Susceptibility of the peppermint shrimp Lysmata wurdemanni to the white spot syndrome virus.

ABSTRACT White Spot Syndrome Virus (WSSV) affects the penaeid shrimp culture industry, but the potential impact on the ornamental shrimp industry has not been studied. Therefore, experiments were conducted to determine the susceptibility of the ornamental peppermint shrimp Lysmata wurdemanni to WSSV. Litopenaeus vannamei, a cultured penaeid shrimp was used for comparison. Three experiments (two per os, one injection) were conducted. Adult shrimp were challenged in the first per os experiment, whereas juveniles were challenged in the remaining two experiments, in both per os challenges, shrimp (n = 15, 10) were fed WSSV infected L. vannamei tissue at 10% of their body weight. In the injection challenge, shrimp (n = 10) were injected with a [10.sup.-3] dilution of freshly prepared WSSV filtrate at 20 [micro]L/g of body weight. Shrimp were individually housed, maintained at 25[degrees]C, and checked daily for mortalities. PCR was used to determine whether challenged shrimp were infected with WSSV. In all three experiments, L. wurdemanni appeared more resistant to WSSV than L. vannamei (40%, 0% mortality versus 60%, 100% mortality, respectively, because of WSSV). Results indicate that adult L. wurdemanni appear somewhat susceptible to the virus, whereas the juveniles appear refractory to the virus (40% versus 0% mortality), however as the adults and juveniles were collected from different geographical areas, genetic variation cannot be discounted. Although there are no reports to date of a natural WSSV infection in Lysmata or other ornamental shrimp species, the finding that adult Lysmata are susceptible to WSSV has implications for the ornamental industry.

KEY WORDS: WSSV, peppermint shrimp, Lysmata wurdemanni, Litopenaeus vannamet

INTRODUCTION

White Spot Syndrome Virus (WSSV) is a large, double stranded DNA virus, recently assigned to the family Nimaviridae (Van Hulten et al. 2000). WSSV has seriously affected the food shrimp culture industry (Lightner 1999, Flegel 1997, Rosenberry 1998, Rosenberry 2003). Important cultured penaeid species such as Penaeus monodon, Fenneropenaeus chinensis, Fenneropenaeus indicus, Marsupenaeus japonicus, Litopenaeus stylirostris, and Litopenaeus vannamei have all been found to be susceptible to the virus (Inouye et al. 1994, Lo et al. 1996, Kasornchandra et al. 1998, Lightner et al. 1998, Nunan et al. 1997, Sudha et al. 1998, Wang et al. 1998, Zhan et al. 1998).

Although WSSV has been found to infect over 40 species of crustaceans, mortality varies among susceptible crustaceans (Lo et al. 1996, Flegel 1997, Chang et al. 1998, Supamattaya et al. 1998, Wang et al. 1998, Otta et al. 1999, Maeda et al. 2000, Hossain et al. 2001, Sahul Hameed et al. 2001). In addition to cultured penaeid shrimp, freshwater crayfish, such as Procambarus clarkii and Pacifastacus leniusculus, are adversely affected by WSSV (Chang et al. 1998, Wang et al. 1998, Jiravanichpaisal et al. 2001, Maeda et al. 2000). Freshwater prawn, such as Macrobrachium rosenbergii were found to be somewhat tolerant to the virus, but other species, such as M. idella and M. lamerrae were susceptible to the virus (Peng et al. 1998, Wang et al. 1998, Sahul Hameed et al. 2000). Likewise tolerance among crabs varies by species (Lo et al. 1996, Chang et al. 1998, Kanchanaphum et al. 1998, Supamattaya et al. 1998, Wang et al. 1998, Otta et al. 1999, Chen et al. 2000, Chang et al. 2001, Sahul Hameed et al. 2001). Lobsters are considered to be carriers only (Chang et al. 1998, Wang et al. 1998).

