- Email: [email protected]
Hatchery culture of the winged pearl oyster, Pteria penguin, without living micro-algae
Aquaculture 451 (2016) 121–124
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Short communication
Hatchery culture of the winged pearl oyster, Pteria penguin, without living micro-algae Paul C. Southgate a,⁎, Andrew C. Beer b, Poasi Ngaluafe c a b c
Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore, QLD, Australia Government of Western Australia, Department of Regional Development, Perth, WA, Australia Aquaculture Section, Fisheries Division, Ministry of Agriculture and Food, Forests and Fisheries, Tonga
a r t i c l e
i n f o
Article history: Received 3 December 2014 Received in revised form 4 September 2015 Accepted 7 September 2015 Available online 10 September 2015 Keywords: Pearl oyster Pteria penguin Hatchery culture Micro-algae concentrates
a b s t r a c t This paper reports on successful hatchery production of the winged pearl oyster, Pteria penguin, without the use of live micro-algae. Larval nutrition was provided by commercially available micro-algae concentrates, Instant Algae® (“Isochrysis 1800®” and “Pavlova 1800®”, Reed Mariculture Inc., San Jose, CA, USA). Larvae were first transferred to settlement tanks on day 17 when their mean antero-posterior measurement (APM) was 240.2 ± 8.6 μm. Approximately 6.4% of larvae survived to day 17 and more than 700,000 eyed pediveligers were transferred to settlement tanks between day 17 and day 25. Approximately 33,000 spat were harvested from spat collectors on day 105, representing a survival rate of 4.7% from the eyed pediveliger stage. Growth and development of larvae in this study were superior to those reported in a prior study that used a ternary live micro-algae diet to feed P. penguin larvae. Our results indicate that the products used in this study proved nutritious for P. penguin larvae and supported normal growth and development through settlement. The use of commercially available micro-algae concentrates as a replacement for live micro-algae in pearl oyster hatcheries supports development of simplified larval rearing protocols, without live micro-algae culture, that are more appropriate to Pacific island nations. Statement of relevance This paper reports for the first time on successful hatchery production of pearl oysters without the use of live micro-algae. Successful replacement of live micro-algae with commercially available micro-algae concentrates as a larval food source supports development of simpler, cheaper hatchery facilities, and larval rearing protocols that are more appropriate to Pacific island nations. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The winged pearl oyster, Pteria penguin, is traditionally used to produce mabé or half-pearls in southern Japan (Southgate et al., 2008). This species was introduced to the Vava'u island group in northern Tonga in 1975 by the Tasaki Pearl Company of Japan (Fa'anunu and Manu, 1996) and a small industry reliant on natural spat collection became established. Unlike round pearl production, mabé production can be achieved by local people with minimal training and it supports an associated handicraft industry that provides significant livelihood benefits. The Tongan pearl industry has considerable potential for development. Japanese specialists visiting Vava'u in the mid-1990s, for example, estimated that an area of approximately 850 ha could be farmed for
⁎ Corresponding author. E-mail address: [email protected] (P.C. Southgate).
http://dx.doi.org/10.1016/j.aquaculture.2015.09.007 0044-8486/© 2015 Elsevier B.V. All rights reserved.
mabé production, generating potential annual revenues of around US$7.5 million (Yamamoto and Tanaka, 1997). A major impediment to the sustainability and expansion of the Tongan pearl industry is a reliable and adequate supply of oysters. Over recent years, poor recruitment of spat has encouraged farmers to harvest adult oysters from the wild, which has further impacted recruitment. Natural spat fall of P. penguin in Vava'u is now extremely limited and the industry relies on hatchery production. A research project was begun in 2007 to establish routine hatchery production of P. penguin and improve spat supply to Tongan pearl farmers. However, south Pacific island nations often lack the technical resources and skilled personnel required for successful hatchery operation, and production of appropriate quantities of high quality live micro-algae as a larval food source is a common bottleneck. Recent years have seen increased availability of commercially available ‘off-the-shelf’ food products that are designed to replace or supplement live aquaculture foods including micro-algae. A number of these
122
P.C. Southgate et al. / Aquaculture 451 (2016) 121–124
