Milky Hemolymph Syndrome Associated with an Intranuclear Bacilliform Virus in Snow Crab Chionoecetes Opilio from the Sea of Japan

Fish Sci (2011) 77:999–1007 DOI 10.1007/s12562-011-0405-0

ORIGINAL ARTICLE Aquaculture

Milky hemolymph syndrome associated with an intranuclear bacilliform virus in snow crab Chionoecetes opilio from the Sea of Japan

Toshi Kon • Tadashi Isshiki • Toshiaki Miyadai • Yoshiharu Honma

Received: 30 May 2011 / Accepted: 18 August 2011 / Published online: 27 September 2011 Ó The Japanese Society of Fisheries Science 2011

Abstract Milky hemolymph syndrome (MHS) is a pre- targeted by the virus differed from those targeted by IBVs. viously undescribed disease in snow crab Chionoecetes This is the first report of a virus from the genus Chion- opilio. Outbreaks of this disease occurred in reared and oecetes. Thus, we have tentatively designated the virus wild populations from the Sea of Japan when the water presumably associated with MHS as Chionoecetes opilio temperature was 2–3°C. The common symptom of the bacilliform virus (CoBV) until its relationship with other disease was a distinct milky or opaque coloration of the crustacean viruses can be clarified. hemolymph. Some severely affected crabs also showed yellow or ivory discoloration on the ventral shell and un- Keywords Snow crab Chionoecetes opilio calcification of the arthrodial membranes of walking legs. Milky hemolymph Bacilliform virus Histopathology Histopathological changes consisted of widespread cellular Electron microscopy Crustacean viral disease CoBV degeneration characterized by enlarged nuclei with marginal hyperchromatosis and basophilic intranuclear inclusions in the interstitial connective tissues of various organs. Introduction Electron microscopy analyses of degenerated cells revealed a nonoccluded, enveloped bacilliform virus within the Crabs in the genus Chionoecetes are widely distributed in nuclei. The virus morphologically resembles white spot cold waters of the northern hemisphere and are important syndrome virus (WSSV) or unassigned intranuclear bacil- fishery resources in the United States, Canada, Russia, liform viruses (IBVs) reported from other crustaceans. Greenland, Japan, and the Koreas. In the Sea of Japan, two However, we could not detect WSSV from diseased tissues Chionoecetes species are caught commercially: snow crab by PCR using WSSV-specific primers, and the tissues C. opilio and red snow crab C. japonicus. There are three main diseases that affect Chionoecetes crabs. These include bitter crab disease (BCD) [1] (bitter T. Kon (&) crab syndrome; BCS [2]), caused by parasitic dinoflagel- The Wakasa Wan Energy Research Center, 37-8-1 Heiwatyou, lates of the genus Hematodinium; black mat syndrome Turuga, Fukui 914-0815, Japan (BMS), caused by the ascomycete fungus Trichomaris e-mail: [email protected] invadens on the exterior of the carapace [3]; and the shell T. Isshiki disease known as black spot (or brown, rust, or burn spot), Graduate School of Bioresources, Mie University, which is caused by chitinolytic bacteria [4]. Among these 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan diseases, BCD [1] and BMS [5] are fatal and have signif- T. Miyadai icant impact on crab population dynamics. Faculty of Marine Biotechnology, Fukui Prefectural University, BCD has been found in snow crab and Tanner crab 1-1 Gakuencho, Obama, Fukui 917-0003, Japan C. bairdi from the southeastern Bering Sea, the Gulf of Alaska, and the Atlantic Ocean off Canada, as well as Y. Honma Department of Anatomy, Niigata University School of Medicine, in many other crab species [6]. Recently, BCD was 1-757 Asahimachi, Chuoh-ku, Niigata, Niigata 951-8510, Japan observed in crabs in the family Lithodidae (red king crab 123 1000 Fish Sci (2011) 77:999–1007