The marine ornamental trade involves a wide variety of crustacean species, both wild and cultured. Ornamental shrimp are popular crustaceans in the aquarium trade and research has been done to establish culture conditions for many species (Lin & Zhang 2001, Simones et al. 2002, Calado et al. 2003). The susceptibility of ornamental shrimp species to WSSV is currently unknown. The possibility of potential transfer of WSSV from susceptible crustaceans to ornamental shrimp and vice versa is a real possibility both in culture systems and the wild. The aim of the present study was to determine the susceptibility of an ornamental shrimp species, Lysmata wurdemanni, to WSSV.

MATERIALS AND METHODS

Experimental Animals

Lysmata wurdemanni used in the first per os challenge experiment (April 2002) were obtained from the company Ocean, Reefs, and Aquariums (ORA, Ft. Pierce, FL). These shrimp were 1 g adults, cultured from broodstock obtained from the Dominican Republic. Lysmata wurdemanni used in the second per os challenge and injection challenge experiments (July 2002) were obtained from Florida Tech (Melbourne, FL). These shrimp were juveniles (0.1-.2 g) cultured from broodstock obtained from north of Tampa Bay, FL. Litopenaeus vannamei used in the first per os challenge experiment were obtained from Harbor Branch Oceanographic Institution, Inc. (HBOI) and were approximately 1 g in weight. Litopenaeus vannamei used in the second per os challenge and injection challenge experiments (July 2002) were 0.1-0.2 g and were obtained from Indian River Aquaculture (Vero Beach, FL). Broodstock from which L. vannamei were obtained had been previously tested for the presence of WSSV, IHHNV, and TSV and found to be free of these viruses.

Viral Filtrate Preparation

WSSV used in the challenge studies was isolated from naturally infected L. vannamei tissue obtained from Ecuador in 2000 that had been stored at -80[degrees]C. This tissue was tested by PCR for the presence of WSSV, IHHNV and TSV prior to use and was found to be positive for WSSV only. Prior to experimental challenges WSSV negative L. vannamei juveniles were fed minced pieces of this frozen tissue. Moribund shrimp were collected, tested for the presence of WSSV and fed to a second group of WSSV negative L. vannamei juveniles. This procedure was repeated and tissue obtained a third time was used for challenge experiments.

The filtrate was prepared by grinding moribund WSSV shrimp with UV-filtered saltwater at 1:10 w/v of tissue to saltwater. The tissue was homogenized for 30 sec and then centrifuged at 5000g for 10 min. The supernatant was removed and centrifuged at 10,000g for 5 min. This supernatant was collected, passed through a 0.2 [micro]m filter, serially diluted with saltwater to [10.sup.-3] and used in injection challenges.

Experimental Setup

Shrimp were housed in individual 250 mL Styrofoam containers containing 100 mL of 1-[micro]m filtered salt well water. Salinity was 27 g/L and water temperature was maintained at 25[degrees]C by placing Styrofoam containers in a temperature-controlled incubator. Twenty percent of the water was exchanged daily. Shrimp were fed once daily with a juvenile 1/16 inch pelleted diet containing 42% protein (Bonney, Laramore & Hopkins, Inc., Ft. Pierce, FL). For per os challenge experiments, shrimp were fed freshly minced WSSV positive abdominal tissue at 10% of their body weight on day zero. Control groups were fed WSSV negative tissue. In the injection challenge experiment shrimp were injected (20 [micro]l/g body weight) with freshly prepared [10.sup.-3] diluted filtrate in the first abdominal segment using a HPLC syringe. Negative controls were injected with filtered physiological saline. Shrimp were checked for mortalities twice each day. Experiments were terminated at either 25 days (experiment 1 and 3) or at 30 days (experiment 2). Because of limited availability, control groups consisting of Lysmata wurdemanni were not used in either the first per os or the injection challenge experiment.

The first per os challenge experiment consisted of two treatment and one negative control groups. The two treatment groups consisted of either 15 challenged 1-g adult L. wurdemanni or 15 challenged l-g juvenile L. vannanwi. The control group consisted of 15 unchallenged 1-g juvenile L. vannamei.

The second per os challenge experiment consisted of two treatment and two control groups. The two treatment groups consisted of either 10 challenged juvenile (0.1-0.2 g) L. wurdemanni or 10 challenged juvenile (0.1-0.2 g) L. vannamei. The two control groups consisted of 10 unchallenged juvenile L. wurdemanni and 10 unchallenged juvenile L. vannamei.

