbiont list mostly consisted of helminths (27% of the known diversity) and protists (23% of the known diversity), followed by bacteria (12%) and microsporidians (12%). Carcinus maenas, the globally invasive and extremely well-studied green crab, harboured the greatest number of symbionts (n = 72). Additional screening is imperative to become more informed on invasive symbiont threats. We reveal that few studies provide truly empirical data that connect biodiversity loss with invasive parasites and suggest that dedicated studies on available systems will help to provide vital case studies. Despite the lack of empirical data, co-invasive parasites of invasive invertebrates appear capable of lowering local biodiversity, especially by causing behavioural change and mortality in native species. Alternatively, several invasive parasites appear to protect ecosystems by controlling the impact and population size of their invasive host. We provide a protocol that could be followed to explore symbiont diversity in invasive groups as part of our case studies. The consequence of limited parasite screening of INNS, in addition to the impacts invasive parasites impart on local ecologies, are explored throughout the review. We conclude in strong support of the ‘One Health’ prerogative and further identify a need to better explore disease in invasion systems, many of which are accountable for economic, human health and ecological diversity impacts.

Recent genomic and metagenomic studies have led to a dramatic expansion of the known diversity of nucleocytoplasmic large DNA viruses (NCLDVs) of eukaryotes, which include giant viruses of protists and important pathogens of vertebrates, such as poxviruses. However, the characterization of viruses from nonmodel hosts still lags behind. We sequenced the complete genomes of three viruses infecting crustaceans, the Caribbean spiny lobster, demon shrimp, and European shore crab. These viruses have the smallest genomes among the known NCLDVs, with losses of many core genes, some of which are shared with iridoviruses. The deterioration of the transcription apparatus is compatible with microscopic and ultrastructural observations indicating that these viruses replicate in the nucleus of infected cells rather than in the cytoplasm. Phylogenomic analysis indicates that these viruses are sufficiently distinct from all other NCLDVs to justify the creation of a separate family, for which we propose the name “Mininucleoviridae” (i.e., small viruses reproducing in the cell nucleus)., Panulirus argus virus 1 (PaV1) is the only known virus infecting the Caribbean spiny lobster (Panulirus argus) from the Caribbean Sea. Recently, related viruses, Dikerogammarus haemobaphes virus 1 (DhV1) and Carcinus maenas virus 1 (CmV1), have been detected in the demon shrimp (Dikerogammarus haemobaphes) and the European shore crab (Carcinus maenas), respectively, from sites in the United Kingdom. The virion morphology of these crustacean viruses is similar to that of iridoviruses. However, unlike iridoviruses and other nucleocytoplasmic large DNA viruses (NCLDVs), these viruses complete their morphogenesis in the host cell nucleus rather than in the cytoplasm. To date, these crustacean viruses have remained unclassified due to a lack of genomic data. Using an Illumina MiSeq sequencer, we sequenced the complete genomes of PaV1, CmV1, and DhV1. Comparative genome analysis shows that these crustacean virus genomes encode the 10 hallmark proteins previously described for the NCLDVs of eukaryotes, strongly suggesting that they are members of this group. With a size range of 70 to 74 kb, these are the smallest NCLDV genomes identified to date. Extensive gene loss, divergence of gene sequences, and the accumulation of low-complexity sequences reflect the extreme degradation of the genomes of these “minimal” NCLDVs rather than any direct relationship with the NCLDV ancestor. Phylogenomic analysis supports the classification of these crustacean viruses as a distinct family, “Mininucleoviridae,” within the pitho-irido-Marseille branch of the NCLDVs.

Invasive and non-native species can pose risks to vulnerable ecosystems by co-introducing bacterial pathogens. Alternatively, co-introduced bacterial pathogens may regulate invasive population size and invasive traits. We describe a novel candidate genus and species of bacteria (‘Candidatus Aquirickettsiella gammari’) found to infect Gammarus fossarum, from its native range in Poland. The bacterium develops intracellularly within the haemocytes and cells of the musculature, hepatopancreas, connective tissues, nervous system and gonad of the host. The developmental cycle of ‘Candidatus Aquirickettsiella gammari’ includes an elementary body (496.73 nm ± 37.56 nm in length, and 176.89 nm ± 36.29 nm in width), an elliptical, condensed spherical stage (737.61 nm ± 44.51 nm in length and 300.07 nm ± 44.02 nm in width), a divisional stage, and a spherical initial body (1397.59 nm ± 21.26 nm in diameter). We provide a partial genome for ‘Candidatus Aquirickettsiella gammari’, which clades phylogenetically alongside environmental 16S rRNA sequences from aquatic habitats, and bacterial symbionts from aquatic isopods (Asellus aquaticus), grouping separately from the Rickettsiella, a genus that includes bacterial pathogens of terrestrial insects and isopods. Increased understanding of the diversity of symbionts carried by G. fossarum identifies those that might regulate host population size, or those that could pose a risk to native species in the invasive range. Identification of ‘Candidatus Aquirickettsiella gammari’ and its potential for adaptation as a biological control agent is explored.

Biological invasions have the capacity to introduce non-native parasites. This study aimed to determine whether the invasive green crab population, Carcinus spp., on the Southwestern Atlantic coast of Argentina harbours any symbionts, and whether these may spillover or spillback between native crabs, Cyrtograpsus altimanus and C. angulatus. Macroscopy, histology, and molecular analyses of some parasites were used to describe and compare their diversity across the three species of crab. We also evaluated the susceptibility of invasive Carcinus spp. to a native digenean, Maritrema madrynense, via experimental infections (exposure and cohabitation). Our results revealed that the green crab pathobiome included similar symbiotic groups to native crabs. This included putative viral, bacterial, and protozoan parasites. Haplosporidium-like observations were recorded in all crab species, and a single green crab was found to be parasitized by an Agmasoma-like microsporidium. Metagenomic analysis of one individual revealed additional symbiotic diversity (46 bacteria, 5 eukaryotic species). The green crabs were infected by more microparasite taxa than the native crabs (5:3). Wild populations of Carcinus spp. were free of metazoan parasites and are shown not to be susceptible to M. madryense under experimental conditions. Our results suggest a reduction/escape of macroparasites (trematode Maritrema madrynense; acanthocephalan Profilicollis chasmagnathi) in invasive Carcinus spp. compared to their native competitors.

Littorina saxatilis is a common intertidal gastropod on shores of the North Atlantic, and an 24 important study species for evolutionary investigations. Its congener L. arcana is much less 25 widely distributed, but both species are common at Old Peak, Yorkshire, UK. The parasite 26 profiles of L. saxatilis and L. arcana from this shore were determined histologically, revealing 27 a ciliated protist, Protophrya ovicola, an unidentified apicomplexan (present in

A single-stranded DNA (ssDNA) virus is presented from a metagenomic data set derived from Alphaproteobacteria -infected hepatopancreatic tissues of the crab Eurypanopeus depressus . The circular virus genome (4,768 bp) encodes 14 hypothetical proteins, some similar to other bacteriophages ( Microviridae ). Based on its relatedness to other Microviridae , this virus represents a member of a novel genus.