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Hematodinium sp. infection of red Paralithodes camtschaticus and blue Paralithodes platypus king crabs from the Sea of Okhotsk, Russia
Journal of Invertebrate Pathology 105 (2010) 329–334
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Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Hematodinium sp. infection of red Paralithodes camtschaticus and blue Paralithodes platypus king crabs from the Sea of Okhotsk, Russia T.V. Ryazanova a, M.G. Eliseikina b, A.D. Kukhlevsky b, V.I. Kharlamenko b,⇑ a b
Kamchatka Research Institute of Fisheries and Oceanography, Petropavlovsk-Kamchatsky 683002, Russia A.V. Zhirmunsky Institute of Marine Biology FEB RAS, Palchevsky str. 17, Vladivostok 690041, Russia
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Article history: Received 8 December 2009 Accepted 29 July 2010 Available online 5 August 2010 Keywords: Hematodinium sp. Paralithodes camtschaticus Paralithodes platypus Sea of Okhotsk Infection Prevalence Parasitic dinoflagellate
a b s t r a c t A disease caused by a parasitic dinoflagellate of the genus Hematodinium was identified in red, Paralithodes camtschaticus, and blue, Paralithodes platypus, king crabs from the north-east region of the Sea of Okhotsk, Russia, during annual stock surveys. No carapace color change was observed even in heavily infected crabs, but diseased crabs possessed creamy-yellow hemolymph, which was visible through the arthrodial membranes of the abdomen and appendages. Several stages of the parasite’s life history, including trophonts, plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. Numerous parasite cells were observed in the lumina of the myocardium, the gills, the connective tissue of antennal glands and the sinuses of nerve ganglia, eyestalks and gastrointestinal tract of king crabs with gross signs of infection. Based on sequencing of the 18S rDNA, it appears that the Hematodinium sp. found in red and blue king crabs is identical or closely related to Hematodinium sp. isolated from crabs of the genera Chionoecetes and Lithodes. Observed prevalences were 0.33% in sublegal male red king crabs, 0.18% in female red king crabs, 0.34% in sublegal male blue king crabs and 0.31% in female blue king crabs. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The red king crab, Paralithodes camtschaticus, followed by the blue king crab, Paralithodes platypus, are the most commercially valuable crab species in the Sea of Okhotsk, Russia. Combined annual landings of these species total 50,000 metric tons and over recent decades, these landings have been stable when compared to other regions (Otto and Jamieson, 2001). However, since 1999, the Sea of Okhotsk king crab stock has declined significantly and remains low (Dolgenkov and Koblikov, 2009). In addition to overfishing, a number of other factors may impact crab population structure, including diseases. In particular, infections caused by the parasitic dinoflagellate Hematodinium sp., may significantly change the size and structure of important crab populations (Stentiford and Shields, 2005). Hematodinium sp. infections in various marine crustacean species have been recorded in many areas of the world. Hematodinium-associated diseases are generally fatal to the host (Messick and Shields, 2000), and its occurrence can reach 100% for some crustacean species (Messick, 1994). The majority of infections are reported in brachyuran crabs (Stentiford and Shields, 2005). In disease transmission studies, Meyers et al. (1987) reported that none of the lithodid king crabs inoculated with infected Tanner ⇑ Corresponding author. E-mail address: [email protected] (V.I. Kharlamenko). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.07.009
crab ( Chionoecetes bairdi) hemolymph developed detectable infections; and hemolymph smears from southeast Alaska crabs, P. camtschaticus, P. platypus, and Lithodes aequispina contained no dinoflagellate forms (Meyers et al., 1987). The first incident of Hematodinium sp. infection was observed on the West Kamchatka shelf in 2002 in snow crabs, Chionoecetes opilio, and this disease was found in red and blue king crabs, P. platypus, from this area four years later. This report describes the results of gross or macroscopic examination as well as microscopic (light and transmission electron microscopy) observations of the infection in king crabs P. camtschaticus and P. platypus, molecular identification of Hematodinium sp. in king crabs, and it also includes information on the distribution of infected crabs in the north-east region Sea of Okhotsk. 2. Materials and methods Lithodid crabs, the red king crab P. camtschaticus and blue king crab P. platypus, were used as material for the present research. The survey took place in the northeastern area of the Sea of Okhotsk during annual stock assessment surveys (Fig. 1). Crab trap and trawl surveys were used. The crab trap survey onboard the RV Ametist from August 25 to December 15 2006 was conducted from the north to the south. Traps were deployed in ‘fleets’ of 100 standard Japanese conical traps baited with 1 kg of frozen herring. Eighty-eight fleets of traps were set between 64 and 322 m depth
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drated in acetone and embedded in Epon-Araldite. Semithin and ultrathin sections were cut using a Leica EM UC6 ultramicrotome. Semithin sections were stained with methylene blue and examined using a Leica DM 4500 microscope. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Zeiss Libra 120 transmission electron microscope. The standard method for total DNA isolation from the tissue of crabs was used (Sambrook et al., 1989). Hematodinium-specific primers Hemat-F-1487 and Hemat-R-1654 were used to detect Hematodinium (Gruebl et al., 2002). PCR was performed using a GenAMP 9600 thermal cycler system (Perkin Elmer). Amplification conditions for the PCR included an initial denaturation step (94 °C for 10 min) and 30 amplification cycles (denaturation, 15 s at 94 °C; annealing, 15 s at 56 °C; elongation, 30 s at 72 °C). Reaction products were checked for size and purity on 1% agarose gels. To confirm the identity of the parasites detected in king crabs, representative 196 bp PCR amplicons were used as templates for sequencing amplification using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Purified sequencing products were analyzed by electrophoresis on a 50 cm capillary array of an ABI Prism 3130 DNA sequencer. Sequences were assembled with SeqScape v2.5 software (Applied Biosystems). In addition, a long segment of 18S rDNA (1682 bp) from the parasite detected in the red king crab was sequenced. Direct sequence determination of each of the obtained amplicons yielded a 1464 bp sequence. The consensus sequences were compared with homologous Hematodinium 18S rDNA sequences available in GeneBank. Calculation of similarity values was conducted using the program MEGA version 4 (Tamura et al., 2007). The density of infected crabs was estimated with the program Chartmaster version 3.1 using the kriging interpolation method. Fig. 1. Map of the study area with sampling stations.