The injection challenge experiment consisted of two treatments and one control group. The two treatment groups consisted of either 10 (0.1-0.2 g) challenged juvenile L. wurdemanni or l0 (0.1-0.2 g) challenged juvenile L. vannanwi. The control group consisted of 10 saline injected juvenile (0.1-0.2 g) L. vannamei.

Mortality data from these experiments were used to calculate an Entrance Point (EP) function (Scarpa & Allen 1992). The time (days) that it took for either a 20% ([EP.sub.20]) or 40% ([EP.sub.40]) mortality to occur was calculated. This was based on the cumulative percentage of shrimp that died.

Dead and survivors were tested for WSSV by PCR. DNA was extracted front the cephalothorax using the Wizard DNA extraction kit from Promega Corp (Madison, WI). The WSSV primer set that was used (sequence proprietary) amplifies a 300 bp region of the WSSV genome and was obtained from DiagXotics, Inc. (Wilton, CT). Twelve [micro]l of a PCR master mix front Promega, containing Mg[Cl.sub.2], nucleotides and Taq polymerase, were added to a 0.5 mL PCR tube along with 1 [micro]l of primers, 1 [micro]l of DNA extracted material and 15 [micro]l of d[H.sub.2]O. Negative controls consisted of all the above with the exclusion of the DNA extracted material. The PCR program used consisted of 35 cycles of 94[degrees]C 30s, 60[degrees]C 30s, 72[degrees]C 45s, followed by 72[degrees]C 10m and a 4[degrees]C hold. Ten [micro]l of amplified material was loaded onto a 2% agarose gel and eletrophorized at 80 V for 90 min using a 1% TBE buffer. Chi-square analysis was used to compare mortality differences between control and treatment groups and between species (P = 95% CI) (Arkin & Colton 1970).

RESULTS

In the initial per os experiment there was no significant difference in mortality between Lysmata adults (40%) and L. vannamei juveniles (60%) that tested positive for WSSV (Fig. 1). However, in the second per os experiment there was a significant difference (P < 0.01) in WSSV induced mortality between Lysmata (0%) and L. vannameijuveniles (100%) (Fig. 1). The injection challenge experiment exhibited a similar significant difference (P < 0.01) to the second per os experiment in WSSV induced mortality between Lysmata (0%) and L. vannamei juveniles (100%) (Fig. 1).

Of the Lysmata that died from experimental infection, only those in the first per os experiment tested positive for WSSV (Table 1). Lysmata challenged in this experiment consisted of adults, whereas those in subsequent experiments were juveniles. All L. vannamei were WSSV positive, whether dead or survivors (Table 1). Only one Lysmata survivor, an adult from the first per os challenge, was positive for WSSV by PCR (Table 1).

Mortalities ([EP.sub.20]) occurred much later in L. wurdemanni (11.3-27 days) than in L. vannamei (1.8-2.3 days) (Table 2). Although an [EP.sub.50] was obtained for L. vannamei it was never reached for L. wurdemanni within the experimental timeframe although an [EP.sub.40] was obtained for adult L. wurdemanni in experiment 1 (Table 2).

[FIGURE 1 OMITTED]

DISCUSSION

There are no published reports available concerning susceptibility of ornamental shrimp species to WSSV, a virus that has a wide host range, including marine shrimp. Results of the present study indicate that the ornamental shrimp species, L. wurdemanni, was susceptible to WSSV, although more tolerant than penaeid shrimp.

Care was taken in the present study to ensure that each shrimp received an equal amount of WSSV infected tissue by housing them individually and feeding tissue or injecting filtrate based on body weight. In most challenge experiments, crustaceans are housed together in tanks containing from 5-200 individuals (Kanchanaphum et al. 1998, Lightner et al. 1998, Supamattaya et al. 1998, Wang et al. 1998, Maeda et al. 2000, Sahul Hameed et al. 2000, Sahul Hameed et al. 2001). The likelihood that some individuals receive a greater viral load than others by ingesting more tissue (per os experiments) or cannibalizing moribund individuals is compounded when animals are housed together.

