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A recurring trend in evidence scrutinized over the past few decades is that disease outbreaks will become more frequent, intense, and widespread on land and in water, due to climate change. Pathogens and the diseases they inflict represent a major constraint on seafood production and yield, and by extension, food security. The risk(s) for fish and shellfish from disease is a function of pathogen characteristics, biological species identity, and the ambient environmental conditions. A changing climate can adversely influence the host and environment, while augmenting pathogen characteristics simultaneously, thereby favoring disease outbreaks. Herein, we use a series of case studies covering some of the world's most cultured aquatic species (e.g., salmonids, penaeid shrimp, and oysters), and the pathogens (viral, fungal, bacterial, and parasitic) that afflict them, to illustrate the magnitude of disease-related problems linked to climate change., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Epizootiologists recurrently encounter symbionts and pathobionts in the haemolymph (blood equivalent) of shellfish. One such group is the dinoflagellate genus Hematodinium , which contains several species that cause debilitating disease in decapod crustaceans. The shore crab Carcinus maenas acts as a mobile reservoir of microparasites, including Hematodinium sp., thereby posing a risk to other co-located commercially important species, e.g. velvet crabs ( Necora puber ). Despite the widespread prevalence and documented seasonality of Hematodinium infection dynamics, there is a knowledge gap regarding host-pathogen antibiosis, namely, how Hematodinium avoids the host's immune defences. Herein, we interrogated the haemolymph of Hematodinium -positive and Hematodinium -negative crabs for extracellular vesicle (EV) profiles (a proxy for cellular communication), alongside proteomic signatures for post-translational citrullination/deimination performed by arginine deiminases, which can infer a pathologic state. Circulating EV numbers in parasitized crab haemolymph were reduced significantly, accompanied by smaller EV modal size profiles (albeit non-significantly) when compared to Hematodinium -negative controls. Differences were observed for citrullinated/deiminated target proteins in the haemolymph between the parasitized and control crabs, with fewer hits identified overall in the former. Three deiminated proteins specific to parasitized crab haemolymph were actin, Down syndrome cell adhesion molecule (DSCAM), and nitric oxide synthase - factors that contribute to innate immunity. We report, for the first time, Hematodinium sp. could interfere with EV biogenesis, and that protein deimination is a putative mechanism of immune-modulation in crustacean- Hematodinium interactions.

The term shell disease subsumes a number of debilitating conditions affecting the outer integument (the carapace) of decapod crustaceans, such as lobsters and crabs. Herein, we seek to find commonality in the aetiology and pathology of such conditions, and those cases that result in the progressive erosion of the cuticle through to the visceral tissues by a cocktail of microbial-derived enzymes including lipases, proteases and chitinases. Aquimarina spp. are involved in shell disease in many different crustaceans across a wide geographical area, but the overall view is that the condition is polymicrobial in nature leading to dysbiosis within the microbial consortium of the damaged cuticle. The role of environment, decapod behaviour and physiology in triggering this disease is also reviewed. Finally, we provide a conceptual model for disease aetiology and suggest several avenues for future research that could improve our understanding of how such factors trigger, or exacerbate, this condition., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Invasion and spread of alien species can drive ecosystem changes, such as, the dynamics of infectious diseases. The non-native, marine gastropod Crepidula fornicata has become established across European coastlines over the last century, but there remains little insight into its disease carrying capacity and potential role as a source/sink of parasites. To address this knowledge gap, we surveyed limpets from two sites in South Wales, UK for signatures of disease/pathology using polymerase chain reaction-based methods (haemolymph) and histology (solid tissue). We encountered trematode-like parasites in ~1% individuals (5 out of 462). Three limpets displayed gross damage in the gonad, i.e. castration, and encysted metacercariae were found in the muscle of two other individuals. On the basis of 28S rDNA and internal transcribed spacer 2 genomic targets, we identified the gonad-infecting trematodes as members of the family Microphallidae – putative novel species related to the genus Longiductotrema. Earlier reports suggest that C. fornicata is not a host for trematode parasites in either its native or alien range but may act as a sink due to its filter feeding lifestyle. We provide clear evidence that C. fornicata is parasitized by at least one trematode species at two sites in Wales, UK, and likely act as a spillback or accidental host among native littorinids.

Host, pathogen, and environment are determinants of the disease triangle, the latter being a key driver of disease outcomes and persistence within a community. The dinoflagellate genus Hematodinium is detrimental to crustaceans globally - considered to suppress the innate defences of hosts, making them more susceptible to co-infections. Evidence supporting immune suppression is largely anecdotal and sourced from diffuse accounts of compromised decapods. We used a population of shore crabs ( Carcinus maenas ), where Hematodinium sp. is endemic, to determine the extent of collateral infections across two distinct environments (open-water, semi-closed dock). Using a multi-resource approach (PCR, histology, haematology, population genetics, eDNA), we identified 162 Hematodinium- positive crabs and size/sex-matched these to 162 Hematodinium- free crabs out of 1191 analysed. Crabs were interrogated for known additional disease-causing agents; haplosporidians, microsporidians, mikrocytids, Vibrio spp., fungi, Sacculina , trematodes, and haemolymph bacterial loads. We found no significant differences in occurrence, severity, or composition of collateral infections between Hematodinium -positive and Hematodinium -free crabs at either site, but crucially, we recorded site-restricted blends of pathogens. We found no gross signs of host cell immune reactivity towards Hematodinium in the presence or absence of other pathogens. We contend Hematodinium sp. is not the proximal driver of co-infections in shore crabs, which suggests an evolutionary drive towards latency in this environmentally plastic host., Competing Interests: CD, JT, SM, FB, AR, CC No competing interests declared, (© 2022, Davies et al.)

Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 μg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80-400 μg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 μg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 μg/kg) followed 24 h later by bacterial inoculation (2 × 10 5 Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 μg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays., (© 2021. The Author(s).)

The slipper limpet Crepidula fornicata is an invasive, non-native, marine species found throughout the coastal waters of southern England and Wales, UK. These limpets are considered to blight commercial shellfish banks, notably oysters, yet little is known about their disease-carrying capacity or their immunobiology. To address the latter, we isolated haemolymph (blood) from limpets and tested for the presence of the immune-enzyme phenoloxidase. Invertebrate phenoloxidases produce melanic polymers from simple phenolic substrates, which are deployed in the presence of pathogens because of their potent microbicidal and microbiostatic properties. We used a series of established substrates (e.g., tyrosine, hydroquinone) and inhibitors (e.g., 4-hexylresorcinol, benzoic acid) to target three distinct enzymes: laccase (para-diphenoloxidase), catecholoxidase (ortho-diphenoloxidase) and tyrosinase (monophenoloxidase). We confirmed laccase and catecholoxidase activities and characterised their kinetic properties across temperature and pH gradients (5-70 °C and 5-10, respectively). Crucially, we demonstrated that products derived from such laccase and catecholoxidase activities reduced significantly the numbers of colony-forming units of both Gram-positive and Gram-negative bacteria in vitro. We further screened limpet tissues for signs of melanin using wax histology, and found cells replete with eumelanin-like pigments and lipofuscin in the digestive gland, connective tissues, barrier epithelia and gills. Our data represent the first account of enzyme-based antibacterial defences, notably laccase, in C. fornicata., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

There is a paucity of knowledge regarding the diversity and impact(s) of disease-causing fungi in marine animals, especially shellfish. In efforts to address this knowledge gap for the shore crab Carcinus maenas , a year-long disease screen was carried out across two sites in Swansea Bay (Wales, UK) with a view to characterising putative fungal infections. Crabs were sampled between November 2017 and October 2018, and screened systematically for disease signatures using haemolymph (blood) preparations, targeted PCR and tissue histopathology. Strikingly, mycosis was confirmed in ~0.4% of total crabs tested ( n = 1191) and restricted to one location only (Mumbles Pier). Clinical infections were observed in four out of four infected crabs. In these animals, the gills and hepatopancreas were congested with fungal morphotypes. In addition, some evidence indicates haemocyte (immune cell) reactivity toward the fungi. Phylogenetic placement of the partial internal transcribed spacer (ITS1) gene regions amplified from three mycotic crabs revealed the causative agent to be related to hypocrealean fungi, thereby representing a novel species.

Sacculina carcini is a common parasite of the European shore crab, Carcinus maenas. Following successful penetration of the host, numerous rootlets are formed that permeate through the hosts' tissues. Ultimately, these form an externa that houses the developing nauplii larvae of the parasite. Most studies have quantified levels of infection by counting the presence of reproductive externae and their breakdown structures, called scars. However, the diagnosis of the disease based only on external features may lead to underreporting the prevalence of the parasite. In the current study, we examined the presence and severity of S. carcini in C. maenas (n = 221) in the Prince of Wales Dock, South Wales, U.K. using a range of diagnostic approaches to give an accurate representation of temporal dynamics of infection. Parasitized crabs were found with a mean prevalence of 24% as determined by histological examination of the hepatopancreas. However, the prevalence of S. carcini based on the presence of externae and scars was only 6.3% and 1.8%, respectively. Overall, parasitism was associated with smaller crabs, crabs later in the moulting cycle that were orange in colour (as opposed to green or yellow), and those with a higher number of bacteria in the haemolymph. Interestingly, only 7.5% of infected crabs showed evidence of distinct host (cellular) response to the presence of rootlets in the hepatopancreas., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mikrocytids are a widespread but rather neglected group of parasites of aquatic invertebrates. One such parasite is Paramikrocytos canceri -discovered to infect the antennal gland of the juvenile edible crab, Cancer pagurus , taken from several intertidal sites across the United Kingdom. To determine if this parasite is also present in other species of decapod crustaceans, we surveyed crabs ( n = 330) across two contrasting sites in Pembrokeshire (UK). Using a histopathological approach, P. canceri infection was confirmed in variable numbers of edible crabs from both survey sites, 7%-44%. No measurable signs of infection were encountered in four other co-located species, including European shore crabs ( Carcinus maenas ), Montagu's crabs ( Xantho hydrophilus ), velvet swimming crabs ( Necora puber ) and broad-clawed porcelain crabs ( Porcellana platycheles ). These data imply that P. canceri has a more limited host range than suggested by molecular diagnosis alone.

