Hematodinium perezi and Hematodinium sp. of Atlantic Crabs
Category 1 (Not Reported in Canada)
Common, generally accepted names of the organism or disease agent
Hematodinium perezi, Hematodinium sp., Dinoflagellate blood disease, Pink crab disease.
Scientific name or taxonomic affiliation
Hematodinium perezi and Hematodinium sp. in the family Syndiniceae, Order Syndinida (Syndiniales to botanists). Hematodinium spp. can be identified as dinoflagellates on the basis of their: typical dinokaryon (or mesokaryon); the alveolate pellicle; the presence of naked, athecate gymnodinoid dinospores (or zoospores); and the classic form of mitosis known as dinomitosis (Stentiford and Shields 2005). Although Hematodinium spp. have been reported from a broad range of brachyuran crabs (Stentiford and Shields 2005, Morado 2011), only two species (H. perezi and Hematodinium australis) have been described because of their lack of distinct characteristics and poorly understood life cycles. The description of the type species, Hematodinium perezi Chatton and Poisson 1931, is based on only hand-drawn figures which were produced from unstained and stained parasite stages resulting in a lack of apparent informative morphological characters required for species comparisons. In addition, this type species was described as infecting two portunid crab species, Carcinus maenas and Liocarcinus (=Portunus) depurator from multiple locations on the coasts of France with no archive of type specimens (Small 2012). Therefore, Small et al. (2012) described the morphological and molecular characteristics of H. perezi from one of the type hosts L. depurator from a site in the English Channel similar to the geographical location of the type description. Further refinements in DNA analysis should help in differentiating between species of Hematodinium, and thus facilitate our understanding of basic biological parameters including the identification of alternate or reservoir hosts.
Reports on Hematodinium from the eastern and western sides of the North Atlantic Ocean are presented below under subsections a) and b), respectively. This separation may be significant because of the possible presence of different species of Hematodinium in crabs along the coasts of the two continents.
- European waters. Initially described off Luc-sur-Mer and Penpoul (near Roscoff) on the English Channel coast of France, off Arcachon on the Atlantic coast of France, and off Banyuls-sur-Mer on the Mediterranean coast of France (Chatton and Poisson 1931, Stentiford 2006). Distribution also includes the coast of the Bay of Biscay, east coast of Denmark, both sides of the English Channel, waters around Ireland including the Irish Sea, the west coast of Scotland including the Clyde Sea, and the southwest coast of Greenland (Latrouite et al. 1988, Ní Chualáin et al. 2009, Hamilton et al. 2009, Eigemann et al. 2010).
- East and Gulf coasts of the United States from Delaware to Texas including high salinity waters of the lower Chesapeake Bay, coastal bays in Maryland and Virginia, Georgia, and Florida (Newman and Johnson 1975, Couch 1983, Messick 1994, Messick and Shields 2000, Shields 2003, Shields and Overstreet 2007). Zimmerman et al. (2011) reported infected Callinectes sapidus from low salinity (3 ppt) waters in the Northern Gulf of Mexico.
- Carcinus maenas, Liocarcinus (=Portunus) depurator, Cancer pagurus, Necora (=Liocarcinus) puber, Portumnus latipes, Pagurus bernhardus, Pagurus prideaux, Hyas araneus and Munida rugosa (Stentiford and Shields 2005, Small et al. 2007c, Hamilton et al. 2009, Eigemann et al. 2010)
- Callinectes sapidus, Callinectes similis, Cancer irroratus, Cancer borealis, Ovalipes oscellatus, Carcinus maenas, Libinia emerginata, Libinia dubia, Menippe mercenaria, Neopanope sayi, Panopeus herbstii, Eurypanopeus depressus, Hexapanopeus angustifrons, Rhithropanopeus spp. and Pagurus pollicaris (Stentiford and Shields 2001, Shields 2003, Sheppard et al. 2003, Shields and Overstreet 2007, Pagenkopp Lohan et al. 2012). In addition, several species of amphipods have been reported with Hematodinium-like infections (e.g., Johnson 1986), resulting in speculations that amphipods may act as reservoir, intermediate or alternate hosts for the parasite (Shields 1994, 2003; Small et al. 2006, Small and Pagenkopp 2011).
