DISEASES OF AQUATIC ORGANISMS Published October 18 - Dis. aquat. Org. Rod-shaped nuclear viruses of crustaceans: gut-infecting species Phyllis T. ~ohnson',Donald V. ~ightner~ ' National Marine Fisheries Service, Northeast Fisheries Center, Oxford, Maryland 21654, USA * University of Arizona, Environmental Research Laboratory, 2601 E. Airport Drive, Tucson, Arizona 85706,USA ABSTRACT: There are 6 baculovirus-related nuclear viruses known to attack the hepatopancreatic epithelium of decapod crustaceans, including penaeid shrimp, brachyuran crabs, and anomuran crabs. Four of the 6 viruses are nonoccluded. One of the occluded species is accepted as a member of the Baculoviridae by the International Commission on Taxonomy of Viruses. The 4 available gut-infecting crustacean viruses are described and all 6 are compared to each other and to viruses from insects and arachnids that share some similar characteristics. Morphological similarities, including among others the presence of a truly cyhdrical nucleocapsid, a unilateral subapical envelope expansion, and a tail- like appendage that arlses from the nucleocapsid and is reflexed withln the envelope expansion, indicate that the crustacean viruses are related to one another. In morphological and some developmen- tal attributes, the crustacean vlruses, including the occluded species, show affinities to the nonoccluded virus of Oryctes, which is currently the type of proposed Subgroup C of the Baculoviridae. INTRODUCTION MATERIALS AND METHODS Six rod-shaped, singly enveloped, nuclear viruses For light microscopy, crab tissues were fixed in Hel- are known which attack endodermally derived tissues, ly's solution (Johnson 1980) and stained by the Feulgen mainly the hepatopancreatlc epithelium, of decapod technique, using picric acid and methyl-blue counter- crustaceans. They are clearly related to the stains (Farley 1969); shrimp tissue was fixed in David- Baculoviridae, although only one has been sufficiently son's fluid (Humason 1967) and stained with hema- characterized to allow its acceptance as a member of toxylin and eosin. Primary fixation for crab tissues used that family by the International Committee on Tax- in electron microscopy was in cold 4 % glutaraldehyde onomy of Viruses (ICTV] (Matthews 1982). Four of the in 0.1 M, pH 7.4, Sorenson's phosphate buffer. Shrimp 6 are nonoccluded. tissues were fixed in cold 6 O/O glutaraldehyde in 0.15 M Study of 1 new virus and 3 others at our disposal adds Millonig's phosphate buffer (Pease 1964), supple- considerable information about the general nature of mented with 1 Oio sodium chloride and 0.5 % sucrose. the gut-infecting viruses and gives some insight into All tissues were postfixed at room temperature in 1 % which developmental and structural characters might osmium tetroxide in their respective buffers, dehy- indicate degrees of relatedness among them. Develop- drated in ethanol and propylene oxide, and embedded ment of the species apparently differs, but virions are in Spurr's low viscosity embedding medium (Spurr morphologically similar and unlike virions of lepi- 1969) or in an Epon/Araldite mixture (1:l). Thin sec- dopteran nuclear polyhedrosis viruses (NPVs). These tions were stained with uranyl acetate and lead citrate. viruses are compared to each other, to the remaining Negative staining was with phosphotungstic acid. crustacean viruses, and to other arthropod viruses that Shrimp tissues were viewed with either a Hitachi H- have some attributes in common. Most of the latter 500, a Phillips 200, or a JEOL lOOCX electron micro- viruses are currently assigned to Subgroups A, B, and scope; crab tissues were viewed with a Zeiss EM 9s 2 proposed Subgroup C of the Baculoviridae (Matthews electron microscope. 1982), and to the Polydnaviridae (Stoltz et al. 1984, Terms used in this paper to describe viral structure Brown 1986). are defined by Johnson (1988: con~panionarticle). O Inter-Research/Printed in F. R. Germany 124 Dis. aquat. Org. 5: 123-141, 1988 OBSERVATIONS this membrane. It is not uncommon for crabs to eat fecal strings, and containment of virions of Baculo-A The 6 viruses that infect the epithelium of the within the string would make for more efficient tram- hepatopancreas (midgut gland), and sometimes that of mission than if virions were released separately into the the midgut proper, can be grouped together, depending water. Transmission through cannibalism probably mainly on nucleocapsid/virion morphology. Early occurs also The focal nature of infection suggests that development apparently differs between the 2 species virions may pass from one ep~thelialcell to another, but that form occlusion bodies. Three of the viruses, includ- how they enter epithelial cells from the gut or pass from ing the occluded ones, are from various species of one epithelia1 cell to another is unknown. penaeid shrimp. Two viruses are from the brachyuran Infected nuclei were up to 2.3 times normal size. The portunid crabs Callinectes sapidus and Carcinus center of the nucleus was evenly and not strongly mediterraneus, and one is from the blue king crab Feulgen positive except that