Background: The parasitic dinoflagellates of the genus Hematodinium represent the causative agent of so-called bitter or pink crab disease in a broad range of shellfish taxa. Outbreaks of Hematodinium-associated disease can devastate local fishing and aquaculture efforts. The goal of our study was to examine the potential role of the common shore (green) crab Carcinus maenas as a reservoir for Hematodinium. Carcinus maenas is native to all shores of the UK and Ireland and the North East Atlantic but has been introduced to, and subsequently invaded waters of, the USA, South Africa and Australia. This species is notable for its capacity to harbour a range of micro- and macro-parasites, and therefore may act as a vector for disease transfer., Methods: Over a 12-month period, we interrogated 1191 crabs across two distinct locations (intertidal pier, semi-closed dock) in Swansea Bay (Wales, UK) for the presence and severity of Hematodinium in the haemolymph, gills, hepatopancreas and surrounding waters (eDNA) using PCR-based methods, haemolymph preparations and histopathology., Results: Overall, 13.6% were Hematodinium-positive via PCR and confirmed via tissue examination. Only a small difference was observed between locations with 14.4% and 12.8% infected crabs in the Dock and Pier, respectively. Binomial logistic regression models revealed seasonality (P < 0.002) and sex (P < 0.001) to be significant factors in Hematodinium detection with peak infection recorded in spring (March to May). Male crabs overall were more likely to be infected. Phylogenetic analyses of the partial ITS and 18S rRNA gene regions of Hematodinium amplified from crabs determined the causative agent to be the host generalist Hematodinium sp., which blights several valuable crustaceans in the UK alone, including edible crabs (Cancer pagurus) and langoustines (Nephrops norvegicus)., Conclusions: Shore crabs were infected with the host generalist parasite Hematodinium sp. in each location tested, thereby enabling the parasite to persist in an environment shared with commercially important shellfish.

Indomethacin is a non-steroidal anti-inflammatory drug that causes gastric ulceration and increased 'leakiness' in rat models, and is used routinely as a toxicology assay to screen novel compounds for repair and restitution properties. We set out to establish conditions for indomethacin-induced gut damage in wax-moth (Galleria mellonella) larvae with a view to reducing the need for rodents in such experimentation. We administered indomethacin (0.5-7.5 µg/larva; 2-30 mg/kg) to G. mellonella via intrahaemocoelic injection and gavage (force-feeding) and monitored survival and development, blood cell (haemocyte) numbers, and changes in gut permeability. Increased levels of gut leakiness were observed within the first 4- to 24 h by tracking fluorescent microspheres in the faeces and haemolymph (blood equivalent). Additionally, we recorded varying levels of tissue damage in histological sections of the insect midgut, including epithelial sloughing and cell necrosis. Degeneration of the midgut was accompanied by significant increases in detoxification-associated activities (superoxide dismutase and glutathione-S-transferase). Herein, we present the first evidence that G. mellonella larvae force-fed indomethacin display broad symptoms of gastric damage similar to their rodent counterparts.

The polyether toxin, okadaic acid, causes diarrhetic shellfish poisoning in humans. Despite extensive research into its cellular targets using rodent models, we know little about its putative effect(s) on innate immunity. We inoculated larvae of the greater wax moth, Galleria mellonella, with physiologically relevant doses of okadaic acid by direct injection into the haemocoel (body cavity) and/or gavage (force-feeding). We monitored larval survival and employed a range of cellular and biochemical assays to assess the potential harmful effects of okadaic acid. Okadaic acid at concentrations ≥ 75 ng/larva (≥ 242 μg/kg) led to significant reductions in larval survival (> 65%) and circulating haemocyte (blood cell) numbers (> 50%) within 24 h post-inoculation. In the haemolymph, okadaic acid reduced haemocyte viability and increased phenoloxidase activities. In the midgut, okadaic acid induced oxidative damage as determined by increases in superoxide dismutase activity and levels of malondialdehyde (i.e. lipid peroxidation). Our observations of insect larvae correspond broadly to data published using rodent models of shellfish-poisoning toxidrome, including complementary LD 50 values: 206-242 μg/kg in mice, ~ 239 μg/kg in G. mellonella. These data support the use of this insect as a surrogate model for the investigation of marine toxins, which offers distinct ethical and financial incentives.

There are few reports of bacterial diseases in crabs. A juvenile edible crab (Cancer pagurus) with a rickettsial-like infection was found in the intertidal zone at Freshwater East in South West Wales in July, 2012. Large numbers of bacteria-like particles were found in the haemolymph and within fixed phagocytes of the hepatopancreas. Molecular sequencing and subsequent phylogenetic analysis showed that the infectious agent was a member of the order Rhizobiales and therefore distinct to bacteria classified as rickettsia., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The prevalence of disease in edible crabs (Cancer pagurus) was assessed at two sites in South West Wales; one estuarine (Pembroke Ferry) and another facing open water (Freshwater East). Diseases included pink crab disease caused by Hematodinium sp., an infection of the antennal gland caused by Paramikrocytos canceri and an idiopathic inflammatory condition of the connective tissue surrounding the anterior ganglionic masses. This latter condition was only found in crabs from Pembroke Ferry. There was a significantly higher prevalence of pink crab disease at Freshwater East than Pembroke Ferry, although both sites had similar levels of infection by P. canceri., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Hematodinium spp. infect over 40 species of crustaceans worldwide, but have not been reported to infect the European lobster, Homarus gammarus. In this study, Hematodinium parasites (a mixture of uni- and multinucleate trophont-like stages) were taken from donor crabs (Cancer pagurus) and injected into juvenile H. gammarus. Juvenile C. pagurus were also injected with the same inoculum. Haemolymph was taken at regular intervals and examined for the presence of Hematodinium using light microscopy and PCR, in two separate experiments of duration 4 and 8months. All lobsters were negative for Hematodinium whilst the C. pagurus challenged became infected. It is concluded that European lobsters are not susceptible to infection with a clade of Hematodinium that infects C. pagurus., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This study reports on the prevalence and severity of infections caused by the parasitic dinoflagellate, Hematodinium in juvenile edible crabs (Cancer pagurus) found in 2 intertidal survey sites (Mumbles Head and Oxwich Bay) in the Bristol Channel, UK. Crabs were assessed for the presence and severity of Hematodinium infections by the histological examination of infected tissues. Such infections were found to exhibit a seasonal trend in the 2 study areas with high numbers of animals (ca. 30%) infected in the spring to summer but with low severity. Conversely, in November only ca. 10% of crabs were infected but these animals had large numbers of parasites in their haemolymph and other tissues. At this time, the carapace and underlying tissues of infected crabs had the chalky, pinkish-orange appearance that is characteristic of this disease. Hematodinium-infected crabs ranged in size from 12 to 74 mm carapace width. Overall, it is concluded that the high prevalence of infection of juvenile crabs in this area may have implications for the sustainability of the edible crab fishery in the Bristol Channel.

