Kelly S. Bateman

Microsporidia: diverse, dynamic, and emergent pathogens in aquatic systems

Microsporidia cause important diseases in aquatic organisms. Susceptible hosts span a remarkable taxonomic spectrum, from protists to mammals. Known genera represent a small fraction of extant taxa in global aquatic systems. Transmitting horizontally or vertically, effects range from cryptic to catastrophic; individual level effects being extrapolated to populations and ecosystems. Debate over positioning within the eukaryotes and internal structuring of the phylum has benefited from molecular phylogenetic approaches to taxonomy. Similar tools offer new perspectives on transmission between hosts of differing trophic status and habitats. Accessible models for host-pathogen interaction are emerging from full genome sequencing projects. Hyperparasitism and the close phylogenetic relationship between taxa infecting invertebrates and vertebrates not only underline a ubiquity in aquatic systems but also potential for zoonotic transfer.

Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships

A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7x1.1microm and contained a single nucleus, 5-6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10nm) and an electron-dense exospore (2nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.

A RANKING SYSTEM FOR THE EVALUATION OF INTERSEX CONDITION IN EUROPEAN FLOUNDER (PLATICHTHYS FLESUS )

Intersex (ovotestis) is the condition whereby an individual possesses oocytes, at varying degrees of severity, within the normal testicular tissue and is regarded as an endpoint of endocrine disruption in male fish. The occurrence of intersex has been linked with exposure to endocrine-disrupting compounds present in the environment, which are thought to mimic the actions of the female sex hormone 17-beta-oestradiol. Using stained histological sections of archived cases of intersex from European flounder (Platichthys flesus), this study developed a grading method to assess the extent of the condition. This was termed the ovotestis severity index (OSI). The OSI utilizes the distribution pattern (focal, diffuse, cluster, zonal) and type of oocyte (previtellogenic, cortical alveolar, vitellogenic) to assign a severity rating to each intersex fish. A high OSI refers to fish with testicular material almost completely replaced with mature (vitellogenic) oocytes, and lower scores indicate those fish with small numbers of immature (previtellogenic) oocytes. The development of this index provides pathologists with a robust tool for the grading of the intersex condition in European flounder and potentially in other fish species.

Morphological and Molecular Characterization of Hematodinium perezi (Dinophyceae: Syndiniales), a Dinoflagellate Parasite of the Harbour Crab, Liocarcinus depurator

Hematodinium perezi Chatton and Poisson 1931 (Dinophyceae: Syndiniales) is reported from one of its type hosts, Liocarcinus depurator, from Rye Bay in the English Channel, a site in a similar geographical location to that of the type description. The histology and ultrastructure of vegetative trophont stages, and rDNA sequences of the parasite infecting this host are reported for the first time. Ultrastructurally, H. perezi was confirmed by the presence of condensed chromatin profiles, trichocysts, an alveolar membrane, and micropores. The pathology of H. perezi was similar to other Hematodinium descriptions with large numbers of parasites present within the haemolymph and host tissues. No host responses against the parasite were observed. Molecular analysis of the ITS rRNA regions from H. perezi infecting L. depurator suggests that Callinectes sapidus from the United States, and Portunus trituberculatus and Scylla serrata from China are infected with different genotypes of H. perezi. The morphological and molecular characterization of H. perezi in one of the type hosts from Europe will allow for a better understanding of the phylogeny of these pathogens of commercially important Crustacea.

Molecular Mechanisms of White Spot Syndrome Virus Infection and Perspectives on Treatments

Since its emergence in the 1990s, White Spot Disease (WSD) has had major economic and societal impact in the crustacean aquaculture sector. Over the years shrimp farming alone has experienced billion dollar losses through WSD. The disease is caused by the White Spot Syndrome Virus (WSSV), a large dsDNA virus and the only member of the Nimaviridae family. Susceptibility to WSSV in a wide range of crustacean hosts makes it a major risk factor in the translocation of live animals and in commodity products. Currently there are no effective treatments for this disease. Understanding the molecular basis of disease processes has contributed significantly to the treatment of many human and animal pathogens, and with a similar aim considerable efforts have been directed towards understanding host–pathogen molecular interactions for WSD. Work on the molecular mechanisms of pathogenesis in aquatic crustaceans has been restricted by a lack of sequenced and annotated genomes for host species. Nevertheless, some of the key host–pathogen interactions have been established: between viral envelope proteins and host cell receptors at initiation of infection, involvement of various immune system pathways in response to WSSV, and the roles of various host and virus miRNAs in mitigation or progression of disease. Despite these advances, many fundamental knowledge gaps remain; for example, the roles of the majority of WSSV proteins are still unknown. In this review we assess current knowledge of how WSSV infects and replicates in its host, and critique strategies for WSD treatment.