3. Results at randomly selected locations in the range of latitudes from 58°510 N to 57°10 N and from 55°410 N to 54°150 N. The standard soak time for traps was 36–48 h, but in some cases varied from 35 to 190 h depending on weather conditions. The 2007 trawl survey was conducted onboard the RV Professor Kaganovsky from July 8 to August 1 beginning in the south and proceeding north. 146 trawls from 51°050 N to 57°420 N were carried out within depth range 14–200 m. The trawl sections were performed in a standard pattern at a speed of 3 knots for 30 min, using a DT 27.1 trawl. All crabs were removed from the catch, sorted by species and sex. Crab size is reported as carapace width excluding spines. Crabs were divided into three groups – legal males (carapace width (cw) greater than or equal to 150 mm and 130 mm for red and blue kings crabs, respectively), sublegal (cw < 150 and 130 mm) for males and females, respectively. Intermolt state of the carapace was assessed and assigned to one of six classes according to specific criteria (Lysenko, 2001). In total, 3189 males and 1601 females of the blue king crab, and 8587 males and 3295 females of the red king crab were examined macroscopically. Randomly selected crabs with external signs of disease and additional individuals that appeared healthy upon gross examination were dissected (700 males, 282 females of the red king crab, 230 males and 147 females of the blue crab). From these, 77 red king crabs and 56 blue king crabs were selected for histological examination. Tissue samples were fixed for 24–48 h in Davidson’s fixative (Bell and Lightner, 1988) prepared in sea water and processed using standard histological techniques. Five-micron sections were stained with Meyer’s hematoxylin–eosin (H&E) and examined using Olympus Al-2 or Leica DM 4500 light microscopes with an automatic camera. Seven infected crabs were studied by TEM. Tissue samples for transmission electron microscopy were fixed with 2.5% glutaraldehyde in sterile sea water, post-fixed with 1% of 0s04 in sea water, dehy-
Overall, 11882 red king crabs and 4790 blue king crabs were subjected to gross examination. Gross signs of infection were found in 14 red king crabs and nine blue king crabs. All infected crabs were females or sublegal males (with the carapace width from 75 to 102 mm) in the third intermolt stage (carapace and chela are hard and cannot be depressed by thumb). No external wounds were visible. None of the collected crabs changed color of their carapace, but infected crabs did possess a creamy-yellow hemolymph, which was visible through the arthrodial membranes of abdomen and appendages (Fig. 2). Their dissection revealed that the hemolymph of suspect crabs appeared as a creamy-yellow fluid mass surrounding the muscles of appendages and all internal organs, filling the pericardial cavity and gill lamellae and stems. The cooked meat of suspect crabs had a bitter and astringent taste. Prevalence was 0.33%, 0.18%, 0.34% and 0.31% in sublegal male and legal female red king crabs, and legal female and sublegal male blue king crabs, respectively. Gross evidence of infections was not found in legal male red or blue king crabs (Table 1). Although the data are limited, prevalence of infection was different in trawl and pot surveys. Infected crabs were found at depths ranging from to 30 to134 m. Although small in number, the majority of infected crabs were located between latitudes 57°000 N and 57°350 N along the western Kamchatka shelf (Fig. 1). Interpolation suggests that maximum density of infected red and blue king crabs was 84 and 108 crabs per km2, respectively. In this area infection prevalence as determined by macroscopic signs was 0.61% in sublegal male red king crabs, 0.83% in female red king crabs, 0.50% in sublegal male blue king crabs and 0.81% in female blue king crabs. Histopathological changes were similar in both crab species with gross signs of infection and all infections appeared advanced, with large numbers of parasite cells distributed throughout the
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Fig. 2. The sublegal males of king crabs infected by Hematodinium sp. (A) The cream-yellow hemolymph (arrow) is visible through the translucent covers of abdomen and appendage joints of blue king crab. (B) The internal organs of a red king crab surrounded by cream-yellow dense hemolymph.
Table 1 Prevalence of visually diagnosed Hematodinium sp. infections in king crabs from north-east region of the Sea of Okhotsk. Paralithodes camtschaticus Legal males
Paralithodes platypus Sublegal males
Females
Legal males
Sublegal males
Females
Total number of crab examined Trawls 4722 Traps 1414
1091 1360
2934 361
153 1843
51 1142
312 1289
Total
2451
3295
1996
1193
1601
Number of crabs infected Trawls 0 Traps 0
6136
5 3
6 0
0 0
0 4
4 1
Total
0
8
6
0
4
5
Prevalence (%) Trawls Traps
0 0
0.46 0.22
0.20 0.00
0 0
0.00 0.35
1.28 0.08
Total
0
0.33
0.18
0
0.34
0.31
tissues. Rounded trophonts (10.7 ± 1.7 lm) with distinct dinokaryotic nuclei and rounded or vermiform plasmodia, containing from 2 to 8 or more nuclei were found in 21 of 23 crabs (Fig. 3A). Three red king crabs had some plasmodia of vermiform shape with 2–5 nuclei located in a row (Fig. 3B). In crabs with advanced infections, identified by high densities of trophonts and plasmodia throughout, numerous parasite cells were observed in the lumen of the myocardium (Fig. 4A), the gill and connective tissue of the antennal gland (Fig. 4B), nerve ganglia, eyestalks, gastrointestinal tract, and testes, as well as between oocytes of females. The hematopoietic tissue of infected animals was mitotically active, however few hemocytes were observed in circulation. The hemal sinuses of the hepatopancreas were dilated and filled by large number of parasite cells, and the connective tissue between the tubules was replaced by these cells. Vermiform plasmodial stages were attached to the basal membrane of hepatopan-
creatic tubules, the wall of the gut and blood vessels. The connective tissue between the muscle fibrils was reduced, and the sarcolemma was breached by the parasite cells (Fig. 4C). Some small melanized nodules in the antennal gland, stomach and hematopoietic tissue were found in three individuals. In the tissues of two of the more heavily infected crabs, the parasite cells were smaller in size compared to the normal trophonts and their cytoplasm had a very small volume (Figs. 3C and 4D). The majority of the cells were uninucleated, but binucleated and other multi-nucleated cells were also observed. The size of those cells was 4.45 ± 0.4 lm (mean ± standard deviation, N = 20), and the size of their nuclei was 3.35 ± 0.4 lm (N = 20). The nuclei of the cells were filled with a granular nucleoplasm along with the polymorphous electron-dense heterochromatin clumps. The multivesicular bodies and vacuoles with homogeneous contents were located in the granular cytoplasm. Nuclear chromatin was dense
Fig. 3. Stages of Hematodinium sp. in king crabs. (A) Semithin section of trophonts and multinucleate plasmodial stages of Hematodinium sp. (methylene blue staining (scale bar = 50 lm)). (B) The vermiform plasmodia attached to the wall of a blood vessel (scale bar = 50 lm). (C) Pre-spore stage (scale bar = 50 lm). (D) Nomarski differential interference contrast microscopy showing the round trophont and goblet-like form cell with beak-like protrusion in fixed hemolymph of sublegal male blue crab (scale bar = 20 lm).
