Detection of the parasitic dinoflagellate Hematodinium in the Norway lobster Nephrops norvegicus by ELISA

Norway lobsters Nephrops norvegicus from the coastal waters of Scotland are seasonally infected by a parasitic dinoflagellate of the genus Hematodinium. An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of the parasite in the haemolymph of N. norvegicus. The ELISA is simple to perform with a detection limit of 5 x 10(4) parasites ml(-1) haemolymph. The ELISA is currently being used to study the prevalence and seasonality of Hematodinium infection in N. norvegicus and other crustacean hosts.     

Molecular detection of Hematodinium spp. in Norway lobster Nephrops norvegicus and other crustaceans

The Norway lobster Nephrops norvegicus (L.) from the coastal waters of Scotland is seasonally infected by a parasitic dinoflagellate of the genus Hematodinium. Methods used to detect infection include a morphological index (pleopod diagnosis) and several immunoassays. The present study describes the development and application of a set of Hematodinium-specific polymerase chain reaction (PCR) primers and DNA probes based on Hematodinium ribosomal DNA (rDNA). In the PCR assay, a diagnostic band of 380 bp was consistently amplified from total genomic DNA isolated from Hematodinium-infected N. norvegicus. The sensitivity of the assay was 1 ng DNA, which is equivalent to 0.6 parasites. The primer pair also detected Hematodinium DNA in preparations of the amphipod Orchomene nanus, indicating that the amphipod may be infected with the same Hematodinium sp. infecting N. norvegicus. DNA probes detected Hematodinium parasites in heart, hepatopancreas and gill tissues from N. norvegicus, and hepatopancreas and gill tissues from Carcinus maenas, confirming Hematodinium infection in the latter.     

Alterations in the biochemistry and ultrastructure of the deep abdominal flexor muscle of the norway lobster Nephrops norvegicus during infection by a parasitic dinoflagellate of the genus Hematodinium

Changes in various biochemical and ultrastructural characteristics of the deep abdominal flexor (DAF) muscles were studied in Norway lobster Nephrops norvegicus (L.) from the Clyde estuary, Scotland, UK, at different stages of infection by a parasitic dinoflagellate of the genus Hematodinium. Abdominal DAF muscles from infected lobsters showed slight, significant increases in total water content, along with greatly depleted glycogen reserves and an altered free amino acid profile. However, protein concentration and composition remained unchanged. Ultrastructurally, parasitic infection of DAF muscle fibres caused alterations in sarcolemmal structure, and localized disruption of myofibrillar bundles around the periphery, but not throughout the centre of the fibres. Overall, the reduction in swimming performance previously reported for N. norvegicus during Hematodinium infection reflect an alteration in carbohydrate supply to the active muscle and some subtle disruption of muscle structure. The altered carbohydrate titre could reflect the Hematodinium parasites acting as a carbohydrate sink in the haemolymph, a disruption of normal tissue glycogenesis, or some alteration in the host's hormonal regulation. These changes could also adversely affect the taste, texture and marketability of infected meat.     

Conservation in the first internal transcribed spacer region (ITS1) in Hematodinium species infecting crustacean hosts found in the UK and Newfoundland

Parasitic dinoflagellates in the genus Hematodinium infect a number of decapod crustaceans in waters off the UK, including the Norway lobster Nephrops norvegicus and the edible crab Cancer pagurus. This study investigated sequence variability in the first internal transcribed spacer (ITS1) region of the ribosomal RNA complex of Hematodinium spp. infecting N. norvegicus, C. pagurus, and Pagurus bernhardus from 4 locations in the UK and from the Hematodinium sp. infecting Chionoecetes opilio from the province of Newfoundland and Labrador, Canada. Phylogenetic analysis of the Hematodinium ITS1 sequences from N. norvegicus, C. pagurus, P. bernhardus and C. opilio suggest that these crustaceans are infected with the same species of Hematodinium. Length variability of the ITS1 region was observed (324 to 345 bp) and attributed to 4 variable microsatellite regions (CATG)n' (GCC)nTCCGC(TG)n' (TA)n' and (GAA)n(GGA)n within the sequenced ITS1 fragment. The observed variation may be due to co-infection of the host crustacean with several different strains of Hematodinium or differences among copies of ITS1 region within the genome of a single parasite cell. The Hematodinium ITS1 sequence from N. norvegicus, C. pagurus, P. bernhardus and C. opilio isolates was sufficiently conserved in primer binding regions targeted by previous molecular diagnostic assays; therefore, we suggest that this assay could be used to screen for Hematodinium infections in these crustacean hosts.     