Even when housed, individually it is possible that viral loads may vary in individual pieces (Dhar et al. 2001, Durand et al. 2003). Per os experiments in this study were conducted on different dates (April and July 2002) and it is possible that the WSSV infected shrimp tissue used in these two challenges may have varied in viral load. Although this may account for the difference in WSSV induced mortality that occurred in L. vannamei in the first and subsequent experiments (60%, 100%, 100%), one would expect to see a corresponding increase in mortality in Lysmata during subsequent experiments, however a decrease occurred instead (40%, 0%, 0%).

Another possible explanation as to why an expected mortality of 100% for L. vannamei was not seen in all experiments may have been because of exposure of experimental shrimp to infected tissue only once, on day zero. Although Wang et al. (1998) fed infected tissue to experimental crustaceans only once, in the majority of published per os challenge experiments crustaceans were fed infected tissue over a period of 3-10 days (Lightner et al. 1998, Maeda et al. 2000, Sahul Hameed et al. 2000, Sahul Hameed et al. 2001). Mortality varied with species. Only Lightner et al. (1998) fed infected tissue to L. vannamei, which showed 100% mortality.

The injection challenge sought to rectify viral load differences that may have occurred between per os experiments by injecting viral filtrate based on body weight. The typical pattern seen in studies comparing oral and injection challenges is that similar mortality is seen between methods (Lightner et al. 1998, Maeda et al. 2000, Sahul Hameed et al.) or a greater percent mortality is seen with injection challenge (Lotz 1997, Supamattaya et al. 1998). Shrimp used were acquired at the same time and from the same source as in the second per os experiment. Previous experiments with L. vannamei had shown that a [10.sup.-6] dilution injected at 20[micro]l/g of body weight caused 100% mortality. Other WSSV injection challenge experiments have used similar dilution factors (Prior et al. 2003). Based on the per os experiments, L. wurdemanni appeared more tolerant to WSSV than L. vannamei. Therefore, a less dilute preparation ([10.sup.-3]) was chosen for this injection experiment. It was expected that less mortality would occur in L. wurdemanni than in L. vannamei. As predicted, 100% mortality occurred in L. vannamei, however no mortality occurred in L. wurdemanni, which was unexpected.

If variation in viral load did not account for the difference in mortality observed among L. wurdemanni in the three experiments then, perhaps the differences seen were caused by the different life stages used in these experiments. All L. wurdemanni that died and tested positive for WSSV by PCR were adults. One Lysmata juvenile died (in the second per os experiment), however it did not test positive for WSSV. One adult Lysmata survivor tested positive for WSSV, whereas none of the surviving Lysmata juveniles did. These results indicate that the adult stage of Lysmata is somewhat susceptible to WSSV infection, whereas the juvenile stage is refractory to WSSV. It is been shown that different life stages of penaeid shrimp have different susceptibility to viral diseases (Lightner et al. 1998, Lightner 1999, Lotz 1997). P. setiferus and P. vannamei postlarvae and juveniles were both susceptible to WSSV and YHV challenge, whereas the juvenile stages of P. aztecus and P. duorarum were much less susceptible to those viruses than the postlarvae stage (Lightner et al. 1998). Juvenile P. vannamei were also shown to be less susceptible to Taura virus than adults (Lotz 1997) although no differences were seen in susceptibility between postlarvae and juveniles (Overstreet et al. 1997).

The role played by inherent genetic variation that exists between shrimp populations must also be considered. All L. vannamei used in these experiments were juveniles of approximately the same size and age, but obtained from two different sources. Less mortality was seen in experiment one (source one) than in experiments two and three (source two). Although mortality differences may be explained by variation in viral load among these experiments they could also be explained by susceptibility among family groups. Most published accounts of experiments with L. vannamei report high mortality rates, approaching 100%. Differences in susceptibility among family groups are known to exist, such that these differences have been the basis for selective breeding of L. vannamei for disease resistance (Fegan & Clifford 2001, Argue et al. 2002). This suggests that genetic variation may play a role in differences seen in WSSV susceptibility between Lysmata adults and juveniles collected from two different sites (Dominican Republic and the gulf coast of Florida). Recent taxonomic evidence suggests that the Lysmata used in these experiments may represent two different species. The adults used are currently described as Lysmata wurdemanni Haiti variety, nov. sp. whereas the juveniles used are currently considered Lysmata wurdemanni Florida Gulf Coast variety, nov. sp. (Rhyne et al. 2004). Species differences in tolerance to WSSV have been described for penaeid shrimp (Lightner et al. 1998, Lightner 1999) as well as for Macrobrachium (Sahul Hameed et al. 2001).