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Fig. 4. Light micrographs tissues from king crabs infected by Hematodinium sp. (A) The trophonts and plasmodium in the myocardium. (B) The parasite in the connective tissue of the antennal gland. (C) The parasite around and inside the muscle fibrils. (D) Transverse section of hepatopancreatic tubules showing replacement of the interstitial connective tissue and hemal sinuses by numerous parasites. Scale bar = 50 lm.
and V-shaped. In the cytoplasm of the polynuclear and mononuclear cells, the electron-dense trichocysts of a rhomb or a square shape were observed with TEM (Fig. 5A and B). Using Nomarski differential interference contrast microscopy, some goblet-like cells, having beak-like and keel-like protrusions, were found in the hemolymph of one male blue crab (Fig. 3D). More severe pathological changes were observed in the tissue of two heavily infected crabs. The connective tissue of all organs was replaced by parasites. The hemal sinuses of the hepatopancreas were dilated and occluded by large numbers of parasites, although few parasite cells were observed above the basement membrane (Fig. 4D). The tubules were often destroyed, and only basal membranes remained. Muscle tissue was surrounded by parasites, nuclei of muscle fibers were absent, and the fibril striation was lost. Muscle fibers had been almost completely replaced by the proliferating parasites and only small islands of muscle fibrils were preserved. Small 18S rDNA fragments amplified from red king crab had a 100% sequence similarity to respective gene fragments of the Hematodinium sp. found in Lithodes couesi and four species of the genus Chionoecetes (Genbank Accession Numbers FJ844413, FJ844414, FJ844418, FJ844423 and FJ844426). Based on this comparison, the red king crab parasite was identified as Hematodinium sp., and the sequence recovered from red king crab was deposited in Genbank (Accession Number EU856717). To confirm that the small 196 bp 18S rDNA PCR amplicon diagnostic for Hematodinium sp. was indeed derived from Hematodinium sp., 18S rDNA large gene fragments were amplified from three red king crabs. All three 1464 bp sequenced amplicons were identical. The sequence
recovered from the red king crab was deposited in Genbank (Accession Number EU856716) and showed 100% base pair similarity to the sequenced Hematodinium sp. (Genbank Accession Numbers FJ844412-FJ844431) isolated from L. couesi, Hyas coarctatus, C. bairdi, C. opilio, C. tanneri, C. angulatus, Callinectes sapidus and Nephrops norvegicus. This implies the presence of Hematodinium sp. in the tissues of red king crab. 4. Discussion In the advanced stages of the disease, most species of crabs infected with Hematodinium spp. develop changes in carapace color (Stentiford and Shields, 2005), and these changes are used as macroscopic criteria for diagnosing Hematodinium spp. infection. The sternae and ventral surfaces of the Australian sand crabs Portunus pelagicus had a chalky, white appearance (Hudson and Shields, 1994). The abdomen of dying velvet swimming crabs Necora puber from several areas in France and Spain had a pale-pink color (Wilhelm and Mialhe, 1996). Changes in carapace color of some species of crabs are even more noticeable. The edible crab Cancer pagurus from UK waters exhibited an altered coloration (pink hyperpigmentation), and this infection is called ‘‘Pink Crab Disease” (Stentiford et al., 2002). The primary macroscopic sign of Hematodinium spp. infection in snow crabs, C. opilio, and Tanner crabs, C. bairdi, was a distinct change in carapace color that resulted in a cooked appearance (Meyers et al., 1987), and this feature was used for macroscopic estimation of disease prevalence (Meyers et al., 1990; Pestal et al., 2003). For red and blue king crabs in Kamchatka
Fig. 5. Transmission electron micrographs of Hematodinium sp. cells with trichocysts in cytoplasm. (A) Bi-nucleate parasitic cell (scale bar = 5 lm). (B) Uninucleate parasitic cell (scale bar = 2 lm). N: nucleus; mb: multivesicular body; t: trichocysts.
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waters, we did not register any color change even in highly infected crabs except for a creamy-yellow color of the hemolymph, visible through the arthrodial membranes of the abdomen and appendages. Dissection showed that the hemolymph of the infected king crabs is creamy-yellow in color and did not clot. Cream color and dense consistency of hemolymph in crabs in late stages of disease, such as registered in heavily infected king crabs, have been described in many species of crustaceans infected with Hematodinium spp. (Meyers et al., 1987; Shields, 1994). In addition, infected Tanner crabs have a bitter, astringent taste of cooked meat (Messick, 1994; Wilhelm and Mialhe, 1996; Stentiford et al., 2002). Several stages of the parasite, including trophonts, plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. The presence of trophonts and multinucleated plasmodial stages of the parasite in internal organs of crustaceans is a major diagnostic attribute of Hematodinium sp. infection (Meyers et al., 1987; Messick, 1994). Unattached vermiform plasmodia probably represent the motile form of the parasite, but the use of only fixed material did not allow us to confirm this suggestion. Vermiform plasmodia have been observed in many decapods (Stentiford and Shields, 2005), but only three king crabs with gross signs of infection had vermiform plasmodia. The reason for the low prevalence of crabs with such plasmodia may be the relatively short duration of this stage (Appleton and Vickerman, 1998). Trichocysts were present in the parasites from heavily infected king crabs. Studies of stages of Hematodinium sp. development in vitro showed that trichocysts are absent in vegetative cells (Appleton and Vickerman, 1998), implying that small cells, found in two very heavily infected red and blue crabs are sporonts. Similar cells were observed in later stages of Hematodinium sp. infection before sporulation (Meyers et al., 1987; Appleton and Vickerman, 1998; Shields and Squyars, 2000; Stentiford and Shields, 2005). In fixed hemolymph of such crabs, goblet-like cells were found which looked like the macrodinospores described by Meyers et al. in Tanner crabs (Meyers et al., 1987). Damage to the muscles, and lysis of the hepatopancreas epithelium found in the infected red and blue king crabs are typical for this