An analysis of swimming performance in the Norway lobster, Nephrops norvegicus L. infected by a parasitic dinoflagellate of the genus Hematodinium

Various components of swimming performance were measured in uninfected Norway lobsters (Nephrops norvegicus) and compared to animals at different stages of infection by a parasitic dinoflagellate (Hematodinium sp.). Animals showed a progressive decline in overall swimming performance as infection severity increased, with reductions in the number of tail-flips performed, the number of swimming bouts and the total distance travelled by swimming. The velocity of the first (giant-fibre mediated) tail flip and average velocity over the swimming bout were also significantly reduced in infected lobsters. Possible reasons for this decreased swimming performance are suggested and the implications of this for predator avoidance of infected lobsters in the benthic habitat, and for capture of Nephrops by trawl rigs are discussed.     

Hematodinium sp. (Alveolata, Syndinea) detected in marine decapod crustaceans from waters of Denmark and Greenland

Five decapod crustacean species were examined for presence of the parasitic dinoflagellate Hematodinium spp. (Alveolata, Syndinea) by morphological methods (colour and pleopod methods) as well as by PCR and nested PCR with Hematodinium-specific primers. Nephrops norvegicus, Pagurus bernhardus and Liocarcinus depurator were sampled by trawling in Danish waters and Chionoecetes opilio and Hyas araneus were sampled by trapping off the west coast of Greenland. The existence of Hematodinium has not previoiusly been documented in Danish waters, but it was detected in all 3 decapod species examined in the present study. Hematodinium sp. was also detected for the first time in H. araneus and the existence of Hematodinium sp. in Greenlandic C. opilio was documented by PCR. Analyses of 26 Hematodinium sp. ITS1 sequences, including sequences from all 5 host species sampled, revealed more than 95% sequence similarity between 24 of the sequences. Two Hematodinium sp. ITS1 sequences from C. opilio were only 81% similar to the 24 other ITS1 sequences. The nested PCR approach resulted in the highest reported percentages of positive samples for Hematodinium sp.in the hosts investigated (between 45 and 87.5%). However, no decapods were found to be infected with Hematodinium sp. based on morphological methods. Consequently, Hematodinium sp. may be more common than previously believed, and, assuming that the DNA found originated from viable and infectious parasite cells, infections may not always be fatal. We suggest that the hosts investigated may have been subject to latent infections that could develop into a fatal disease only if the hosts were physiologically stressed due to other factors.     

Infection by a Hematodinium-like parasitic dinoflagellate causes Pink Crab Disease (PCD) in the edible crab Cancer pagurus

The edible crab (Cancer pagurus) supports a large and valuable fishery in UK waters. Much of the catch is transported live to continental Europe in specially designed live-well ('vivier') vehicles. During the winter of 2000/2001, many trap-caught crabs from Guernsey, Channel Islands, UK, were reportedly moribund and pink in colour. These crabs generally died before and during vivier transportation. We provide histological, immunological, and molecular evidence that this condition is associated with infection by a Hematodinium-like dinoflagellate parasite similar to that previously reported in C. pagurus and to an infection causing seasonal mass mortalities of the Norway lobster (Nephrops norvegicus). Pathologically, every altered host bore the infection, which was characterised by very large numbers of plasmodial and vegetative stages in the haemolymph and depletion of reserve cells in the hepatopancreas. Due to the hyperpigmentation of the carapace and appendages, we have called this infection 'Pink Crab Disease' (PCD). Similar Hematodinium infections cause 'Bitter Crab Disease' in tanner and snow crabs, which has had a negative effect on their marketability. At present, little is known about the seasonality, transmission, and market impact of this infection in C. pagurus.     