The ectoparasitic copepod, Nicothoë astaci (the 'lobster louse'), infests the gills of the European lobster, Homarus gammarus. There have been limited studies on this haematophagous species; therefore knowledge of this parasite is rudimentary. The current study examines the surface morphology of this parasitic copepod, detached from the host, concentrating on adaptations of the suctorial mouthpart, the oral disc. Cryo-scanning electron microscopy revealed structural adaptations that facilitate attachment of these parasites to the gill filaments of their lobster host. The aperture of the feeding channel, through which host haemolymph is drawn, is only ca. 5μm in diameter. The edge of the oral disc is lined with numerous setae, whilst the surface of the disc is covered with large numbers of small (<1μm in diameter) teeth-like structures, which presumably pierce through, and grip, the cuticle lining of the host's gill. Overall, these structures are thought to provide a 'vacuum seal' to assist in pumping of blood, via peristalsis, into the alimentary canal of the copepod host., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Juvenile edible crabs, Cancer pagurus L., were surveyed from Mumbles Head and Oxwich Bay in South Wales, UK, and the number of heterotrophic bacteria and vibrios in the hemolymph was determined. The percentage of crabs with hemolymph containing bacteria was variable over the survey with higher numbers of animals affected in summer than in winter. Post-moult crabs contained significantly higher numbers of heterotrophic bacteria in the hemolymph than pre- and intermoult animals. Crabs with cuticular damage to the gills also had significantly higher numbers of bacteria in the hemolymph. Crabs were found to have a high prevalence of infection by the dinoflagellate, Hematodinium. Such animals had significantly fewer bacteria in the blood in comparison with Hematodinium-free animals. Of the 463 crabs surveyed, only 3 individuals had hemolymph containing 2000 + CFU mL(-1). Based on 16S rRNA gene sequences, two of these crabs contained a Vibrio pectenicida-like isolate, while the other had a mixed assemblage of vibrios. Although 59% of the crabs surveyed had culturable bacteria in the hemolymph, the majority only had small numbers (<2000 CFU mL(-1) ), suggesting that such infections may be of limited importance to the sustainability of the crab fishery in this region., (© 2013 John Wiley & Sons Ltd.)