disease (Messick, 1994; Meyers et al., 1987; Field and Appleton, 1995; Stentiford et al., 2002; Sheppard et al., 2003). The reason for the lack of pronounced inflammatory reaction of the organism to the Hematodinium sp. infection is the depletion of hemocytes in the hemolymph of the host in the course of disease development (Field and Appleton, 1995; Sheppard et al., 2003). Parasite cells multiply rapidly and cause hemocytopenia (Field and Appleton, 1995; Shields and Squyars, 2000). Infiltration of the connective tissue of most organs occurs at the later stages of infection. Comparison of the sequences of fragments of the 18S rDNA implies that the Hematodinium sp. found in red king crabs is the same or closely related to Hematodinium sp. isolated from crabs of the genera Chionoecetes and Lithodes (Genbank Accession Numbers FJ844412- FJ844431). Sequence similarity of our data (deposited in GenBank; EU856716) with other 18S rDNA sequences available in GenBank allows us to consign the Hematodinium sp. from Paralithodes camtschaticus within the clade Hematodinium that infects many decapod hosts in the North Pacific and North Atlantic Oceans (Jensen et al., 2010). The prevalence of Hematodinium sp. was variable in P. camtschaticus and P. platypus populations in the northeastern part of the Sea of Okhotsk, with the most but not all regions exhibiting very low prevalence. The largest number of Hematodinium sp. infected crabs was detected in the area with a maximum density of P. camtschaticus and P. platypus. A positive correlation between the number of infected crabs and their total density was observed for C. sapidus and Libinia emarginata (Sheppard et al., 2003). Gross inspection allows us to determine the disease only when it reached its maximum development, therefore many infected crabs could be
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missed. The accuracy of macroscopic diagnosis based on carapace color of C. opilio was around 0.53 (Pestal et al., 2003), and gross inspection based on hemolymph changes is less accurate. For more precise assessment it is necessary to use other methods, such as microscopic analysis of hemolymph or molecular methods (see Jensen et al., 2010). Infection of crustaceans with the Hematodinium spp. dinoflagellates has a strongly pronounced seasonal pattern (Stentiford and Shields, 2005). The occurrence of the disease in the Tanner crab C. bairdi in Alaska decreased in the autumn, as infected crabs died by that time (Love et al., 1993). In our study, gross signs of infection were absent in king crabs caught starting from the second half of October. Hematodinium infections have been reported in many brachyuran crabs (Stentiford and Shields, 2005) and in the present study we described this infection in anomuran crabs using a diverse approach. Acknowledgments We thank the anonymous reviewers for valuable comments and suggestions to this manuscript. This work was supported, in part, by FEB RAS (Project 09-III-A-06-210). References Appleton, P.L., Vickerman, K., 1998. In vitro cultivation and development cycle in culture of a parasitic dinoflagellate (Hematodinium sp.) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters. Parasitology 116, 115–130. Bell, T.A., Lightner, D.V., 1988. A Handbook of Normal Penaeid Shrimp Histology. The World aquaculture society, Baton Rouge. Dolgenkov, V.N., Koblikov, V.N., 2009. Current status of fisheries resources of important species of crabs and prospects of their industrial development. In: Alimov, A.F., Adrianov, A.V. (Eds.), Abstracts of X Congress of Hydrobiological Society of Russian Academy of Sciences. Dalnauka, Vladivostok, p. 125. Field, R.H., Appleton, P.L., 1995. A Hematodinium-like dinoflagellate infection of the Norway lobster Nephrops norvegicus: observations on pathology and progression of infection. Dis. Aquat. Organ. 22, 115–128. Gruebl, T., Frischer, M.E., Sheppard, M., Neumann, M., Maurer, A.N., Lee, R.F., 2002. Development of an 18S rRNA gene targeted PCR based diagnostic for the blue crab parasite Hematodinium sp. Dis. Aquat. Organ. 49, 61–70. Hudson, D.A., Shields, J.D., 1994. Hematodinium Australis n. sp., a parasitic dinoflagellate of the sand crab Portunus pelagicus from Moreton Bay, Australia. Dis. Aquat. Organ. 19, 109–119. Jensen, P.C., Califf, K., Lowe, V., Hauser, L., Morado, J.F., 2010. Molecular detection of Hematodinium sp. in Northeast Pacific Chionoecetes spp. and evidence of two species in the Northern Hemisphere. Dis. Aquat. Organ. 89, 155–166. Love, D.C., Rice, S.D., Moles, D.A., Eaton, W.D., 1993. Seasonal prevalence and intensity of bitter crab dinoflagellate infection and host mortality in Alaskan Tanner crabs Chionoecetes bairdi from Auke Bay, Alaska, USA. Dis. Aquat. Organ. 15, 1–7. Lysenko, V.N., 2001. Specific biological traits of males of the blue crab Paralithodes platypus in the northeast part of the Sea of Okhotsk. Russ. J. Mar. Biol. 27, 135– 142. Messick, G.A., 1994. Hematodinium perezi infections in adult and juvenile blue crabs Callinectes sapidus from coastal bays of Maryland and Virginia, USA. Dis. Aquat. Organ. 19, 77–82. Messick, G.A., Shields, J.D., 2000. Epizootiology of the parasitic dinoflagellate Hematodinium sp. in the American blue crab Callinectes sapidus. Dis. Aquat. Organ. 43, 139–152. Meyers, T.R., Koeneman, T.M., Botelho, C., Short, S., 1987. Bitter crab disease: a fatal dinoflagellate infection and marketing problem for Alaskan Tanner crabs Chionoecetes bairdi. Dis. Aquat. Organ. 3, 195–216. Meyers, T.R., Botelho, C., Koeneman, T.M., Short, S., Imamura, K., 1990. Distribution of bitter crab dinoflagellate syndrome in southeast Alaskan Tanner crabs Chionoecetes bairdi. Dis. Aquat. Organ. 9, 37–43. Otto, R.S., Jamieson, G.S., 2001.Commercially Important Crabs, Shrimps and Lobsters of the North Pacific Ocean. PICES Sci. Rep. No. 19. Pestal, G.P., Taylor, D.M., Hoenig, J.M., Shields, J.D., Pickavance, R., 2003. Monitoring the prevalence of parasitic dinoflagellate Hematodinium sp. in snow crabs Chionoecetes opilio from Conception Bay, Newfoundland. Dis. Aquat. Organ. 53, 67–75. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sheppard, M., Walker, A., Frischer, M.E., Lee, R.F., 2003. Histopathology and prevalence of the parasitic dinoflagellate, Hematodinium sp., in crabs (Callinectes sapidus, Callinectes similis, Neopanope sayi, Libinia emarginata, Menippe mercenaria) from a Georgia estuary. J. Shellfish Res. 22, 873–880.