products, such as dried and concentrated micro-algae, have been developed specifically for bivalves. However, while some prior studies have assessed the potential of such products for pearl oyster larvae (e.g. Southgate et al., 1998; Teitelbaum and Fale, 2008), successful hatchery production of pearl oysters has not previously been reported without the use of live micro-algae. A range of phototrophically grown, highlyconcentrated marine micro-algae are now available commercially and have potential in many aspects of bivalve production including hatchery culture (Reed and Henry, 2014). This paper reports on successful hatchery production of P. penguin in Tonga using commercially available micro-algae concentrates as the sole food source. 2. Material and methods Adult oysters with a mean (±SE) dorso-ventral shell measurement of 192 ± 15 mm were sourced from pearl farmers and transported to the government aquaculture facility and hatchery at Sopu on the island of Tongatapu, where they were cleaned and placed into 1 μm filtered seawater (FSW). Spawning was induced using thermal stimulation following ‘cold conditioning’ (Southgate and Beer, 1997). Broodstock were held overnight in a small tank filled with lightly aerated FSW in an airconditioned room at 20–21 °C, and then placed directly into tanks containing FSW at 30–31 °C. Tanks were drained to expose the oysters to air and then refilled with FSW at 30–31 °C on a 60 min cycle; spawning began after four such cycles. Spawning individuals were removed into separate containers and were allowed to complete spawning. Fertilised eggs were collected on a 25 μm mesh screen and incubated at a density of 30 mL− 1 in gently aerated 500 L tanks containing FSW. Antibiotic (streptomycin sulphate) was added to egg incubation tanks at a concentration of 10 mg L−1. After 24 h, D-stage veliger larvae were removed from the incubation tanks using a 25 μm mesh screen, counted and placed into larval rearing tanks. 2.1. Larval rearing Four 500 L tanks and two 1000 L tanks were filled with FSW and stocked (on day 1) with 24 h old P. penguin veligers at a density of 2.75 mL−1. Tanks were provided with gentle aeration and were drained, washed and refilled with clean FSW every two days. Larvae were retained on a suitably sized mesh screen and held in 20 L plastic aquaria containing FSW, before being returned to the cleaned, newly refilled tank. Water temperature and salinity ranged from 24.5–30.1 °C and from 33.2–34.4 g L−1, respectively, during larval culture. Larvae were fed commercially available micro-algae concentrates, Instant Algae® (Reed Mariculture Inc., San Jose, CA, USA). The two
Fig. 1. Change in the daily ration of micro-algae concentrates fed to P. penguin larvae and post-larvae during the hatchery culture period. The ration was composed of a 1:1 mixture (by cell numbers) of ‘Isochrysis 1800®’ and ‘Pavlova 1800®’ (Reed Mariculture Inc., San Jose, CA, USA). The maximum ration provided to larval culture tanks was 20,000 cells mL−1 and larvae were transferred from larval culture tanks to settlement tanks on days 17, 19, 21, 23 and 25 post-fertilisation.
products used in this study were “Isochrysis 1800®” and “Pavlova 1800®,” which were obtained from an Australian distributor of the products. Concentrates were stored in their original bottles in a refrigerator at 4 °C for the duration of the study. Prior to use, a 5 mL aliquot of each concentrate was added to approximately 2 L of FSW in a separate container and gently hand-shaken to disperse the micro-algae cells. The cell density in each micro-algal suspension was determined using a haemocytometer and the volume needed to obtain the required ration in each larval tank was determined. Micro-algae suspensions were poured into larval culture tanks through a 20 μm mesh sieve to remove or break up any clumps of micro-algae cells that might be present. “Isochrysis 1800®” and “Pavlova 1800®” were fed at a 1:1 ratio (on the basis of cell numbers) as part of a total daily ration shown in Fig. 1. The maximum micro-algae cell density provided to larval culture tanks was 20,000 cells mL−1 (Fig. 1) and the daily ration was split between a morning and evening feed. 2.2. Settlement and nursery culture On days 17, 19, 21, 23 and 25 post-fertilisation, larvae large enough to be retained on a 170 μm mesh screen were removed from larval culture tanks and placed into 500 L settlement tanks containing strongly aerated FSW. Spat collectors measuring approximately 30 × 15 cm and consisting of an outer 5 mm pore size mesh bag filled with 0.5 m2 of 50% woven shade mesh were suspended in the settlement tanks from a plastic frame placed on top of the tank. Instant Algae® (prepared as described above) was added to the settlement tanks at the rate shown in Fig. 1. Water in the settlement tanks was completely exchanged on a daily basis using periodic flow-through, and water temperature in the settlement tanks ranged from 22.8 to 29.0 °C during the study. Spat collectors were removed from the settlement tanks on day 30 and placed inside plastic mesh trays (55 × 30 × 10 cm) with lids. Four collectors were tied into each tray. Trays were then weighted and transferred to the ocean where they were suspended from a surface long line at Sopu (21°07′02.19″S 175°13′20.92″W) at a depth of 6 m. Surviving spat were removed from spat collectors and counted on day 105. 3. Results Of the 70 broodstock induced for spawning, 20 females collectively spawned 95 million eggs. Fertilised eggs had a mean (±SE) diameter of 48.7 ± 1.7 μm. Hatch rate was around 20% and yielded 15 million D-stage larvae. Changes in the mean antero-posterior measurement (APM) of P. penguin larvae during development are shown in Fig. 2. Larvae had reached the D-stage by 20–24 h after fertilisation and had a mean (± SE) APM of 75.3 ± 1.6 μm. Umbonal larvae were first seen on day 7, however the majority of larvae were umbonal on day 9 when the mean APM was 121.5 ± 3.1 μm. The growth of umbonal larvae
Fig. 2. Change in mean (±SE) antero-posterior measurement (APM) of P. penguin larvae during hatchery culture when fed micro-algae concentrates showing line of best fit (y = 0.6038x2 − 1.4123x + 83.359, R2 = 0.9897).
