The effects of egg stocking density and antibiotic treatment on survival and development of winged pearl oyster (Pteria penguin, Roding 1798) embryos.
KEY WORDS: pearl oyster, Pteria penguin, embryo, incubation, stocking density, antibiotics, survival, development
Hatchery propagation of pearl oysters (Bivalvia: Pteriidae) has become increasingly important as a source of stock for the cultured pearl industry (Southgate 2008) and is now particularly valuable given recent research interest in selective breeding of pearl oysters for genetically desirable traits (Kvingedal et al. 2010). Maximizing hatchery output requires an understanding of the conditions that support development and survival of larvae to ensure an adequate supply of juveniles for commercial use. Pearl oyster eggs incubated in captivity typically require 1824 h to undergo embryogenesis and pass through the trochophore stage to become free-swimming D-stage veligers with a calcified shell (Wada 1953, Alagarswami et al. 1982, Rose & Baker 1994, Araya-Nunez & Ganning 1995, Yu et al. 2000, Doroudi & Southgate 2003). The similarity in development between species has resulted in a standard period for pearl oyster incubation of 24 h (Southgate 2008). This phase is characterized by high rates of mortality (Southgate 2008) and is a part of the life cycle of pearl oysters for which our knowledge of optimal culture conditions is lacking.
Pearl oyster embryos have been shown to tolerate a wide range of water temperatures and salinities (Doroudi et al. 1999, O'Connor & Lawler 2004), suggesting that mortality during incubation is primarily attributed to poor water quality and the proliferation of harmful bacteria. Hatcheries typically expose incoming seawater to filtration and UV radiation to eliminate pathogens (Minaur 1969, Rose & Baker 1994, Araya-Nunez & Ganning 1995), leaving the eggs themselves as the main route for bacterial contamination. Stocking incubation tanks with a high density of eggs is important to ensure an adequate number of larvae for the next stage of production, but doing so may compromise survival and development by introducing excessive decaying matter on the surface of eggs (Blaxter 1956, Gruffydd & Beaumont 1970) and by transferring bacterial infections from parental gonad tissue (Riquelme et al. 1994, Jorquera et al. 2001). For pearl oysters of the genus Pinctada, the proportion of fertilized eggs to reach D-stage during egg incubation generally decreases with increasing stocking density (Southgate et al. 1998).
The assumption that mass mortality of embryos is linked to pathogenic infection has resulted in incubation protocols for pearl oysters often including the application of a broad-spectrum antibiotic (Southgate 2008). In research conducted on the culture of fish eggs, Peck et al. (2004) demonstrated that, when compared with other chemical disinfectants, antibiotic solution is the most effective antimicrobial for maximizing egg hatch rate. Furthermore, controlling bacterial growth in seawater used for bivalve culture is best achieved with the addition of two or more antibiotics at the same time (Walne 1958, Fitt et al. 1992, Doroudi 2001, Stoeckel et al. 2004). The antibiotics most appropriate for promoting survival in a given aquaculture species are dependent on their physiological response to treatment of the culture medium and the nature of the bacteria to which they are exposed.
The winged pearl oyster, Pteria penguin (Roding 1798), has a wide Indo-Pacific distribution (Wada & Temkin 2008), where it is used in the commercial production of half-pearls or "mabe" (Strack 2006, Southgate et al. 2008). The cultured pearl industry based on P. penguin has traditionally relied on oysters collected from the wild, either as adults or juveniles (spat), as a source of culture stock. However, in regions with poor spat recruitment or depleted wild populations, the industry has become increasingly reliant on hatchery production (Teitelbaum & Fale 2008). Currently, efficient hatchery production of P. penguin is constrained by our lack of knowledge regarding optimal conditions for the culture of eggs, larvae, and juveniles of this species. This study assesses the impact of egg density and antibiotics on (1) the survival of newly fertilized eggs and (2) the proportion of surviving embryos that undergo normal development to achieve D-stage within 24 h.