Diseases of lobster shells have a significant impact on fishing industries but the risk of disease transmission between different lobster species has yet to be properly investigated. This study compared bacterial biofilm communities from American (Homarus americanus) and European lobsters (H. gammarus), to assess both healthy cuticle and diseased cuticle during lesion formation. Culture-independent molecular techniques revealed diversity in the bacterial communities of cuticle biofilms both within and between the two lobster species, and identified three bacterial genera associated with shell lesions plus two putative beneficial bacterial species (detected exclusively in healthy cuticle or healing damaged cuticle). In an experimental aquarium shared between American and European lobsters, heterospecific transmission of potentially pathogenic bacteria appeared to be very limited; however, the claws of European lobsters were more likely to develop lesions when reared in the presence of American lobsters. Aquarium biofilms were also examined but revealed no candidate pathogens for environmental transmission. Aquimarina sp. 'homaria' (a potential pathogen associated with a severe epizootic form of shell disease) was detected at a much higher prevalence among American than European lobsters, but its presence correlated more with exacerbation of existing lesions rather than with lesion initiation., (© 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Both wild and cultured mussels (Mytilus edulis, Mytilus galloprovincialis and hybrids), are found along most of the Irish coastline. M. edulis is widespread along all Irish coasts and is the only mussel species present on both the east coast of Ireland and the Welsh coast in the Irish Sea. M. galloprovincialis and hybrids are found along the Irish coastline except for the east coast. Samples of Mytilus spp. were collected from twenty-four sites, encompassing all coasts of Ireland and the Welsh coast, at different times of the year and over several years (2008-2011). In total, 841 mussels were examined histologically to assess their health status and the presence of any parasites or commensals. Mussels from 14 of the 24 sites were screened using polymerase chain reaction (PCR) to determine which mytilid species were present. A range of parasites were observed, generally at low levels. The most diverse community of parasites was observed at a sheltered site with poor water quality. Of significance, a previously undescribed haplosporidian was detected in a single mussel sample in the Menai Strait, Wales, by PCR and was confirmed by direct sequencing and is most closely related to Minchina chitonis and a haplosporidian of the Florida marsh clam Cyrenoida floridana. While M. edulis were infected by a variety of micro- and macro-parasites, only trematodes were observed in M. galloprovincialis and hybrids. Habitat description and the environmental factors influencing the study sites, including water quality and exposure, were recorded., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The integument of arthropods is an important first-line defence against the invasion of parasites and pathogens. Once damaged, this can be subject to colonisation by microbial agents from the surrounding environment, which in crustaceans can lead to a condition termed shell disease syndrome. This condition has been reported in several crustacean species, including crabs and lobsters. The syndrome is a progressive condition where the outer cuticle becomes pitted and eroded, and in extreme cases is compromised, leaving animals susceptible to septicaemia. This study examined the susceptibility of juvenile American (Homarus americanus) and European (Homarus gammarus) lobsters to shell disease, as a result of mechanical damage. Scanning electron microscopy was used as a method to identify differences in the cuticle structure and consequences of mechanical damage. Claw regions were aseptically punctured, whilst carapaces were abraded using sterile sandpaper, to mimic natural damage. After a period of between 10 and 12 weeks, lobsters were sacrificed, fixed and stored for later examination. The carapace and claws of juvenile American lobsters were shown to be thinner and more vulnerable to abrasion damage than their European counterparts. In addition, the number and distribution of setal pits and pore canal openings also differed between the two species of lobster. Mechanical damage resulted in the formation of shell disease lesions on the claw and carapace of both lobster species. However, American lobsters, unlike their European counterparts, had extensive bacterial colonisation on the margins of these lesions. Overall, it is concluded that the cuticle of the American lobster is more susceptible to damage and resulting microbial colonisation. This may have implications for susceptibility of both species of lobster to shell disease syndrome., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This study aimed to examine the pathobiology of a haplosporidian-like infection in juvenile (pre-recruit) edible crabs (Cancer pagurus) from two locations in South West Wales, UK. Infected crabs showed no external symptoms of the disease but dissection revealed an infected and hypertrophic antennal gland. Histological examination showed extensive parasitisation of the antennal gland overlying the hepatopancreas. Heavily infected crabs also showed the presence of parasites with morphological similarities to Haplosporidia in the labyrinth of the antennal gland and in the gills. The spread of the infection from the antennal gland to the gills suggests that these parasites are released into the haemolymph. Attempts to characterise the haplosporidian-like organism using several primers previously shown to amplify members of the phylum Haplosporidia failed. The prevalence of infection in juvenile edible crabs varied throughout the sampling period of November 2011 to July 2012 with the lowest level of ca. 15% in November peaking at 70% in March. This parasite may represent a threat to the sustainability of edible crab fisheries in this region if the damage observed in the antennal gland and gills results in host mortality. The identification of these parasites as members of the phylum Haplosporidia based on morphology alone must be seen as tentative in the absence of sequence data., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This study reports on an emerging fungal disease of the edible crab, Cancer pagurus. Juvenile (prerecruit) crabs were found to be subject to this disease condition during the months of May to September at two intertidal sites in South Wales, United Kingdom. Histopathology revealed that the fungi overwhelm the host response in the tissues, leading to progressive septicemia. The causative agent of this infection was isolated and grown in pure culture and was identified as a member of the Ophiocordyceps clade by sequencing of the small subunit of the fungal ribosomal DNA (rDNA). Of the crabs naturally infected with the fungus, 94% had a coinfection with the parasitic dinoflagellate Hematodinium species. To determine if there was any interaction between the two disease-causing agents, apparently fungus-free crabs, both with and without natural Hematodinium infections, were challenged with the fungal isolate. The presence of Hematodinium caused a significant reduction in fungal multiplication in the hemocoel of the crabs in comparison to that in Hematodinium-free individuals. Histopathology of coinfected crabs showed a systemic multiplication of Hematodinium within host tissues, leading to a rapid death, while Hematodinium-free crabs experimentally infected with the fungal isolate died due to fungal sepsis (septicemia) with the same characteristic pathology as seen in natural infections.

Marine reserves (or No-Take Zones) are implemented to protect species and habitats, with the aim of restoring a balanced ecosystem. Although the benefits of marine reserves are commonly monitored, there is a lack of insight into the potential detriments of such highly protected waters. High population densities attained within reserves may induce negative impacts such as unfavourable trophic cascades and disease outbreaks. Hence, we investigated the health of lobster populations in the UK's Marine Conservation Zone (MCZ) at Lundy Island. Comparisons were made between the fished, Refuge Zone (RZ) and the un-fished, No-Take Zone (NTZ; marine reserve). We show ostensibly positive effects such as increased lobster abundance and size within the NTZ; however, we also demonstrate apparent negative effects such as increased injury and shell disease. Our findings suggest that robust cost-benefit analyses of marine reserves could improve marine reserve efficacy and subsequent management strategies.