Paralithodes camtschaticus; blue king crab P. platyus)in January to May 2009 when the rearing temperature was the West Kamchatka subarea in the Sea of Okhotsk [7]. 2–3°C. The juvenile crabs were collected by a deep-sea However, BCD has never been reported in crabs in the Sea motor trawl net, operated by Fukui Prefectural Fisheries of Japan. BMS has been found in stocks of snow crab and Experimental Station, from the Sea of Japan in June 2009 grooved Tanner crab C. tanneri from the Bering Sea and when the inhabiting ambient water temperature was 2–3°C. the Gulf of Alaska [8]. This disease has also been found in As the control, apparently healthy crabs (1 male, carapace stocks of snow crab [9] and red snow crab [10] from the width 80 mm; 5 females, carapace width 75–86 mm) were Sea of Japan. The occurrence of BMS in snow crabs is very also sampled from the catch in June 2009. rare in the western waters of the Sea of Japan [9]; therefore, it was thought that this disease barely affects crab repro- Light and electron microscopy duction in these waters. Black spot has been found in snow crab in the Atlantic Ocean [4] and in grooved Tanner crab After external and internal observations, the hepatopan- in the Canadian Pacific [11], and a shell disease clinically creas, gonads (ovaries, seminal receptacles, testes, and vas resembling black spot has been found in crabs in the Sea of deferens), gut, heart, meropodite musculature, and gills of Japan [9]; however, black spot is usually nonfatal [11]. the crabs were fixed in Bouin’s solution for light micro- Many kinds of marine crabs can be infected by viruses scopy. The fixed tissues were routinely embedded in par- [12], but no viral infections have been found in crabs in the affin and cut into 5-lm sections. Tissue sections were then genus Chionoecetes. Consequently, there are no infectious stained with hematoxylin and eosin (H&E) and Giemsa. diseases associated with large-scale decreases of Chion- For electron microscopy, small pieces of the remaining oecetes crab populations in the Sea of Japan. tissues from the adult crabs were fixed in a mixture of 2.5% In the early 2000s, a previously undescribed disease was glutaraldehyde and 2% paraformaldehyde in phosphate observed in snow crabs reared at a commercial aquarium buffer (pH 7.3), post-fixed in 2% osmium tetroxide, and facility on the coast of the Sea of Japan when the rearing embedded in Epon epoxy resin. Ultrathin sections were temperature was 2–3°C. The major symptom of the disease stained with uranyl acetate and lead citrate and observed at in infected crabs was a distinct milky or opaque coloration of 80 kV with a JEM-1010 transmission electron microscope. the hemolymph. In some severe cases, a yellow or ivory discoloration developed on the ventral shell, and the Polymerase chain reaction (PCR) examination arthrodial membranes of walking legs were uncalcified. The diseased crabs had listless or lethargic behavior, but no To detect white spot syndrome virus (WSSV), PCR analyses extensive losses were recorded. In June 2008, the disease was were carried out on the hemolymph, gill, appendage mus- again observed among wild-caught snow crabs off the Sea of culature, and ovary tissues from the diseased crabs. PCR Japan that were inhabiting water with an ambient tempera- analyses were conducted according to Kimura et al. [13]. ture of 2–3°C. Fishermen were able to recognize affected live Briefly, DNA was extracted from each tissue sample with crabs by a discolored carapace or milky hemolymph and ISOGEN (Japan Gene Company) according to the manu- referred to them as ‘‘milky crabs.’’ Such crabs were culled facturer’s instructions and submitted to a two-step PCR from harvests at the fishing grounds because they were examination using two specific primer sets: primers P1 unmarketable. Therefore, the occurrence of this new disease (50-ATCATGGCTGCTTCACAGAC-30) and P2 (50-GTGG has been economically significant. In this paper, we describe CTGGAGAGGACAAGAC-30) for one-step PCR, and P3 the pathological features of this new disease, named milky (50-TCTTCATCAGATGCTACTGC-30) and P4 (50-TAAC hemolymph syndrome (MHS), and present the electron GCTATCCAGTATCACG-30) for nested PCR. After 30 microscopic findings to define the etiological agent of MHS. cycles of amplification for each primer set at 93°C for 60 s, 57°C for 90 s, and 72°C for 60 s, the amplified products were analyzed by electrophoresis on 1.5% agarose gel. Materials and methods Purified WSSV particle was similarly processed as a positive control. Snow crabs

