Infections of the Norway lobster, Nephrops norvegicus (L.) by dinoflagellate and ciliate parasites

Parasitic dinoflagellates of the genus Hematodinium have been reported from a number of commercially important crustacean hosts, including the Norway lobster, Nephrops norvegicus, from the coastal waters of Scotland. Several methods for detection of the parasite have been developed but each has associated drawbacks. As part of this study, an enzyme linked immunoabsorbent assay (ELISA) has been developed for the detection of the parasite in the haemolymph of N. norvegicus and other crustaceans. The ELISA is a simple, sensitive, reproducible assay, with a detection limit of 5x104 parasites ml-1 haemolyph. To further investigate low-level hematodinium infection in n. norvegicus and other crustacean hosts, a set of Hematodinium-specific polymerase chain reaction (PCR) primers and DNA probes have been developed, based on Hematodinium ribosomal DNA (rDNA) regions. In the PCR assay, a diagnostic band of 380 bp is produced in the presence of parasite DNA. The limit of detection of the assay was found to be 1 ng DNA, which is equivalent to 6 parasites. The DNA probes detected Hematodinium cells in a range of tissues from N. norvegicus and from the crab Carcinus maenas. The level of genetic similarity between nine isolates of Hematodinium originating from several species of Crustacea from the Uk was examined. PCR was used to amplify and sequence the 3' of the small sub-unit (SSU) and the first internal transcribed spacer (ITS1) of the parasite rDNA complex. Analysis of the SSU and ITS 1 sequences revealed that both the regions are highly conserved (92.2%) or greater) between isolates examined, and that there is no apparent geographical separation of isolates. the results suggested that the same species of Hematodinium infects a number of crustacean species from different geographical locations. Hematodinium perezi, the parasitic dinoflagellate of the blue crab Callinectes sapidus, has been successfully isolated and cultured in vitro. Although the in vivo form of this parasite is morphologically and mollecularly very different from that of the Hematodinium sp. infecting N. norvegicus, a number of similar life cycle stages were observed in vitro. These included syncytial networks, filamentous trophonts, and gorgonlock colonies. The isolation and in vitro culture of H. perezi and the Hematodinium sp. infecting n. norvegicus allowed the internal and external enzyme profiles of both species to be examined using the API ZYM system and biochemical assays, leading to the identification of several enzymes that may have pathogenic importance during Hematodinium infections. Differences in the secrretion of acid phosphate and leucine arylamidase by the two Hematodium sp. studied may account for their different levels of virulence and infectivity. A ciliate infection of the wild and laboratory-held N. norvegicus was discovered during the course of this project. Extensive damage to heart muscle tissue was observed in affected lobsters. The ciliate was identified as belonging to the genus Mesanophyrs, based on silver carbonate impregnation of oral structures. However, molecular sequence data (ITS1 and ITS2) indicated that the ciliate sequences have 100% identity with rDNA sequences from Orchitophyra stellarum, a ciliate parasite of sea stars. Since the morphology of O. stellarum differs from that of Mesanophyrs, the possibility arises that the previous studies have misreported the molecular data. Otherwise, it must be concluded that morphological features cannot be used to discriminate between closely related ciliate species. The initiation of in vitro cultures of the ciliate isolated from N. norvegicus allowed the investigation of proteolytic factors that may be involved in the initiation and progression of its infection. The ciliate was found to secrete a number of proteases into the culture medium, and these are exclusively of the metallo type They have gelatinolytic and azocaseinolytic activities and are active at the physiological temperature and haemolyph pH of the host. Secreted proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a common structural component of all lobster muscle tissues. Consequently, these proteases may have multiple roles in the invasion and progression of this ciliate infection, or in assisting nutrient uptake by the ciliate. The results of these studies are discussed in terms of the thechnical development of diagnostic assays for Hematodium, their potential application in examining the prevalence and transmission of this parasite in N. norvegicus and other crustaceans, and the potential pathogenic mechanisms involved in parasitic infections of N. norvegicus

Immunofluorescence Visualization of Polycyclic Aromatic Hydrocarbon Mixtures in the Eastern Oyster Crassostrea virginica

Bivalve mollusks including oysters have low metabolic potential and are therefore susceptible to accumulating high levels of lipophilic organic contaminants such as polycyclic aromatic hydrocarbons (PAHs). Human exposure to PAHs via consumption of this important commercial shellfish can be a serious public health concern in areas where high PAH contamination exists. Previous PAH immunohistochemical studies have been limited to laboratory-based exposures focusing on one or a few individual PAH compounds. To date, such studies have yet to explore PAH accumulation in oysters, known to have some of the highest levels of PAHs across different food products. Using a monoclonal antibody selective for a range of three- to five-ring PAHs, we present a method to detect and localize complex mixtures of PAHs in oyster tissues via fluorescent immunohistochemistry. Observed immunofluorescence intensity followed a similar trend as measured levels of PAHs in oyster interstitial fluid from PAH-contaminated sites and oysters exposed to the water accommodated fraction of crude oil. This method will be valuable in understanding internal partitioning mechanisms of PAH-exposed oysters and will have important applications in studies on PAH distribution in the tissues of additional organisms for environmental, medical, or veterinary purposes