Both eicosanoid generation and the complement system have long evolutionary histories predating the emergence of the vertebrates over 500 myr ago. This study investigated the interplay between these two systems in an example of a bony fish, the rainbow trout (Oncorhynchus mykiss). Specifically, it examined whether purified complement fragments including C3a-1 and zymosan-activated serum, stimulate the biosynthesis of any of these eicosanoids by trout macrophages. Incubation of macrophages with zymosan pre-incubated with normal trout serum resulted in the phagocytosis of such particles and the generation of both intra- and extra-cellularly located lipoxygenase and cyclooxygenase products. Both eicosanoid generation and phagocytosis levels were significantly elevated following incubation of zymosan in trout serum in comparison with heat-inactivated (60°C for 30 min) trout serum and saline alone. A combined mass spectrometry/high performance liquid chromatography approach was employed to conclusively demonstrate the presence of the cyclooxygenase product, prostaglandin E (PGE) in the culture supernatants of ionophore-challenged macrophages. Incubation of trout macrophages with zymosan-activated trout serum (i.e. no zymosan present) failed to stimulate PGE generation. Similarly, incubation of these cells for up to 60 min with C3a-1 (4 or 50 nM) failed to generate significant amounts of PGE or lipoxygenase products such as leukotriene B(4/5) or lipoxin A(4/5). Longer term (6 & 24h) incubation of macrophages with C3a-1 (4 nM) resulted in a time dependent increase in the generation of PGE but not leukotriene B in culture supernatants. No conclusive evidence that the increase in PGE generation was caused by changes in the expression of either cyclooxygenase-1 or -2 was found., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ectoparasitic copepods have been reported in a wide range of aquatic animals, including crustacean shellfish. However, with the exception of the salmon louse, Lepeophtheirus salmonis, our knowledge of such parasites in commercial species is rudimentary. The current study examines the morphology and pathology of the parasitic copepod, Nicothoë astaci (the 'lobster louse') in its host, the European lobster, Homarus gammarus. Lobsters were sampled from waters surrounding Lundy Island (Bristol Channel, UK) and all individuals collected were found to harbour female adult N. astaci in their gills, with a mean of 47·3 parasites/lobster. The majority of N. astaci were found in the basal region of pleurobranch gills. The parasite was found to attach to gill filaments via its oral sucker, maxillae and maxillipeds, and to feed on host haemolymph (blood) through a funnel-like feeding channel. It caused varying degrees of damage to the host gill, including occlusion of gill filaments and disruption to the vascular system in the central axis. Although there was evidence of extensive host response (haemocytic infiltration) to the parasite, it was displaced from the parasite attachment site and thus was observed in the central gill axis below. The region of gill filament immediately underlying the parasite feeding channel was devoid of such activity suggesting that the parasite interferes with the cellular defence and haemostatic mechanisms of the lobster in order to maintain invasion of the host.

This study was conducted to determine if exposure of shrimp, Litopenaeus vannamei, to a commercial anti-vibrio vaccine caused changes in antibacterial and cellular (phagocytosis) defences. Shrimp post-larvae were administered either Vibromax™ vaccine or a blank preparation. Whole body homogenates were prepared before (day 0), during (day 10) and after (day 20) vaccination and incubated with a selection of pathogenic vibrios. Homogenate from day 0 animals showed natural antibacterial activity towards Vibrioanguillarum which was significantly enhanced for bacteria-exposed shrimp at 10 days post-challenge. This effect of the vaccine was short-term in its duration. No antibacterial activity was observed in day 0 shrimp homogenate against Vibrio alginolyticus but it was significantly enhanced for both vaccinated and blank-vaccinated shrimp by day 10. No natural or inducible antibacterial activity was observed against Vibrio harveyi at 0, 10 or 20 days post-challenge. To determine if prior exposure of shrimp to inactivated vibrios results in elevated hemocyte phagocytic activity, juveniles were injected with either a mixture of formalin-inactivated vibrios or saline. Hemocyte monolayers made from these shrimp were overlaid with a 1:1 mix of Bacillus subtilis and these vibrios. Hemocytes from vibrio-exposed animals showed elevated levels of internalised vibrios compared with those from the saline injected group. These studies show selectively enhanced cellular defences of shrimp following 'vaccination'., (Copyright © 2011 Elsevier Inc. All rights reserved.)

It has long been viewed that invertebrates rely exclusively upon a wide variety of innate mechanisms for protection from disease and parasite invasion and lack any specific acquired immune mechanisms comparable to those of vertebrates. Recent findings, however, suggest certain invertebrates may be able to mount some form of specific immunity, termed 'specific immune priming', although the mechanism of this is not fully understood (see Textbox S1). In our initial experiments, either formalin-inactivated Vibrio harveyi or sterile saline were injected into the main body cavity (haemocoel) of juvenile shrimp (Litopenaeus vannamei). Haemocytes (blood cells) from V. harveyi-injected shrimp were collected 7 days later and incubated with a 1:1 mix of V. harveyi and an unrelated gram positive bacterium, Bacillus subtilis. Haemocytes from 'vaccinated' shrimp showed elevated levels of phagocytosis of V. harveyi, but not B. subtilis, compared with those from saline-injected (non-immunised) animals. The increased phagocytic activity was characterised by a significant increase in the percentage of phagocytic cells. When shrimp were injected with B. subtilis rather than vibrio, there was no significant increase in the phagocytic activity of haemocytes from these animals in comparison to the non-immunised (saline injected) controls. Whole haemolymph (blood) from either 'immunised' or non-immunised' shrimp was shown to display innate humoral antibacterial activity against V. harveyi that was absent against B. subtilis. However, there was no difference in the potency of antibacterial activity between V. harveyi-injected shrimp and control (saline injected) animals showing that 'vaccination' has no effect on this component of the shrimp's immune system. These results imply that the cellular immune system of shrimp, particularly phagocytosis, is capable of a degree of specificity and shows the phenomenon of 'immune priming' reported by other workers. However, in agreement with other studies, this phenomenon is not universal to all potential pathogens.