MATERIALS AND METHODS
This study was conducted at the Aquaculture Facility of the Ministry of Agriculture and Food, Forests & Fisheries at Sopu in the Kingdom of Tonga (21[degrees]07'21" S, 175[degrees]13'36" W). Fifty broodstock were collected from a longline located 500 in offshore and were cleaned before being induced to spawn via repeated air exposure (Victor et al. 2001). The method for spawning induction involved placing the oysters in a shallow spawning tank for 1 h before completely draining the tank and leaving the oysters exposed to direct sunlight for 10 min. The tank was then refilled with UV-treated 1-[micro]m filtered sea water (FSW) at a temperature of 28[degrees]C, and the oysters were left undisturbed for 30 min. This process was repeated twice before several males began releasing sperm, prompting the females to release eggs 3-5 min later. Spawning oysters were removed from the tank and placed in individual 8-L plastic aquaria to continue releasing gametes. Within 10 min of the initial male spawning, gametes from 10 males were combined separately with gametes from 10 females in two 20-L aquaria. Twenty milliliters of sperm mixture was added to the 20-L dilution of eggs and mixed gently to achieve an even distribution (Southgate et al. 2008).
Forty minutes was allowed for fertilization to take place, by which time more than 80% of eggs displayed the first polar body. Fertilized eggs remained submerged as they were collected onto a 20-[micro]m sieve and rinsed with FSW. Eggs were then stocked in 5-L plastic aquaria with lids. This study used a factorial design combining 3 egg stocking densities (10, 50, and 100/mL) and 3 antibiotic treatments: (1) the absence of any antibiotic (control), (2) 5 mg/L standard antibiotic applied during pearl oyster incubation (streptomycin-sulfate (Southgate 2008)), and (3) 5 mg/L antibiotic combination shown to benefit pearl oyster larvae in a prior study (tetracycline-erythromycin, 2.5:2.5 mg/L (Doroudi 2001)). Antibiotics ([greater than or equal to] 98% potency), manufactured by Sigma-Aldrich (St. Louis, MO), were diluted with FSW to a concentration of 1 g/L before being added to incubation aquaria prior to stocking with eggs. A relatively low concentration of antibiotics was used in an effort to avoid the deformities seen in pearl oyster larvae exposed to high antibiotic concentrations (>5 mg/L (Doroudi 2001)). The treatment combinations were conducted in triplicate, resulting in 27 aquaria, maintained at a temperature of 27 [+ or -] 1[degrees]C and given continuous gentle aeration.
The experiment was terminated after 24 h, which has been shown to be sufficient time for fertilized eggs from P. penguin to develop into shelled veligers or D-stage larvae (Yu et al. 2000). The contents of each aquarium were collected on a 25-[micro]m nylon sieve mesh and preserved in separate 50-mL vials containing 4% formaldehyde solution in buffered seawater. Triplicate 1-mL subsamples were removed from each vial, and the number of larvae in each was counted using a high-power optical microscope at 20X magnification. Embryos or larvae showing signs of decay were classified as deceased and were not included in the data set. Embryos that survived 24 h without having formed a calcified shell were assumed to have experienced some degree of slowed or impaired development. Counts were used to estimate (1) the total proportion of fertilized eggs that had "survived" 24 h of incubation (trochophores + D-stage) and (2) the proportion of surviving embryos that had undergone "normal development" and were therefore D-stage larvae with a calcified shell. Data were square root arcsine transformed prior to analysis by 2-way ANOVA. Significant differences between treatment means were determined using a post hoc Tukey test at an alpha level of 0.05.
Two-way ANOVA examining the effects of stocking density and antibiotic treatment on the survival (including all stages of development) of P. penguin eggs during incubation revealed no significant interaction between the two factors. The addition of antibiotics to incubation tanks significantly influenced mortality (F = 11.279, df = 2, P < 0.001), whereby mean percentage survival was approximately 20% greater in aquaria treated with antibiotics and maximized with the application of tetracycline erythromycin (Fig. 1A). Egg stocking density did not have an overall significant effect on survival; however, the antibiotic combination of tetracycline-erythromycin was significantly (F = 4.180, df = 2, P = 0.028) more effective ([approximately equal to] 13.5%) at enhancing survival at low and intermediate egg densities than at a high egg density (Fig. 1B). Furthermore, when egg density was high, the addition of streptomycin-sulfate did not increase survival when compared with larvae in control aquaria without antibiotic.