Differences in enzyme activities between two species of Hematodinium, parasitic dinoflagellates of crustaceans

Parasitic dinoflagellates of the genus Hematodinium infect several commercially important decapod crustaceans. Different species of Hematodinium have different levels of virulence in their respective hosts. Enzyme activities were studied from two species of Hematodinium, one isolated from the Norway lobster (Nephrops norvegicus) and the other from the American blue crab (Callinectes sapidus). We report the identification of differences in secretion of acid phosphatase (AP) and leucine arylamidase from two parasite species. Leucine arylamidase was only contained and secreted by the species infecting the blue crab. Both parasite species contained AP, but only the species infecting the Norway lobster secreted this enzyme. In this species, AP activity was predominantly in the soluble fraction (69.5%). AP activity was localized to cytoplasmic granules and on the membranes surrounding the cell nucleus. In addition to providing information on the cellular metabolism of the parasite, the pattern of activities of these enzymes may also be useful in distinguishing among different species of Hematodinium.     

Alteration of burrow‐related behaviour of the norway lobster,nephrops norvegicus during infection by the parasitic dinoflagellate hematodinium

This study has used time‐lapse video recordings to analyze the burrow‐related behaviour of N. norvegicus under a 12 h light: dark regime in aquarium conditions, and compared this with the behaviour of lobsters infected by the parasitic dinoflagellate Hematodinium. Uninfected lobsters performed a higher number of burrow departures, of longer duration, during the hours of darkness than during the light. Infected lobsters performed a relatively constant number of departures of similar‐duration in the light and the dark periods. However, the mean number of departures per day performed by infected lobsters (70 day^‐1) was more than double that of uninfected lobsters (30.1 day^‐1), while the mean duration of burrow departures performed by infected lobsters (258.5s departure^‐1) was more than six times greater than in uninfected lobsters (38.7s ? departure^‐1). This led to a more than ten‐times increase (from 1.7 to 19.4%) in the percentage of the day spent out of the burrow by infected lobsters. This altered burrow‐related behaviour could be due to the nutritional demands of the parasites on infected lobsters, causing an increased requirement to forage, or alternatively to a ‘functional hypoxia’, due to the additional respiratory demands of the parasite, causing the host to emerge from the burrow onto the sediment surface to seek oxygen. Implications for the increased time spent out of the burrow are discussed in relation to availability of infected lobsters by trawlers and predators, and to the use of infection prevalence data from trawl‐caught samples in stock assessment models for N. norvegicus.     

A review of the parasitic dinoflagellates Hematodinium species and Hematodinium-like infections in marine crustaceans

Parasitic dinoflagellates in the genus Hematodinium are important parasites of marine Crustacea. Outbreaks of these parasites have damaged commercial stocks of Norway lobster Nephrops norvegicus, snow crab Chionoecetes opilio, Tanner crab C. bairdi, American blue crab Callinectes sapidus, and velvet swimming crab Necora puber. Species of Hematodinium can reach high enough levels to regulate their host populations, but mortalities are also centred on the unfished juveniles and females, hosts not normally sampled by fisheries; hence impacts are often underreported. Seasonal prevalences of up to 85 % occur annually in many host populations; in effect, these parasites form cryptic blooms in the water column with crabs and other crustaceans at risk of disease. We review the biology and ecology of Hematodinium spp. infections in crustaceans. Included is a comparison of the different infections, a synthesis of what is known, and an attempt to highlight fruitful areas for continued research.     

Ensuring crustacean product quality in the post-harvest phase

Recent studies of the fisheries for the Norway lobster, Nephrops norvegicus (L.), have illustrated the negative effects of pathogens and of the physiological stresses of capture processes on the exploitation of live animals and their products, and have identified mitigating measures. Firstly, having established that trawl capture of N. norvegicus is highly stressful, but that these animals have powerful physiological mechanisms of recovery, procedures for on-board recovery of animals destined for vivier transport to distant European markets have been implemented commercially, with significant improvements in survival rates. Such procedures also mitigate against the initiation of a stress-induced muscle necrosis. Secondly, measurements of post-mortem autolytic and spoilage processes have identified the existence of a post-capture 'handling window' of several hours which allows the whole or tailed products to be preserved, by icing or freezing, without detriment to quality. Commercial consortia of Scottish fishermen are exploiting this opportunity to extend product shelf-life by freezing at sea within this handling window. Thirdly, the well-documented infections of Scottish N. norvegicus populations by the dinoflagellate Hematodinium sp. not only provide examples of pathogen-induced mortality, but also have effects on post-harvest products including vivier transport losses and changes in post-mortem degradation leading to earlier organoleptic rejection. Under commercial conditions these effects can be mitigated by post-capture visual screening, but only during the periods of peak patent infection when parasitised animals are visually identifiable. Wider implementation of such mitigating procedures during the harvesting of wild-caught crustaceans will contribute to a more sustainable exploitation of these valuable marine resources.     