De novo assembly of the Carcinus maenas transcriptome and characterization of innate immune system pathways

BackgroundThe European shore crab, Carcinus maenas, is used widely in biomonitoring, ecotoxicology and for studies into host-pathogen interactions. It is also an important invasive species in numerous global locations. However, the genomic resources for this organism are still sparse, limiting research progress in these fields. To address this resource shortfall we produced a C. maenas transcriptome, enabled by the progress in next-generation sequencing technologies, and applied this to assemble information on the innate immune system in this species.ResultsWe isolated and pooled RNA for twelve different tissues and organs from C. maenas individuals and sequenced the RNA using next generation sequencing on an Illumina HiSeq 2500 platform. After de novo assembly a transcriptome was generated encompassing 212,427 transcripts (153,699 loci). The transcripts were filtered, annotated and characterised using a variety of tools (including BLAST, MEGAN and RSEM) and databases (including NCBI, Gene Ontology and KEGG). There were differential patterns of expression for between 1,223 and 2,741 transcripts across tissues and organs with over-represented Gene Ontology terms relating to their specific function. Based on sequence homology to immune system components in other organisms, we show both the presence of transcripts for a series of known pathogen recognition receptors and response proteins that form part of the innate immune system, and transcripts representing the RNAi, Toll-like receptor signalling, IMD and JAK/STAT pathways.ConclusionsWe have produced an assembled transcriptome for C. maenas that provides a significant molecular resource for wide ranging studies in this species. Analysis of the transcriptome has revealed the presence of a series of known targets and functional pathways that form part of their innate immune system and illustrate tissue specific differences in their expression patterns.

Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. infecting invasive Chinese mitten crabs (Eriocheir sinensis) in Europe.

We describe a microsporidian parasite infecting non-native Chinese mitten crabs (Eriochier sinensis) from Europe. Electron microscopy revealed merogonic and sporogonic life stages bound within a plasmalemma. The crab parasite develops polar tube precursors at the sporont stage but does not complete formation of the intact spore extrusion apparatus at the stage of the sporogonial plasmodium like Enterocytozoon bienuesi and other representatives of the Enterocytozoonidae. Its presence within an aquatic crustacean host, and a distinct molecular phylogeny based on partial small subunit ribosomal RNA (SSU rRNA) gene sequences also place it relatively close, though distinct to, existing genera within the Enterocytozoonidae. Consideration of morphological and phylogenetic characteristics of other hepatopancreas-infecting microsporidia from crustaceans suggests that certain ones (e.g. Enterospora canceri) are retained within the clade corresponding to the existing family Enterocytozoonidae, while others, including the parasite described here, may eventually be grouped in a sister taxon potentially of family rank. Based upon morphological and host similarity, it is likely that the parasite described here is the same as Endoreticulatus eriocheir (Wang and Chen, 2007), previously described from Chinese mitten crabs in Asia. However, using a combined taxonomic approach based upon morphological and phylogenetic data, we propose the formation of a new genus (Hepatospora) to replace the previous generic classification of the Asian parasite as Endoreticulatus. The microsporidian from the hepatopancreas of E. sinensis is named Hepatospora eriocheir (Wang and Chen, 2007) gen. et comb. nov. It is assumed that the parasite was introduced during initial invasions of this crab to Europe during the early 20th Century.

Susceptibility to infection and pathogenicity of White Spot Disease (WSD) in non-model crustacean host taxa from temperate regions.