Hematodinium spp. have been reported from other marine crustaceans including other species of crabs from the vicinity of Australia and China and Chionecetes spp. from the North Pacific and North Atlantic oceans and Lithodidae crabs and Hyas coarctatus from the North Pacific Ocean, as well as from Norway lobsters (Stentford 2006, Morado 2011, Small 2012). A morphologically similar but genetically distinct parasite, that was initially thought to be a species of Hematodinium, was reported from shrimp.
Impact on the host
a) Infections in crabs in European waters are usually low in prevalence. For example, H. perezi originally described from C. maenas in 1930 (Chatton and Poisson 1930) was not reported from this species of crab until 2005 when infection was detected in 1 of 162 C. maenas from six estuaries in the United Kingdom (Stentiford and Feist 2005). However, exceptions to low prevalences of infection have been reported. For example, Hamilton et al. (2009) detected Hematodinium sp. in 14.6% (30/206) of the C. maenas from the Clyde Sea, Scotland sampled between June 2004 and May 2006. The parasite was problematic in other species of crabs in Europe. During the winter of 2000/2001, many trap-caught Cancer pagurus from Guernsey, Channel Islands, UK, were reportedly moribund, pink in colour and generally died before and during live (vivier) transportation to markets (Stentiford et al. 2002) and prevalence of about 50%, with associated mortalities, were reported in C. pagurus from the vicinity of Portsall, France. Also, prevalences up to 87% were observed in Necora puber from Brittany, France and was suspected of causing mass mortalities of N. puber in Mor-Braz (south Brittany) (Wilhelm and Mialhe 1996). Outbreaks in C. pagurus and Necora puber fisheries in the English Channel were associated with embayments or partially restricted coastal systems presumably with entrained water masses (Latrouite et al. 1988, Wilhelm and Mialhe 1996). Between 25 and 100% of Hematodinium-infected C. pagurus and N. puber from 2 sites in the English Channel (south coast of Guernsey and Newton’s Cove, Weymouth, UK) also harboured a secondary (possibly opportunistic) systemic infection of budding yeast-like cells within haemocytes and free in the host plasma (Stentiford et al. 2003).
In heavily infected crabs, Hematodinium is systemic with plasmodial forms infiltrating all major organs and tissues, severely damaging the reproductive organs and causing castration through the disruption of the testis or ovary of infected hosts (Stentiford 2006). Ní Chualáin et al. (2009) postulated that seawater temperature or a temperature-linked process was a key factor in triggering the final stages of infection in C. pagurus from waters around Ireland, because significant autumn peaks were followed by a reduction in infection intensity as temperature decreased in the late winter to early spring months with no increase in intensity again until the following autumn. Physiological responses to Hematodinium, varied profoundly between the crabs Carcinus maenas and Cancer pagurus (Brachyura) and also differed to those of Pagurus bernhardus (Anomura). Specifically, osmoregulatory capacity was reduced significantly in Hematodinium-infected C. maenas, haemolymph pH increased in parasitised C. pagurus and P. bernhardus, L-lactate concentration decreased in infected P. bernhardus and changes to tissues and exoskeletons were observed in C. pagurus, but not in C. maenas and P. bernhardus (Hamilton et al. 2010a).