Two-way ANOVA examining the effects of stocking density and antibiotic treatment on the percentage of surviving P. penguin embryos that developed normally to become D-stage veligers showed no significant interaction between the two factors. Both factors had an overall significant effect on successful development to D-stage (density: F = 14.418, df = 2, P < 0.001; antibiotic: F = 58.372, df = 2, P < 0.001). The mean proportion of normally developed D-stage larvae was significantly reduced at the highest egg density (Fig. 2A), although the difference was small in magnitude ([approximately equal to] 5%). At each level of egg stocking density, the antibiotic combination of tetracycline-erythromycin caused significant (10/mL: F = 19.465, df = 2, P < 0.001; 50/mL: F = 26.888, df = 2, P < 0.001; 100/mL: F = 18.123, df = 2, P < 0.001) developmental impairment, resulting in a lower proportion of D-stage larvae ([approximately equal to] 11.5%) and corresponding higher proportion of trochophores (Fig. 2B). The impaired development associated with tetracycline-erythromycin became more pronounced with increasing egg density.
Hatchery production of pearl oyster species belonging to the genus Pinctada has shown that the proportion of eggs that survive and develop normally to become D-stage veligers, when using a typical egg density of 20-50/mL for an incubation period of 24 h, can vary greatly, ranging from 6% to 75-80% (Southgate et al. 1998). A disparity in egg quality (i.e., the amount and condition of endogenous reserves available as an energy source during embryogenesis) (Utting & Millican 1997) is thought to be the primary reason for differences in the performance of embryos between multiple spawning events from the same species (Southgate et al. 1998). When a controlled spawning event is achieved in a hatchery, the focus is to minimize mortality of eggs and maximize normal development of the available embryos.
[FIGURE 1 OMITTED]
Bivalve larvae are susceptible to mortality caused by pathogenic bacteria, which are known to destroy juveniles via direct invasion or contact (Guillard 1959). Douillet and Langdon (1993) tested the effects of 21 bacterial strains on larvae of the oyster species Crassostrea gigas and found most to be detrimental to survival. Bacteria from the genus Vibrio have been identified as being particularly dangerous for oyster species (DiSalvo et al. 1978, Jones 2007). A number of prior studies on early life culture of bivalves have reported the benefits associated with implementing broad-spectrum antibiotics to control bacterial contamination and therefore decrease mortality (e.g., Walne 1958, Fitt et al. 1992, Stoeckel et al. 2004). The benefits of antibiotic application for increasing survival of shelled larvae have been demonstrated for the pearl oyster Pinctada margaritifera (Doroudi 2001), and the current study has confirmed such benefits during the incubation of newly fertilized Pteria penguin eggs.
It is common for pearl oyster incubation tanks to be treated with a broad-spectrum antibiotic, such as streptomycin-sulfate (Southgate & Beer 1997, Southgate 2008), but until now there has been no formal comparison with survival under control conditions without antibiotic. The results of this study show that the absence of streptomycin-sulfate reduced survival of P. penguin embryos over an incubation period of 24 h. Doroudi (2001) found tetracycline-erythromycin (1:1) to be beneficial in promoting survival of Pinctada margaritifera larvae that had already formed a calcified shell, but prior to the current study, this combination of antibiotics was yet to be tested on newly fertilized pearl oyster eggs. Our results indicate that survival of P. penguin embryos during the initial 24 h postfertilization is enhanced in the presence of tetracycline-erythromycin, but this treatment did not significantly improve survival when compared with the widely adopted streptomycin-sulfate.
