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Inhibitory Effects of Ovoglobulins on Bacillary Necrosis in Larvae of the Pacific Oyster, Crassostrea gigas
Journal of Invertebrate Pathology 75, 212–217 (2000) doi:10.1006/jipa.1999.4922, available online at http://www.idealibrary.com on
Inhibitory Effects of Ovoglobulins on Bacillary Necrosis in Larvae of the Pacific Oyster, Crassostrea gigas Keisuke G. Takahashi, Akifumi Nakamura, and Katsuyoshi Mori Laboratory of Aquacultural Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori Amamiyamachi, Aoba-ku, Sendai 981-8555, Miyagi, Japan Received May 26, 1999; accepted December 14, 1999
In order to develop an alternative method to antibiotics for preventing bacillary necrosis in bivalve mollusc larvae, we examined the effects of ovoglobulins (proteins derived from the whites of hens’ eggs) on the survival of larvae of the Pacific oyster Crassostrea gigas. The pathogenic Vibrio tubiashii (ATCC 19106) was used to infect larvae of the Pacific oyster. V. tubiashii showed strong pathogenicity to oyster larvae, causing 100% mortality after experimental exposure for 24 h at a concentration of 10 5 cfu (colony-forming units)/ml. In contrast, the addition of ovoglobulins at a concentration of 10 g/ml to larval oysters, challenged with V. tubiashii at 10 5 cfu/ml, led to a marked increase in larval survival of 96.5% at 24 h after infection. The V. tubiashii culture supernatant was also shown to be pathogenic to larval oysters; however, its pathogenicity was completely inhibited by the addition of 10 g/ml of ovoglobulins. Larval oysters infected by V. tubiashii showed typical symptoms of bacillary necrosis including anomalous swimming and detachment of cilia and/or vela. In contrast, live larvae were actively motile, and their cilia and vela were not necrotized in the ovoglobulins-added group. The addition of ovoglobulins clearly suppressed the growth of V. tubiashii in gelatin–sea water broth, but the number of viable V. tubiashii 24 h after incubation did not decrease to the initial dose level. Findings obtained in this study indicate that ovoglobulins almost completely protect larval oysters from V. tubiashii infection by nonbactericidally inhibiting the growth of V. tubiashii without affecting survival of the oysters. ©
2000 Academic Press
Key Words: Crassostrea gigas; Vibrio tubiashii; ovoglobulins; bacillary necrosis; larvae; bivalve molluscs.
INTRODUCTION
Many species of bivalve molluscs are cultured widely around the world due to their commercial importance. Hatchery-rearing systems for the production of bivalve seeds have been established in the United States, Eu0022-2011/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
rope, and Japan (Korringa, 1952; Imai and Sakai, 1961; Loosanoff and Davis, 1963). At present, many hatcheries are being established worldwide for the production of various bivalve molluscs. As a result of the increase in intensive culture of bivalve larvae, mass larval mortalities induced by bacterial infection have been reported (Tubiash et al., 1965; DiSalvo et al., 1978; Elston et al., 1981; Garland et al., 1983; Lodeiros et al., 1987; Nicolas et al., 1992, 1996; Elston, 1993). In many cases, marine vibrios have been found to be the causative agents (Sindermann, 1988; Elston, 1993). Larval vibriosis or bacillary necrosis results in mortalities often exceeding 90% within 24 h of infection, and production in hatcheries has been frequently limited by outbreaks of this disease in many marine bivalve species (Sindermann, 1988). Therefore, bacillary necrosis can be considered the most serious disease of hatchery-reared larvae. Recently in Japan, mass mortalities caused by bacillary necrosis outbreaks have been reported from hatcheries of the cockle Fulvia mutica (Fujiwara et al., 1993) and the Pacific oyster Crassostrea gigas (Sugumar et al., 1998a). The use of antibiotics such as chloramphenicol (Tubiash et al., 1965; Jeffries, 1982; Lodeiros et al., 1987) and streptomycin (Tubiash et al., 1965; Sugumar et al., 1998a) has been shown to be a very effective method for controlling bacillary necrosis and improving the survival of bivalve larvae. However, antibiotic treatment as a preventive procedure is not recommended because it can affect bacterial flora in the culture sea water and can also result in the selection of resistant pathogenic strains (Nicolas et al., 1996). Recent attempts have been made to apply biocontrol (biological control) techniques to bivalve larvae rearing systems as a method for preventing infectious larval diseases. One such method uses live bacteria to suppress the growth of pathogenic bacteria and to control the microflora in culture sea water (Nogami and Maeda, 1992). In the case of rearing bivalve larvae, this biocontrol technique was experimentally adapted
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for scallop Argopecten purpuratus culture (Riquelme et al., 1997) and for C. gigas cultures (Gibson et al., 1998; Nakamura et al., 1999). However, the efficacy of this biocontrol technique in bivalve larvae hatchery-rearing systems remains to be clarified. We have been carrying out research on effective techniques against bacillary necrosis without the use of antibiotics or bactericidal drugs since 1996. In this study, we examined the effect of ovoglobulins (ovoGs), which are proteins derived from the whites of hens’ eggs, on bacillary necrosis in C. gigas larvae infected experimentally by pathogenic Vibrio tubiashii. We determined the survival rate of larval oysters inoculated with V. tubiashii in the presence or absence of ovoGs. We also examined the effect of ovoGs on the growth of V. tubiashii in gelatin–sea water broth. To our knowledge, this paper is the first report concerning the inhibitory effect of nonbactericidal proteins such as ovoGs on bacillary necrosis in larval oysters. MATERIALS AND METHODS
Collection and Rearing of Oyster Larvae Cultivation of eggs and larvae of the Pacific oyster, C. gigas, under small-scale laboratory conditions was performed according to Loosanoff and Davis (1963). Ripe eggs and sperm were obtained by stripping mature adult oysters harvested from hanging cultures in Onagawa Bay, Miyagi Prefecture, Japan. Fertilized eggs were gently rinsed in sand-filtered sea water and then placed in 100-L culture aquaria at densities of 20 –30 eggs/ml of sand-filtered sea water at 23°C. Two days after hatching, the fertilized eggs developed into straight-hinge larvae of about 90 m in length. Larvae were collected from the culture aquaria with 50-m mesh sieves and gently transferred to 10-L aquaria at a density of 3 individuals/ml of sand-filtered sea water at 23°C. Effects of Ovoglobulins on Survival of Larval Oysters Challenged with V. tubiashii Two-day-old larval oysters were placed in 3-L flasks filled with 0.22 m filtered sea water (sterile sea water) at 23°C at a density of 1 larva/ml. Virulence assays were performed to determine the pathogenicity of a V. tubiashii strain isolated as a strong pathogen of bivalve molluscs (Tubiash et al., 1970). The strain tested, V. tubiashii strain ATCC 19106, was obtained from the American Type Culture Collection (Rockville, MD). This strain was cultured in marine broth 2216 (Difco, Detroit, MI) at room temperature for 24 h and was harvested by centrifugation at 2450g for 20 min. The organisms were washed twice with sterile sea water and resuspended in sterile sea water containing 0.3% glucose. Three milliliters of the