P.C. Southgate et al. / Aquaculture 451 (2016) 121–124 Table 1 Comparison of culture conditions and the timing of developmental stages of P. penguin larvae fed live micro-algae and micro-algae concentrates. Culture conditions and larval development Temperature (°C) Diet
26–28 Live micro-algae: 1:1 mixture of T-ISO and Pavlova sp. until day 12, then 1:1:1 mixture of T-ISO, Pavlova sp. and Chaetoceros muelleri; daily ration 1800–24,000 cells mL−1.
24.5–30.1 Micro-algae concentrates: 1:1 mixture of “Isochrysis 1800®” and “Pavlova 1800®”; daily ration 1000–20,000 cells mL−1
Stage
Time (hours/days)
Time (hours/days)
Size (APM)
D-stage Early Umbone Umbone Eye spot Pediveliger Source
18–22 h 83 8 days 107 12 days 129 20 days 226 22 days 233 Wassnig and Southgate (2012)
20–24 h 7 days 9 days 15 days 17 days This study
75 105 121 196 240
a
Size (APM)a
Antero-posterior measurement.
was rapid and the mean APM was 196.1 ± 6.2 μm on day 15 (Fig. 2) when ‘eyed’ larvae were first seen. The first larvae were removed into settlement tanks on day 17 when the mean APM was 240.2 ± 8.6 (Fig. 2). Larval mortality was steady during the first half of the larval rearing period and survival was 26% by day 9. However, only 11% of larvae survived to day 15 and 6.4% to day 17. A total of more than 700,000 eyed pediveligers were removed from larval culture tanks and placed into settlement tanks between days 17 and 25. When nursery trays were opened for the final count and grading of juveniles, few juveniles remained associated with the settlement substrate; most had relocated and attached directly to the inside surfaces of the plastic trays holding the spat collectors. A total of 33,000 spat were removed from collectors, representing a survival rate of 4.7% from the eyed pediveliger stage. They had a mean (± SE) dorsoventral measurement (DVM) and APM of 14.2 (± 2.0) mm and 27.4 (±3.5) mm, respectively (n = 100). 4. Discussion While acknowledging earlier studies in Japan and China to develop hatchery culture methods for P. penguin (e.g. Shinmura et al., 1959; Kozuka et al., 1961; Yu et al., 2000; Liang et al., 2001), similar reports in the English language literature are very limited. Spawning was readily induced using the thermal stimulation method used successfully with other species of pearl oysters (e.g. Southgate and Beer, 1997) and our results also show that the general larval culture techniques developed for other species of pearl oysters (Southgate, 2008) are appropriate for P. penguin. Wassnig and Southgate (2012) reported in detail on embryonic and larval development of P. penguin, which is similar to that described for other species of pearl oysters (Rose and Baker, 1994; Araya-Nunez and Ganning, 1995; Doroudi and Southgate, 2003) from both a morphological and timing perspective. However, P. penguin larvae are readily distinguished from those of Pinctada spp. by shell shape which is skewed towards the posterior end of the deepest shell valve (Wassnig and Southgate, 2012). Although earlier research in Tonga reported successful hatchery production of P. penguin following significant partial replacement of live micro-algae with commercial micro-algae concentrates (Teitelbaum and Fale, 2008), this is the first time that successful larval rearing of pearl oyster larvae has been reported without the use of live microalgae. While there have been previous reports of the successful use of commercially available micro-algae concentrates for larviculture of bivalves (Rikard and Walton, 2012), and their use as a total replacement for live micro-algae in bivalve hatcheries (Reed and Henry, 2014), we are unaware of any reports in peer reviewed literature, of hatchery
123
production of a bivalve at an industry-relevant scale, using only micro-algae concentrates. The products used in this study proved nutritious for P. penguin larvae and supported normal growth and development through settlement. The larvae in this study showed more rapid development than that reported for P. penguin larvae fed a live ternary micro-algae diet (Table 1) in an earlier study in Australia (Wassnig and Southgate, 2012). Additionally, Wassnig and Southgate (2012) reported a mean increase in APM of 7.2 mm day−1 over a 22 day larval culture period compared to an average daily increase in APM of 10.3 μm over the 17 day larval culture period in this study. Although intact, individual cells of Instant Algae® are non-motile and negatively buoyant. Despite this they remain in the water column for an extended period, and this can be assisted by gentle aeration in culture tanks. Uneaten algae cells eventually settled onto the bottom of culture tanks and were evident on mesh screens when tanks were drained. Careful observation of these factors as well as micro-algae cells evident in the water column, allows adjustment of feeding rates as required. Under the conditions of this study, a micro-algae density of 20,000 cells mL−1 was considered appropriate as the highest larval ration. At times during this study small clumps or strands of microalgae cells were evident at the water's surface in larval culture tanks. The cause of this is unknown and the clumps were removed using a fine mesh hand scoop. However, significant settling, clumping or flocking of micro-algae cells in larval culture tanks was not seen. The results of this study show that live micro-algae are not required for successful hatchery culture of P. penguin. Indeed, successful hatchery production of P. penguin spat, using commercially available micro-algae concentrates as the sole larval food source, has occurred routinely in Tonga two or three times a year since 2008. Hatchery production in Tonga occurs between November and May and uses farm-collected oyster broodstock. Larval survival and spat production vary between hatchery runs with survival of eyed larvae to harvested spat (3.5 months old) ranging from around 4% to 15%. The micro-algae concentrates used in this study provide appropriate nutrition to P. penguin larvae and support spat production at a level that satisfies current industry demand. Our results provide a basis for further research to investigate the relative nutritional value of other Instant Algae® products, such as diatoms, with a view to optimising a dietary protocol for P. penguin larvae based on these products. This, in turn, will support improved growth and survival of larvae and increased hatchery production. A considerable proportion of the infrastructure and cost associated with establishing a pearl oyster hatchery in the Pacific are attributed to micro-algae culture (Ito, 1999). The use of commercially available micro-algae concentrates as a replacement for live micro-algae in pearl oyster hatcheries supports development of simpler, cheaper hatchery facilities, and larval rearing protocols that are more appropriate to Pacific island nations.