occasionally somewhat Paralithodesplatypus, which is an anomuran. Most of the diffuse and more deeply staining areas occurred there. viruses have been extensively studied histologically with Usually the center of the nucleus was surrounded by an the light microscope and evidence of infection outside intensely Feulgen-positive marginal band (Fig. 1). the gut has not been found. It is likely, therefore, that they Nucleolar remnants were common, espec~allyin F-celis normally attack only endodermally derived cells. (Fibrillar cells), which typically have large, multiple The shrimp viruses vary considerably in their abhty nucleoli. The remnants were needle-shaped, spherical, to cause disease, depending on the viral species and or irregular. the life-history stage of the host (Lightner 1983, John- son 1984). The viruses from Callinectes sapidus and Paralithodes platypus are apparently nonpathogenic in Electron microscopy juveniles and adults, the only stages that have been studied. The virus from Carcinus mediterraneus causes Because Baculo-A does not cause disease or grossly fatal disease in mature crabs (Pappalardo & Bonami visible changes in fresh tissues, infected nuclei could 1979, Pappalardo et al. 1986). be found only by chance in hepatopancreatic tissues. The species reviewed here are the occluded virus The few infected nuclei available for ultrastructural first found in Penaeus monodon (Lightner & Redman study were from 4 crabs. Tissue from 1 crab was tipped 1981),the occluded virus from American and Hawaiian into icewater shortly after being immersed in glutaral- Penaeus spp. (Couch 1974a, Brock et al. 1986), the dehyde, causing damage to cellular components. The nonoccluded virus of Callinectes sapidus (Johnson most obvious damage was shrinkage and loss of inter- 1976), and a previously undescribed nonoccluded virus nal cell membranes; positions of plasma membranes from Alaskan Paralithodes platypus. remained visible. Tissue from another crab had been embedded in paraffin and was re-embedded for elec- tron-microscopic exammation as described by Johnson Hepatopancreatic virus of Callinectes sapidus (1977). (Baculo-A) Because of insufficient material, progressive assem- bly of virions could not be studied. An early infection Baculo-A is a nonoccluded virus that infects differ- was found in one properly fixed nucleus. This nucleus entiating cells of the hepatopancreatic epithelium had finely fibrillar material through the center and (Johnson 1976, 1983, 1984). Prevalence in groups of marginated chromatin (Fig. 2). (The fibrillar material crabs collected from Chesapeake and Chincoteague had a more granular appearance in ~nfectednuclei Bays and their tributaries (Maryland and Virginia, from the damaged tissue.) In the early infection virions USA) usually varied from 4 to 20 % ; overall prevalence were scattered at random through the nucleus, and was 6 % in l500 crabs. Baculo-A has not been observed there were no envelope precursors in the central por- to cause bsease in the blue crab. Although infected tion, which was presumed to 'be the viral stroma. Nu- cells undoubtedly do not perform normal functions, cleocaps~ds were cyl~ndrical and squared apically they are almost always few in number and in isolated (Fig. 3). A boundary was not visible between core and groups. Baculo-A does not attack stem cells; infection capsid. The nucleoprotein of the core had distinctly occurs only in those cell populations which are under- lucent areas. The reticulate appearance was more pro- going constant replacement. nounced in virions from the damaged tissue. Naked Transmission is presumably per 0s. Materials from (unenveloped) nucleocapsids were not seen except that the hepatopancreas and stomach are enclosed in a particles in the re-embedded tissue lacked, any indica- peritrophic membrane as they pass through the tion of envelopes. Envelopes were tnlaminar in all rnidgut, and feces are expelled in a string enclosed by other preparations, including the glutaraldehyde-fixed, lupuural leloapnu nu uT1 L&.[ = leg (shiolle) jl~dIequas ur sprsdes dldura '6~01aIoN ~ii~~eur6leu1alelnurnssp suou!~,uorlsajur pasuehpv m prsdesoa~snu JU !ado[a~ua.a Iprsdes 6uol :s .mu g01 = lea .(~olle) prsdesoalsnu suluasxa ue qlrM suou~pauoqsas-ss013 I ur palesrpur os[e sr qsrq~'(s~olle) uo!suedxa adolahua ~es~deqnsaql a$oN .sprsdes lildua '6~01pue suour~m uti L& L leg .uoqJajur A11ex uru,l 01 = leg ua61nad .(MOII~ uado) ~uasaid -[ .p 1 osle alp lapnu IeuloN (.nol~e)nas aieapnurq e ur !a[snu qloq 30 uoqsajur Bu~~oqsqdeldo~srw 1q6g .v-oTnsea 01 .s6y 126 Dis. aquat. Org. 5: 123-141, 1988 damaged tissues where nuclear and other internal cell Pnbilof Islands in June 1982. Baculo-PP was not membranes were missing. The envelope was 8 to detected in tissues of 25 crabs each from St Matthews 11 nm thick. It was unilaterally expanded subapically Island, July 1982, and St Lawrence Island, September and around the apex of one end of many virions (Fig. 3). 1982, or in 9 crabs from the Pribilof Islands, February The area between the nucleocapsid and the envelope, 1983.
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