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bacterial suspension was added to larval oysters in 3-L flasks. The final concentration of V. tubiashii used was approximately 10 5 cfu (colony-forming units)/ml. To examine the inhibitory effects of ovoglobulins on vibrio infection of oyster larvae, we inoculated 3 ml of V. tubiashii suspension into 3 L of sterile sea water containing larvae and ovoglobulins (Sigma Chemical Co., St. Louis, MO) at final concentrations of 1.0, 3.0, 5.0, 10.0, and 20.0 g/ml. Unexposed controls were subjected to the same procedure as exposed larvae but without the inoculation with V. tubiashii and the addition of ovoGs. The ovoGs used in this study consisted of two globulin fractions which were purified from whites of hens’ eggs according to Rhodes et al. (1958). Therefore, the ovoGs products did not contain any detectable amounts of ovomucoid, ovalbumin, conalbumin, and lysozyme (⬎99% of total globulins; refer to the certificate of product analysis from Sigma Chemical Co.). These assays were performed in duplicate at 23°C. Larval survival was recorded at intervals of 3 h for 24 h after exposure. The number of live and dead larvae from each flask was counted under light microscopy, until a total of 300 larvae was reached. Moribund larvae were counted as dead. Effects of OvoGs on Survival of Larval Oysters Exposed to Cell-Free Supernatant of V. tubiashii Culture Cultures of V. tubiashii were centrifuged at 3500g for 20 min. The supernatant was collected and filtered through 0.22-m pore-size membranes (Millipore Co., Bedford, MA). To examine the effects of ovoGs on the survival of larvae exposed to bacterial supernatant, 100 ml of filtered supernatant was added to each flask containing larval oysters in 900 ml of sterile sea water. OvoGs were then added at final concentrations of 1 and 10 g/ml. After inoculation with the supernatant, each flask containing larval oysters was maintained at 23°C for 48 h, and larval survival was determined at intervals of 6 h. Unexposed larvae were also reared as controls in 100 ml of marine broth 2216 and 900 ml of sterile sea water without ovoGs. Effects of ovoGs on Growth of V. tubiashii in Culture Medium V. tubiashii was cultured overnight in marine broth 2216 at 26°C and was harvested by centrifugation at 2450g for 20 min. Bacterial cells were washed with 10 mM phosphate-buffered 0.45 M saline (marine PBS, pH 7.6) and resuspended in marine PBS at a concentration of 10 5 cfu/ml. This bacterial suspension (0.5 ml) was inoculated into 4.5 ml of gelatin– glucose–marine medium (GGMM, 5.0 g gelatin dialyzed overnight against sterile sea water and 3.0 g glucose in 1 L sterile sea water, pH 7.4) containing ovoGs at final concentra-
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tions of 0, 100, and 200 g/ml. The cultures were incubated at 26°C for 24 h. After incubation, the number of viable V. tubiashii cells was determined by counting colonies formed on plates of marine agar 2216 (Difco). Statistical Analysis of Data To test for differences in the mean survival rate of larval oysters at various concentrations of ovoGs, we subjected the data to one-way analysis of variance (ANOVA) and Duncan’s multiple range test (Duncan’s test). Differences in the mean number of viable V. tubiashii at different concentrations of ovoGs were also assessed using one-way ANOVA and Duncan’s test. In all cases, the level for significant differences was set at P ⬍ 0.01. RESULTS
Effects of OvoGs on Survival of Larval Oysters Challenged with V. tubiashii Larval oysters started to die 12 h after challenge with V. tubiashii, and the survival rate markedly decreased 15 h after challenge, reaching 0% survival at 21 h (Fig. 1). In contrast, we found a remarkable inhibitory activity of ovoGs against larval infection by V. tubiashii. The survival rate in the ovoGs-added group was 96.5% 24 h after challenge with V. tubiashii. This value was not different from the larval survival in the control group (97.1% at 24 h). Under microscopic observation, V. tubiashii induced both loss of larval swimming (larvae settled on the bottom) and anoma-
FIG. 2. Percentage of larval survival challenged with V. tubiashii in flasks containing different concentrations of ovoGs. V. tubiashii was inoculated into each flask at a concentration of 10 5 cfu/ml. The data represent the mean percentage of larval survival after 24 h of exposure to V. tubiashii. The vertical lines represent standard errors of six separate experiments. Different lowercase letters denote significant differences (P ⬍ 0.01, Duncan’s multiple range test).
lies in the function and structure of the velum in the early phase of infection (9 h after challenge). Thereafter, the velum and the internal organs became severely necrotized in the acute phase of infection. In contrast, no anomalies were observed in either the motility or the morphology of larval oysters in groups with only ovoGs added. Suppression by ovoGs of larval infection by V. tubiashii exhibited concentration-dependent effects in the range of 1.0 to 5.0 g/ml of ovoGs (Fig. 2). Namely, survival of larval oysters treated with ovoGs increased significantly at higher concentrations of ovoGs. The percentage of larval survival at 5.0 g/ml of ovoGs was 96.5 ⫾ 1.3% and showed no significant difference between larval survival at ovoGs concentrations of 10.0 (97.8 ⫾ 0.6%) or 20.0 (97.4 ⫾ 0.9%) g/ml. Survival rates in all ovoGs-added groups were significantly higher than those of the control group (0%). Effects of OvoGs on Survival of Larval Oysters Exposed to Cell-Free Supernatant of V. tubiashii Culture
FIG. 1. Changes in survival rate of C. gigas larvae experimentally challenged with V. tubiashii, and the effect of ovoglobulins (ovoGs) in suppressing the V. tubiashii infection. The percentage of larval survival is plotted under conditions of no challenge with V. tubiashii and no addition of ovoGs (control, 䡬); challenged with V. tubiashii at a concentration of 10 5 cfu/ml (䡺); challenged with V. tubiashii at 10 5 cfu/ml in the presence of ovoGs at a concentration of 10.0 g/ml (f).