Molecular Detection Of Hematodinium Sp Infecting The Blue Crab, Callinectes Sapidus

Species of Hematodinium are endoparasitic dinoflagellates of crustaceans. Certain stages of the parasites can be very difficult to detect in the hemolymph of their hosts, because the trophic stages resemble hemocytes, and they can occur at relatively low densities, making diagnosis by microscopy difficult. We developed a polymerase chain reaction (PCR) assay to detect the Hernatodinium sp. infecting the blue crab, Callinectes sapidus, based on the amplification of the parasite\u27s first internal transcribed spacer region (ITS1) of the ribosomal RNA (rRNA) gene complex. The PCR assay was combined with a restriction endonucleases digestion (Bsg I) of the amplification products to differentiate between different forms of Hematodinium from different hosts. The assay had a limit of detection equivalent to 0.3 parasites per 100-mu L hemolymph. In addition, two oligonucleotide DNA probes were designed to target the 18S rRNA gene sequence of the parasite, facilitating detection in situ in crustacean tissues. These probes appear to target several, if not all species within the genus, because they labeled all isolates of Hematodinium tested in this study, whereas they were not hybridizing to other parasite species. The PCR-RFLP assay will be invaluable for future studies investigating parasite prevalence, the existence of secondary hosts or environmental reservoirs, and modes of transmission, whereas the DNA probes will be useful for confirming and localizing Hematodinium parasites in crustacean tissues

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

Quantifying the behavioral and economic effects of regulatory change in a recreational cobia fishery

Fisheries economists typically assume recreational anglers make decisions that maximize individual angler utility, which may depend on fishery and regulatory conditions. Under this framework, changes in regulations can lead to target species substitution by anglers in response to shifts in expectations of trip utility. A stated preference survey was developed and distributed to recreational cobia (Rachycentron canadum) anglers in Virginia to explore the effects of regulatory change on angler decision-making, species targeting, and resulting economic outcomes. The survey included a series of hypothetical choice scenarios, where respondents were asked to select their most preferred alternative after being presented with different fishing trips targeting cobia,red drum (Sciaenops ocellatus), or summer flounder (Paralichthys dentatus). Seven regulatory treatments of the survey were distributed, providing anglers a variety of species targeting tradeoffs. A mixed logit model was usedto estimate angler preferences associated with hypothetical trip attributes and regulatory environment. Changes in angler welfare resulting from changes in cobia regulations were then assessed. Anglers were found to prefer targeting cobia to red drum or summer flounder under status quo management. Increases in catch, average weight of catch, and legal harvest of cobia were also found to provide anglers greater improvements in triputility compared to increases in these attributes for trips targeting red drum or summer flounder. The economic effects of regulatory change were asymmetric because restrictive regulations were found to reduce angler welfare whereas liberalizing regulations had no significant effects. Increased availability of alternative target species was found to dampen the negative welfare effects of restrictive cobia regulations due to predicted target species substitution by anglers

Penaeid Shrimp in Chesapeake Bay: Population Growth and Black Gill Disease Syndrome

Since 1991, the number of penaeid shrimp occurring in Virginia waters of Chesapeake Bay has steadily increased, prompting an interest in developing a fishery. Although development of a shrimp fishery in the Chesapeake Bay region could bring economic benefits, the fishery may be hampered by the presence of a disease syndrome known as shrimp black gill (sBG). The objectives of our study were to (1) describe the spatial distribution and abundance patterns of shrimp in Chesapeake Bay, (2) relate relative abundance of shrimp to habitat characteristics, and (3) determine the presence and seasonality of sBG to better understand disease dynamics in the region. Subadult penaeid shrimp were collected monthly from Virginia waters by trawl from 1991 to 2017, and individuals were identified to species and counted. White shrimp Litopenaeus setiferus were the most numerous species captured, followed by brown shrimp Farfantepenaeus aztecus and pink shrimp F. duorarum. Shrimp were captured primarily from July to December. White shrimp were the only species that exhibited visible signs of sBG, which was first observed in October 2016 (13.4% prevalence); the condition continued into November and recurred the following year. Shrimp with visible signs of gill disease were examined by microscopy, histology, and PCR assay and were diagnosed with infections of a histophagous apostome ciliate, presumably Hyalophysa lynni. Any impacts of sBG on shrimp survival or marketability should be considered in fishery management plans to ensure sustainability of the resource

New insight into the transmission dynamics of the crustacean pathogen Hematodinium perezi (Dinoflagellata) using a novel sentinel methodology

