Development and application of an immunoassay diagnostic technique for studying Hematodinium infections in Nephrops norvegicus populations.

Patent Hematodinium infections of the Norway lobster Nephrops norvegicus can be detected with a morphological method (pleopod diagnosis), but this fails to identify low-level haemolymph (sub-patent) and any tissue-based (latent) infections. The current study describes the development and application of an immunoassay for the detection of antigens of the parasite Hematodinium in the Norway lobster N. norvegicus. Infected tissue and haemolymph samples were detected as multiple-band reactions to a polyclonal antibody (anti-Hematodinium). The sensitivity limit of the method was 204 parasites mm(-3), approximately 10 times more sensitive than the pleopod diagnosis method. Use of the immunoassay on tissue samples taken from catches in the Clyde Sea area, Scotland, UK, showed that the pleopod method considerably under-diagnosed infection prevalence in the early part of the season, though this under-diagnosis decreased as infected lobsters in the field progressed from latent and sub-patent to patent infections. However, the immunoassay failed to detect any infected lobsters during the summer months, suggesting that infection may not be carried over from one season to the next. The data presented suggest that this immunoassay allows for the accurate estimation of Hematodinium infection prevalence in the field and should be employed, where possible, for the routine monitoring of infection prevalence in N. norvegicus.     

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.     

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.     

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.     

Effects of emersion and re-immersion on physiological and immunological variables in creel-caught and trawled Norway lobster Nephrops norvegicus

Immune defence in creel-caught and trawled Nephrops norvegicus was investigated to assess a possible relationship between phenoloxidase (PO) activation and the total haemocyte count (THC). Capture, capture method and emersion evoked physiological and immunological responses that may have implications for the ability of N. norvegicus to survive the effects of such stressors. Haemolymph THC was always negatively related to PO activity in the trawled samples, suggesting a decreased level of the plasma serine proteinase inhibitors which reportedly regulate the ProPO system (Le Moullac et al. 1998; Fish shellfish Immunol 8:621-629). In contrast, creel-caught samples showed increased levels of both PO and THC (cf. control N. norvegicus), after a 12 h emersion period. Trawling and emersion evoked progressive and significant increases (p < 0.05) in the mean levels of haemolymph L-lactate, glucose and total ammonia. The evidence of overt activity and measured haemolymph parameters suggest that creel fishing yields N. norvegicus that are more likely to survive post-harvest treatments than those that are trawled.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The role of alternate hosts in the ecology and life history of Hematodinium sp., a parasitic dinoflagellate of the blue crab (Callinectes sapidus)

Hematodinium sp. infections are relatively common in some American blue crab (Callinectes sapidus) populations in estuaries of the western Atlantic Ocean. Outbreaks of disease caused by Hematodinium sp. can be extensive and can cause substantial mortalities in blue crab populations in high salinities. We examined several species of crustaceans to determine if the same species of Hematodinium that infects C. sapidus is found in other crustaceans from the same localities. Over a 2-yr period, 1,829 crustaceans were collected from the Delmarva Peninsula, Virginia, examined for the presence of infections. A portion of the first internal transcribed spacer (ITS1) region of the ribosomal RNA (rRNA) gene complex from Hematodinium sp. was amplified and sequences were compared among 35 individual crustaceans putatively infected with the parasite, as determined by microscopic examination, and 4 crustaceans putatively infected based only on PCR analysis. Of the 18 crustacean species examined, 5 were infected with Hematodinium sp. after microscopic examination and PCR analysis, including 3 new host records, and an additional species was positive only via PCR analysis. The ITS1 rRNA sequences of Hematodinium sp. from the infected crustaceans were highly similar to each other and to that reported from C. sapidus (>98%). The similarity among these ITS1 sequences and similarities in the histopathology of infected hosts is evidence that the same species of Hematodinium found in C. sapidus infects a broad range of crustaceans along the Delmarva Peninsula. Our data indicate that the species of Hematodinium found in blue crabs from estuaries along the east coast of North America is a host generalist, capable of infecting hosts in different families within the Order Decapoda. Additionally, evidence indicates that it may be capable of infecting crustaceans within the Order Amphipoda.     

The development and characterization of an anti‐haemolymph antiserum for the detection of mollusc remains within carabid beetles

Molluscan haemolymph was evaluated as an antigen for the detection, by enzyme‐linked immunosorbent assay (ELISA), of mollusc proteins within the crop contents of carabid beetles, using an anti‐haemolymph antiserum. The value of using a narrow range of prey‐specific immunogens, rather than whole‐body macerates, was discussed. Predators and potential prey were tested by a quantitative indirect ELISA, with separate evaluation of antigen reactions with non‐specific and specific antibodies. The assay proved to be capable of detecting less than 1 ng of haemolymph protein and the antiserum reacted strongly with all molluscs tested. Initial cross‐reactions with earthworms were effectively eliminated by absorption. A range of invertebrates was tested, and a significance level established relative to the invertebrates giving the strongest cross‐reaction, which proved to be wolf spiders Pardosa sp. Harvestmen (Opiliones), that initially gave strong reactions to the antiserum, were shown to have fed upon molluscs. Laboratory tests on the crop contents of slug‐fed beetles demonstrated clear detection for at least 4 days after feeding.     

Development of an 18S rRNA gene-targeted PCR-based diagnostic for the blue crab parasite Hematodinium sp.

The 18S rRNA gene from Hematodinum sp., a parasitic dinoflagellate that infects blue crabs, was amplified, cloned, and sequenced. The sequence showed a high similarity (95% at the nucleotide level) to sequences obtained from other dinoflagellate species, including both free-living and symbiotic species. Sequence similarity was much lower when compared with parasites of other marine invertebrates with similar life histories and with the 18S rRNA gene from the blue crab. Based on comparison of sequence alignments between Hematodinium, other dinoflagellate species, protozoan pathogens of oysters, and blue crab 18S rRNA gene sequences, 2 sets of PCR primers that specifically amplified fragments of the Hematodinium 18S rRNA gene were developed and tested. One of these primer sets (Hemat-F-1487 and Hemat-R-1654) amplified a 187 bp fragment that could be used routinely as a diagnostic test for the presence of Hematodinium in hemolymph from blue crabs. This fragment was consistently amplified from genomic DNA extracted from hemolymph of Hematodinium infected blue crabs. Comparison between the PCR technique and standard histological examination indicated that the PCR technique was reliable and provided 1000 times more sensitivity than the histological methods. The sensitivity of the PCR diagnostic was estimated to be one parasite cell among 300,000 crab hemocytes. Preliminary studies using the PCR diagnostic technique suggest that Hematodinium sp. is absent in crabs collected from waters with low salinity (5 to 10 ppt), but common in crabs from higher salinity environments in estuarine waters from southeastern Georgia (USA).