[FIGURE 2 OMITTED]
At the highest egg density used in this study of 100/mL, streptomycin-sulfate did not support improved survival compared with that in control aquaria. Also, the antibiotic combination of tetracycline erythromycin was significantly less effective at enhancing survival at an egg density of 100/mL when compared with 50/mL. Bivalve eggs are capable of carrying decaying matter--in particular, superfluous sperm--on their surfaces (Blaxter 1956, Gruffydd & Beaumont 1970) and, given the contoured exterior of pearl oyster eggs (Doroudi & Southgate 2003), it is unlikely that simply rinsing with filtered sea water prior to incubation will eliminate all potentially harmful bacteria. Total egg surface area increases proportionally with density, providing greater refuge for dangerous bacteria at higher egg densities. Furthermore, infections transferred from the parental gonad tissue (Riquelme et al. 1994, Jorquera et al. 2001) would be expected to increase with egg density. The low antibiotic dosage of 5 mg/mL applied to incubation aquaria in this study may have been insufficient to deal with bacterial loading at a high egg density and, on this basis, antibiotics were not as effective in promoting survival compared with lower densities.
Across each of the egg densities tested, the proportion of embryos with impaired development was significantly greater in aquaria treated with tetracycline-erythromycin when compared with control aquaria and aquaria that received the same dosage of streptomycin sulfate. This was most likely caused by interference in early shell development--in particular, initial calcification, which occurs in the final transition from the trochophore to veliger stages (Eyster 1986, Moueza et al. 2006). Tetracycline is known to interfere with calcification and cause shell deformities in bivalve larvae (Fitt et al. 1992), but the effect on embryos has not been investigated. Doroudi (2001) observed some deformity in pearl oyster larvae (P. margaritifera) exposed to tetracycline-erythromycin (1:1) at a concentration of 10 mg/mL, but not at 5 mg/mL. The results of this study suggest that the antibiotic combination of tetracycline-erythromycin (1:1) is capable of disrupting early shell formation prior to D-stage, even when applied at a dosage that is safe for shelled larvae. Doroudi (2001) found that increasing the concentration of neomycin-sulfate-streptomycin-sulfate (1:1) from 5-10 mg/mL did not cause deformity in shelled larvae, but was not recommended because it failed to improve survival.
Rose (1990) recommended that Pinctada maxima eggs be incubated at a maximum density of 30/mL; however, Southgate et al. (1998) observed high rates of survival to D-stage of both P. margaritifera and P. maxima at densities [greater than or equal to] 100/mL. The results of the current study show no significant reduction in the survival of P. penguin embryos when doubling stocking density from 50-100/mL. The impaired development observed at the highest density may be attributed to elevated bacterial levels or mechanical interference between eggs (Blaxter, 1956), but was small enough in magnitude to have little effect on the number of normally developed D-stage larvae. On this basis, a high stocking density is recommended if the tank space available for incubation is limiting adequate production of D-stage larvae for the next phase of production.
In summary, the antibiotic combination tetracycline-erythromycin (1:1) is effective in enhancing survival of Pteria penguin embryos during a 24 h period of incubation, but hinders development during the transition from trochophore to D-stage. In contrast, streptomycin sulfate supports greater survival without significantly compromising development. Additional research is required to determine whether a longer period of incubation would allow P. penguin embryos with impaired development to eventually reach D-stage. If this were the case, we would expect tetracycline-erythromycin (1:1) to support a superior hatch rate of D-stage larvae. Incubating P. penguin eggs at a density [greater than or equal to] 100/mL can impede normal development and hinder the efficacy of antibiotics when applied at a low dosage. However, increasing stocking density from 50-100/mL did not significantly reduce mean survival and therefore resulted in greater production of D-stage larvae. Further research is warranted to determine whether higher concentrations of streptomycin sulfate than those tested in this study can be used to enhance survival when utilizing a high egg density.
We thank Andrew Beer and Scott Mactier of James Cook University for facilitating this research. We also thank the staff of the Aquaculture Section of the MAFFF in the Kingdom of Tonga--in particular, Poasi Ngaluafe, Vea Kava, and Tonga Tuiano--for their expertise and assistance with this study. This study was jointly funded by the Australian Centre for International Agricultural Research (ACIAR) and the James Cook University Graduate Research School, and was conducted as part of ACIAR Project FIS/2006/172 "Winged Oyster Pearl Industry Development in Tonga."