We examined a total of 10 diseased snow crabs, including Results 5 adults (3 females, carapace width 72–84 mm; 2 males, carapace width 118–130 mm) and 5 juveniles (4 females, Clinical symptoms carapace width 46–71 mm; 1 male, carapace width 83 mm). The adult crabs were obtained from a commercial Diseased crabs did not usually show any specific clinical aquarium facility on the coast of the Sea of Japan from signs but were listless or lethargic, and some died after 123 Fish Sci (2011) 77:999–1007 1001 being caught in the trawl net. The severely affected crabs Light microscopy sometimes exhibited a yellow or ivory discoloration on the ventral shell (Fig. 1a). Their walking legs had General histopathological changes of all the diseased crabs uncalcified arthrodial membranes (on both sides of the were observed systemically in the interstitial connective coxopodite) with a milky or ivory coloration, in contrast tissues and were characterized by widespread cellular to normal crabs with pale pink or white coloration degeneration. The degenerated cells had a pale and (Fig. 1b, c). Internally, the hemolymph of diseased crabs enlarged nucleus with marginal hyperchromatosis, which was frequently milky or opaque (Fig. 1d). The color tone was frequently replaced by basophilic granules. In mark- of the milky hemolymph differed among individuals, edly degenerated cells, the nucleus was more enlarged and from faintly semitranslucent white to deep milky white or was filled with a basophilic inclusion. Eventually, the pale pink. Some diseased crabs also showed pale color- intranuclear inclusion occupied most of the cytoplasm of ation of the gills (Fig. 1e). The disease prevalence was the affected cells, which ranged from 13 to 25 lminthe independent of age or sex among all specimens exam- long axis, and residual chromatin remained on the inner ined. No ectoparasitic organisms were detected in any nuclear membrane. The cytoplasm eventually became organs. The meat from severely affected crabs was lucent and surrounded the enlarged nucleus. Furthermore, indistinguishable from that of healthy crabs in tasting these degenerated cells occasionally contained deep baso- trials. philic intracytoplasmic inclusions. These inclusions were

Fig. 1 Clinical symptoms of milky hemolymph syndrome (MHS) in healthy crab showing pale pink or white coloration. Scale bar 1 cm. snow crab Chionoecetes opilio. a Ventral view of female juvenile d Internally, color of hemolymph (Hl) in slightly affected crab (left)is snow crab prior to terminal molt. Diseased crab (top) exhibits yellow faintly semitranslucent white, in contrast to transparent in a healthy or ivory discoloration on ventral shell compared with healthy crab crab (right). Scale bar 3 cm. e Severely affected crab showing deep (bottom). Scale bar 2 cm. b Walking legs of diseased crab have milky coloration of hemolymph (Hl) and a pale coloration of gills uncalciﬁed membranes (Um) on both sides of a coxopodite (Cp) (Gi). Scale bar 2cm showing milky or ivory coloration. Scale bar 1 cm. c Same part of

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Fig. 2 Histopathological changes in milky hemolymph syndrome–affected snow crab Chionoecetes opilio. a Interstitial connective tissues surrounding the seminal receptacle show enlarged nuclei with basophilic inclusions (large arrows) and marginal hyperchromatosis (arrowheads). Note the lucent cytoplasm (small arrows) surrounding the enlarged nuclei and the deep basophilic intracytoplasmic inclusion (asterisk). H&E. Scale bar 40 lm. b Deep basophilic intracytoplasmic inclusions (asterisks) in degenerated cells of interstitial connective tissues surrounding the gut. Inclusion is delimited by membrane and contains basophilic granules. H&E. Scale bar 40 lm. c–f Interstitial connective tissues surrounding or extending into hepatopancreas (Hp), ovary (Ov), vas deferens (Vd), and gill stem (Gs) show the same cellular degeneration as shown in a. Arrows indicate enlarged nuclei. H&E. Scale bars 100 lm. g In the heart (He), hemolymph with basophilic granules (Hl) accumulates in the trabecular channels within the myocardium. H&E. Scale bar 200 lm. h In the gill lamellae (Gl), branchial sinuses lack hemocytes, and epithelium thickness is decreased. H&E. Scale bar 100 lm