The culture supernatant of V. tubiashii induces mortality of larval oysters; however, its toxicity was inhibited by the addition of ovoGs in a dose-dependent manner (Fig. 3). With exposure to 10% culture supernatant, the survival rate of larval oysters markedly decreased 18 h after addition and reached 34.3% survival at 48 h. The toxicity of the culture supernatant was completely inhibited by the addition of ovoGs at a concentration of 10.0 g/ml, and the survival rate in the ovoGs-added
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FIG. 3. Changes in survival rate of larval oysters experimentally exposed to culture supernatant of V. tubiashii, and suppressive effects of ovoGs on toxicity of the supernatant to larval oysters. The percentage of larval survival is plotted under conditions of no exposure to culture filtrate of V. tubiashii and no addition of ovoGs (control, 䡬); exposed to 10% (volume per volume) of culture supernatant of V. tubiashii (䡺); exposed to 10% of culture supernatant of V. tubiashii in the presence ovoGs at concentrations of 1.0 g/ml (‘) and 10.0 g/ml (f).
group remained high for 48 h. Under microscopic observation, the vela of supernatant-exposed larval oysters 48 h after challenge were grossly deformed, and the internal organs of 60% or more of the larvae were necrotized. These symptoms of infection were qualitatively similar to those of larval oysters infected with V. tubiashii cells. Effects of OvoGs on Growth of V. tubiashii in Culture Medium OvoGs partially suppressed the growth of V. tubiashii in GGMM (Table 1). After 24 h of incubation, the number of V. tubiashii in the group with 200 g ovoGs/ml reached 1.10 ⫻ 10 8 cfu/ml, which was significantly lower than the value of 4.65 ⫻ 10 8 cfu/ml in the group without ovoGs. No significant difference was found between the number of viable V. tubiashii treated with 100 and 200 g ovoGs/ml.
effects to antibiotics in the protection of larval oysters against bacillary necrosis and in their subsequent survival. However, ovoGs are simply proteins derived from the whites of hens’ eggs and are not antibiotics or bactericidal substances. This is the first report showing that ovoglobulin proteins are capable of preventing larval infection by pathogenic Vibrio species. V. tubiashii showed very strong pathogenicity to oyster larvae and induced 100% mortality of oyster larvae by exposure at a concentration of 10 5 cfu/ml (Figs. 1 and 2). Infected larvae also showed typical symptoms of bacillary necrosis including anomalous swimming and detachment of cilia and/or vela. In the acute phase of infection, moreover, V. tubiashii could be seen swarming and invading the inside of moribund larvae and subsequently proliferated within the larvae. These observations are very similar to those reported by Tubiash et al. (1965), Brown and Losee (1978), Elston and Leibovitz (1980), and Jeffries (1982). However, the addition of ovoGs at a concentration of 10 g/ml led to an increase in the survival rate of larval oysters inoculated with V. tubiashii to 96.5%, 24 h after challenge (Fig. 1). In the ovoGs-added groups, no observations were made of any massive bacterial invasion and proliferation within or around the larvae. In addition, no changes in the motility and the morphology of larval oysters were observed in the ovoGs-added groups. These results indicate that ovoGs almost completely protect larval oysters from V. tubiashii infection and that ovoGs do not affect the survival of larval oysters. Sugumar et al. (1998b) reported that both extracellular products and intracellular components of V. splendidus biovar II were lethal to larvae of C. gigas. Lodeiros et al. (1987) and Riquelme et al. (1995) also indicated that extracellular products or exotoxins produced by invading vibrios were pathogenic to larvae of Ostrea edulis and A. purpuratus, respectively. In other studies concerning the details of pathogenic factors of the culture supernatants of Vibrio sp. NCMB 1338 and
TABLE 1 Inhibitory Effects of OvoGs on Growth of V. tubiashii Viable V. tubiashii
DISCUSSION
In this study, we have demonstrated that ovoGs possess a remarkable inhibitory effect on bacillary necrosis in larval oysters infected with V. tubiashii. Use of antibiotics such as chloramphenicol and streptomycin for improving the survival of larvae of bivalve molluscs has been reported by previous investigators (Tubiash et al., 1965; Brown and Losee, 1978; DiSalvo et al., 1978; Jeffries, 1982; Lodeiros et al., 1987; Sugumar et al., 1998a). In this study, ovoGs showed almost equal
OvoGs (g/ml)
Initial number (10 4 cfu/ml)
Number grown after 24 h (10 8 cfu/ml)
0 100 200
2.45 ⫾ 0.05 2.48 ⫾ 0.06 2.45 ⫾ 0.04
4.65 ⫾ 0.07 a 1.26 ⫾ 0.08 b 1.10 ⫾ 0.12 b
Note. V. tubiashii was cultured in gelatin– glucose–marine medium at 26°C. Each number of viable V. tubiashii represents the mean ⫾ standard error of five separate experiments performed in triplicate. Different lowercase letters denote significant differences (P ⬍ 0.01, Duncan’s multiple range test).