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Hematodinium sp. infection of red Paralithodes camtschaticus and blue Paralithodes platypus king crabs from the Sea of Okhotsk, Russia T.V. Ryazanova a, M.G. Eliseikina b, A.D. Kukhlevsky b, V.I. Kharlamenko b,⇑ a b
Kamchatka Research Institute of Fisheries and Oceanography, Petropavlovsk-Kamchatsky 683002, Russia A.V. Zhirmunsky Institute of Marine Biology FEB RAS, Palchevsky str. 17, Vladivostok 690041, Russia
a r t i c l e
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Article history: Received 8 December 2009 Accepted 29 July 2010 Available online 5 August 2010 Keywords: Hematodinium sp. Paralithodes camtschaticus Paralithodes platypus Sea of Okhotsk Infection Prevalence Parasitic dinoflagellate
a b s t r a c t A disease caused by a parasitic dinoflagellate of the genus Hematodinium was identified in red, Paralithodes camtschaticus, and blue, Paralithodes platypus, king crabs from the north-east region of the Sea of Okhotsk, Russia, during annual stock surveys. No carapace color change was observed even in heavily infected crabs, but diseased crabs possessed creamy-yellow hemolymph, which was visible through the arthrodial membranes of the abdomen and appendages. Several stages of the parasite’s life history, including trophonts, plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. Numerous parasite cells were observed in the lumina of the myocardium, the gills, the connective tissue of antennal glands and the sinuses of nerve ganglia, eyestalks and gastrointestinal tract of king crabs with gross signs of infection. Based on sequencing of the 18S rDNA, it appears that the Hematodinium sp. found in red and blue king crabs is identical or closely related to Hematodinium sp. isolated from crabs of the genera Chionoecetes and Lithodes. Observed prevalences were 0.33% in sublegal male red king crabs, 0.18% in female red king crabs, 0.34% in sublegal male blue king crabs and 0.31% in female blue king crabs. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The red king crab, Paralithodes camtschaticus, followed by the blue king crab, Paralithodes platypus, are the most commercially valuable crab species in the Sea of Okhotsk, Russia. Combined annual landings of these species total 50,000 metric tons and over recent decades, these landings have been stable when compared to other regions (Otto and Jamieson, 2001). However, since 1999, the Sea of Okhotsk king crab stock has declined significantly and remains low (Dolgenkov and Koblikov, 2009). In addition to overfishing, a number of other factors may impact crab population structure, including diseases. In particular, infections caused by the parasitic dinoflagellate Hematodinium sp., may significantly change the size and structure of important crab populations (Stentiford and Shields, 2005). Hematodinium sp. infections in various marine crustacean species have been recorded in many areas of the world. Hematodinium-associated diseases are generally fatal to the host (Messick and Shields, 2000), and its occurrence can reach 100% for some crustacean species (Messick, 1994). The majority of infections are reported in brachyuran crabs (Stentiford and Shields, 2005). In disease transmission studies, Meyers et al. (1987) reported that none of the lithodid king crabs inoculated with infected Tanner ⇑ Corresponding author. E-mail address: [email protected] (V.I. Kharlamenko). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.07.009
crab ( Chionoecetes bairdi) hemolymph developed detectable infections; and hemolymph smears from southeast Alaska crabs, P. camtschaticus, P. platypus, and Lithodes aequispina contained no dinoflagellate forms (Meyers et al., 1987). The first incident of Hematodinium sp. infection was observed on the West Kamchatka shelf in 2002 in snow crabs, Chionoecetes opilio, and this disease was found in red and blue king crabs, P. platypus, from this area four years later. This report describes the results of gross or macroscopic examination as well as microscopic (light and transmission electron microscopy) observations of the infection in king crabs P. camtschaticus and P. platypus, molecular identification of Hematodinium sp. in king crabs, and it also includes information on the distribution of infected crabs in the north-east region Sea of Okhotsk. 2. Materials and methods Lithodid crabs, the red king crab P. camtschaticus and blue king crab P. platypus, were used as material for the present research. The survey took place in the northeastern area of the Sea of Okhotsk during annual stock assessment surveys (Fig. 1). Crab trap and trawl surveys were used. The crab trap survey onboard the RV Ametist from August 25 to December 15 2006 was conducted from the north to the south. Traps were deployed in ‘fleets’ of 100 standard Japanese conical traps baited with 1 kg of frozen herring. Eighty-eight fleets of traps were set between 64 and 322 m depth
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drated in acetone and embedded in Epon-Araldite. Semithin and ultrathin sections were cut using a Leica EM UC6 ultramicrotome. Semithin sections were stained with methylene blue and examined using a Leica DM 4500 microscope. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Zeiss Libra 120 transmission electron microscope. The standard method for total DNA isolation from the tissue of crabs was used (Sambrook et al., 1989). Hematodinium-specific primers Hemat-F-1487 and Hemat-R-1654 were used to detect Hematodinium (Gruebl et al., 2002). PCR was performed using a GenAMP 9600 thermal cycler system (Perkin Elmer). Amplification conditions for the PCR included an initial denaturation step (94 °C for 10 min) and 30 amplification cycles (denaturation, 15 s at 94 °C; annealing, 15 s at 56 °C; elongation, 30 s at 72 °C). Reaction products were checked for size and purity on 1% agarose gels. To confirm the identity of the parasites detected in king crabs, representative 196 bp PCR amplicons were used as templates for sequencing amplification using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Purified sequencing products were analyzed by electrophoresis on a 50 cm capillary array of an ABI Prism 3130 DNA sequencer. Sequences were assembled with SeqScape v2.5 software (Applied Biosystems). In addition, a long segment of 18S rDNA (1682 bp) from the parasite detected in the red king crab was sequenced. Direct sequence determination of each of the obtained amplicons yielded a 1464 bp sequence. The consensus sequences were compared with homologous Hematodinium 18S rDNA sequences available in GeneBank. Calculation of similarity values was conducted using the program MEGA version 4 (Tamura et al., 2007). The density of infected crabs was estimated with the program Chartmaster version 3.1 using the kriging interpolation method. Fig. 1. Map of the study area with sampling stations.
