Ultimate opportunists - The emergent Enterocytozoon group Microsporidia

This is the final version. Available on open access from Public Library of Science via the DOI in this recordDefraBritish Council Newton FundUniversity of Exete

Cucumispora ornata n. sp. (Fungi: Microsporidia) infecting invasive 1 ‘demon shrimp’

Dikerogammarus haemobaphes, the ‘demon shrimp’, is an amphipod native to the Ponto-Caspian region. This species invaded the UK in 2012 and has become widely established. Dikerogammarus haemobaphes has the potential to introduce non-native pathogens into the UK, creating a potential threat to native fauna. This study describes a novel species of microsporidian parasite infecting 72.8% of invasive D. haemobaphes located in the River Trent, UK. The microsporidium infection was systemic throughout the host; mainly targeting the sarcolemma of muscle tissues. Electron microscopy revealed this parasite to be diplokaryotic and have 7-9 turns of the polar filament. The microsporidium is placed into the ‘Cucumispora’ genus based on host histopathology, fine detail parasite ultrastructure, a highly similar life-cycle and SSU rDNA sequence phylogeny. Using this data this novel microsporidian species is named Cucumispora ornata, where ‘ornata’ refers to the external beading present on the mature spore stage of this organism. Alongside a taxonomic discussion, the presence of a novel Cucumispora sp. in the United Kingdom is discussed and related to the potential control of invasive Dikerogammarus spp. in the UK and the health of native species which may come into contact with this parasite

Disease will limit future food supply from the global crustacean fishery and aquaculture sectors

Seafood is a highly traded food commodity. Farmed and captured crustaceans contribute a significant proportion with annual production exceeding 10 M metric tonnes with first sale value of $40bn. The sector is dominated by farmed tropical marine shrimp, the fastest growing sector of the global aquaculture industry. It is significant in supporting rural livelihoods and alleviating poverty in producing nations within Asia and Latin America while forming an increasing contribution to aquatic food supply in more developed countries. Nations with marine borders often also support important marine fisheries for crustaceans that are regionally traded as live animals and commodity products. A general separation of net producing and net consuming nations for crustacean seafood has created a truly globalised food industry. Projections for increasing global demand for seafood in the face of level or declining fisheries requires continued expansion and intensification of aquaculture while ensuring best utilisation of captured stocks. Furthermore, continued pressure from consuming nations to ensure safe products for human consumption are being augmented by additional legislative requirements for animals (and their products) to be of low disease status. As a consequence, increasing emphasis is being placed on enforcement of regulations and better governance of the sector; currently this is a challenge in light of a fragmented industry and less stringent regulations associated with animal disease within producer nations. Current estimates predict that up to 40$3bn) is lost annually, mainly due to viral pathogens for which standard preventative measures (e.g. such as vaccination) are not feasible. In light of this problem, new approaches are urgently required to enhance yield by improving broodstock and larval sourcing, promoting best management practices by farmer outreach and supporting cutting-edge research that aims to harness the natural abilities of invertebrates to mitigate assault from pathogens (e.g. the use of RNA interference therapeutics). In terms of fisheries losses associated with disease, key issues are centred on mortality and quality degradation in the post-capture phase, largely due to poor grading and handling by fishers and the industry chain. Occurrence of disease in wild crustaceans is also widely reported, with some indications that climatic changes may be increasing susceptibility to important pathogens (e.g. the parasite Hematodinium). However, despite improvements in field and laboratory diagnostics, defining population-level effects of disease in these fisheries remains elusive. Coordination of disease specialists with fisheries scientists will be required to understand current and future impacts of existing and emergent diseases on wild stocks. Overall, the increasing demand for crustacean seafood in light of these issues signals a clear warning for the future sustainability of this global industry. The linking together of global experts in the culture, capture and trading of crustaceans with pathologists, epidemiologists, ecologists, therapeutics specialists and policy makers in the field of food security will allow these issues to be better identified and addressed

A nested PCR assay to avoid false positive detection of the microsporidian enterocytozoon hepatopenaei (EHP) in environmental samples in shrimp farms

PublishedJournal Article© 2016 Jaroenlak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is an important disease of cultivated shrimp. Heavy infections may lead to retarded growth and unprofitable harvests. Existing PCR detection methods target the EHP small subunit ribosomal RNA (SSU rRNA) gene (SSU-PCR). However, we discovered that they can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms. This is problematic for investigating and monitoring EHP infection pathways. To overcome this problem, a sensitive and specific nested PCR method was developed for detection of the spore wall protein (SWP) gene of EHP (SWP-PCR). The new SWP-PCR method did not produce false positive results from closely related microsporidia. The first PCR step of the SWP-PCR method was 100 times (104 plasmid copies per reaction vial) more sensitive than that of the existing SSU-PCR method (106 copies) but sensitivity was equal for both in the nested step (10 copies). Since the hepatopancreas of cultivated shrimp is not currently known to be infected with microsporidia other than EHP, the SSU-PCR methods are still valid for analyzing hepatopancreatic samples despite the lower sensitivity than the SWP-PCR method. However, due to its greater specificity and sensitivity, we recommend that the SWP-PCR method be used to screen for EHP in feces, feed and environmental samples for potential EHP carriers.OI acknowledges support from Agricultural Research Development Agency under project CRP5905020530 and Mahidol University. KS received funding from National Research Council Thailand, Division of Plan Administration and Research Budget/2557-79. PJ is supported by the Science Achievement Scholarship of Thailand (SAST). GDS acknowledges support of DG SANCO of the European Commission, and the UK Department of Environment, Food and Rural Affairs under project FB002. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

New Paradigms to Help Solve the Global Aquaculture Disease Crisis.