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V. alginolyticus NCMB 1339, proteinases (proteases), a ciliostatic toxin, and a heat-labile protein (which was designated as a spat toxin) were suggested to be involved in the pathogenicity of O. edulis and C. gigas spats (Nottage and Birkbeck, 1986, 1987a). The V. tubiashii culture supernatant, containing extracellular products, was also shown to be pathogenic to larval oysters; however, its pathogenicity was completely inhibited by the addition of ovoGs (Fig. 3). Moreover, the addition of ovoGs clearly suppressed the growth of V. tubiashii, although the number of viable V. tubiashii after 24 h of incubation did not decrease to the initial dose level (Table 1). These data indicate that V. tubiashii could not grow effectively because of an inability to utilize nitrogen and carbon sources from gelatin when potent and broad-spectrum protease inhibitors are present. Although the ovoGs used in this study are apparently separated in a high purity of ⬎99% of total globulins (data from Sigma Chemical Co.), the ovoGs are considered to consist of eight or nine proteins including ovomicroglobulin, ovomacroglobulin, and ovoinhibitor as strong inhibitors of bacterial proteases (Takahashi and Mori, unpublished data). These results also suggest that the inhibition of extracellular protease activity by ovoGs in the vibrio infection may suppress not only the pathogenic mechanism of proteases but also bacterial growth. However, biological activities of these individual proteins will need to be further studied. Nottage and Birkbeck (1987b) indicated that anti-V. alginolyticus protease antiserum reduced the toxicity of purified V. alginolyticus protease to O. edulis larvae, whereas it had no effect on the toxicity of a crude culture supernatant of V. alginolyticus. As described above, however, ovoGs had a complete inhibitory effect on bacillary necrosis in larval oysters exposed to the culture supernatant of V. tubiashii (Fig. 3). Therefore, the data indicate that the activity of some other toxic factor(s) as well as that of proteases could be inhibited by the administration of ovoGs. However, there is not enough evidence to clarify the mechanism by which ovoGs suppress pathogenic Vibrio strains. Nicolas et al. (1996) indicated that alternative methods to the use of antibiotics must be developed for the prevention of bacillary necrosis. Although the data described above were obtained only under experimental conditions, we consider that the addition of ovoGs has potential as a method for controlling bacillary necrosis in larval oysters. We will need to address many additional issues concerning the applicability of the method to large-scale culture and the mechanism of inhibition of bacillary necrosis. ACKNOWLEDGMENTS We thank the staff of the Education and Research Center of Marine Bioresources at Onagawa, Tohoku University, for their help in
our experiments. We are also grateful to Dr. Harriet Baillie for thorough reading and correction the manuscript. This study was partly supported by a Grant-in-Aid for Encouragement of Young Scientists (11760130) from the Ministry of Education, Science, Sports, and Culture, Japanese Government. REFERENCES Brown, C., and Losee, E. 1978. Observations on natural and induced epizootics of vibriosis in Crassostrea virginica larvae. J. Invertebr. Pathol. 31, 41– 47. DiSalvo, L. H., Blecka, J., and Zebal, R. 1978. Vibrio anguillarum and larval mortality in a California coastal shellfish hatchery. Appl. Environ. Microbiol. 35, 219 –221. Elston, R. A., and Leibovitz, L. 1980. Pathogenesis of experimental vibriosis in larval American oysters, Crassostrea virginica. Can. J. Fish. Aquat. Sci. 37, 964 –978. Elston, R. A., Leibovitz, L., Relyea, D., and Zatila, J. 1981. Diagnosis of vibriosis in a commercial oyster hatchery epizootic: Diagnostic tools and management features. Aquaculture 24, 53– 62. Elston, R. A. 1993. Infectious diseases of the Pacific oyster, Crassostrea gigas. Annu. Rev. Fish Dis. 3, 259 –276. Fujiwara, M., Uyeno, Y., and Iwao, A. 1993. A Vibrio sp. associated with mortalities in cockle larvae Fulvia mutica (Mollusca: Cardiidae). Fish Pathol. 28, 83– 89. (in Japanese with English summary) Garland, C. D., Nash, G. V., Summer, C. E., and McMeekin, T. A. 1983. Bacterial pathogens of oyster larvae (Crassostrea gigas) in a Tasmanian hatchery. Aust. J. Mar. Freshw. Res. 34, 483– 487. Gibson, L. F., Woodworth, J., and George, A. M. 1998. Probiotic activity of Aeromonas media on the Pacific oyster, Crassostrea gigas, when challenged with Vibrio tubiashii. Aquaculture 169, 111–120. Imai, T., and Sakai, S. 1961. Study of breeding Japanese oyster, Crassostrea gigas. Tohoku J. Agr. Res. 12, 125–163. Jeffries, V. E. 1982. Three Vibrio strains pathogenic to larvae of Crassostrea gigas and Ostrea edulis. Aquaculture 29, 201–226. Korringa, P. 1952. Recent advances in oyster biology. Q. Rev. Biol. 27, 339 –365. Lodeiros, C., Bolinches, J., Dopazo, C. P., and Toranzo, A. E. 1987. Bacillary necrosis in hatcheries of Ostrea edulis in Spain. Aquaculture 65, 15–29. Loosanoff, V. L., and Davis, H. C. 1963. Rearing of bivalve mollusks. In “Advances in Marine Biology” (F. S. Russell, Ed.), Vol. 1, pp. 1–136. Academic Press, London. Nakamura, A., Takahashi, K. G., and Mori, K. 1999. Vibriostatic bacteria isolated from rearing seawater of oyster brood stock: Potentiality as biocontrol agents for vibriosis in oyster larvae. Fish Pathol. 34, 139 –144. Nicolas, J. L., Ansquer, D., and Cochard, J. C. 1992. Isolation and characterization of a pathogenic bacterium specific to Manila clam Tapes philippinarum larvae. Dis. Aquat. Org. 14, 153–159. Nicolas, J. L., Corre, S., Gauthier, G., Robert, R., and Ansquer, D. 1996. Bacterial problems associated with scallop Pecten maximus larval culture. Dis. Aquat. Org. 27, 67–76. Nogami, K., and Maeda, M. 1992. Bacteria as biocontrol agents for rearing larvae of the crab Portunus trituberculatus. Can. J. Fish Aquat. Sci. 49, 2373–2376. Nottage, A. S., and Birkbeck, T. H. 1986. Toxicity to marine bivalves of culture supernatant fluids of the bivalve-pathogenic Vibrio stain NCMB 1338 and other marine vibrios. J. Fish Dis. 9, 249 –256. Nottage, A. S., and Birkbeck, T. H. 1987a. Purification of a proteinase produced by the bivalve pathogen Vibrio alginolyticus NCMB 1339. J. Fish Dis. 10, 211–220. Nottage, A. S., and Birkbeck, T. H. 1987b. Production of proteinase during experimental infection of Ostrea edulis L. larvae with
EFFECTS OF OVOGLOBULINS ON BACILLARY NECROSIS Vibrio alginolyticus NCMB 1339 and the antigenic relationship between proteinases produced by marine vibrios pathogenic for fish and shellfish. J. Fish Dis. 10, 265–273. Rhodes, M. B., Azari, P. R., and Feeney, R. E. 1958. Analysis, fractionation, and purification of egg white proteins with cellulosecation exchanger. J. Biol. Chem. 230, 399 – 408. Riquelme, C., Hayashida, G., Toranzo, A. E., Vilches, J., and Chavez, P. 1995. Pathogenicity studies on a Vibrio anguillarum-related (VAR) strain causing an epizootic in Argopecten purpuratus larvae cultured in Chile. Dis. Aquat. Org. 22, 135–141. Riquelme, C., Araya, R., Vergara, N., Rojas, A., Guaita, M., and Candia, M. 1997. Potential probiotic strains in the culture of the Chilean scallop Argopecten purpiratus (Lamarck, 1819). Aquaculture 154, 17–26. Sindermann, C. J. 1988. Vibriosis of larval oysters. In “Disease Diagnosis and Control in North American Aquaculture” (C. J.