Although a more thorough study of Lysmata wurdemanni needs to be undertaken, for instance a comparison of juvenile and adult stages of both putative species, it appears that Lysmata are more resistant to WSSV than L. vannamei, but that life stage or subspecies variation may exist. Although there are no reports to date of a natural WSSV infection in Lysmata or other ornamental shrimp species, the finding that some varieties of Lysmata at least at the adult stage may be susceptible to WSSV has implications for the ornamental industry.

ACKNOWLEDGMENTS

The author thanks Kevin Gaines of ORA (Ft. Pierce, FL), Dr. Junda Lin and Andy Rhyne of Florida Tech (Melbourne, FL) for providing the Lysmata used; Joe Mountain of HBOI Shrimp (Ft. Pierce, FL) and Donovan Schumann of Indian River Aquaculture (Vero Beach, FL) for providing the L. vannamei used; Harbor Branch Oceanographic Institution for providing the facilities in which to conduct these experiments; and Dr. John Scarpa of Harbor Branch Oceanographic Institution for editing this manuscript. This is HBOI contribution #1667.

LITERATURE CITED

Argue, B. J., S. M. Arce, J. M. Lotz & S. M. Moss. 2002. Selective breeding of Pacific white shrimp (Litopenaeus vannamei) for growth and resistance to Taura Syndrome Virus. Aquaculture 204:447-460.

Arkin, H. & R. Colton. (1970) Statistical Methods, 5th ed. Barnes and Noble Inc. New York, NY.

Calado, R., L. Narciso, S. Morais, A. L. Rhyne & J. Lin. 2003. A rearing system for the culture of ornamental decapod crustacean larvae. Aquaculture 218:329-339.

Chang, P. S., H. C. Chen & Y. C. Wang. 1998. Detection of white spot syndrome associated baculovirus in experimentally infected wild shrimp, crab and lobsters by in situ hybridization. Aquaculture 164:233-242.

Chang, Y. S., S. E. Peng, H. C. Wang, H. C. Hsu, C. H. Ho, C. H. Wang, S. Y. Wang, C. F. Lo & G. H. Kou. 2001. Sequencing and amplified restriction fragment length polymorphism analysis of ribonucleotide reductase large subunit gene of the white spot syndrome virus in blue crab (Callinectes sapidus) from American coast waters. Mar. Biotechnol. 3:163-171.

Chen, L. L., C. F. Lo, Y. L. Chiu, C. F. Chang & G. H. Kou. 2000. Natural and experimental infection of white spot syndrome virus (WSSV) in benthic larvae of mud crab Scylla serrata. Dis. Aquat. Organ. 40:157-161.

Dhar, A. K., M. M. Roux & K. R. Klimpel. 2001. Detection and quantification of infectious hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR green chemistry. J. Clin. Microbiol. 39:2835-2845.

Durand, S. V., R. M. Redman, L. L. Mohney, K. Tank-Nelson, J. R. Bonami & D. V. Lightner. 2003. Qualitative and quantitative studies on the relative virus load of tails and heads of shrimp acutely infected with WSSV. Aquaculture 216:9-18.

Fegan, D.F. & H.C. Clifford. (2001) Health management for viral diseases in shrimp farms. In: C.L. Browdy & D.E. Jory. editors. The new wave, proceedings of the special session on sustainable shrimp farming, Aquaculture 2001. Baton Rouge, Louisiana, USA: The World Aquaculture Society.

Flegel, T. W. 1997. Special Topic review: major viral disease of the black tiger prawn (Penaeus monodon) in Thailand. World J. Microbiol. Biotechnol. 13:433-442.