Despite almost two decades since its discovery, White Spot Disease (WSD) caused by White Spot Syndrome Virus (WSSV) is still considered the most significant known pathogen impacting the sustainability and growth of the global penaeid shrimp farming industry. Although most commonly associated with penaeid shrimp farmed in tropical regions, the virus is also able to infect, cause disease and kill a wide range of other decapod crustacean hosts from temperate regions, including lobsters, crabs, crayfish and shrimp. For this reason, WSSV has recently been listed in European Community Council Directive 2006/88. Using principles laid down by the European Food Safety Authority (EFSA) we applied an array of diagnostic approaches to provide a definitive statement on the susceptibility to White Spot Syndrome Virus (WSSV) infection in seven ecologically or economically important crustacean species from Europe. We chose four marine species: Cancer pagurus, Homarus gammarus, Nephrops norvegicus and Carcinus maenas; one estuarine species, Eriocheir sinensis and two freshwater species, Austropotamobius pallipes and Pacifastacus leniusculus. Exposure trials based upon natural (feeding) and artificial (intra-muscular injection) routes of exposure to WSSV revealed universal susceptibility to WSSV infection in these hosts. However, the relative degree of susceptibility (measured by progression of infection to disease, and mortality) varied significantly between host species. In some instances (Type 1 hosts), pathogenesis mimicked that observed in penaeid shrimp hosts whereas in other examples (Types 2 and 3 hosts), infection did not readily progress to disease, even though hosts were considered as infected and susceptible according to accepted principles. Results arising from challenge studies are discussed in relation to the potential risk posed to non-target hosts by the inadvertent introduction of WSSV to European waters via trade. Furthermore, we highlight the potential for susceptible but relatively resistant hosts to serve as models to investigate natural mitigation strategies against WSSV in these hosts. We speculate that these non-model hosts may offer a unique insight into viral handling in crustaceans.

Mikrocytids Are a Broadly Distributed and Divergent Radiation of Parasites in Aquatic Invertebrates

Microcell parasites have independently evolved in several eukaryotic lineages and are increasingly recognized as important and emerging pathogens of diverse hosts, including species of economic importance subject to international legislation concerning the trading of aquatic animals [1-3]. The microcell Mikrocytos mackini causes Denman Island disease of oysters and represents one of the most genetically divergent eukaryotes known. Mikrocytos has remained an isolated lineage with a limited distribution. We investigated two emerging diseases of juvenile crabs and oysters from the UK using massively parallel sequencing and targeted primer approaches to reveal that their causative agents are highly divergent lineages related to M. mackini (Paramikrocytos canceri n. gen. et n. sp. and M. mimicus sp. nov., respectively). We demonstrate a major new globally distributed parasite radiation (Mikrocytida ord. nov.) with phylogenetic affinities to the commercially important haplosporidian parasites of invertebrates. Mikrocytids have eluded detection because of their small size, intracellular habit, and extreme sequence divergence. P. canceri was frequently detected in a range of shoreline invertebrates, demonstrating that these newly recognized parasites are in fact common, diverse, and widespread and should be considered when assessing the risks of aquaculture activities, invasive species spread, and movements of ballast water and sediments with associated invertebrates.

Sphaerothecum destruens pathology in cyprinids

Sphaerothecum destruens is a significant intracellular parasite of fish which has caused disease and mortalities in cultured north American Chinook salmon Oncorhynchus tshawytscha and Atlantic salmon Salmo salar. Several hosts for S. destruens have been identified within the Salmonidae family, and the histopathology of the infection can differ between hosts. Recently, S. destruens has been associated with the most invasive cyprinid species in Europe, topmouth gudgeon Pseudorasbora parva. Accurate disease identification based on thorough descriptions of clinical signs and histopathology in this new range of hosts is thus paramount to support further epizootiological studies. In this study, the associated histopathology of S. destruens infection is described along with its pathogenesis in the endangered cyprinid sunbleak Leucaspius delineatus. Histological examination of 100 L. delineatus in a wild population in the south of England revealed the presence of S. destruens infections, with a prevalence of 5% with S. destruens, suggesting an over-dispersed distribution within the L. delineatus sample. Clinical signs of the infection were absent, but histological examination revealed the presence of both disseminated and nodular lesions in several organs.