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Sindermann and D. V. Lightner, Ed.), pp. 271–274. Elsevier, Amsterdam. Sugumar, G., Nakai, T., Hirata, Y., Matsubara, D., and Muroga, K. 1998a. Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae. Dis. Aquat. Org. 33, 111–118. Sugumar, G., Nakai, T., Hirata, Y., Matsubara, D., and Muroga, K. 1998b. Pathogenicity of Vibrio splendidus biovar II, the causative bacterium of bacillary necrosis of Japanese oyster larvae. Fish Pathol. 33, 79 – 84. Tubiash, H. S., Chanley, P. E., and Leifson, E. 1965. Bacillary necrosis, a disease of larval and juvenile bivalve mollusks. I. Etiology and epizootiology. J. Bacteriol. 90, 1036 –1044. Tubiash, H. S., Colwell, R. R., and Sakazaki, R. 1970. Marine vibrios associated with bacillary necrosis, a disease of larval and juvenile bivalve mollusks. J. Bacteriol. 103, 271–272.
Inhibitory Effects of Ovoglobulins on Bacillary Necrosis in Larvae of the Pacific Oyster, Crassostrea gigas Keisuke G. Takahashi, Akifumi Nakamura, and Katsuyoshi Mori Laboratory of Aquacultural Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori Amamiyamachi, Aoba-ku, Sendai 981-8555, Miyagi, Japan Received May 26, 1999; accepted December 14, 1999
In order to develop an alternative method to antibiotics for preventing bacillary necrosis in bivalve mollusc larvae, we examined the effects of ovoglobulins (proteins derived from the whites of hens’ eggs) on the survival of larvae of the Pacific oyster Crassostrea gigas. The pathogenic Vibrio tubiashii (ATCC 19106) was used to infect larvae of the Pacific oyster. V. tubiashii showed strong pathogenicity to oyster larvae, causing 100% mortality after experimental exposure for 24 h at a concentration of 10 5 cfu (colony-forming units)/ml. In contrast, the addition of ovoglobulins at a concentration of 10 g/ml to larval oysters, challenged with V. tubiashii at 10 5 cfu/ml, led to a marked increase in larval survival of 96.5% at 24 h after infection. The V. tubiashii culture supernatant was also shown to be pathogenic to larval oysters; however, its pathogenicity was completely inhibited by the addition of 10 g/ml of ovoglobulins. Larval oysters infected by V. tubiashii showed typical symptoms of bacillary necrosis including anomalous swimming and detachment of cilia and/or vela. In contrast, live larvae were actively motile, and their cilia and vela were not necrotized in the ovoglobulins-added group. The addition of ovoglobulins clearly suppressed the growth of V. tubiashii in gelatin–sea water broth, but the number of viable V. tubiashii 24 h after incubation did not decrease to the initial dose level. Findings obtained in this study indicate that ovoglobulins almost completely protect larval oysters from V. tubiashii infection by nonbactericidally inhibiting the growth of V. tubiashii without affecting survival of the oysters. ©
2000 Academic Press
Key Words: Crassostrea gigas; Vibrio tubiashii; ovoglobulins; bacillary necrosis; larvae; bivalve molluscs.
INTRODUCTION
Many species of bivalve molluscs are cultured widely around the world due to their commercial importance. Hatchery-rearing systems for the production of bivalve seeds have been established in the United States, Eu0022-2011/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
rope, and Japan (Korringa, 1952; Imai and Sakai, 1961; Loosanoff and Davis, 1963). At present, many hatcheries are being established worldwide for the production of various bivalve molluscs. As a result of the increase in intensive culture of bivalve larvae, mass larval mortalities induced by bacterial infection have been reported (Tubiash et al., 1965; DiSalvo et al., 1978; Elston et al., 1981; Garland et al., 1983; Lodeiros et al., 1987; Nicolas et al., 1992, 1996; Elston, 1993). In many cases, marine vibrios have been found to be the causative agents (Sindermann, 1988; Elston, 1993). Larval vibriosis or bacillary necrosis results in mortalities often exceeding 90% within 24 h of infection, and production in hatcheries has been frequently limited by outbreaks of this disease in many marine bivalve species (Sindermann, 1988). Therefore, bacillary necrosis can be considered the most serious disease of hatchery-reared larvae. Recently in Japan, mass mortalities caused by bacillary necrosis outbreaks have been reported from hatcheries of the cockle Fulvia mutica (Fujiwara et al., 1993) and the Pacific oyster Crassostrea gigas (Sugumar et al., 1998a). The use of antibiotics such as chloramphenicol (Tubiash et al., 1965; Jeffries, 1982; Lodeiros et al., 1987) and streptomycin (Tubiash et al., 1965; Sugumar et al., 1998a) has been shown to be a very effective method for controlling bacillary necrosis and improving the survival of bivalve larvae. However, antibiotic treatment as a preventive procedure is not recommended because it can affect bacterial flora in the culture sea water and can also result in the selection of resistant pathogenic strains (Nicolas et al., 1996). Recent attempts have been made to apply biocontrol (biological control) techniques to bivalve larvae rearing systems as a method for preventing infectious larval diseases. One such method uses live bacteria to suppress the growth of pathogenic bacteria and to control the microflora in culture sea water (Nogami and Maeda, 1992). In the case of rearing bivalve larvae, this biocontrol technique was experimentally adapted
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for scallop Argopecten purpuratus culture (Riquelme et al., 1997) and for C. gigas cultures (Gibson et al., 1998; Nakamura et al., 1999). However, the efficacy of this biocontrol technique in bivalve larvae hatchery-rearing systems remains to be clarified. We have been carrying out research on effective techniques against bacillary necrosis without the use of antibiotics or bactericidal drugs since 1996. In this study, we examined the effect of ovoglobulins (ovoGs), which are proteins derived from the whites of hens’ eggs, on bacillary necrosis in C. gigas larvae infected experimentally by pathogenic Vibrio tubiashii. We determined the survival rate of larval oysters inoculated with V. tubiashii in the presence or absence of ovoGs. We also examined the effect of ovoGs on the growth of V. tubiashii in gelatin–sea water broth. To our knowledge, this paper is the first report concerning the inhibitory effect of nonbactericidal proteins such as ovoGs on bacillary necrosis in larval oysters. MATERIALS AND METHODS