Aims: The objective of the work was to determine whether known strains of nonpathogenic vibrios can act as probiotics for the control of Vibrio infections in the Pacific white shrimp, Litopenaeus vannamei., Methods and Results: Of the ten species tested, only Vibrio alginolyticus (NCIMB 1339) and Vibrio gazogenes (NCIMB 2250) showed antagonistic activity towards a panel of shrimp pathogenic vibrios. In the case of V. alginolyticus, this activity depended on the presence of live bacteria while in V. gazogenes both live and dead bacteria showed anti-Vibrio activity. Injection of shrimp with either V. alginolyticus or V. gazogenes at 3 × 10(7) or 3 × 10(5) total bacteria per shrimp resulted in mortality with higher levels in the case of V. alginolyticus (100% mortality 18 h postinjection of 3 × 10(7) bacteria). Juvenile shrimp were fed commercial diets top-coated with either chitin (an immune stimulant) or chitin + V. gazogenes. Both chitin and V. gazogenes caused a significant decline in the number of Vibrio-like bacteria in the fore and hind gut, and changes were also seen in the hepatosomatic index (a measure of digestive health) and the total number of blood cells in circulation. Analysis of mid/hindgut and faecal samples obtained using terminal restriction fragment length polymorphism showed that the gut microbiota of shrimp has limited bacterial diversity and that after 8 weeks exposure to the experimental diets there were significant changes in the microbial flora of the GI tract of shrimp as a result of the presence of V. gazogenes., Conclusions: Of the vibrios tested, V. gazogenes has potential as a probiotic for the control of bacterial diseases in shrimp., Significance and Impact of the Study: Overall, this study shows the promise of V. gazogenes together with chitin to improve the health and welfare of shrimp under aquaculture conditions., (© 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.)

The exoskeletons of aquatic crustaceans and other arthropods contain chitin, a biopolymer of beta-(1,4)-linked N-acetylglucosamine together with associated proteins. Despite the vast amounts of chitin within such animals little is found in sediments and open water because microorganisms rapidly degrade this following its loss after moulting or upon the animals' death. Shell disease syndrome is a worldwide disease condition that affects a wide range of crustaceans. It comes about as a result of bacterial degradation of the exoskeleton leading to unsightly lesions and even death if the underlying tissues become infected. There are at least two potential forms of the disease; one that appears to centre around chitin degradation and an additional form termed 'epizootic' shell disease, in which chitin degradation is of less significance. This account reviews our current understanding of the causative agents of this syndrome, assesses the potential economic consequences of the disease, and critically examines whether it is associated with anthropogenic disturbances including pollution. Overall, despite extensive studies during the last few decades, the potential links between faecal, heavy metal and insecticide pollution and shell disease are still unclear.

Until recently, it was widely accepted that invertebrates fail to show a high degree of specificity and memory in their immune strategies. Recent reports have challenged this view such that our understanding of the capabilities of the invertebrate immune systems needs to be reassessed. This account critically reviews the available evidence that suggests the existence of a high degree of memory and specificity in some invertebrates and seeks mechanistic explanations of such observations. It is postulated that elevated levels of phagocytosis may be a partial explanation for this phenomenon.

Several rickettsia-like diseases have been reported in arthropods (insects and crustaceans), some of which result in significant losses of economically important species such as shrimp and crabs. This study reports on the molecular pathology of a recently emerged disease of the European shore crab, Carcinus maenas, termed milky disease - named as a result of the unusual milky appearance of the haemolymph (blood). This disease was more prevalent (>26 %) during summer months when the water temperature in a pilot crab farm was approximately 19 degrees C. The putative causative agent of the disease was a Gram-negative bacterium that could not be cultured on a range of agar-based growth media. Diseased crabs showed significant reductions in free blood cell numbers and total serum protein. Such animals also displayed raised levels of glucose and ammonium in blood. Ultrastructural and in situ hybridization studies revealed that the causative agent associated with milky disease multiplied in the fixed phagocytes of the hepatopancreas (digestive gland), ultimately to be released into the haemolymph, where the circulating blood cells showed little response to the presence of these agents. Attempts to induce the infection by short-term temperature stress failed, as did transmission experiments where healthy crabs were fed infected tissues from milky disease affected individuals. Sequence analysis of the 16S rRNA gene from the milky disease bacteria indicated that they are a previously undescribed species of alpha-proteobacteria with little phylogenetic similarity to members of the order Rickettsiales.

Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acids. The potential of the coelomocytes of the starfish, Asterias rubens, to generate eicosanoids through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways was investigated using reverse-phase high performance liquid chromatography, enzyme immunoassay and gas chromatography-mass spectrometry. The principal LOX product was identified as 8-hydroxyeicosatetraenoic acid (8-HETE) with 8-hydroxyeicosapentaenoic acid (8-HEPE) synthesised at significantly lower levels. No classical prostaglandins (PG), such as PGE2 or PGD2, were found to be generated by ionophore-challenged coelomocytes. Incubation of coelomocytes with lipopolysaccharides from either Escherichia coli or Salmonella abortus failed to induce an increase in generation of LOX products and the presence of 8-HETE (0-25 microM) had no significant effect on the in vitro phagocytic activity of Asterias coelomocytes. Neither indomethacin (a COX inhibitor) or esculetin (a LOX inhibitor) had any effect on the clearance of the bacterium, Vibrio splendidus, from the coelomic cavity of starfish suggesting that products of these enzymes are not involved in such coelomocyte responses to foreign particles.

Adult male shore crabs (Carcinus maenas) were maintained on a fish-based diet supplemented with 0, 5 or 10% chitin for 11 weeks. Significantly greater mortality was found during this period in the control group (0% chitin) than those fed 10% chitin. Crabs fed 5 or 10% chitin had lower numbers of cultivatable bacteria in the hepatopancreas than those on the basal diet alone. The addition of chitin had no significant effect on the serum concentrations of protein and glucose, and the levels of glycogen in the hepatopancreas. The total number of circulating hemocytes in the blood was unaffected by the addition of chitin to the diet, however, at week 6 there were significantly more hyaline hemocytes in those crabs fed 10% chitin than the control group. The in vitro phagocytic activity of hemocytes was unaffected by chitin supplementation and crabs challenged with Vibrio alginolyticus showed a similar pattern of susceptibility in the three dietary groups (0, 5 or 10% chitin). Overall although crabs on a chitin-supplemented diet showed greater survival, this was not explained in terms of elevation in the cellular defences of these animals. The enhanced survival of crabs-fed chitin is probably as a result of the removal of potentially pathogenic bacteria from the hepatopancreas. Because chitin appears to 'purge' bacteria from the gut, this may prove to be a useful addition to diets on animals undergoing oral probiotic treatment.