delimited by a membrane and contained basophilic gran- receptacles (Fig. 2a), gut (Fig. 2b), hepatopancreatic ules. Similar intracytoplasmic inclusions were also present tubules (Fig. 2c), ovary (Fig. 2d), vas deferens (Fig. 2e), in granular cells (Fig. 2a, b). These histopathological gill stems (Fig. 2f), and other organs such as the testes and changes were observed commonly in the interstitial con- myomeres. In heart tissues, these histopathological changes nective tissues surrounding or extending into the seminal were slight, but we observed accumulation of hemolymph

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Fig. 3 Electron micrographs of interstitial connective tissues in gut from milky hemolymph syndrome–affected snow crab Chionoecetes opilio. a Enlarged nuclei (n) contain numerous virus particles in a paracrystalline arrangement, and heterochromatin is deposited on inner nuclear membrane. Scale bar 10 lm. b High power view of virus particles at different stages of morphogenesis; almost spherical capsid originators (arrowheads) (approx. 50 nm in diameter), capsids developing in association with membrane, and helical ﬁlament-shaped nucleocapsids (arrows) extending the full length of the envelope. Scale bar 250 nm. c High power view of mature virions in nucleus. Virions are ovoid or ellipsoid to bacilliform in shape and consist of an inner, rod-shaped nucleocapsid (nc) surrounded by a loose-ﬁtting outer envelope (e). Scale bar 250 nm

with many basophilic granules and little hemocyte in the Electron microscopy trabecular channels within the myocardium (Fig. 2g). There was little or no evidence of obvious histopatholo- In the interstitial connective tissue specimens from all the gical changes in the parenchyma of various organs, except diseased crabs, numerous virus particles were found in a for the gills. In the gill lamellae, the epithelium was thinner paracrystalline arrangement or scattered inside the enlarged than that in healthy crabs. In addition, the branchial sinuses nucleus, with a small amount of heterochromatin deposited of diseased crabs lacked hemocytes and granular cells, on the inner nuclear membrane (Fig. 3a). The virus indicating a state of hypocytosis in the gills (Fig. 2h). No aggregations consisted of virions at different stages of bacteria, fungi, or parasites were observed in any tissues. morphogenesis. The capsid originator was almost round

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Fig. 4 Electron micrographs of interstitial connective tissues of ovary and hepatopancreas from milky hemolymph syndrome– affected snow crab Chionoecetes opilio showing intracytoplasmic locations of virus particles. a Membrane- delimited intracytoplasmic structure (asterisk) in the ovary. Structure is located close to the enlarged virus-infected nucleus (n) and contains viral envelope- like materials and enveloped capsids. Scale bar 2 lm. m Mitochondria. b Another membrane-delimited intracytoplasmic structure (asterisk) in the ovary, located beside enlarged virus-infected nucleus (n). Structure is packed with mature virions and a ﬁne granular matrix with high electron density. Scale bar 2 lm. c Intracytoplasmic viroplasm-like structure (v) associated with virions in ovary. Structure consists of electron-dense granules and ﬁlaments. Scale bar 500 nm. d Membrane-delimited intracytoplasmic structures (asterisks) containing virions in granular cell in the hepatopancreas. Note the apparently normal nucleus (n) of the granular cell, and presumably degenerated virions (spherical viral envelopes and U-shaped nucleocapsids) within structures. Scale bar 5 lm