3. Results at randomly selected locations in the range of latitudes from 58°510 N to 57°10 N and from 55°410 N to 54°150 N. The standard soak time for traps was 36–48 h, but in some cases varied from 35 to 190 h depending on weather conditions. The 2007 trawl survey was conducted onboard the RV Professor Kaganovsky from July 8 to August 1 beginning in the south and proceeding north. 146 trawls from 51°050 N to 57°420 N were carried out within depth range 14–200 m. The trawl sections were performed in a standard pattern at a speed of 3 knots for 30 min, using a DT 27.1 trawl. All crabs were removed from the catch, sorted by species and sex. Crab size is reported as carapace width excluding spines. Crabs were divided into three groups – legal males (carapace width (cw) greater than or equal to 150 mm and 130 mm for red and blue kings crabs, respectively), sublegal (cw < 150 and 130 mm) for males and females, respectively. Intermolt state of the carapace was assessed and assigned to one of six classes according to specific criteria (Lysenko, 2001). In total, 3189 males and 1601 females of the blue king crab, and 8587 males and 3295 females of the red king crab were examined macroscopically. Randomly selected crabs with external signs of disease and additional individuals that appeared healthy upon gross examination were dissected (700 males, 282 females of the red king crab, 230 males and 147 females of the blue crab). From these, 77 red king crabs and 56 blue king crabs were selected for histological examination. Tissue samples were fixed for 24–48 h in Davidson’s fixative (Bell and Lightner, 1988) prepared in sea water and processed using standard histological techniques. Five-micron sections were stained with Meyer’s hematoxylin–eosin (H&E) and examined using Olympus Al-2 or Leica DM 4500 light microscopes with an automatic camera. Seven infected crabs were studied by TEM. Tissue samples for transmission electron microscopy were fixed with 2.5% glutaraldehyde in sterile sea water, post-fixed with 1% of 0s04 in sea water, dehy-
Overall, 11882 red king crabs and 4790 blue king crabs were subjected to gross examination. Gross signs of infection were found in 14 red king crabs and nine blue king crabs. All infected crabs were females or sublegal males (with the carapace width from 75 to 102 mm) in the third intermolt stage (carapace and chela are hard and cannot be depressed by thumb). No external wounds were visible. None of the collected crabs changed color of their carapace, but infected crabs did possess a creamy-yellow hemolymph, which was visible through the arthrodial membranes of abdomen and appendages (Fig. 2). Their dissection revealed that the hemolymph of suspect crabs appeared as a creamy-yellow fluid mass surrounding the muscles of appendages and all internal organs, filling the pericardial cavity and gill lamellae and stems. The cooked meat of suspect crabs had a bitter and astringent taste. Prevalence was 0.33%, 0.18%, 0.34% and 0.31% in sublegal male and legal female red king crabs, and legal female and sublegal male blue king crabs, respectively. Gross evidence of infections was not found in legal male red or blue king crabs (Table 1). Although the data are limited, prevalence of infection was different in trawl and pot surveys. Infected crabs were found at depths ranging from to 30 to134 m. Although small in number, the majority of infected crabs were located between latitudes 57°000 N and 57°350 N along the western Kamchatka shelf (Fig. 1). Interpolation suggests that maximum density of infected red and blue king crabs was 84 and 108 crabs per km2, respectively. In this area infection prevalence as determined by macroscopic signs was 0.61% in sublegal male red king crabs, 0.83% in female red king crabs, 0.50% in sublegal male blue king crabs and 0.81% in female blue king crabs. Histopathological changes were similar in both crab species with gross signs of infection and all infections appeared advanced, with large numbers of parasite cells distributed throughout the
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Fig. 2. The sublegal males of king crabs infected by Hematodinium sp. (A) The cream-yellow hemolymph (arrow) is visible through the translucent covers of abdomen and appendage joints of blue king crab. (B) The internal organs of a red king crab surrounded by cream-yellow dense hemolymph.
Table 1 Prevalence of visually diagnosed Hematodinium sp. infections in king crabs from north-east region of the Sea of Okhotsk. Paralithodes camtschaticus Legal males
Paralithodes platypus Sublegal males
Females
Legal males
Sublegal males
Females
Total number of crab examined Trawls 4722 Traps 1414
1091 1360
2934 361
153 1843
51 1142
312 1289
Total
2451
3295
1996
1193
1601
Number of crabs infected Trawls 0 Traps 0
6136
5 3
6 0
0 0
0 4
4 1
Total
0
8
6
0
4
5
Prevalence (%) Trawls Traps
0 0
0.46 0.22
0.20 0.00
0 0
0.00 0.35
1.28 0.08
Total
0
0.33
0.18
0
0.34
0.31
tissues. Rounded trophonts (10.7 ± 1.7 lm) with distinct dinokaryotic nuclei and rounded or vermiform plasmodia, containing from 2 to 8 or more nuclei were found in 21 of 23 crabs (Fig. 3A). Three red king crabs had some plasmodia of vermiform shape with 2–5 nuclei located in a row (Fig. 3B). In crabs with advanced infections, identified by high densities of trophonts and plasmodia throughout, numerous parasite cells were observed in the lumen of the myocardium (Fig. 4A), the gill and connective tissue of the antennal gland (Fig. 4B), nerve ganglia, eyestalks, gastrointestinal tract, and testes, as well as between oocytes of females. The hematopoietic tissue of infected animals was mitotically active, however few hemocytes were observed in circulation. The hemal sinuses of the hepatopancreas were dilated and filled by large number of parasite cells, and the connective tissue between the tubules was replaced by these cells. Vermiform plasmodial stages were attached to the basal membrane of hepatopan-
creatic tubules, the wall of the gut and blood vessels. The connective tissue between the muscle fibrils was reduced, and the sarcolemma was breached by the parasite cells (Fig. 4C). Some small melanized nodules in the antennal gland, stomach and hematopoietic tissue were found in three individuals. In the tissues of two of the more heavily infected crabs, the parasite cells were smaller in size compared to the normal trophonts and their cytoplasm had a very small volume (Figs. 3C and 4D). The majority of the cells were uninucleated, but binucleated and other multi-nucleated cells were also observed. The size of those cells was 4.45 ± 0.4 lm (mean ± standard deviation, N = 20), and the size of their nuclei was 3.35 ± 0.4 lm (N = 20). The nuclei of the cells were filled with a granular nucleoplasm along with the polymorphous electron-dense heterochromatin clumps. The multivesicular bodies and vacuoles with homogeneous contents were located in the granular cytoplasm. Nuclear chromatin was dense
Fig. 3. Stages of Hematodinium sp. in king crabs. (A) Semithin section of trophonts and multinucleate plasmodial stages of Hematodinium sp. (methylene blue staining (scale bar = 50 lm)). (B) The vermiform plasmodia attached to the wall of a blood vessel (scale bar = 50 lm). (C) Pre-spore stage (scale bar = 50 lm). (D) Nomarski differential interference contrast microscopy showing the round trophont and goblet-like form cell with beak-like protrusion in fixed hemolymph of sublegal male blue crab (scale bar = 20 lm).