Hossain, S., A. Chakraborty, B. Joseph, S. K. Otta, I. Karunasagar & I. Karunasagar. 2001. Detection of new hosts for white spot syndrome virus of shrimp using nested polymerase chain reaction. Aquaculture 198:1-11.

Inouye, K., S. Miwa, N. Oseko, H. Nakano, T. Kimura, K. Momoyama & M. Hiraoka. 1994. Mass mortalities of cultured kuruma shrimp Penaeus japonicus in Japan in 1993: Electron microscopic evidence of the causative virus. Fish Pathol. 29:149-158.

Jiravanichpaisal, P., E. Bangyeekhun, K. Soderhall & I. Soderhall. 2001. Experimental infection of white spot syndrome virus in freshwater crayfish Pacfastacus leniusculus. Dis. Aquat. Organ. 47: 151-157.

Kanchanaphum, P., C. Wongteerasupaya, N. Sitidilokratana, V. Boonsaeng, S. Panyim, A. Tassanakajon, B. Withyachumnarnkul & T. W. Flegel. 1998. Experimental transmission of white spot syndrome virus (WSSV) from crabs to shrimp Penaeus monodon. Dis. Aquat. Organ. 34:1-7.

Kasornchandra, J., S. Boonyaratpalin & T. Itami. 1998. Detection of white-spot syndrome in cultured penaeid shrimp in Asia: microscopic observation and polymerase chain reaction. Aquaculture 164:243-251.

Lightner, D. V., K. W. Hasson, B. L. White & R. M. Redman. 1998. Experimental infection of Western hemisphere penaeid shrimp with Asian White Spot Syndrome Virus and Asian Yellow Head Virus. J. Aquat. Anita. Health 10:271-281.

Lightner, D. V. 1999. The penaeid shrimp viruses TSV, IHHNV, WSSV, and YHV Current status in the Americas, available diagnostic methods and management strategies. J. Appl. Aquacult. 9:27-52.

Lin, J. & D. Zhang. 2001. Effect of broodstock diet on reproductive performance of the peppermint shrimp, Lysmata wurdemanni. J. Shellfish Res. 20:361-363.

Lo, C. F., C. H. Ho, S. E. Peng, C. H. Chen, H. C. Hsu, Y. L. Chiu, C. F. Chang, K. F. Liu, M. S. Su, C. H. Wang & G. H. Kou. 1996. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis. Aquat. Organ. 27:215-225.

Lotz, J. M. 1997. Effect of host size on virulence of Taura Virus to the marine shrimp Penaeus vannamei (Crustacea: Penaeidae). Dis. Aquat. Organ. 30:45-51.

Maeda, M., T. Itami, E. Mizuki, R. Tanaka, Y. Yoshizu, K. Doi, C. Yasunaga-Aoki, Y. Takahashi & T. Kawarabata. 2000. Red swamp crawfish (Procambarus clarkii): an alternative experimental host in the study of white spot syndrome virus. Acta Virol. 44:371 374.

Nunan, L. M., B. T. Poulos & D. V. Lightner. 1997. The detection of white spot syndrome virus (WSSV) and yellow head virus (YHV) in imported commodity shrimp. Aquaculture 160:19-30.

Otta, S. K., G. Shubha, B. Joseph, A. Chakraborty, I. Karunasagar & I. Karunasagar. 1999. Polymerase chain reaction (PCR) detection of white spot syndrome virus (WSSV) in cultured and wild crustaceans in India. Dis. Aquat. Organ. 38:67-70.

Overstreet, R. M., D. V. Lightner, K. W. Hasson, S. McIlwain & J. M. Lotz. 1997. Susceptibility To TSV of some penaeid shrimps native to the Gulf of Mexico and Southeastern US. J. Invertebr. Pathol. 69:165-176.

Peng, S. E., C. F. Lo, C. H. Ho, C. F. Chang & G. H. Kou. 1998. Detection of white spot baculovirus (WSBV) in giant freshwater prawn, Macrobrachium rosenbergii, using polymerase chain reaction. Aquaculture 164:253-262.

Prior, S., C. L. Browdy, E. F. Sheppard, R. Laramore & P. Parnell. 2003. Controlled bioassay Systems for determination of lethal infective doses of tissue homogenates containing Taura syndrome or white spot syndrome virus. Dis. Aquat. Organ. 54:89-96.