b) Prevalences up to 40% were detected in Callinectes sapidus from Florida, USA (Messick and Shields 2000) and have reached 100% prevalence in focal outbreaks in Virginia and Maryland (Messick 1994) with most of the diseased crabs likely dying of the infection (Messick and Shields 2000, Shields and Squyars 2000). Hematodinium can cause high mortalities among C. sapidus (from salinities above 11 ppt) in enzootic areas along the east coast of North America (Stentiford and Shields 2005), is usually more abundant in juvenile than adult hosts, and outbreaks have been associated with embayments, shallow backwaters and lagoons (Messick 1994, Messick and Shields 2000). The parasite is rarely reported below salinities of 18 ppt (Newman and Johnson 1975, Messick and Sinderman 1992, Messick and Shields 2000). Results from preliminary surveys using a PCR diagnostic technique suggested that Hematodinium sp. was absent in C. sapidus from low salinity waters (5 to 10 ppt) but common in C. sapidus from higher salinity estuarine waters in southeastern Georgia, USA (Gruebl et al. 2002, Lee and Frischer 2004). This parasite is believed to represent a significant threat to C. sapidus fisheries in high salinity estuaries and may have a greater effect on mature females that move to higher salinities to breed (Lee and Frischer 2004). However, in the Northern Gulf of Mexico, infected C. sapidus occurred at all survey sites regardless of salinity and reached a prevalence of 90% at a low salinity (3 ppt) site (Zimmerman et al. 2011). In addition to salinity, temperature-related environmental factors also influences the ability of the parasite to proliferate within crab haemolymph. Hematodinium exhibits a strong peak in prevalence in C. sapidus during the fall and rapidly decline in winter (Messick et al. 1999, Shields and Overstreet 2007). Sheppard et al. (2003) suggested that Hematodinium sp. was responsible for the disappearance of C. sapidus from Wassaw Sound, Georgia, USA in the summer months allowing other opportunist crab species to invade the niche vacated by C. sapidus.
In Callinectes sapidus, haemocyanin concentration, serum proteins and tissue glycogen were all more heavily depleted in infected males versus infected females (Shields et al. 2003, Shields and Overstreet 2007). The logarithmic proliferation of the parasites coupled with their metabolic requirements during rapid growth drains the protein and carbohydrate constituents of the host. This metabolic drain coupled with lethargy and cessation of feeding in heavily infected hosts usually leads to host morbidity and death (Shields et al. 2003, Stentiford and Shields 2005). Shields (2003) speculated that the sudden mortalities in Hematodinium-infected crabs could be related to hypoxic events, especially given the oxygen demands of the parasite and the moribund host.
Infection can be artificially transmitted between Callinectes sapidus by injection (Shields and Squyars 1999, 2000, Pagenkopp Lohan et al. 2012). In experimentally infected C. sapidus, haemocyte densities declined rapidly, approaching an 80% decrease within the first week of infection. In these experiments, survival analysis indicated that inoculated crabs had a mean time to death of 30 days and were 7 to 8 times more likely to die than uninfected crabs with a mortality rate of 87% over 40 days. During challenge studies, small numbers of C. sapidus were refractory to infection and exhibited significant relative and absolute increases in granulocytes (Shields and Squyars 2000, Shields and Overstreet 2007). Such ‘immune’ crabs did not develop infections after serial challenges with infectious doses of H. perezi which may explain the fact that mature hosts appear less prone to developing Hematodinium infection compared to their juvenile counterparts (Stentiford and Shields 2005). Although Hematodinium can be transmitted through the water, the rapid spread of the disease during an epizootic is probably due to cannibalism (Lee and Frischer 2004). Walker et al. (2009) reported the experimental transmission of Hematodinium sp. into C. sapidus that were fed pieces of infected crab tissues with 73% (11 of 15) of the experimental crabs showing evidence of infection by 48 hours after feeding and four of the infected crabs were dead by day four. However, Li et al. (2011a) showed that Hematodinium sp. was not effectively transmitted through ingestion of diseased tissues and indicated that cannibalism may not be a major route of transmission for Hematodinium sp. in C. sapidus
Unlike the Hematodinium sp. from the Norway lobster (Nephrops norvegicus), the Hematodinium sp. from Callinectes sapidus contains and secrets the enzyme leucine arylamidase and although it also contains the enzyme acid phosphatase it does not secret this enzyme (Small et al. 2007a). Small et al. (2007a) suggested that the pattern of activities of these enzymes may be useful in distinguishing among different species of Hematodinium.