Acknowledgements This study was funded by the Australian Centre for International Agricultural Research (ACIAR) through project FIS/2009/057 “Pearl industry development in the western Pacific” for which the University of the Sunshine Coast (USC) is the commissioned organisation. We thank Vea Kava and Martin Finau (MAFFF, Tonga), and Scott Mactier, Matt Wassnig (James Cook University) and Max Wingfield (USC) for technical support, and Dr Eric Henry of Reed Mariculture Inc. for valuable comments on the manuscript.
References Araya-Nunez, O., Ganning, B., 1995. Embryonic development, larval culture, and settling of the American pearl-oyster (Pteria sterna, Gould) spat. Calif. Fish Game 81, 10–21. Doroudi, M., Southgate, P.C., 2003. Embryonic and larval development of Pinctada margaritifera (Linnaeus, 1758). Molluscan Res. 23, 101–107.
124
P.C. Southgate et al. / Aquaculture 451 (2016) 121–124
Fa'anunu, U., Manu, N., 1996. Aquaculture development in Tonga. Integrated Fisheries Survey Report. Ministry of Fisheries and Japan International Cooperation Agency (JICA), Nuku'alofa, Tonga, pp. 70–75. Ito, M., 1999. Technical guidance on pearl hatchery development in the Kingdom of Tonga. South Pacific Aquaculture Development Project (phase II). Food and Agriculture Organisation of the United Nations. Field Document No. 10. Suva, Fiji. Kozuka, M., Yamaguchi, A., Deshimaru, O., Toyoda, M., 1961. Studies on the propagation of Mabe Pteria penguin (Röding) [IV] larval rearing and growth of spat (in Japanese) Investigational Report of the Oshima Branch, Fisheries Experimental Station, Kagoshima Prefecture, Japan, pp. 1–12. Liang, F., Fu, S., Yu, X., 2001. Study on the method of culture and spawning induction of Pteria penguin. Trans. Oceanol. Limnol. 2, 41–45. Reed, T., Henry, E., 2014. The case for concentrates. Hatch. Int. 15 (4), 24–25. Rikard, F.S., Walton, W.C., 2012. Use of micro-algae concentrates for rearing oyster larvae, Crassostrea virginica. Mississippi–Alabama Sea Grant Publication No.: MASGP-12-048. Rose, R.A., Baker, S.B., 1994. Larval and spat culture of the Western Australian silver- or goldlip pearl oyster, Pinctada maxima Jameson (Mollusca: Pteriidae). Aquaculture 126, 35–50. Shinmura, I., Toyoda, M., Kozuka, M., 1959. Studies on the propagation of Mabe Pteria penguin (Röding) [III] larval rearing and development (in Japanese) Investigational Report of the Oshima Branch, Fisheries Experimental Station, Kagoshima Prefecture, Japan, pp. 1–13. Southgate, P.C., 2008. Pearl oyster culture. In: Southgate, P.C., Lucas, J.S. (Eds.), The Pearl Oyster. Elsevier, Oxford, pp. 231–272.
Southgate, P.C., Beer, A.C., 1997. Hatchery and early nursery culture of the blacklip pearl oyster Pinctada margaritifera (L.). J Shellfish Res 16 (2), 561–568. Southgate, P.C., Beer, A.C., Duncan, P.F., Tamburri, R., 1998. Assessment of the nutritional value of three species of tropical micro-algae, dried Tetraselmis and a yeast-based diet for larvae of the blacklip pearl oyster, Pinctada margaritifera (L.). Aquaculture 162, 247–257. Southgate, P.C., Strack, E., Hart, A., Wada, K.T., Monteforte, M., Carino, M., Langy, S., Lo, C., Acosta-Salmon, H., Wang, A., 2008. Exploitation and culture of major commercial species. In: Southgate, P.C., Lucas, J.S. (Eds.), The Pearl Oyster. Elsevier, Oxford, pp. 303–355. Teitelbaum, A., Fale, P., 2008. Support for the Tongan pearl industry. SPC Pearl Oyster Inf. Bull. 18, 11–14. Wassnig, M., Southgate, P.C., 2012. Embryonic and larval development of Pteria penguin (Röding, 1798) (Bivalvia: Pteriidae). J Molluscan Stud 78 (1), 134–141. Yamamoto, T., Tanaka, H., 1997. Potential of commercial development of mabe pearl farming in Vava'u islands, Kingdom of Tonga (field document no. 5). South Pacific Aquaculture Development Project (Phase II), Food and Agriculture Organization of the United Nations (GCP/RAS/116/JPN), Suva, Fiji (22 pp.). Yu, X., Wang, M., Yie, F., 2000. Development and artificial propagation of Pteria (Magnavicula) penguin Roding. Natural Science Journal of Hainan University 3, 54–67.