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Fig. 4. Light micrographs tissues from king crabs infected by Hematodinium sp. (A) The trophonts and plasmodium in the myocardium. (B) The parasite in the connective tissue of the antennal gland. (C) The parasite around and inside the muscle fibrils. (D) Transverse section of hepatopancreatic tubules showing replacement of the interstitial connective tissue and hemal sinuses by numerous parasites. Scale bar = 50 lm.
and V-shaped. In the cytoplasm of the polynuclear and mononuclear cells, the electron-dense trichocysts of a rhomb or a square shape were observed with TEM (Fig. 5A and B). Using Nomarski differential interference contrast microscopy, some goblet-like cells, having beak-like and keel-like protrusions, were found in the hemolymph of one male blue crab (Fig. 3D). More severe pathological changes were observed in the tissue of two heavily infected crabs. The connective tissue of all organs was replaced by parasites. The hemal sinuses of the hepatopancreas were dilated and occluded by large numbers of parasites, although few parasite cells were observed above the basement membrane (Fig. 4D). The tubules were often destroyed, and only basal membranes remained. Muscle tissue was surrounded by parasites, nuclei of muscle fibers were absent, and the fibril striation was lost. Muscle fibers had been almost completely replaced by the proliferating parasites and only small islands of muscle fibrils were preserved. Small 18S rDNA fragments amplified from red king crab had a 100% sequence similarity to respective gene fragments of the Hematodinium sp. found in Lithodes couesi and four species of the genus Chionoecetes (Genbank Accession Numbers FJ844413, FJ844414, FJ844418, FJ844423 and FJ844426). Based on this comparison, the red king crab parasite was identified as Hematodinium sp., and the sequence recovered from red king crab was deposited in Genbank (Accession Number EU856717). To confirm that the small 196 bp 18S rDNA PCR amplicon diagnostic for Hematodinium sp. was indeed derived from Hematodinium sp., 18S rDNA large gene fragments were amplified from three red king crabs. All three 1464 bp sequenced amplicons were identical. The sequence
recovered from the red king crab was deposited in Genbank (Accession Number EU856716) and showed 100% base pair similarity to the sequenced Hematodinium sp. (Genbank Accession Numbers FJ844412-FJ844431) isolated from L. couesi, Hyas coarctatus, C. bairdi, C. opilio, C. tanneri, C. angulatus, Callinectes sapidus and Nephrops norvegicus. This implies the presence of Hematodinium sp. in the tissues of red king crab. 4. Discussion In the advanced stages of the disease, most species of crabs infected with Hematodinium spp. develop changes in carapace color (Stentiford and Shields, 2005), and these changes are used as macroscopic criteria for diagnosing Hematodinium spp. infection. The sternae and ventral surfaces of the Australian sand crabs Portunus pelagicus had a chalky, white appearance (Hudson and Shields, 1994). The abdomen of dying velvet swimming crabs Necora puber from several areas in France and Spain had a pale-pink color (Wilhelm and Mialhe, 1996). Changes in carapace color of some species of crabs are even more noticeable. The edible crab Cancer pagurus from UK waters exhibited an altered coloration (pink hyperpigmentation), and this infection is called ‘‘Pink Crab Disease” (Stentiford et al., 2002). The primary macroscopic sign of Hematodinium spp. infection in snow crabs, C. opilio, and Tanner crabs, C. bairdi, was a distinct change in carapace color that resulted in a cooked appearance (Meyers et al., 1987), and this feature was used for macroscopic estimation of disease prevalence (Meyers et al., 1990; Pestal et al., 2003). For red and blue king crabs in Kamchatka
Fig. 5. Transmission electron micrographs of Hematodinium sp. cells with trichocysts in cytoplasm. (A) Bi-nucleate parasitic cell (scale bar = 5 lm). (B) Uninucleate parasitic cell (scale bar = 2 lm). N: nucleus; mb: multivesicular body; t: trichocysts.
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waters, we did not register any color change even in highly infected crabs except for a creamy-yellow color of the hemolymph, visible through the arthrodial membranes of the abdomen and appendages. Dissection showed that the hemolymph of the infected king crabs is creamy-yellow in color and did not clot. Cream color and dense consistency of hemolymph in crabs in late stages of disease, such as registered in heavily infected king crabs, have been described in many species of crustaceans infected with Hematodinium spp. (Meyers et al., 1987; Shields, 1994). In addition, infected Tanner crabs have a bitter, astringent taste of cooked meat (Messick, 1994; Wilhelm and Mialhe, 1996; Stentiford et al., 2002). Several stages of the parasite, including trophonts, plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. The presence of trophonts and multinucleated plasmodial stages of the parasite in internal organs of crustaceans is a major diagnostic attribute of Hematodinium sp. infection (Meyers et al., 1987; Messick, 1994). Unattached vermiform plasmodia probably represent the motile form of the parasite, but the use of only fixed material did not allow us to confirm this suggestion. Vermiform plasmodia have been observed in many decapods (Stentiford and Shields, 2005), but only three king crabs with gross signs of infection had vermiform plasmodia. The reason for the low prevalence of crabs with such plasmodia may be the relatively short duration of this stage (Appleton and Vickerman, 1998). Trichocysts were present in the parasites from heavily infected king crabs. Studies of stages of Hematodinium sp. development in vitro showed that trichocysts are absent in vegetative cells (Appleton and Vickerman, 1998), implying that small cells, found in two very heavily infected red and blue crabs are sporonts. Similar cells were observed in later stages of Hematodinium sp. infection before sporulation (Meyers et al., 1987; Appleton and Vickerman, 1998; Shields and Squyars, 2000; Stentiford and Shields, 2005). In fixed hemolymph of such crabs, goblet-like cells were found which looked like the macrodinospores described by Meyers et al. in Tanner crabs (Meyers et al., 1987). Damage to the muscles, and lysis of the hepatopancreas epithelium found in the infected red and blue king crabs are typical for this disease (Messick, 1994; Meyers et al., 