Published onlineJournal ArticleThis is the final version of the article. Available from Public Library of Science via the DOI in this record.n/aThe authors (GDS, KS) acknowledge funding administered by the British Council under the Newton Fund Researcher Links Programme, for a UK-Thailand bilateral workshop entitled "Scientific, technological and social solutions for sustainable aquaculture in Thailand: a key player in global aquatic food supply," Bangkok, March 2016. Further funding support is acknowledged from the European Commission (EC) and the UK Department for Environment, Food and Rural Affairs (Defra) under contracts C6928 and FB002 (to GDS and DB); from the Royal Society under a University Research Fellowship (to BAPW); and to the Agricultural Research Development Agency (ARDA) and National Research Council of Thailand (NRCT) (to KS, TWF, and OI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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

Published onlineJournal ArticleReviewSince 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.This work was funded by the Open Innovation Platform at the University of Exeter (Open Innovation Fund Initiative PHSW029) and by the Centre for Environment, Fisheries and Aquaculture Science (Cefas) (under seedcorn project DP318 to GDS) under the Strategic Alliance partnership between the University of Exeter and Cefas

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

Journal ArticleCopyright © 2015 Verbruggen et al.BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: We 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.Cefas Seedcorn Contract #DP318University of Exeter’s Open Innovation PlatformWellcome Trust Institutional Strategic Support Awar

Halioticida noduliformans infection in eggs of lobster ( Homarus gammarus ) reveals its generalist parasitic strategy in marine invertebrates

publisher: Elsevier articletitle: Halioticida noduliformans infection in eggs of lobster (Homarus gammarus) reveals its generalist parasitic strategy in marine invertebrates journaltitle: Journal of Invertebrate Pathology articlelink: http://dx.doi.org/10.1016/j.jip.2018.03.002 content_type: article copyright: © 2018 The Authors. Published by Elsevier Inc.0000-0002-6719-5565The file attached is the Published/publisher’s pdf version of the articleThis is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/)

Halioticida noduliformans infection in eggs of lobster (Homarus gammarus) reveals its generalist parasitic strategy in marine invertebrates

This is the final version of the article. Available from the publisher via the DOI in this record.A parasite exhibiting Oomycete-like morphology and pathogenesis was isolated from discoloured eggs of the European lobster (Homarus gammarus) and later found in gill tissues of adults. Group-specific Oomycete primers were designed to amplify the 18S ribosomal small subunit (SSU), which initially identified the organism as the same as the ‘Haliphthoros’ sp. NJM 0034 strain (AB178865.1) previously isolated from abalone (imported from South Australia to Japan). However, in accordance with other published SSU-based phylogenies, the NJM 0034 isolate did not group with other known Haliphthoros species in our Maximum Likelihood and Bayesian phylogenies. Instead, the strain formed an orphan lineage, diverging before the separation of the Saprolegniales and Pythiales. Based upon 28S large subunit (LSU) phylogeny, our own isolate and the previously unidentified 0034 strain are both identical to the abalone pathogen Halioticida noduliformans. The genus shares morphological similarities with Haliphthoros and Halocrusticida and forms a clade with these in LSU phylogenies. Here, we confirm the first recorded occurrence of H. noduliformans in European lobsters and associate its presence with pathology of the egg mass, likely leading to reduced fecundity.This work was conducted within the Centre for Sustainable Aquaculture Futures, a joint initiative between the University of Exeter and the Centre for Environment, Fisheries and Aquaculture Science (Cefas) and funded by a Cefas-Exeter University Alliance PhD Studentship to CH. Work was also supported through the Agri-Tech Catalyst, Industrial Stage Awards, Lobster Grower 2 project funded by Innovate UK (102531) and BBSRC (BB/N013891/1) and Defra contracts C6560 and C7277 to D

Pathogens of Dikerogammarus haemobaphes regulate host activity and survival, but also threaten native amphipod populations in the UK

Dikerogammarus haemobaphes is a non-native amphipod in UK freshwaters. Studies have identified this species as a low-impact invader in the UK, relative to its cousin Dikerogammarus villosus. It has been suggested that regulation by symbionts (such as Microsporidia) could explain this difference in impact. The effect of parasitism on D. haemobaphes is largely unknown. This was explored herein using 2 behavioural assays measuring activity and aggregation. First, D. haemobaphes were screened histologically post-assay, identifying 2 novel viruses (D. haemobaphes bi-facies-like virus [DhbflV], D. haemobaphes bacilliform virus [DhBV]), Cucumispora ornata (Microsporidia), Apicomplexa, and Digenea, which could alter host behaviour. DhBV infection burden increased host activity, and C. ornata infection reduced host activity. Second, native invertebrates were collected from the invasion site at Carlton Brook, UK, and tested for the presence of C. ornata. PCR screening identified that Gammarus pulex and other native invertebrates were positive for C. ornata. The host range of this parasite, and its impact on host survival, was additionally explored using D. haemobaphes, D. villosus, and G. pulex in a laboratory trial. D. haemobaphes and G. pulex became infected by C. ornata, which also lowered survival rate. D. villosus did not become infected. A PCR protocol for DhbflV was also applied to D. haemobaphes after the survival trial, associating this virus with decreased host survival. In conclusion, D. haemobaphes has a complex relationship with parasites in the UK environment. C. ornata likely regulates populations by decreasing host survival and activity, but despite this benefit, the parasite threatens susceptible native wildlife