Identification and partial characterisation of metalloproteases secreted by a Mesanophrys-like ciliate parasite of the Norway lobster Nephrops norvegicus

A ciliate parasite, tentatively identified as Mesanophrys sp. of Norway lobsters Nephrops norvegicus, is demonstrated to secrete several proteases into the culture medium (modified Nephrops saline). Analyses using substrate-impregnated sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 12 activity bands differing greatly in mobility in the gels. The complete inhibition of proteolytic activity by 1,10-phenanthroline indicated that the proteases are of the metallo class. The proteases were active at the physiological temperature (8 degrees C) and haemolymph pH (7.8) of the host. The proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a major structural component of lobster muscle. Consequently, these proteases may have important roles in several aspects of the host-parasite interaction including invasion, nutrient uptake by the ciliate, and pathogenesis.     

Ecological and physiological effects of PaV1 infection on the Caribbean spiny lobster (Panulirus argus Latreille)

Pathogens can profoundly impact the ecology of the organisms they infect through changes in host behavior that influence demographic processes. For example, juvenile Caribbean spiny lobsters (Panulirus argus Latreille) infected with the PaV1 virus (Panulirus argus Virus 1) are avoided by their normally social conspecifics, which alters local spatial distributions and presumably rates of disease transmission. PaV1 infections are nearly always lethal, but prior to succumbing to the disease, infection may impact other host dynamics (e.g., movement, growth, or survival) that effect transmission of the virus. We used mark-recapture surveys and laboratory studies to determine the impact of PaV1 infection on lobster movement and physiological condition, and in turn, the effect of condition on susceptibility to infection. Significantly more healthy lobsters were recaptured than heavily diseased lobsters in 5-d mark-recapture surveys, indicating either greater emigration or greater mortality of infected lobsters. Results of a laboratory bioassay suggest that lobsters with early-stage infections moved at an equivalent rate to healthy lobsters, however, as infection progressed, lobsters inoculated with PaV1 moved less than healthy lobsters ultimately remaining sedentary. Infected lobsters captured in nature also had significantly lower hemolymph (blood) serum protein values, indicating poor physiological condition. However, poor condition did not predispose lobsters to greater risk of infection because prevalence was not different between starved lobsters and healthy lobsters challenged with PaV1. Although lobsters infected with PaV1 eventually become sedentary and of poor nutritional condition, during the early stages of infection they remain active and are thus capable of dispersing the virus throughout the population.     

Partial characterization and amino acid composition of a high molecular weight peptide with salmon calcitonin immunoreactivity in a crustacean: Nephrops norvegicus

Anti-salmon calcitonin antibodies were used to follow the purification of a high molecular weight peptide present both in the haemolymph and in the hepatopancreas of the Norway lobster Nephrops norvegicus. An apparent molecular weight of 22 kDa has been measured in electrophoresis on SDS gels and amino acid composition compared to salmon calcitonin. The amount determined by the immunoreactivity assay corresponds to about 1/40 and 1/140 of that which is based on direct protein measurement for the hepatopancreas and the haemolymph respectively. The total amount of this peptide could be estimated as 3.5 mg/g fresh weight for the hepatopancreas and 140 ug/ml for the haemolymph. The function of this peptide is still unknown.     