a & b) Developmental stages of Hematodinium within crustacean hosts occur primarily as plasmodial forms that rapidly divide and grow until they undergo sporogony to produce a motile dinospore stage. The vermiform, multinucleate plasmodia occur in early infections and probably arise from an infectious dinospore (Shields and Overstreet 2007). In Callinectes sapidus, the vermiform-like plasmodium (filamentous trophont) undergoes budding (or merogany) to reproduce more plasmodia. The plasmodial stage has no chloroplasts and obtains nutrition via osmotrophy during the trophic phase, where lipid and polysaccharide inclusions suggest active feeding at the expense of the host (Stentiford and Shields 2001). The vegetative amoeboid trophonts separate during segmentation of the plasmodia, and in turn undergo fission to produce additional trophonts. At some point in their development (perhaps as a result of high cell densities), the amoeboid trophonts undergo a final fission to produce a rounded trophont (possibly a sporoblast) that then undergoes an apparent sporogonal division to produce dinospores (Shields and Overstreet 2007). Sporulation is rapid and occurs over two to four days in C. sapidus (Shields and Squyars 2000). Dinospores have been shown to exit the infected host via the gills in C. sapidus and Cancer pagurus (Stentiford and Shields 2005). Sheppard et al. (2003) have reported successful transmission of disease to naïve C. sapidus via ingestion. Callinectes sapidus is an avid cannibal; conspecifics represent up to 25% of their diet. Thus, cannibalism may be an efficient alternate mode of transmission for Hematodinium, for some host species (Stentiford and Shields 2005, Walker et al. 2009), a speculation contrary to experimental results obtained by Li et al. (2011a). In all of these systems, infections seem to undergo times when prevalence is extremely low or even undetectable in host populations. These nadirs in prevalence are suggestive of either a latency of infection or an external reservoir for these parasites. Hamilton et al. (2011) used DNA analysis techniques on environmental samples from the Clyde Sea, Scotland to determine 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. Here again, the role of amphipods or other crustaceans as reservoirs or a resting cyst as a stage in the life cycle cannot be discounted (Stentiford and Shields 2005).
Gross Observations: Lethargy. Haemolymph of heavily infected crabs is opalescent or with a creamy consistency and colouration and slow to clot possibly caused by the massive numbers of Hematodinium in the haemolymph (Stentiford and Shields 2005). Infection with the Hematodinium-like parasite in Cancer pagurus caused pink hyperpigmentation of the carapace (Ní Chualáin and Robinson 2011) and appendages with discolouration (yellowing) of the arthrodial membranes and the genital pores, and was called ‘Pink Crab Disease’ by Stentiford et al. (2002). In some cases, the haemolymph and muscle of affected crabs assumed a pink colouration, with the meat having an irregular texture and a bitter taste when cooked (Stentiford et al. 2002).
Wet Mounts: In heavy infections, the haemolymph is usually devoid of haemocytes and filled with Hematodinium. In lighter infections, fresh haemolymph wet mounts containing Hematodinium parasites can be difficult to diagnose. The motile, vermiform, multinucleate plasmodium (filamentous trophont about 15 to 100 µm in length) is the most easily identified form, but the non-motile, vegetative, trophont stage (amoeboid or rounded and 9 to 22 µm in diameter) is more prevalent and, to the inexperinced observer, is easily confused with a haemocyte. Indeed, Hematodinium infections in Callinectes sapidus have been described as a ‘neoplastic granulocytemia’ (Newman 1970). However, Neutral Red vital stain applied to a wet mount can be used to differentiate between Hematodinium and haemocytes because the dye is taken up by the lysosomes of the parasite and host haemocytes generally do not acquire the stain (Shields and Overstreet 2007). Neutral Red has the advantage of providing a visual contrast and can thus be used for diagnostic purposes (Stentiford and Shields 2005).
Smears: For diagnosis of Hematodinium infection in crabs, the most reliable, cost-effective, permanent method is microscopic examination of prepared haemolymph smears for the presence of parasites. A standard air-dried haemolymph smear followed by methanol fixation and staining with a suitable histological stain such as Giemsa or haematoxylin and eosin provides consistent results (Messick 1994, Wilhelm and Mialhe 1996, Messick and Shields 2000). Preferred, however, is a wet smear prepared on a poly-L-lysine-coated slide, that is rapidly fixed in Bouins solution or 10% neutral buffered formalin, then processed through a routine haematoxylin and eosin procedure or a modified Giemsa stain (Messick 1994; Messick and Shields 2000, Shields and Squyars 2000). Hematodinium vegetative stages (5.8-6.4 µm in diameter) containing one or more nuclei that are large in proportion to cytoplasmic volume with either condensed or diffused chromosomes of the characteristic dinokaryon nucleus. Hematodinium sp. cells are distinguishable from haemocytes by the lack of a defined nuclear membrane (Nagle et al. 2009).