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Short communication
Hatchery culture of the winged pearl oyster, Pteria penguin, without living micro-algae Paul C. Southgate a,⁎, Andrew C. Beer b, Poasi Ngaluafe c a b c
Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore, QLD, Australia Government of Western Australia, Department of Regional Development, Perth, WA, Australia Aquaculture Section, Fisheries Division, Ministry of Agriculture and Food, Forests and Fisheries, Tonga
a r t i c l e
i n f o
Article history: Received 3 December 2014 Received in revised form 4 September 2015 Accepted 7 September 2015 Available online 10 September 2015 Keywords: Pearl oyster Pteria penguin Hatchery culture Micro-algae concentrates
a b s t r a c t This paper reports on successful hatchery production of the winged pearl oyster, Pteria penguin, without the use of live micro-algae. Larval nutrition was provided by commercially available micro-algae concentrates, Instant Algae® (“Isochrysis 1800®” and “Pavlova 1800®”, Reed Mariculture Inc., San Jose, CA, USA). Larvae were first transferred to settlement tanks on day 17 when their mean antero-posterior measurement (APM) was 240.2 ± 8.6 μm. Approximately 6.4% of larvae survived to day 17 and more than 700,000 eyed pediveligers were transferred to settlement tanks between day 17 and day 25. Approximately 33,000 spat were harvested from spat collectors on day 105, representing a survival rate of 4.7% from the eyed pediveliger stage. Growth and development of larvae in this study were superior to those reported in a prior study that used a ternary live micro-algae diet to feed P. penguin larvae. Our results indicate that the products used in this study proved nutritious for P. penguin larvae and supported normal growth and development through settlement. The use of commercially available micro-algae concentrates as a replacement for live micro-algae in pearl oyster hatcheries supports development of simplified larval rearing protocols, without live micro-algae culture, that are more appropriate to Pacific island nations. Statement of relevance This paper reports for the first time on successful hatchery production of pearl oysters without the use of live micro-algae. Successful replacement of live micro-algae with commercially available micro-algae concentrates as a larval food source supports development of simpler, cheaper hatchery facilities, and larval rearing protocols that are more appropriate to Pacific island nations. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The winged pearl oyster, Pteria penguin, is traditionally used to produce mabé or half-pearls in southern Japan (Southgate et al., 2008). This species was introduced to the Vava'u island group in northern Tonga in 1975 by the Tasaki Pearl Company of Japan (Fa'anunu and Manu, 1996) and a small industry reliant on natural spat collection became established. Unlike round pearl production, mabé production can be achieved by local people with minimal training and it supports an associated handicraft industry that provides significant livelihood benefits. The Tongan pearl industry has considerable potential for development. Japanese specialists visiting Vava'u in the mid-1990s, for example, estimated that an area of approximately 850 ha could be farmed for
⁎ Corresponding author. E-mail address: [email protected] (P.C. Southgate).
http://dx.doi.org/10.1016/j.aquaculture.2015.09.007 0044-8486/© 2015 Elsevier B.V. All rights reserved.
mabé production, generating potential annual revenues of around US$7.5 million (Yamamoto and Tanaka, 1997). A major impediment to the sustainability and expansion of the Tongan pearl industry is a reliable and adequate supply of oysters. Over recent years, poor recruitment of spat has encouraged farmers to harvest adult oysters from the wild, which has further impacted recruitment. Natural spat fall of P. penguin in Vava'u is now extremely limited and the industry relies on hatchery production. A research project was begun in 2007 to establish routine hatchery production of P. penguin and improve spat supply to Tongan pearl farmers. However, south Pacific island nations often lack the technical resources and skilled personnel required for successful hatchery operation, and production of appropriate quantities of high quality live micro-algae as a larval food source is a common bottleneck. Recent years have seen increased availability of commercially available ‘off-the-shelf’ food products that are designed to replace or supplement live aquaculture foods including micro-algae. A number of these
122
P.C. Southgate et al. / Aquaculture 451 (2016) 121–124