Collection and Rearing of Oyster Larvae Cultivation of eggs and larvae of the Pacific oyster, C. gigas, under small-scale laboratory conditions was performed according to Loosanoff and Davis (1963). Ripe eggs and sperm were obtained by stripping mature adult oysters harvested from hanging cultures in Onagawa Bay, Miyagi Prefecture, Japan. Fertilized eggs were gently rinsed in sand-filtered sea water and then placed in 100-L culture aquaria at densities of 20 –30 eggs/ml of sand-filtered sea water at 23°C. Two days after hatching, the fertilized eggs developed into straight-hinge larvae of about 90 m in length. Larvae were collected from the culture aquaria with 50-m mesh sieves and gently transferred to 10-L aquaria at a density of 3 individuals/ml of sand-filtered sea water at 23°C. Effects of Ovoglobulins on Survival of Larval Oysters Challenged with V. tubiashii Two-day-old larval oysters were placed in 3-L flasks filled with 0.22 m filtered sea water (sterile sea water) at 23°C at a density of 1 larva/ml. Virulence assays were performed to determine the pathogenicity of a V. tubiashii strain isolated as a strong pathogen of bivalve molluscs (Tubiash et al., 1970). The strain tested, V. tubiashii strain ATCC 19106, was obtained from the American Type Culture Collection (Rockville, MD). This strain was cultured in marine broth 2216 (Difco, Detroit, MI) at room temperature for 24 h and was harvested by centrifugation at 2450g for 20 min. The organisms were washed twice with sterile sea water and resuspended in sterile sea water containing 0.3% glucose. Three milliliters of the
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bacterial suspension was added to larval oysters in 3-L flasks. The final concentration of V. tubiashii used was approximately 10 5 cfu (colony-forming units)/ml. To examine the inhibitory effects of ovoglobulins on vibrio infection of oyster larvae, we inoculated 3 ml of V. tubiashii suspension into 3 L of sterile sea water containing larvae and ovoglobulins (Sigma Chemical Co., St. Louis, MO) at final concentrations of 1.0, 3.0, 5.0, 10.0, and 20.0 g/ml. Unexposed controls were subjected to the same procedure as exposed larvae but without the inoculation with V. tubiashii and the addition of ovoGs. The ovoGs used in this study consisted of two globulin fractions which were purified from whites of hens’ eggs according to Rhodes et al. (1958). Therefore, the ovoGs products did not contain any detectable amounts of ovomucoid, ovalbumin, conalbumin, and lysozyme (⬎99% of total globulins; refer to the certificate of product analysis from Sigma Chemical Co.). These assays were performed in duplicate at 23°C. Larval survival was recorded at intervals of 3 h for 24 h after exposure. The number of live and dead larvae from each flask was counted under light microscopy, until a total of 300 larvae was reached. Moribund larvae were counted as dead. Effects of OvoGs on Survival of Larval Oysters Exposed to Cell-Free Supernatant of V. tubiashii Culture Cultures of V. tubiashii were centrifuged at 3500g for 20 min. The supernatant was collected and filtered through 0.22-m pore-size membranes (Millipore Co., Bedford, MA). To examine the effects of ovoGs on the survival of larvae exposed to bacterial supernatant, 100 ml of filtered supernatant was added to each flask containing larval oysters in 900 ml of sterile sea water. OvoGs were then added at final concentrations of 1 and 10 g/ml. After inoculation with the supernatant, each flask containing larval oysters was maintained at 23°C for 48 h, and larval survival was determined at intervals of 6 h. Unexposed larvae were also reared as controls in 100 ml of marine broth 2216 and 900 ml of sterile sea water without ovoGs. Effects of ovoGs on Growth of V. tubiashii in Culture Medium V. tubiashii was cultured overnight in marine broth 2216 at 26°C and was harvested by centrifugation at 2450g for 20 min. Bacterial cells were washed with 10 mM phosphate-buffered 0.45 M saline (marine PBS, pH 7.6) and resuspended in marine PBS at a concentration of 10 5 cfu/ml. This bacterial suspension (0.5 ml) was inoculated into 4.5 ml of gelatin– glucose–marine medium (GGMM, 5.0 g gelatin dialyzed overnight against sterile sea water and 3.0 g glucose in 1 L sterile sea water, pH 7.4) containing ovoGs at final concentra-
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tions of 0, 100, and 200 g/ml. The cultures were incubated at 26°C for 24 h. After incubation, the number of viable V. tubiashii cells was determined by counting colonies formed on plates of marine agar 2216 (Difco). Statistical Analysis of Data To test for differences in the mean survival rate of larval oysters at various concentrations of ovoGs, we subjected the data to one-way analysis of variance (ANOVA) and Duncan’s multiple range test (Duncan’s test). Differences in the mean number of viable V. tubiashii at different concentrations of ovoGs were also assessed using one-way ANOVA and Duncan’s test. In all cases, the level for significant differences was set at P ⬍ 0.01. RESULTS
Effects of OvoGs on Survival of Larval Oysters Challenged with V. tubiashii Larval oysters started to die 12 h after challenge with V. tubiashii, and the survival rate markedly decreased 15 h after challenge, reaching 0% survival at 21 h (Fig. 1). In contrast, we found a remarkable inhibitory activity of ovoGs against larval infection by V. tubiashii. The survival rate in the ovoGs-added group was 96.5% 24 h after challenge with V. tubiashii. This value was not different from the larval survival in the control group (97.1% at 24 h). Under microscopic observation, V. tubiashii induced both loss of larval swimming (larvae settled on the bottom) and anoma-
FIG. 2. Percentage of larval survival challenged with V. tubiashii in flasks containing different concentrations of ovoGs. V. tubiashii was inoculated into each flask at a concentration of 10 5 cfu/ml. The data represent the mean percentage of larval survival after 24 h of exposure to V. tubiashii. The vertical lines represent standard errors of six separate experiments. Different lowercase letters denote significant differences (P ⬍ 0.01, Duncan’s multiple range test).