Histology: Numerous, extracellular vegetative stages (mononuclear trophonts, binucleated and multinucleated forms) and few haemocytes in the enlarged haemal spaces of all tissues. Degeneration of hepatopancreatic tubules and infiltration and extensive destruction of muscle fibres may occur (Stentiford et al. 2002, Sheppard et al. 2003). In infected Carcinus maenas, the parasite invaded the lumen and haemal spaces of the myocardium where filamentous and rounded plasmodia were in different stages of nuclear division, showing basophilic nuclei and reduced, frothy cytoplasm. In some parasites, fibrillar chromosomes were visible, and plasmodia and putative trophonts were detected adjacent (or possibly attached) to muscle fibres (Hamilton et al. 2007, Li et al. 2011a). In the gills of Hematodinium-positive C. maenas, the haemal sinuses became enlarged and filled with trophonts and plasmodia. Also, there was a scarcity of haemocytes, the distal tips of gill lamellae became swollen, the epithelial layer thinned and there was destruction of the pillar cells (Hamilton et al. 2007). Histopathological alterations in the tissues of Callinectes sapidus and Cancer pagurus include pressure necrosis caused by oedema and resulting dilation of the haemal sinuses in the soft connective tissues of the hepatopancrease and other organs with loss of this tissue in heavy infections (Wheeler and Shields 2011, Ní Chualáin and Robinson 2011). In C. sapidus, varied damage to the gills such as apparent thinning of the cuticular layers and loss of host epithelial cells also occured and crab death during acute infections was attributed to tissue disruption and not metabolic wasting (Wheeler and Shields 2011). Usually, no host responses against the parasite were reported (Small et al. 2012). Pagenkopp Lohan et al. (2012) found similarities in the histopathology of a broad range of infected Decapod crustaceans along the Delmarva Peninsula, Virginia, USA.
Electron Microscopy: Plasmodia typically have condensed chromatin profiles (up to 5 nuclei per plasmodia), abundant lipid droplets, membrane-bound trichocysts, mitochondria, a surrounding alveolar membrane (alveolate pellicle) and a centriolar apparatus. The ultrastructure of H. perezi includes the presence of condensed chromatin profiles, trichocysts, and an alveolar membrane with micropores (Small et al. 2012). Remnants of degenerated host tissue (such as atrophied mitochondria, myelin bodies, and membranous material) were often found surrounding plasmodia at the periphery of the remaining tissue (Stentiford et al. 2002).
Immunological assays: Polyclonal antibodies produced against Hematodinium isolated from Nephrops norvegicus showed a clear positive reaction to protein extracts from the hepatopancreas of infected Cancer pagurus via western blots (Stentiford et al. 2002).
DNA Probes: For Hematodinium spp., all published efforts have exclusively focused upon segments of the ribosomal RNA gene complex, which is present in the nuclear genome of eukaryotes as tandemly repeated clusters of highly conserved genes encoding the small subunit (SSU or 18S), 5.8S, and large subunit (LSU) genes, which are separated by highly variable spacer sequences, the first and second internal transcribed spacers (ITS1 and ITS2) (Small 2012). Hudson and Adlard (1994) developed a generic polymerase chain reaction (PCR)-based diagnostic test that amplified ITS1 and the flanking 3-prime end of the SSU of Hematodinium parasites in Cancer pagurus, Callinectes sapidus, Nephrops norvegicus, Chionoecetes opilio, and Chionoecetes bairdi (Stentiford et al. 2002; Small et al. 2006, 2007b,c; Hamilton et al. 2009). Small et al. (2007b) identified other primers that were specifically designed to amplify a 302 segment of the ITS1 region of Hematodinium in the western Atlantic C. sapidus, the European harbour crab Liocarcinus depurator, and the Chinese swimming/sand crab Portunus trituberculatus. Application of a restriction endonucleases digestion (Bsg I) to the resulting amplification products differentiated the Hematodinium of C. sapidus from the Hematodinium of the other two species of crabs (Small et al. 2007b). Small et al. (2007c) suggested that the Hematodinium from N. norvegicus, C. pagurus, and Pagurus bernhardus from the United Kingdom and the Hematodinium