A New Pathogenic Virus in the Caribbean Spiny Lobster Panulirus Argus from the Florida Keys
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DISEASES OF AQUATIC ORGANISMS Vol. 59: 109–118, 2004 Published May 5 Dis Aquat Org A new pathogenic virus in the Caribbean spiny lobster Panulirus argus from the Florida Keys Jeffrey D. Shields1,*, Donald C. Behringer Jr2 1Virginia Institute of Marine Science, The College of William & Mary, Gloucester Point, Virginia 23062, USA 2Department of Biological Sciences, Old Dominion University, Norfolk, Virginia 23529, USA ABSTRACT: A pathogenic virus was diagnosed from juvenile Caribbean spiny lobsters Panulirus argus from the Florida Keys. Moribund lobsters had characteristically milky hemolymph that did not clot. Altered hyalinocytes and semigranulocytes, but not granulocytes, were observed with light microscopy. Infected hemocytes had emarginated, condensed chromatin, hypertrophied nuclei and faint eosinophilic Cowdry-type-A inclusions. In some cases, infected cells were observed in soft con- nective tissues. With electron microscopy, unenveloped, nonoccluded, icosahedral virions (182 ± 9 nm SD) were diffusely spread around the inner periphery of the nuclear envelope. Virions also occurred in loose aggregates in the cytoplasm or were free in the hemolymph. Assembly of the nucleocapsid occurred entirely within the nucleus of the infected cells. Within the virogenic stroma, blunt rod-like structures or whorls of electron-dense granular material were apparently associated with viral assembly. The prevalence of overt infections, defined as lethargic animals with milky hemolymph, ranged from 6 to 8% with certain foci reaching prevalences of 37%. The disease was transmissible to uninfected lobsters using inoculations of raw hemolymph from infected animals. Inoculated animals became moribund 5 to 7 d before dying and they began dying after 30 to 80 d post-exposure. The new virus is apparently widespread, infectious, and lethal to the Caribbean spiny lobster. Given the pathogenic nature of the virus, further characterization of the disease agent is warranted. KEY WORDS: Crustacea · Disease · Decapoda · Hemocyte · Pathology · Viral assembly · Herpes-like · Iridovirus Resale or republication not permitted without written consent of the publisher INTRODUCTION focused on diseases of the spiny lobster or the potential for negative impacts on their populations. The Caribbean spiny lobster Panulirus argus sup- Palinurid lobsters in the genera Panulirus spp., Pal- ports the single most valuable fishery in Florida and is inurus spp. and Jasus spp. have few reported diseases heavily exploited by both commercial and recreational (for review see Evans & Brock 1994, Evans et al. 2000). fishers. In 1999, 7.5 million pounds (~US$20 to 23 mil- Until now, viral infections have never been conclu- lion) of spiny lobsters were landed in Florida with sively demonstrated. Shell disease from chitinoclastic approximately 90% of the fishery occurring within the bacteria can cause lesions around the tail and uropods Florida Keys (Florida Marine Research Institute 2001: of infected animals resulting in poor marketability Spiny lobster fact sheet; http://floridamarine.org/ (Sinderman & Rosenfield 1967, Alderman 1973, Iver- features). The recreational fishery may account for an sen & Beardsley 1976, Booth 1988). Systemic infections additional 20% of the annual catch in a short 2 d open- of Vibrio spp. have occasionally developed in lobsters ing prior to the start of the commercial season. For subjected to increased temperature, holding stress, or years, concerns about the sustainability of this impor- poor water quality (Chong & Chao 1986, Diggles et al. tant resource have focused on over-fishing and deteri- 2000). A presumed bacterial infection called hepato- oration of nursery habitats. Yet, few studies have pancreatic disease occurred in larval lobsters used in *Email: [email protected] © Inter-Research 2004 · www.int-res.com 110 Dis Aquat Org 59: 109–118, 2004 life history studies, and the condition was treated with site was located in hard-bottom habitat, the preferred streptomycin (Kittaka & Abrunhosa 1997). Filamentous nursery habitat of juvenile spiny lobsters in the Florida bacteria, presumably Leucothrix mucor, indicative of Keys (Butler et al. 1995, Herrnkind et al. 1997). Sites poor water quality or stress, have been observed on the were surveyed by 2 divers seasonally during the win- gills and eggs of Jasus edwardsii (J.D.S. unpubl. data). ter (January to March) and summer (June to August). Additionally, in experimental infections, Aerococcus Survey and site details will be reported elsewhere viridans, the causative agent of gaffkemia in clawed D.C.B et al. unpubl. data). During the surveys, all lob- lobsters, is pathogenic to Panulirus interruptus sters were captured