lies in the function and structure of the velum in the early phase of infection (9 h after challenge). Thereafter, the velum and the internal organs became severely necrotized in the acute phase of infection. In contrast, no anomalies were observed in either the motility or the morphology of larval oysters in groups with only ovoGs added. Suppression by ovoGs of larval infection by V. tubiashii exhibited concentration-dependent effects in the range of 1.0 to 5.0 g/ml of ovoGs (Fig. 2). Namely, survival of larval oysters treated with ovoGs increased significantly at higher concentrations of ovoGs. The percentage of larval survival at 5.0 g/ml of ovoGs was 96.5 ⫾ 1.3% and showed no significant difference between larval survival at ovoGs concentrations of 10.0 (97.8 ⫾ 0.6%) or 20.0 (97.4 ⫾ 0.9%) g/ml. Survival rates in all ovoGs-added groups were significantly higher than those of the control group (0%). Effects of OvoGs on Survival of Larval Oysters Exposed to Cell-Free Supernatant of V. tubiashii Culture
FIG. 1. Changes in survival rate of C. gigas larvae experimentally challenged with V. tubiashii, and the effect of ovoglobulins (ovoGs) in suppressing the V. tubiashii infection. The percentage of larval survival is plotted under conditions of no challenge with V. tubiashii and no addition of ovoGs (control, 䡬); challenged with V. tubiashii at a concentration of 10 5 cfu/ml (䡺); challenged with V. tubiashii at 10 5 cfu/ml in the presence of ovoGs at a concentration of 10.0 g/ml (f).
The culture supernatant of V. tubiashii induces mortality of larval oysters; however, its toxicity was inhibited by the addition of ovoGs in a dose-dependent manner (Fig. 3). With exposure to 10% culture supernatant, the survival rate of larval oysters markedly decreased 18 h after addition and reached 34.3% survival at 48 h. The toxicity of the culture supernatant was completely inhibited by the addition of ovoGs at a concentration of 10.0 g/ml, and the survival rate in the ovoGs-added
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FIG. 3. Changes in survival rate of larval oysters experimentally exposed to culture supernatant of V. tubiashii, and suppressive effects of ovoGs on toxicity of the supernatant to larval oysters. The percentage of larval survival is plotted under conditions of no exposure to culture filtrate of V. tubiashii and no addition of ovoGs (control, 䡬); exposed to 10% (volume per volume) of culture supernatant of V. tubiashii (䡺); exposed to 10% of culture supernatant of V. tubiashii in the presence ovoGs at concentrations of 1.0 g/ml (‘) and 10.0 g/ml (f).
group remained high for 48 h. Under microscopic observation, the vela of supernatant-exposed larval oysters 48 h after challenge were grossly deformed, and the internal organs of 60% or more of the larvae were necrotized. These symptoms of infection were qualitatively similar to those of larval oysters infected with V. tubiashii cells. Effects of OvoGs on Growth of V. tubiashii in Culture Medium OvoGs partially suppressed the growth of V. tubiashii in GGMM (Table 1). After 24 h of incubation, the number of V. tubiashii in the group with 200 g ovoGs/ml reached 1.10 ⫻ 10 8 cfu/ml, which was significantly lower than the value of 4.65 ⫻ 10 8 cfu/ml in the group without ovoGs. No significant difference was found between the number of viable V. tubiashii treated with 100 and 200 g ovoGs/ml.
effects to antibiotics in the protection of larval oysters against bacillary necrosis and in their subsequent survival. However, ovoGs are simply proteins derived from the whites of hens’ eggs and are not antibiotics or bactericidal substances. This is the first report showing that ovoglobulin proteins are capable of preventing larval infection by pathogenic Vibrio species. V. tubiashii showed very strong pathogenicity to oyster larvae and induced 100% mortality of oyster larvae by exposure at a concentration of 10 5 cfu/ml (Figs. 1 and 2). Infected larvae also showed typical symptoms of bacillary necrosis including anomalous swimming and detachment of cilia and/or vela. In the acute phase of infection, moreover, V. tubiashii could be seen swarming and invading the inside of moribund larvae and subsequently proliferated within the larvae. These observations are very similar to those reported by Tubiash et al. (1965), Brown and Losee (1978), Elston and Leibovitz (1980), and Jeffries (1982). However, the addition of ovoGs at a concentration of 10 g/ml led to an increase in the survival rate of larval oysters inoculated with V. tubiashii to 96.5%, 24 h after challenge (Fig. 1). In the ovoGs-added groups, no observations were made of any massive bacterial invasion and proliferation within or around the larvae. In addition, no changes in the motility and the morphology of larval oysters were observed in the ovoGs-added groups. These results indicate that ovoGs almost completely protect larval oysters from V. tubiashii infection and that ovoGs do not affect the survival of larval oysters. Sugumar et al. (1998b) reported that both extracellular products and intracellular components of V. splendidus biovar II were lethal to larvae of C. gigas. Lodeiros et al. (1987) and Riquelme et al. (1995) also indicated that extracellular products or exotoxins produced by invading vibrios were pathogenic to larvae of Ostrea edulis and A. purpuratus, respectively. In other studies concerning the details of pathogenic factors of the culture supernatants of Vibrio sp. NCMB 1338 and
TABLE 1 Inhibitory Effects of OvoGs on Growth of V. tubiashii Viable V. tubiashii
DISCUSSION
In this study, we have demonstrated that ovoGs possess a remarkable inhibitory effect on bacillary necrosis in larval oysters infected with V. tubiashii. Use of antibiotics such as chloramphenicol and streptomycin for improving the survival of larvae of bivalve molluscs has been reported by previous investigators (Tubiash et al., 1965; Brown and Losee, 1978; DiSalvo et al., 1978; Jeffries, 1982; Lodeiros et al., 1987; Sugumar et al., 1998a). In this study, ovoGs showed almost equal
OvoGs (g/ml)
Initial number (10 4 cfu/ml)
Number grown after 24 h (10 8 cfu/ml)
0 100 200
2.45 ⫾ 0.05 2.48 ⫾ 0.06 2.45 ⫾ 0.04
4.65 ⫾ 0.07 a 1.26 ⫾ 0.08 b 1.10 ⫾ 0.12 b
Note. V. tubiashii was cultured in gelatin– glucose–marine medium at 26°C. Each number of viable V. tubiashii represents the mean ⫾ standard error of five separate experiments performed in triplicate. Different lowercase letters denote significant differences (P ⬍ 0.01, Duncan’s multiple range test).