products, such as dried and concentrated micro-algae, have been developed specifically for bivalves. However, while some prior studies have assessed the potential of such products for pearl oyster larvae (e.g. Southgate et al., 1998; Teitelbaum and Fale, 2008), successful hatchery production of pearl oysters has not previously been reported without the use of live micro-algae. A range of phototrophically grown, highlyconcentrated marine micro-algae are now available commercially and have potential in many aspects of bivalve production including hatchery culture (Reed and Henry, 2014). This paper reports on successful hatchery production of P. penguin in Tonga using commercially available micro-algae concentrates as the sole food source. 2. Material and methods Adult oysters with a mean (±SE) dorso-ventral shell measurement of 192 ± 15 mm were sourced from pearl farmers and transported to the government aquaculture facility and hatchery at Sopu on the island of Tongatapu, where they were cleaned and placed into 1 μm filtered seawater (FSW). Spawning was induced using thermal stimulation following ‘cold conditioning’ (Southgate and Beer, 1997). Broodstock were held overnight in a small tank filled with lightly aerated FSW in an airconditioned room at 20–21 °C, and then placed directly into tanks containing FSW at 30–31 °C. Tanks were drained to expose the oysters to air and then refilled with FSW at 30–31 °C on a 60 min cycle; spawning began after four such cycles. Spawning individuals were removed into separate containers and were allowed to complete spawning. Fertilised eggs were collected on a 25 μm mesh screen and incubated at a density of 30 mL− 1 in gently aerated 500 L tanks containing FSW. Antibiotic (streptomycin sulphate) was added to egg incubation tanks at a concentration of 10 mg L−1. After 24 h, D-stage veliger larvae were removed from the incubation tanks using a 25 μm mesh screen, counted and placed into larval rearing tanks. 2.1. Larval rearing Four 500 L tanks and two 1000 L tanks were filled with FSW and stocked (on day 1) with 24 h old P. penguin veligers at a density of 2.75 mL−1. Tanks were provided with gentle aeration and were drained, washed and refilled with clean FSW every two days. Larvae were retained on a suitably sized mesh screen and held in 20 L plastic aquaria containing FSW, before being returned to the cleaned, newly refilled tank. Water temperature and salinity ranged from 24.5–30.1 °C and from 33.2–34.4 g L−1, respectively, during larval culture. Larvae were fed commercially available micro-algae concentrates, Instant Algae® (Reed Mariculture Inc., San Jose, CA, USA). The two
Fig. 1. Change in the daily ration of micro-algae concentrates fed to P. penguin larvae and post-larvae during the hatchery culture period. The ration was composed of a 1:1 mixture (by cell numbers) of ‘Isochrysis 1800®’ and ‘Pavlova 1800®’ (Reed Mariculture Inc., San Jose, CA, USA). The maximum ration provided to larval culture tanks was 20,000 cells mL−1 and larvae were transferred from larval culture tanks to settlement tanks on days 17, 19, 21, 23 and 25 post-fertilisation.
products used in this study were “Isochrysis 1800®” and “Pavlova 1800®,” which were obtained from an Australian distributor of the products. Concentrates were stored in their original bottles in a refrigerator at 4 °C for the duration of the study. Prior to use, a 5 mL aliquot of each concentrate was added to approximately 2 L of FSW in a separate container and gently hand-shaken to disperse the micro-algae cells. The cell density in each micro-algal suspension was determined using a haemocytometer and the volume needed to obtain the required ration in each larval tank was determined. Micro-algae suspensions were poured into larval culture tanks through a 20 μm mesh sieve to remove or break up any clumps of micro-algae cells that might be present. “Isochrysis 1800®” and “Pavlova 1800®” were fed at a 1:1 ratio (on the basis of cell numbers) as part of a total daily ration shown in Fig. 1. The maximum micro-algae cell density provided to larval culture tanks was 20,000 cells mL−1 (Fig. 1) and the daily ration was split between a morning and evening feed. 2.2. Settlement and nursery culture On days 17, 19, 21, 23 and 25 post-fertilisation, larvae large enough to be retained on a 170 μm mesh screen were removed from larval culture tanks and placed into 500 L settlement tanks containing strongly aerated FSW. Spat collectors measuring approximately 30 × 15 cm and consisting of an outer 5 mm pore size mesh bag filled with 0.5 m2 of 50% woven shade mesh were suspended in the settlement tanks from a plastic frame placed on top of the tank. Instant Algae® (prepared as described above) was added to the settlement tanks at the rate shown in Fig. 1. Water in the settlement tanks was completely exchanged on a daily basis using periodic flow-through, and water temperature in the settlement tanks ranged from 22.8 to 29.0 °C during the study. Spat collectors were removed from the settlement tanks on day 30 and placed inside plastic mesh trays (55 × 30 × 10 cm) with lids. Four collectors were tied into each tray. Trays were then weighted and transferred to the ocean where they were suspended from a surface long line at Sopu (21°07′02.19″S 175°13′20.92″W) at a depth of 6 m. Surviving spat were removed from spat collectors and counted on day 105. 3. Results Of the 70 broodstock induced for spawning, 20 females collectively spawned 95 million eggs. Fertilised eggs had a mean (±SE) diameter of 48.7 ± 1.7 μm. Hatch rate was around 20% and yielded 15 million D-stage larvae. Changes in the mean antero-posterior measurement (APM) of P. penguin larvae during development are shown in Fig. 2. Larvae had reached the D-stage by 20–24 h after fertilisation and had a mean (± SE) APM of 75.3 ± 1.6 μm. Umbonal larvae were first seen on day 7, however the majority of larvae were umbonal on day 9 when the mean APM was 121.5 ± 3.1 μm. The growth of umbonal larvae
Fig. 2. Change in mean (±SE) antero-posterior measurement (APM) of P. penguin larvae during hatchery culture when fed micro-algae concentrates showing line of best fit (y = 0.6038x2 − 1.4123x + 83.359, R2 = 0.9897).