Alagarswami, K., S. Dharmaraj, T. S. Velayudhan, A. Cheliam & A. C. C. Victor. 1982. Embryonic and early larval development of pearl oyster Pinctada fucata (Gould). Presented at the Proceedings of the Symposium on Coastal Aquaculture, Part 2, MBAI, January, 12-18, 1980, Cochin.
Araya-Nunez, O. & B. Ganning. 1995. Embryonic development, larval culture and settling of the American pearl oyster (Pteria sterna, Gould) spat. Calif. Fish Game 81:10-21.
Blaxter, J. H. S. 1956. Herring rearing. II. The effect of temperature and other factors on development. Mar. Res. 5:1-19.
DiSalvo, L. H., J. Blecka & R. Zebal. 1978. Vibrio anguillarum and larval mortality in a California coastal shellfish hatchery. Appl. Environ. Microbiol. 35:219-221.
Doroudi, M. 2001. Development and culture of black-lip pearl oyster, Pinctada margaritifera (Linnaeus), larvae. PhD diss., School of Marine Biology and Aquaculture, James Cook University. pp. 153.
Doroudi, M., P. C. Southgate & R. Mayer. 1999. The combined effects of temperature and salinity on embryos and larvae of the black-lip pearl oyster, Pinctada margaritifera (L.). Aquacult. Res. 30:1-7.
Doroudi, M. & P. C. Southgate. 2003. Embryonic and larval development of Pinctada margaritifera. (Linnaeus, 1758). Molluscan Research 23:101-107.
Douillet, P. & C. J. Langdon. 1993. Effects of marine bacteria on the culture axenic oyster Crassostrea gigas (Thunberg) larvae. Biol. Bull. 184:36-51.
Eyster, L. S. 1986. Shell inorganic composition and onset of shell mineralisation during bivalve and gastropod embryogenesis. Biol. Bull. 170:211-231.
Fitt, W. K., G. A. Heslinga & T. C. Watson. 1992. Use of antibiotics in the mariculture of giant clams. Aquaculture 104:1-10.
Gruffydd, L. D. & A. R. Beaumont. 1970. Determination of the optimum concentration of eggs and spermatozoa for the production of normal larvae in Pecten maximus (Mollusca, Lamellibranchia). Helgol. Wiss. Meeresunters. 20:486-497.
Guillard, R. L. 1959. Further evidence of the destruction of bivalve larvae by bacteria. Biol. Bull. 117:258-266.
Jones, J. B. 2007. Review of pearl oyster mortalities and disease problems. In: M. G. Bondad-Reantaso, S. E. McGladdery & F. C. J. Berthe, editors. Pearl oyster health management: a manual. FAO fisheries technical paper no. 503. Rome: FAO. p. 120.
Jorquera, M. A., F. R. Silva & C. E. Riquelme. 2001. Bacteria in the culture of the scallop Argopecten purpuratus (Lamarck, 1819). Aquacult. Int. 9:285-303.
Kvingedal, R., B. S. Evans, C. E. Lind, J. J. U. Taylor, M. Dupont-Nivet & D. Jerry. 2010. Population and family growth response to different rearing location, heritability, estimates and genotype* environment interaction in the silver-lip pearl oyster (Pinctada maxima). Aquaculture 304:1-6.
Minaur, J. 1969. Experiments on the artificial rearing of the larvae of Pinctada maxima (Jameson) Lamellibranchia. Aust. J. Mar. Freshw. Res. 20:175-187.
Moueza, M., O. Gros & L. Frenkial. 2006. Embryonic development and shell differentiation in Chione cancellata (Bivalvia: Veneridae): an ultrastructural analysis. Invertebr. Biol. 125:21-33.
O'Connor, W. A. & N. F. Lawler. 2004. Salinity and temperature tolerance of embryos and juveniles of the pearl oyster, Pinctada imbricata Roding. Aquaculture 22:493-506.
Peck, M. A., L. J. Buckley, L. M. O'Bryan, E. J. Davies & A. E. Lapolla. 2004. Efficacy of egg surface disinfectants in captive spawning Atlantic cod Gadus morhua L. and haddock Melanogrammus aeglefinus L. Aquacult. Res. 35:992-996.