Hematodinium perezi causes disease and mortality in several decapod crustaceans along the eastern seaboard and Gulf coast of the USA. The route of transmission of the parasite is unknown, but infections exhibit a sharp seasonal cycle in its primary host, the blue crab Callinectes sapidus, that indicates the possibility of a short transmission period in its life cycle. We developed a sentinel methodology based on the use of naïve, uninfected, early benthic juvenile crabs (instars C1 to C10) to investigate the transmission of H. perezi. Crabs were collected from a non-endemic site, held for a short period for evaluation, and then deployed in a highly endemic site for 14 d. Transmission of the pathogen was successful; 12.7 to 25.7% of the crabs deployed at the endemic site became infected over this period. Infections developed rapidly, with 25% of new infections developing into heavy infections during the deployment. The large number of infections that developed using the sentinel methodology allowed for the first estimates of incidence (the proportion of new infections in a population over time) in this system. Incidence varied from 0.9 to 1.8% of the resident crab population per day and accounts for the high prevalence levels observed in the endemic coastal bays of the Delmarva Peninsula. The development of this sentinel methodology has broad application for studying disease ecology in this system and in other pathogens that infect decapods

Anti-lipopolysaccharide factors in the American lobster Homarus americanus: Molecular characterization and transcriptional response to Vibrio fluvialis challenge

Two partial mRNA sequences predicted to encode anti-lipopolysaccharide factors (ALFs) were identified among expressed sequence tags generated from the American lobster Homarus americanus and complete cDNA sequences were obtained from library clones. Comparison of the translated amino acid sequences to those publicly available confirmed similarity to arthropod anti-lipopolysaccharide factors. Both protein sequences, designated ALFHa-1 and ALFHa-2, contained an N-terminal signal peptide and two half-cysteines participating in a disulfide bridge, features conserved in other ALFs. Predicted secondary structures were similar to that described for the ALF from the horseshoe crab Limulus polyphemus. As part of an exploratory study of immunity in H. americanus, lobsters were injected with the bacterium Vibrio fluvialis and gill, hematopoietic, and hepatopancreas tissues were sampled for analysis of gene expression of ALFHa-1 and ALFHa-2 by quantitative PCR. The relative abundance of ALFHa-2 mRNA was not significantly affected by Vibrio injection in any of the three tissues tested. In contrast, ALFHa-1 mRNA levels in gills were increased by the treatment some 17-fold. Our results support a molecularly specific regulation of antimicrobial proteins in response to bacterial infection in H. americanus. (C) 2008 Elsevier Inc. Ail rights reserved

Characterization of microsporidian Ameson herrnkindi sp. nov. infecting Caribbean spiny lobsters Panulirus argus

The Caribbean spiny lobster Panulirus argus supports a large and valuable fishery in the Caribbean Sea. In 2007-2008, a rare microsporidian parasite with spore characteristics typical of the Ameson genus was detected in 2 spiny lobsters from southeast Florida (FL). However, the parasite species was not confirmed by molecular analyses. To address this deficiency, reported here are structural and molecular data on single lobsters displaying comparable ‘cotton-like’ abdominal muscle containing ovoid microsporidian spores found at different locations in FL in 2014 and 2018 and in Saint Kitts and Nevis Islands in 2017. In the lobster from 2014, multiple life stages consistent with an Ameson-like monokaryotic microsporidian were detected by transmission electron microscopy. A partial (1228 bp) small subunit (SSU) rRNA gene sequence showed each microsporidia to be identical and positioned it closest phylogenetically to Ameson pulvis in a highly supported clade also containing A. michaelis, A. metacarcini, A. portunus, and Nadelspora canceri. Using ecological, pathological, ultrastructural, and molecular data, the P. argus microsporidian has been assigned to a distinct species: Ameson herrnkindi

Laboratory studies on the effect of temperature on epizootic shell disease in the American lobster, Homarus americanus

Epizootic shell disease (ESD) is a persistent threat to the population of American lobsters, Homarus americanus H. Milne-Edwards, 1837, in Long Island Sound and off southern New England, USA. ESD is caused by a bacterial dysbiosis that occurs in association with increased water temperature and exposure to anthropogenic stressors. Temperature is a leading factor driving the severity and incidence of ESD. Our objective was to quantify disease progression and dynamics in relation to host molting and mortality at three rigorously controlled temperatures (6, 12, and 18 °C) over a 5–6-mo period. Lobsters were photographed at various time points and image analysis was used to examine changes in lesion development over time. The disease progressed at all three experimental temperatures, but it had a significantly faster growth rate at 18 °C. Mean progression rates varied from 8.6–10.4 mm2 d–1 at the lower temperatures to \u3e25.6 mm2 d–1 at 18 °C. The mean daily growth rates give conservative estimates for individual progression from light to moderate disease states; i.e., approximately 233 d at 6 °C and 95 d at 18 °C. We show that increased temperature leads to rapid progression of ESD, but individual variation, presumably modulated through immune defenses, can slow the disease and possibly enhance survival of affected lobsters