(approx. 50 nm in diameter) and had a less dense center 144 nm in diameter and 338 nm in length, and the mean and evenly spaced rays or granules on its surface. The size of nucleocapsids was 76 nm in diameter and 293 nm capsid appeared to develop in association with the mem- in length (Fig. 3c). brane. One or two capsids were surrounded by linear or The cytoplasm contained membranous, granular, or circular membrane fragments, and there were many vesi- filamentous structures associated with the virus. The cles enclosing empty capsids and/or nucleocapsids, possi- membrane-delimited structure, comprising viral envelope- bly reflecting the development of enveloped capsids. A like materials and the enveloped capsids, was often present helical filament-shaped nucleocapsid sometimes occurred near the virus-infected nucleus (Fig. 4a). Another similar within the envelope (Fig. 3b). The putative mature virion membrane-delimited structure was packed with mature was ovoid or ellipsoid to bacilliform in shape and consisted virions and a fine granular matrix with high electron of an inner, rod-shaped nucleocapsid surrounded by a density (Fig. 4b). Virions were diffusely located within loose-fitting outer envelope. The mean size of virions was inter- and extracellular spaces. Some were observed in

123 Fish Sci (2011) 77:999–1007 1005 intracytoplasmic viroplasm-like structures comprising function, similar to that observed in Hematodinium-infec- electron-dense granules and filaments (Fig. 4c). The gran- ted snow crabs [14]. It is likely that the resulting decreased ular cell also had intracytoplasmic membrane-delimited gill function caused listless and lethargic behavior of structures containing virions, while the envelopes within affected crabs. The gills of diseased crabs lacked hemo- the structure were spherical and the nucleocapsids were cytes and granular cells, which would also affect respira- apparently forced to assume a U-shape. This may reflect a tory function. The mechanism of hypocytosis in the gills viral degeneration process. In these granular cells, the should be further explored to elucidate the pathogenesis of nucleus was not infected by the virus, nor did it show any MHS in snow crabs. enlargement (Fig. 4d). Our observations of sections stained Electron microscopy revealed accumulation of virus with toluidine blue confirmed that these intracytoplasmic particles within the enlarged nuclei of affected cells in the structures associated with the virus corresponded to the interstitial connective tissues. We did not observe occlu- deep basophilic intracytoplasmic inclusions observed under sion body formations. Virions at different stages of mor- light microscopy (data not shown). No occlusion bodies phogenesis were found in the nucleus. Our preliminary were found in any specimens. electron microscopic observations of the hemolymph showed that the hemolymph contains many virus particles PCR examination resulting from viremia (data not shown), although no electron microscopic finding of hemocytes in the hemo- We conducted PCR analyses to detect WSSV using specific lymph has been obtained and the mechanism of discolor- primers. We did not detect WSSV in any of the tested ation of the hemolymph is not clear. These results suggest tissues. From the positive control, PCR products with the that the virus in the nucleus was associated with MHS and expected size (approx. 1,000 or 570 bp) were amplified. that virus replication takes place at least in the nucleus. The virus shows some similarities to other nonoccluded and intranuclear replicated viruses. The morphology and size of Discussion the nucleocapsid and the mature virion resemble those of viruses in the family Nimaviridae [15] or unassigned The new disease described here, provisionally designated intranuclear bacilliform viruses (IBVs) reported from other as MHS, was found in snow crabs in the genus Chion- crustaceans [16–21]. All of these morphologically similar oecetes. Crabs in this genus are affected by epizootics such viruses can cause diseases in crustaceans. The histopa- as BCD [6], BMS [8, 9], and black spot [4]. MHS was thological changes associated with those diseases include readily distinguishable from BMS and black spot by its the appearance of enlarged nuclei with marginal hyper- characteristic symptoms. The symptoms of MHS, espe- chromatosis, similar to those observed in MHS. The family cially the milky coloration of the hemolymph, resemble Nimaviridae comprises only one genus, Whispovirus, of those of BCD. However, the MHS-affected crabs were free which WSSV is assigned as the only member [15]. Our from any parasites, including the dinoflagellate that causes PCR examinations using WSSV-specific primers con- BCD (Hematodinium sp.), as well as from bacteria and firmed that the present virus differs genetically from fungi (data not shown). Severely affected crabs sometimes WSSV. Since WSSV is the etiological agent of white spot showed a yellow or ivory discoloration on the ventral shell disease (WSD) [15], we can also draw an epidemiological and uncalcification of the arthrodial membranes of walking distinction between the present virus and WSSV because legs. These symptoms are considered to be specific for WSD typically occurs in crustaceans when the water MHS, as they have not been reported for other snow crab temperature ranges from 20 to 30°C[22–28], but MHS diseases. occurs at lower temperatures of 2–3°C. This suggests that Histopathological studies on the diseased crabs revealed the optimal temperature for virus proliferation may differ the pathognomonic symptoms of MHS, which include between the two viruses. Although WSSV infects diverse cellular degeneration characterized by nuclear enlargement target tissues including the epidermis, gills, foregut, hind- with marginal hyperchromatosis and basophilic intranu- gut [29, 30], muscle, eye-stalk, heart, connective tissue, clear inclusions in the interstitial connective tissues of gonad [31, 32], hematopoietic cells, and cells associated various organs. These histopathological changes have not with the nervous system [33, 34], the virus in the present been observed in any other snow crab diseases, and study specifically infected cells of the interstitial connec- therefore, histopathological examinations will be effective tive tissue rather than those in other tissues. For IBVs, the for diagnosis of MHS if Hematodinium sp. is not detected hemocyte, hematopoietic tissue, connective tissue [16, 18, from the milky hemolymph. Another noticeable histologi- 35], hepatopancreas [19, 20], and gut [17, 19] are targets cal change was the decreased thickness of the epithelium of for infection. However, the reports on IBVs did not men- the gill lamellae. This may directly affect respiratory tion the systemically targeted interstitial connective tissues 123 1006 Fish Sci (2011) 77:999–1007 where the typical histopathological changes were observed Chionoecetes, which is an important fishery resource in the present study. Based on these differences in the worldwide. The results of our ongoing infection experi- genetic properties, temperature dependence, and target ments to determine infectivity and virulence of CoBV in tissues for infection, the present virus appears to differ snow crab will be published in the near future. Epidemi- from WSSV or IBVs. In addition, this is the first report of a ological research on MHS should be started as soon as virus from the crab genus Chionoecetes. Thus, we tenta- possible, and preventive strategies to minimize the damage tively designate the virus presumably associated with MHS to fisheries should be discussed. The development of a as Chionoecetes opilio bacilliform virus (CoBV) until more diagnostic method based on genetic analysis of CoBV is is known about its relationships with other crustacean currently underway. viruses. Further virological characterization will be nec- essary to determine the taxonomic position of CoBV. Acknowledgments We thank Dr. Frank Morado (US National Although CoBV appears to replicate within the nucleus Oceanic and Atmospheric Administration), Dr. Richard Cawthorn (Atlantic Veterinary College, University of Prince Edward Island, as described above, we observed virus particles in intra- Canada), and Mikio Moriyasu (Department of Fisheries and Oceans, cytoplasmic locations within membrane-delimited struc- Canada) for their suggestions and advice about crab diseases. We tures produced in either interstitial connective tissue cells thank the staff of the Snow Crab Museum and the Fukui Prefectural or granular cells. In the interstitial connective tissue cells, Fisheries Experimental Station for collecting diseased crabs. the membrane-delimited structures were located near the virus-infected nucleus and contained granular or filamen- References tous materials as well as the virus particles at different stages of morphogenesis. Cells of crustaceans infected by 1. Meyers TR, Koeneman TM, Botelho C, Short S (1987) Bitter the intranuclear-replicated virus Baculovirus penaei often crab disease: a fatal dinoflagellate infection and marketing show a virally modified host cell structure known as the problem for Alaskan Tanner crabs, Chionoecetes bairdi. Dis membranous labyrinth, which is located in the cytoplasm Aquat Org 3:195–216 2. Meyers TR, Morado JF, Sparks AK, Bishop GH, Pearson T, near or against the nuclear envelope [36, 37]. 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