Riquelme, C., P. Chavez, Y. Morales & G. Hayashida. 1994. Evidence for parental bacterial transfer to larvae in Argopecten purpuratus (Lamarck, 1819). Biol. Res. 27:129-134.
Rose, R. A. 1990. A manual for the artificial propagation of the silver-lip or goldlip pearl oyster Pinctada maxima Jameson from Western Australia. Perth: Western Australian Fisheries Department. 41 pp.
Rose, R. A. & S. B. Baker. 1994. Larval and spat culture of the western Australian silver or gold-lip pearl oyster, Pinctada maxima (Jameson) (Mollusca: Pteriidae). Aquaculture 126:35-50.
Southgate, P. C. 2008. Pearl oyster culture. In: P. C. Southgate & J. S. Lucas, editors. The pearl oyster: biology and culture. Oxford: Elsevier. pp. 231-272.
Southgate, P. C. & A. C. Beer. 1997. Hatchery and nursery culture of the black-lip pearl oyster (Pinctada margaritifera L.). J. Shellfish Res. 16:561-567.
Southgate, P. C., E. Strack, A. Hart, K. T. Wada, M. Monteforte, M. Carino, S. Langy, C. Lo, H. Acosta-Salmon & A. Wang. 2008. Exploitation and culture of major commercial species. In: P. C. Southgate & J. S. Lucas, editors. The pearl oyster: biology and culture. Oxford: Elsevier. pp. 303-355.
Southgate, P. C., J. J. Taylor & M. Ito. 1998. The effect of egg density on hatch rate of pearl oyster (Pinctada maxima and Pinctada margaritifera) larvae. Asian Fish. Sci. 10:265-268.
Stoeckel, J. A., D. K. Padilla, D. W. Schneider & C. R. Rehmann. 2004. Laboratory culture of Dreissena polymorpha larvae: spawning success, adult fecundity, and larval mortality patterns. Can. J. Zool. 82:1436-1443.
Strack, S. 2006. Natural pearls: pearl-producing molluscs. In: S. Strack, editor. Pearls. Stuttgart: Ruhle-Diebener-Verlag Publishing. pp. 41-112.
Teitelbaum, A. & P.N. Fale. 2008. Support for the Tongan pearl industry. SPC Pearl Oyster Inform. Bull. 18:11-14.
Utting, S. D. & P. F. Millican. 1997. Techniques for the hatchery conditioning of bivalve broodstocks and the subsequent effect on egg quality and larval viability. Aquaculture 155:45-54.
Victor, A. C. C., D. Kandasami, I. Jagadis, B. Ignatius, A. Chellum, G. Chitra, P. Villan & M. Rajkumar. 200l. Hatchery, seed production and nursery rearing of Indian pearl oyster Pinctada fucata (Gould) under onshore and offshore conditions at Mandapam, Tamil Nadu. In: N. G. Menon & P. P. Pillai, editors. Perspectives in mariculture. Cochin: The Marine Biological Association of India. pp. 241-250.
Wada, S. 1953. Biology of the silver-lip pearl oyster Pinctada maxima (Jameson). J. Artif. Fertil. Dev. 1:3-15.
Wada, K. T. & I. Temkin. 2008. Taxonomy and phylogeny. In: P. C. Southgate & J. S. Lucas, editors. The pearl oyster: biology and culture. London: Elsevier. pp. 37-76.
Walne, P. R. 1958. The importance of bacteria in laboratory experiments on rearing the larvae of Ostrea edulis (L.). J. Mar. Biol. Assoc. UK 37:415-425.
Yu, X. Y., M. F. Wang & F. L. Yie. 2000. Development and artificial propagation of Pteria (Magnavicula) penguin Roding. Nat. Sci. J. Hainan Univ. 18:265-269.
MATTHEW WASSNIG * AND PAUL C. SOUTHGATE
Pearl Oyster Research Group, School of Marine & Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia
* Corresponding author. E-mail: Matthew.Wassnig@jcu.edu.au
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|Author:||Wassnig, Matthew; Southgate, Paul C.|
|Publication:||Journal of Shellfish Research|
|Date:||Apr 1, 2011|
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