and brought aboard the research (Schapiro et al. 1974) and may occur naturally in P. vessel. Moribund animals were returned to the labora- argus (Bobes et al. 1988). The later stages of gaffkemia tory for observation and confirmation of disease. infection cause ‘red tail’ in clawed lobsters, a syndrome Healthy animals were returned to their habitat. To quite different from that observed in viral infections. verify the presence of the virus, hemolymph and other Fungal infections have been reported on the carapace tissues from several lobsters were fixed and processed (Alderman 1973, McAleer 1983, Evans et al. 2000), for histology as described below. gills (cf. Didymaria spp., Penicillium spp.; Sordi 1958, At present there are no crustacean cell lines avail- B. Diggles, NIWA, New Zealand, pers. comm.) and able for viral culture. Therefore, the virus was main- larvae (Kitancharoen & Hatai 1995). A microsporidian tained in the laboratory by serial passage of infected was pathogenic in the muscles of P. argus, P. cygnus hemolymph into uninfected lobsters. Uninfected lob- and P. ornatus, but infections were extremely rare sters used for experiments were collected from outside (Bach & Beardsley 1976, Dennis & Munday 1994). At the designated survey sites and were held for up to least 3 helminths use spiny lobsters as intermediate 1wk prior to treatment to insure acclimation and hosts: a microphallid trematode infects the ovaries of absence of overt diseases. During the experiments, adult P. cygnus (Deblock et al. 1990), a tetraphyllidean lobsters were fed shrimp and squid ad libitum every 2 cestode occurs in the foregut of several species of spiny to 3 d and held individually in 38 l aquaria with flow- lobsters from the Great Barrier Reef (J.D.S. unpubl. through ambient seawater. Only juvenile lobsters, 25 data), and a nematode infects the larvae and juveniles to 55 mm carapace length (CL), were used in inocula- of J. edwardsii (Brett cited in Booth 1988). Finally, at tion trials. Infected lobsters were housed separately least 2 egg predatory nemerteans, Carcinonemertes and used as hemolymph donors to inject naïve hosts spp. (Campbell et al. 1989, Shields & Kuris 1990) and (0.1 to 0.2 ml hemolymph/host). Injections were given amphipods, cf. Parapleustes spp. (J.D.S. pers. obs.), in the arthrodial membrane at the juncture of the basis infest the egg clutches of at least 3 species of spiny and ischium of the 5th walking leg. We have main- lobsters. tained the virus for over 2 yr using this method with no Here we report the first naturally occurring pathogenic apparent loss of pathogenicity. virus to be identified from a lobster. In 1999 and 2000, Two inoculation trials consisted of injecting unin- while sampling juvenile spiny lobster populations in the fected lobsters with hemolymph from infected donors. Florida Keys, one of us (D.C.B.) discovered lethargic, In Trial I, 0.1 ml infected hemolymph was inoculated moribund animals whose hemolymph appeared ‘thin’ separately into one of the limb joints of 10 uninfected and ‘milky,’ rather than its normally transparent color, lobsters using a sterile 27 gauge needle. In Trial II, and which did not clot. The hemolymph was negative 0.2 ml infected hemolymph was inoculated separately for Gram-negative bacteria, but the histopathology into 11 uninfected lobsters. In all cases, a 70% ethanol showed nuclear hypertrophy with diffuse Cowdry-type spray was used to sterilize the area around the injec- A viral inclusions in infected hemocytes. In heavily in- tion site. To verify the presence of the virus, hemo- fected individuals, virtually all of the host’s hyalinocytes lymph and other tissues from the donor lobsters were and semigranulocytes were destroyed; granulocytes fixed and processed for histology as described below. were not infected. Our objectives were to identify the For the control group, uninfected hemolymph was col- causative agent, describe the histopathology of the in- lected from healthy lobsters and treated as above for fection, and report initial findings on the prevalence of injection into uninfected lobsters. In both trials, hemo- the disease in the Florida Keys. lymph smears were taken biweekly from challenge and control lobsters. Animals were monitored daily for morbidity and mortality. MATERIALS AND METHODS Dissections were performed on uninfected control animals and animals showing increasing signs of mor- Juvenile spiny lobsters were collected from 14 bidity. For histology, hepatopancreas, heart, gill, mus- (25 m2) sites located throughout western Florida Bay cle, foregut, hindgut, and, in some cases, hemopoietic adjacent to the middle and lower Florida Keys. Each tissues were dissected and fixed in 10% neutral Shields & Behringer: A new pathogenic virus from a lobster 111 buffered Formalin, with the exception of a few animals staining