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V. alginolyticus NCMB 1339, proteinases (proteases), a ciliostatic toxin, and a heat-labile protein (which was designated as a spat toxin) were suggested to be involved in the pathogenicity of O. edulis and C. gigas spats (Nottage and Birkbeck, 1986, 1987a). The V. tubiashii culture supernatant, containing extracellular products, was also shown to be pathogenic to larval oysters; however, its pathogenicity was completely inhibited by the addition of ovoGs (Fig. 3). Moreover, the addition of ovoGs clearly suppressed the growth of V. tubiashii, although the number of viable V. tubiashii after 24 h of incubation did not decrease to the initial dose level (Table 1). These data indicate that V. tubiashii could not grow effectively because of an inability to utilize nitrogen and carbon sources from gelatin when potent and broad-spectrum protease inhibitors are present. Although the ovoGs used in this study are apparently separated in a high purity of ⬎99% of total globulins (data from Sigma Chemical Co.), the ovoGs are considered to consist of eight or nine proteins including ovomicroglobulin, ovomacroglobulin, and ovoinhibitor as strong inhibitors of bacterial proteases (Takahashi and Mori, unpublished data). These results also suggest that the inhibition of extracellular protease activity by ovoGs in the vibrio infection may suppress not only the pathogenic mechanism of proteases but also bacterial growth. However, biological activities of these individual proteins will need to be further studied. Nottage and Birkbeck (1987b) indicated that anti-V. alginolyticus protease antiserum reduced the toxicity of purified V. alginolyticus protease to O. edulis larvae, whereas it had no effect on the toxicity of a crude culture supernatant of V. alginolyticus. As described above, however, ovoGs had a complete inhibitory effect on bacillary necrosis in larval oysters exposed to the culture supernatant of V. tubiashii (Fig. 3). Therefore, the data indicate that the activity of some other toxic factor(s) as well as that of proteases could be inhibited by the administration of ovoGs. However, there is not enough evidence to clarify the mechanism by which ovoGs suppress pathogenic Vibrio strains. Nicolas et al. (1996) indicated that alternative methods to the use of antibiotics must be developed for the prevention of bacillary necrosis. Although the data described above were obtained only under experimental conditions, we consider that the addition of ovoGs has potential as a method for controlling bacillary necrosis in larval oysters. We will need to address many additional issues concerning the applicability of the method to large-scale culture and the mechanism of inhibition of bacillary necrosis. ACKNOWLEDGMENTS We thank the staff of the Education and Research Center of Marine Bioresources at Onagawa, Tohoku University, for their help in
our experiments. We are also grateful to Dr. Harriet Baillie for thorough reading and correction the manuscript. This study was partly supported by a Grant-in-Aid for Encouragement of Young Scientists (11760130) from the Ministry of Education, Science, Sports, and Culture, Japanese Government. REFERENCES Brown, C., and Losee, E. 1978. Observations on natural and induced epizootics of vibriosis in Crassostrea virginica larvae. J. Invertebr. Pathol. 31, 41– 47. DiSalvo, L. H., Blecka, J., and Zebal, R. 1978. Vibrio anguillarum and larval mortality in a California coastal shellfish hatchery. Appl. Environ. Microbiol. 35, 219 –221. Elston, R. A., and Leibovitz, L. 1980. Pathogenesis of experimental vibriosis in larval American oysters, Crassostrea virginica. Can. J. Fish. Aquat. Sci. 37, 964 –978. Elston, R. A., Leibovitz, L., Relyea, D., and Zatila, J. 1981. Diagnosis of vibriosis in a commercial oyster hatchery epizootic: Diagnostic tools and management features. Aquaculture 24, 53– 62. Elston, R. A. 1993. Infectious diseases of the Pacific oyster, Crassostrea gigas. Annu. Rev. Fish Dis. 3, 259 –276. Fujiwara, M., Uyeno, Y., and Iwao, A. 1993. A Vibrio sp. associated with mortalities in cockle larvae Fulvia mutica (Mollusca: Cardiidae). Fish Pathol. 28, 83– 89. (in Japanese with English summary) Garland, C. D., Nash, G. V., Summer, C. E., and McMeekin, T. A. 1983. Bacterial pathogens of oyster larvae (Crassostrea gigas) in a Tasmanian hatchery. Aust. J. Mar. Freshw. Res. 34, 483– 487. Gibson, L. F., Woodworth, J., and George, A. M. 1998. Probiotic activity of Aeromonas media on the Pacific oyster, Crassostrea gigas, when challenged with Vibrio tubiashii. Aquaculture 169, 111–120. Imai, T., and Sakai, S. 1961. Study of breeding Japanese oyster, Crassostrea gigas. Tohoku J. Agr. Res. 12, 125–163. Jeffries, V. E. 1982. Three Vibrio strains pathogenic to larvae of Crassostrea gigas and Ostrea edulis. Aquaculture 29, 201–226. Korringa, P. 1952. Recent advances in oyster biology. Q. Rev. Biol. 27, 339 –365. Lodeiros, C., Bolinches, J., Dopazo, C. P., and Toranzo, A. E. 1987. Bacillary necrosis in hatcheries of Ostrea edulis in Spain. Aquaculture 65, 15–29. Loosanoff, V. L., and Davis, H. C. 1963. Rearing of bivalve mollusks. In “Advances in Marine Biology” (F. S. Russell, Ed.), Vol. 1, pp. 1–136. Academic Press, London. Nakamura, A., Takahashi, K. G., and Mori, K. 1999. Vibriostatic bacteria isolated from rearing seawater of oyster brood stock: Potentiality as biocontrol agents for vibriosis in oyster larvae. Fish Pathol. 34, 139 –144. Nicolas, J. L., Ansquer, D., and Cochard, J. C. 1992. Isolation and characterization of a pathogenic bacterium specific to Manila clam Tapes philippinarum larvae. Dis. Aquat. Org. 14, 153–159. Nicolas, J. L., Corre, S., Gauthier, G., Robert, R., and Ansquer, D. 1996. Bacterial problems associated with scallop Pecten maximus larval culture. Dis. Aquat. Org. 27, 67–76. Nogami, K., and Maeda, M. 1992. Bacteria as biocontrol agents for rearing larvae of the crab Portunus trituberculatus. Can. J. Fish Aquat. Sci. 49, 2373–2376. Nottage, A. S., and Birkbeck, T. H. 1986. Toxicity to marine bivalves of culture supernatant fluids of the bivalve-pathogenic Vibrio stain NCMB 1338 and other marine vibrios. J. Fish Dis. 9, 249 –256. Nottage, A. S., and Birkbeck, T. H. 1987a. Purification of a proteinase produced by the bivalve pathogen Vibrio alginolyticus NCMB 1339. J. Fish Dis. 10, 211–220. Nottage, A. S., and Birkbeck, T. H. 1987b. Production of proteinase during experimental infection of Ostrea edulis L. larvae with
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