P.C. Southgate et al. / Aquaculture 451 (2016) 121–124 Table 1 Comparison of culture conditions and the timing of developmental stages of P. penguin larvae fed live micro-algae and micro-algae concentrates. Culture conditions and larval development Temperature (°C) Diet
26–28 Live micro-algae: 1:1 mixture of T-ISO and Pavlova sp. until day 12, then 1:1:1 mixture of T-ISO, Pavlova sp. and Chaetoceros muelleri; daily ration 1800–24,000 cells mL−1.
24.5–30.1 Micro-algae concentrates: 1:1 mixture of “Isochrysis 1800®” and “Pavlova 1800®”; daily ration 1000–20,000 cells mL−1
Stage
Time (hours/days)
Time (hours/days)
Size (APM)
D-stage Early Umbone Umbone Eye spot Pediveliger Source
18–22 h 83 8 days 107 12 days 129 20 days 226 22 days 233 Wassnig and Southgate (2012)
20–24 h 7 days 9 days 15 days 17 days This study
75 105 121 196 240
a
Size (APM)a
Antero-posterior measurement.
was rapid and the mean APM was 196.1 ± 6.2 μm on day 15 (Fig. 2) when ‘eyed’ larvae were first seen. The first larvae were removed into settlement tanks on day 17 when the mean APM was 240.2 ± 8.6 (Fig. 2). Larval mortality was steady during the first half of the larval rearing period and survival was 26% by day 9. However, only 11% of larvae survived to day 15 and 6.4% to day 17. A total of more than 700,000 eyed pediveligers were removed from larval culture tanks and placed into settlement tanks between days 17 and 25. When nursery trays were opened for the final count and grading of juveniles, few juveniles remained associated with the settlement substrate; most had relocated and attached directly to the inside surfaces of the plastic trays holding the spat collectors. A total of 33,000 spat were removed from collectors, representing a survival rate of 4.7% from the eyed pediveliger stage. They had a mean (± SE) dorsoventral measurement (DVM) and APM of 14.2 (± 2.0) mm and 27.4 (±3.5) mm, respectively (n = 100). 4. Discussion While acknowledging earlier studies in Japan and China to develop hatchery culture methods for P. penguin (e.g. Shinmura et al., 1959; Kozuka et al., 1961; Yu et al., 2000; Liang et al., 2001), similar reports in the English language literature are very limited. Spawning was readily induced using the thermal stimulation method used successfully with other species of pearl oysters (e.g. Southgate and Beer, 1997) and our results also show that the general larval culture techniques developed for other species of pearl oysters (Southgate, 2008) are appropriate for P. penguin. Wassnig and Southgate (2012) reported in detail on embryonic and larval development of P. penguin, which is similar to that described for other species of pearl oysters (Rose and Baker, 1994; Araya-Nunez and Ganning, 1995; Doroudi and Southgate, 2003) from both a morphological and timing perspective. However, P. penguin larvae are readily distinguished from those of Pinctada spp. by shell shape which is skewed towards the posterior end of the deepest shell valve (Wassnig and Southgate, 2012). Although earlier research in Tonga reported successful hatchery production of P. penguin following significant partial replacement of live micro-algae with commercial micro-algae concentrates (Teitelbaum and Fale, 2008), this is the first time that successful larval rearing of pearl oyster larvae has been reported without the use of live microalgae. While there have been previous reports of the successful use of commercially available micro-algae concentrates for larviculture of bivalves (Rikard and Walton, 2012), and their use as a total replacement for live micro-algae in bivalve hatcheries (Reed and Henry, 2014), we are unaware of any reports in peer reviewed literature, of hatchery
123
production of a bivalve at an industry-relevant scale, using only micro-algae concentrates. The products used in this study proved nutritious for P. penguin larvae and supported normal growth and development through settlement. The larvae in this study showed more rapid development than that reported for P. penguin larvae fed a live ternary micro-algae diet (Table 1) in an earlier study in Australia (Wassnig and Southgate, 2012). Additionally, Wassnig and Southgate (2012) reported a mean increase in APM of 7.2 mm day−1 over a 22 day larval culture period compared to an average daily increase in APM of 10.3 μm over the 17 day larval culture period in this study. Although intact, individual cells of Instant Algae® are non-motile and negatively buoyant. Despite this they remain in the water column for an extended period, and this can be assisted by gentle aeration in culture tanks. Uneaten algae cells eventually settled onto the bottom of culture tanks and were evident on mesh screens when tanks were drained. Careful observation of these factors as well as micro-algae cells evident in the water column, allows adjustment of feeding rates as required. Under the conditions of this study, a micro-algae density of 20,000 cells mL−1 was considered appropriate as the highest larval ration. At times during this study small clumps or strands of microalgae cells were evident at the water's surface in larval culture tanks. The cause of this is unknown and the clumps were removed using a fine mesh hand scoop. However, significant settling, clumping or flocking of micro-algae cells in larval culture tanks was not seen. The results of this study show that live micro-algae are not required for successful hatchery culture of P. penguin. Indeed, successful hatchery production of P. penguin spat, using commercially available micro-algae concentrates as the sole larval food source, has occurred routinely in Tonga two or three times a year since 2008. Hatchery production in Tonga occurs between November and May and uses farm-collected oyster broodstock. Larval survival and spat production vary between hatchery runs with survival of eyed larvae to harvested spat (3.5 months old) ranging from around 4% to 15%. The micro-algae concentrates used in this study provide appropriate nutrition to P. penguin larvae and support spat production at a level that satisfies current industry demand. Our results provide a basis for further research to investigate the relative nutritional value of other Instant Algae® products, such as diatoms, with a view to optimising a dietary protocol for P. penguin larvae based on these products. This, in turn, will support improved growth and survival of larvae and increased hatchery production. A considerable proportion of the infrastructure and cost associated with establishing a pearl oyster hatchery in the Pacific are attributed to micro-algae culture (Ito, 1999). The use of commercially available micro-algae concentrates as a replacement for live micro-algae in pearl oyster hatcheries supports development of simpler, cheaper hatchery facilities, and larval rearing protocols that are more appropriate to Pacific island nations.