Rhyne, A. L., J. Lin & K. J. Deal. 2004. Biological control of aquarium pest anemone Aiptasia pallida Verrill by peppermint shrimp Lysmata Risso. J. Shellfish Res. 28:227-229.

Rosenberry, B. 1998. World Shrimp Farming. Shrimp News International 11:328.

Rosenberry, B. 2003. World Shrimp Farming. Shrimp News International 16:276.

Sahul Hameed, A. S., M. X. Charles & M. Anilkumar. 2000. Tolerance of Macrobrachium rosenbergii to white spot syndrome virus. Aquaculture 183:207-213.

Sahul Hameed, A. S., K. Yoganandhan, S. Sathish, M. Rasheed, V. Murugan & K. Jayaraman. 2001. White spot syndrome virus (WSSV) in two species of freshwater crabs (Paratelphusa hydrodomous and P. pulvinata). Aquaculture 201 : 179-186.

Scarpa, J. & S. Allen. 1992. Comparative kinetics of meiosis in hybrid crosses of Pacific oyster Crassostrea gigas and Suminoe oyster C. rivularis with the American oyster C. virginica. J. Exp. Zool. 263:316-322.

Simones, F., F. Ribeiro & D. A. Jones. 2002. Feeding early larval stages of fire shrimp, Lysmata debelius (Caridea Hippolytidae). Aquaculture International 10:349-360.

Sudha, P. M., C. V. Mohan, K. M. Shankar & A. Hegde. 1998. Relationship between White spot syndrome virus infection and clinical manifestation in Indian cultured penaeid shrimp. Aquaculture 167:95-101.

Supamattaya, K., R. W. Hoffmann, S. Boonyaratpalin & P. Kanchanaphum. 1998. Experimental transmission of white spot syndrome virus (WSSV) from black tiger shrimp Penaeus monodon to the sand crab Portunus pelagieus, mud crab Scylla serrata and krill Acetes sp. Dis. Aquat. Organ. 32:79-85.

Van Hulten, M. C. W., M. F. Tsai, C. A. Schipper, C. F. Lo, G. H. Kou & J. M. Vlak. 2000. Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions. J. Gen. Virol. 81:307 316.

Wang, Y. C., C. F. Lo, P. S. Chang & G. H. Kou. 1998. Experimental infection of white spot baculovirus in some cultured and wild decapods in Taiwan. Aquaculture 164:221-231.

Zhan, W. B., Y. H. Wang, J. L. Fryer, K. K. Yu, H. Fukuda & Q. X. Meng. 1998. White Spot syndrome virus infection of cultured shrimp in China. J. Aquat. Anim. Health 10:405-410.

SUSAN E. LARAMORE

Harbor Branch Oceanographic Institution, Inc., 5600 U.S. 1 North, Fort Pierce, FL 34946

E-mail: [e-mail]

TABLE 1.
Number of challenged shrimp in each experiment determined
by PCR to be WSSV positive (n = 15, 10, 10).

Challenge Experiment   # Dead   # PCR +   # Survivors   # PCR +

per os Exp 1
  L. wurdemanni          7         6           8           1
  L. vannamei            9         9           6           6
per os Exp 2
  L. wurdemanni          2         0           8           0
  L. vannamei           10        10           0          NA
Injection Exp 3
  L. wurdemanni          0        NA          10           0
  L. vannamei           10        10           0          NA

* NA = not applicable, no dead or surviving shrimp to test by PCR.

TABLE 2.
Entrance Point Functions ([EP.sub.20], [EP.sub.40]) calculated
for each challenge experiment, denoting the length of time
of percent mortality.

Challenge Experiment   [EP.sub.20]   [EP.sub.40]

per os Exp 1
  L. wurdemanni            11.3          23
  L. vannamei               2.3           4

per os Exp 2
  L. wurdemanni            27             NA
  L. vannamei               2             3.8

Injection Exp 3
  L. wardemanni             NA            NA
  L. vannamei               1.8           3.5

* NA = not applicable, no death occurred.