Post-copulatory behaviour in barnacles involves a violent rocking movement of the opercular valves, which is thought to contribute to the expulsion of oocytes through the oviduct into the mantle cavity where they are fertilised. We demonstrate in this study that the seminal vesicles/testis of the subtidal barnacle Balanus balanus produce a biologically active factor, barnacle muscle stimulatory factor (BMSF), which causes a significant increase in cirral and body muscular activity. BMSF was identified using a combination of high performance liquid chromatography and mass spectrometry as a novel eicosanoid/oxylipin, 8,13-dihydroxyeicosapentaenoic acid. This is rapidly inactivated under mild acid conditions to form a complex range of triene and pentaene chromophore-containing products that have only been partially identified. Injection of purified BMSF into the mantle cavity of barnacles caused the rocking movements of the opercular valves as reported following fertilisation. In excised barnacles, it also caused muscular contractions of the whole body mass. The breakdown products of BMSF, however, were without such activities. The function of BMSF in facilitating fertilisation in barnacles is comparable to the role of other eicosanoids in human reproduction, reinforcing the view that these compounds have conserved activities in both invertebrates and vertebrates.

Docosahexaenoic acid (DHA; C22:6 n-3) is an abundant fatty acid in fish phospholipids. In the present study, we employed liquid chromatography-ultraviolet spectrometry-tandem mass spectrometry and dissociated rainbow trout (Oncorhynchus mykiss) brain cells to determine whether fish utilize endogenous DHA to produce the recently uncovered novel lipid mediators termed the resolvins and protectins, generated by mammalian cells [Serhan CN, Hong S, Gronert K, et al. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med 2002; 196:1025-37; Hong S, Gronert K, Devchand P, Moussignac R-L, Serhan, CN. Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. J Biol Chem 2003;278:14677-87]. Trout brain cells biosynthesize a range of recently identified di- and tri-hydroxy-containing bioactive products from endogenous sources of DHA when challenged in vitro. We identified neuroprotectin D1, resolvin D5, resolvin D1 and resolvin D2 from trout brain cells. Each compound was identified on the basis of its characteristic physical chemical properties that included MS, MS-MS, UV spectra and chromatographic behavior. The monohydroxy products from DHA, signatures of DHA conversion by lipoxygenases, were also identified. These included both 14S-hydroxy-docosahexaenoic acid and 17S-hydroxy-docosahexaenoic acid. The biosynthesis of these novel bioactive lipid mediators, namely resolvins and protectins, by fish cells provides the first evidence for the conservation of these structures from fish to humans as chemical signals in diverse biological systems.

The nature and quantity of the principal lipoxygenase (LO) products generated by the intertidal barnacle Balanus perforatus were examined at monthly intervals and their potential involvement in reproductive events was investigated. The main mono-hydroxy products generated were found to be formed through the action of an 8-lipoxygenase (8-LO) activity and were the mono-hydroxy fatty acids, 8-hydroxyeicosapentaenoic acid (HEPE) and 8-hydroxyeicosatetraenoic acid. Generation of these products was found to be correlated with the environmental seawater temperature, although no change in either 8-LO activity or the precursor fatty acid levels in total phospholipids was found with the time of the year. Changes in fatty acid composition measured in animals collected from summer and winter conditions were found to follow the pattern expected by homeoviscous adaptation of a cold-acclimated animal. Oogenesis was found to occur in August and was linked to a significant reduction in 8-HEPE generation. Spermatozoa were found to be present year-round in the seminal vesicles although the testes became atrophic during the winter months., ((c) 2005 Wiley-Liss, Inc.)

Prostaglandins (PG) are oxygenated derivatives of C20 polyunsaturated fatty acids including arachidonic and eicosapentaenoic acids. In mammals, these compounds have been shown to play key roles in haemostasis, sleep-wake regulation, smooth muscle tone, and vaso-, temperature and immune regulation. In invertebrates, PGs have been reported to perform similar roles and are involved in the control of oogenesis and spermatogenesis, ion transport and defence. Although there is often a detailed understanding of the actions of these compounds in invertebrates such as insects, knowledge of their mechanism of biosynthesis is often lacking. This account provides a critical review of our current knowledge on the structure and modes of biosynthesis of PGs in invertebrates, with particular reference to aquatic invertebrates. It emphasises some of the most recent findings, which suggest that some PGs have been misidentified. Prostaglandins in invertebrates can be categorised into two main types; the classical forms, such as PGE2 and PGD2 that are found in mammals, and novel forms including clavulones, bromo- and iodo-vulones and various PGA2 and PGE2 esters. A significant number of reports of PG identification in invertebrates have relied upon methods such as enzyme immunoassay that do not have the necessary specificity to ensure the validity of the identification. For example, in the barnacle Balanus amphitrite, although there are PG-like compounds that bind to antibodies raised against PGE2, mass spectrometric analysis failed to confirm the presence of this and other classical PGs. Therefore, care should be taken in drawing conclusions about what PGs are formed in invertebrates without employing appropriate analytical methods. Finally, the recent publication of the Ciona genome should facilitate studies on the nature and mode of biosynthesis of PGs in this advanced deuterostomate invertebrate.