Advances in our understanding of the global diversity and distribution of Hematodinium spp. - significant pathogens of commercially exploited crustaceans

Hematodinium species are parasitic dinoflagellates known to infect a growing number of marine crustacean genera from around the world, many of which support important commercial fisheries. Affected hosts undergo dramatic pathological alterations to their organs, tissues and hemolymph. There are no known control measures for this disease. Economically important wild fished hosts known to be susceptible to Hematodinium spp. include Tanner crabs Chionoecetes bairdi and snow crabs Chionoecetes opilio in the Northeast Pacific and Atlantic Oceans, blue crabs Callinectes sapidus from the Atlantic and Gulf coasts of the United States, and Norway lobsters Nephrops norvegicus and Edible crabs Cancer pagurus from European waters. In recent years, several farmed aquatic crustaceans in China have also been negatively impacted by Hematodinium-associated diseases, likely representing an emerging issue for that expanding industry. Molecular sequence data indicates that there are two species, Hematodinium perezi, and a second species, currently unnamed, infecting hosts from the Northern Hemisphere. Three subtly different H. perezi genotypes have been identified infecting hosts from different geographical locations: the English Channel, the eastern seaboard of the United States and Gulf of Mexico, and eastern China. Genotypic variability between isolates of the Hematodinium sp. infecting hosts from the North Atlantic and North Pacific has also been reported, though it is unclear whether there is any correlation with host or location. Identification of Hematodinium species (and genotypes of H. perezi) is largely dependent upon geographical location, rather than host species. However this is not exclusive, as both Hematodinium species can be found infecting multiple species from same location, as is the case in the English Channel.     

A parasitic scuticociliate infection in the Norway lobster (Nephrops norvegicus)

A histophagous ciliate infection was discovered in a number of Norway lobsters (Nephrops norvegicus) from the Clyde Sea Area, Scotland. Silver-carbonate staining of cultured ciliates revealed an oral apparatus and additional structural features that are morphologically similar to scuticociliates in the genus Mesanophrys, which are known to parasitize crustaceans. However, ribosomal DNA sequences (ITS1/5.8S/ITS2) of the ciliate were identical to Orchitophyra stellarum, a parasitic scuticociliate of sea stars with a different morphology from Mesanophrys spp. and to the ciliate from N. norvegicus. Associated pathology included degeneration and necrosis of the myocardial heart muscle, and large numbers of ciliates in the gill filaments.     

Occurrence of the parasite genus Hematodinium (Alveolata: Syndinea) in the water column

Crustaceans worldwide are infected with alveolate parasites of the genus Hematodinium, causing substantial losses to langoustine and crab fisheries. The distinct seasonality in Hematodinium occurrence in their decapod hosts, as well as unsuccessful attempts at transmission, suggest the existence of life stages outside their benthic crustacean hosts. We used a nested polymerase chain reaction method to detect Hematodinium rDNA in the environment and in potential alternative hosts. Environmental samples from the Clyde Sea, Scotland, were screened during the April release of dinospores and during June and August, when infection prevalence is rare in benthic crustaceans. Hematodinium rDNA was amplified in 15% (14/94) of isolated langoustine larvae, and in 12% (13/111) of crab larvae. In addition, Hematodinium rDNA was present in mixed plankton samples devoid of decapod larvae, but including the 2 μm-10 mm fraction of particulate organic matter in the water column, containing phytoplankton and other zooplankton. These results indicate that Hematodinium occurs in the water column and is harboured by planktonic organisms, including larval stages of the crustacean hosts, when infections are at their lowest in adult hosts.     

Reverse transcriptase loop-mediated isothermal amplification assay for infectious hematopoietic necrosis virus in Oncorhynchus keta

Shell disease (SD) has been observed in lobster populations for almost a hundred years, but recently, rates of an epizootic form of shell disease (ESD) have increased in the southern New England (USA) area. A large proportion of fish in the diet of American lobsters Homarus americanus has been linked to increased rates of SD. Therefore, the use of fish as lobster bait may be linked to increased ESD rates in lobsters. Lobsters from the western portion of Martha's Vineyard, MA (41 degrees N, 71 degrees W), were randomly divided into 3 groups of 16 and exposed to dietary treatments (100% herring; 48% crab, 48% blue mussel and 4% plant matter; or 50% herring, 24% crab, 24% mussel, 2% plant matter) to determine if lobster tissue delta15N levels reflected diet. The results of the feeding experiment confirmed that differences in diet are observed in the delta15N levels of lobster muscle tissue. The delta15N levels of tissue samples from 175 wild lobsters with varying degrees of ESD were unrelated to ESD severity but did indicate lobsters were eating large amounts of fish (bait). This result does not support the speculation that fish used as bait is contributing to ESD outbreaks in portions of the southern New England area.