sp. infecting C. opilio from Conception Bay, Newfoundland, Canada were the same species of Hematodinium based on phylogenetic analysis of the ITS1 sequences. In agreement, Hamilton et al. (2007) indicated that Hematodinium from Carcinus maenas and Nephrops norvegicus caught on the west coast of Scotland (Clyde Sea) belong to the same species based on PCR analysis of conserved and variable regions of the ribosomal RNA gene (including the 18S, 5.8S, ITS1 and ITS2). Based on these publications, Small and Pagenkopp (2011) agreed that this parasite species is a host generalist with a broad geographic range. Conversley, Hamilton et al. (2010b) analysed the secondary structures of the ITS1 and ITS2 sequences to show that Hematodinium from the east and west North-Atlantic is comprised of distinct ribotypes or clades. For example, a Hematodinium ‘Langoustine’ clade was only found in N. norvegicus, whereas other clades were specific to crabs or seem to be generalist parasites based on the structural analysis of the ITS2 whereas, structural analysis of ITS1 indicated a further clade found only in the anomurans Munida rugosa and Pagurus prideaux (Hamilton et al. 2010b). Subsequently, based on molecular analysis of the ITS rRNA regions, Small et al. (2012) suggested that the Hematodinium sp. in C. sapidus from the United States, and in Portunus trituberculatus and Scylla serrata from China are different genotypes of H. perezi which infects L. depurator from Rye Bay in the English Channel. Small (2012) proposed that the ITS1 region be used to designate three different H. perezi genotypes in portunid crabs from the following locations and hosts: genotype I (English Channel, L. depurator); genotype II (east China, P. trituberculatus and S. serrata), and genotype III (east coast US, C. sapidus).
Polymerase chain reaction (PCR)-based assays that target the 18S ribosomal DNA gene of Hematodinium sp. in Callinectes sapidus were developed (Gruebl et al. 2002, Frischer et al. 2006, Small et al. 2007b). Ní Chualáin and Robinson (2011) found that the PCR assay described by Gruebl et al. (2002) provided virtually equivalent accuracy in gauging infection prevalence in asymptomatic Cancer pagurus as haemolymph smears and histological sections of gill, heart, midgut, hepatopancreas, muscle, and gonad, regardless of season. Using a nested PCR analysis, Eigemann et al. (2010) found between 45 and 87.5% of the decapods with no morphological signs of disease were infected with Hematodinium and the nested PCR approach was more sensitive than conventional PCR in detecting the parasite. The PCR assay described by Gruebl et al. (2002) was used by Sheppard et al. (2003) to determine that the Hematodinium sp. in the spider crab Libinia emarginata and the stone crab Menippe mercenaria were the same or very closely related to the Hematodinium sp. from C. sapidus. Also, Pagenkopp Lohan et al. (2012) determined that the same species of Hematodinium found in C. sapidus infects a broad range of crustaceans along the Delmarva Peninsula, Virginia, USA and suggested that this parasite is a host generalist, capable of infecting hosts in different families within the Order Decapoda and possibly crustaceans within the Order Amphipoda. Whereas, the comparison of sequences from several infected hosts outside the natural geographic range of C. sapidus indicated that Hematodinium in C. sapidus was different from those in Nephrops norvegicus, Chionoecetes bairdi and Chionoecetes opilio (Stentiford and Shields 2005). Frischer et al. (2006) used a quantitative Real Time PCR assay (qPCR or RT PCR) for the detection of Hematodinium sp. inside and outside of the host C. sapidus indicating evidence for a free-living life stage for the parasite. The 18S ribosomal DNA was also the target for a fluorescene based qPCR (Steven et al. 2003). This assay was found to be four times as sensitive in detecting Hematodinium sp. in field collections of C. sapidus as identified as positive using traditional haemolymph microscopic examination (Nagle et al. 2009). Because the assays used by Frischer et al. (2006) and Nagle et al. (2009) would amplify the DNA of all Hematodinium and Hematodinium-like spp., Li et al. (2010) developed and validated a qPCR assay targeting the ITS1 region of the rRNA gene for the Hematodinium in C. sapidus.