1987; Field and Appleton, 1995; Stentiford et al., 2002; Sheppard et al., 2003). The reason for the lack of pronounced inflammatory reaction of the organism to the Hematodinium sp. infection is the depletion of hemocytes in the hemolymph of the host in the course of disease development (Field and Appleton, 1995; Sheppard et al., 2003). Parasite cells multiply rapidly and cause hemocytopenia (Field and Appleton, 1995; Shields and Squyars, 2000). Infiltration of the connective tissue of most organs occurs at the later stages of infection. Comparison of the sequences of fragments of the 18S rDNA implies that the Hematodinium sp. found in red king crabs is the same or closely related to Hematodinium sp. isolated from crabs of the genera Chionoecetes and Lithodes (Genbank Accession Numbers FJ844412- FJ844431). Sequence similarity of our data (deposited in GenBank; EU856716) with other 18S rDNA sequences available in GenBank allows us to consign the Hematodinium sp. from Paralithodes camtschaticus within the clade Hematodinium that infects many decapod hosts in the North Pacific and North Atlantic Oceans (Jensen et al., 2010). The prevalence of Hematodinium sp. was variable in P. camtschaticus and P. platypus populations in the northeastern part of the Sea of Okhotsk, with the most but not all regions exhibiting very low prevalence. The largest number of Hematodinium sp. infected crabs was detected in the area with a maximum density of P. camtschaticus and P. platypus. A positive correlation between the number of infected crabs and their total density was observed for C. sapidus and Libinia emarginata (Sheppard et al., 2003). Gross inspection allows us to determine the disease only when it reached its maximum development, therefore many infected crabs could be
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missed. The accuracy of macroscopic diagnosis based on carapace color of C. opilio was around 0.53 (Pestal et al., 2003), and gross inspection based on hemolymph changes is less accurate. For more precise assessment it is necessary to use other methods, such as microscopic analysis of hemolymph or molecular methods (see Jensen et al., 2010). Infection of crustaceans with the Hematodinium spp. dinoflagellates has a strongly pronounced seasonal pattern (Stentiford and Shields, 2005). The occurrence of the disease in the Tanner crab C. bairdi in Alaska decreased in the autumn, as infected crabs died by that time (Love et al., 1993). In our study, gross signs of infection were absent in king crabs caught starting from the second half of October. Hematodinium infections have been reported in many brachyuran crabs (Stentiford and Shields, 2005) and in the present study we described this infection in anomuran crabs using a diverse approach. Acknowledgments We thank the anonymous reviewers for valuable comments and suggestions to this manuscript. This work was supported, in part, by FEB RAS (Project 09-III-A-06-210). References Appleton, P.L., Vickerman, K., 1998. In vitro cultivation and development cycle in culture of a parasitic dinoflagellate (Hematodinium sp.) associated with mortality of the Norway lobster (Nephrops norvegicus) in British waters. Parasitology 116, 115–130. Bell, T.A., Lightner, D.V., 1988. A Handbook of Normal Penaeid Shrimp Histology. The World aquaculture society, Baton Rouge. Dolgenkov, V.N., Koblikov, V.N., 2009. Current status of fisheries resources of important species of crabs and prospects of their industrial development. In: Alimov, A.F., Adrianov, A.V. (Eds.), Abstracts of X Congress of Hydrobiological Society of Russian Academy of Sciences. Dalnauka, Vladivostok, p. 125. Field, R.H., Appleton, P.L., 1995. A Hematodinium-like dinoflagellate infection of the Norway lobster Nephrops norvegicus: observations on pathology and progression of infection. Dis. Aquat. Organ. 22, 115–128. Gruebl, T., Frischer, M.E., Sheppard, M., Neumann, M., Maurer, A.N., Lee, R.F., 2002. Development of an 18S rRNA gene targeted PCR based diagnostic for the blue crab parasite Hematodinium sp. Dis. Aquat. Organ. 49, 61–70. Hudson, D.A., Shields, J.D., 1994. Hematodinium Australis n. sp., a parasitic dinoflagellate of the sand crab Portunus pelagicus from Moreton Bay, Australia. Dis. Aquat. Organ. 19, 109–119. Jensen, P.C., Califf, K., Lowe, V., Hauser, L., Morado, J.F., 2010. Molecular detection of Hematodinium sp. in Northeast Pacific Chionoecetes spp. and evidence of two species in the Northern Hemisphere. Dis. Aquat. Organ. 89, 155–166. Love, D.C., Rice, S.D., Moles, D.A., Eaton, W.D., 1993. Seasonal prevalence and intensity of bitter crab dinoflagellate infection and host mortality in Alaskan Tanner crabs Chionoecetes bairdi from Auke Bay, Alaska, USA. Dis. Aquat. Organ. 15, 1–7. Lysenko, V.N., 2001. Specific biological traits of males of the blue crab Paralithodes platypus in the northeast part of the Sea of Okhotsk. Russ. J. Mar. Biol. 27, 135– 142. Messick, G.A., 1994. Hematodinium perezi infections in adult and juvenile blue crabs Callinectes sapidus from coastal bays of Maryland and Virginia, USA. Dis. Aquat. Organ. 19, 77–82. Messick, G.A., Shields, J.D., 2000. Epizootiology of the parasitic dinoflagellate Hematodinium sp. in the American blue crab Callinectes sapidus. Dis. Aquat. Organ. 43, 139–152. Meyers, T.R., Koeneman, T.M., Botelho, C., Short, S., 1987. Bitter crab disease: a fatal dinoflagellate infection and marketing problem for Alaskan Tanner crabs Chionoecetes bairdi. Dis. Aquat. Organ. 3, 195–216. Meyers, T.R., Botelho, C., Koeneman, T.M., Short, S., Imamura, K., 1990. Distribution of bitter crab dinoflagellate syndrome in southeast Alaskan Tanner crabs Chionoecetes bairdi. Dis. Aquat. Organ. 9, 37–43. Otto, R.S., Jamieson, G.S., 2001.Commercially Important Crabs, Shrimps and Lobsters of the North Pacific Ocean. PICES Sci. Rep. No. 19. Pestal, G.P., Taylor, D.M., Hoenig, J.M., Shields, J.D., Pickavance, R., 2003. Monitoring the prevalence of parasitic dinoflagellate Hematodinium sp. in snow crabs Chionoecetes opilio from Conception Bay, Newfoundland. Dis. Aquat. Organ. 53, 67–75. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sheppard, M., Walker, A., Frischer, M.E., Lee, R.F., 2003. Histopathology and prevalence of the parasitic dinoflagellate, Hematodinium sp., in crabs (Callinectes sapidus, Callinectes similis, Neopanope sayi, Libinia emarginata, Menippe mercenaria) from a Georgia estuary. J. Shellfish Res. 22, 873–880.
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