Hudson et al. (2001) and Small et al. (2007b) identified DNA probes for in situ hybridisation of Hematodinium in paraffin embedded host tissues. Small et al. (2008) indicated that laser-assisted microdisection could be used to isolate Hematodinium spp. from formalin-fixed paraffin-embedded tissue sections and partial regions (up to 300 base pairs) of the SSU and ITS1 rRNA gene complex of the isolates could be amplified using diagnostic primers. Fluorescence in situ hybridization of Hematodinium dinospores in solution was used to determine that Hematodinium survived up to 7 days in aquaria outside of the crab host resulting in the speculation that Hematodinium sp. dinospores have a short free-living stage in the water column and that transmission to new hosts must occur relatively quickly in estuarine habitats (Li et al. 2010). Twelve microsatellite markers designed by Pagenkopp et al. (2011) were used to identify strain variation in Hematodinium from Callinectes sapidus Analysis of resulting data indicated that: Hematodinium in the host are primarily haploid; multiple genetic strains can occur simultaneously in the same host; the level of variation suggested that a sexual reproductive stage may be present in the life cycle of the parasite; and the high level of genotypic variation was comparable to that reported in free-living dinoflagellates (Pagenkopp et al. 2011). Hamilton et al. (2011) used a nested PCR assay, based on previously identified primers, to detect Hematodinium rDNA in the environment and in potential alternative hosts.
Culture: Vegetative stages of Hematodinium from Callinectes sapidus retained their infectivity after one week of culture in the medium described by Appleton and Vickerman (1998) modified by using less fetal bovine serum (Stentiford and Shields 2005). This modified medium was used as a buffer for challenge studies with the parasite (Shields and Squyars 2000). In culture medium, several stages of the Hematodinium from C. sapidus appear similar to those described from the hepatopancrease of the Nephrops norvegicus including vermiform-like plasmodial stages and the apparent arachnoid-like plasmodia (trophonts or clump colony) (Stentiford and Shields 2005). Li et al. (2011b) also used the medium described by Appleton and Vickerman (1998) but modified it by adding 5% (v /v) of C. sapidus serum. They established several continuous in vitro cultures of Hematodinium sp. isolated from the haemolymph of infected C. sapidus. One isolate was continuously maintained for over 12 months through serial subcultivation and was capable of infecting new hosts when inoculated into healthy crabs. In vitro, the parasite underwent characteristic developmental changes consistent with the identifiable stages of Hematodinium sp. including filamentous trophonts, amoeboid trophonts, arachnoid trophonts and sporonts, sporoblasts, prespores and dinospores (macrospores and microspores) (Li et al. 2011b).
Methods of control
No known methods of prevention or control. Anecdotal evidence suggests that some fishing practices may help to spread diseases. Such practices include the culling or disassembly of the catch at sea, re-baiting with infected animals, moving animals between locations (culling while underway), and in some cases using crabs as ‘attractants’ to bait for additional animals (e.g., using male Callinectes sapidus to attract premoult females for the softshell industry). In Cancer pagurus from Irish fisheries, the prevalence of Hematodinium sp. had no distinct seasonal trend (prevalence ranged from 0 to 51%). However, infection intensity was seasonal with significantly higher peaks occurring in late autumn/early winter months which corresponded to industry reports of moribund and dead pink-shelled crabs in commercial catches. Thus, Ní Chualáin et al. (2009) proposed that infection intensity, rather than prevalence, was a more appropriate indication of the period when there is greatest potential for biological and economic impacts. Control strategies include discouraging the discarding of late-stage infected individuals back into the water while at sea, culling or removing moribund/dead animals to onshore fertilizer processing plants, limiting transportation of live animals, and changing baiting practices by not leaving carcasses and terminally infected individuals in pots as attractants for the next fishing period nor retaining moribund, poor quality individuals as bait in other fisheries (Stentiford and Shields 2005, Ní Chualáin et al. 2009).
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Date last revised: February 2013
Comments to Susan Bower
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