Acknowledgements This study was funded by the Australian Centre for International Agricultural Research (ACIAR) through project FIS/2009/057 “Pearl industry development in the western Pacific” for which the University of the Sunshine Coast (USC) is the commissioned organisation. We thank Vea Kava and Martin Finau (MAFFF, Tonga), and Scott Mactier, Matt Wassnig (James Cook University) and Max Wingfield (USC) for technical support, and Dr Eric Henry of Reed Mariculture Inc. for valuable comments on the manuscript.
References Araya-Nunez, O., Ganning, B., 1995. Embryonic development, larval culture, and settling of the American pearl-oyster (Pteria sterna, Gould) spat. Calif. Fish Game 81, 10–21. Doroudi, M., Southgate, P.C., 2003. Embryonic and larval development of Pinctada margaritifera (Linnaeus, 1758). Molluscan Res. 23, 101–107.
124
P.C. Southgate et al. / Aquaculture 451 (2016) 121–124
Fa'anunu, U., Manu, N., 1996. Aquaculture development in Tonga. Integrated Fisheries Survey Report. Ministry of Fisheries and Japan International Cooperation Agency (JICA), Nuku'alofa, Tonga, pp. 70–75. Ito, M., 1999. Technical guidance on pearl hatchery development in the Kingdom of Tonga. South Pacific Aquaculture Development Project (phase II). Food and Agriculture Organisation of the United Nations. Field Document No. 10. Suva, Fiji. Kozuka, M., Yamaguchi, A., Deshimaru, O., Toyoda, M., 1961. Studies on the propagation of Mabe Pteria penguin (Röding) [IV] larval rearing and growth of spat (in Japanese) Investigational Report of the Oshima Branch, Fisheries Experimental Station, Kagoshima Prefecture, Japan, pp. 1–12. Liang, F., Fu, S., Yu, X., 2001. Study on the method of culture and spawning induction of Pteria penguin. Trans. Oceanol. Limnol. 2, 41–45. Reed, T., Henry, E., 2014. The case for concentrates. Hatch. Int. 15 (4), 24–25. Rikard, F.S., Walton, W.C., 2012. Use of micro-algae concentrates for rearing oyster larvae, Crassostrea virginica. Mississippi–Alabama Sea Grant Publication No.: MASGP-12-048. Rose, R.A., Baker, S.B., 1994. Larval and spat culture of the Western Australian silver- or goldlip pearl oyster, Pinctada maxima Jameson (Mollusca: Pteriidae). Aquaculture 126, 35–50. Shinmura, I., Toyoda, M., Kozuka, M., 1959. Studies on the propagation of Mabe Pteria penguin (Röding) [III] larval rearing and development (in Japanese) Investigational Report of the Oshima Branch, Fisheries Experimental Station, Kagoshima Prefecture, Japan, pp. 1–13. Southgate, P.C., 2008. Pearl oyster culture. In: Southgate, P.C., Lucas, J.S. (Eds.), The Pearl Oyster. Elsevier, Oxford, pp. 231–272.
Southgate, P.C., Beer, A.C., 1997. Hatchery and early nursery culture of the blacklip pearl oyster Pinctada margaritifera (L.). J Shellfish Res 16 (2), 561–568. Southgate, P.C., Beer, A.C., Duncan, P.F., Tamburri, R., 1998. Assessment of the nutritional value of three species of tropical micro-algae, dried Tetraselmis and a yeast-based diet for larvae of the blacklip pearl oyster, Pinctada margaritifera (L.). Aquaculture 162, 247–257. Southgate, P.C., Strack, E., Hart, A., Wada, K.T., Monteforte, M., Carino, M., Langy, S., Lo, C., Acosta-Salmon, H., Wang, A., 2008. Exploitation and culture of major commercial species. In: Southgate, P.C., Lucas, J.S. (Eds.), The Pearl Oyster. Elsevier, Oxford, pp. 303–355. Teitelbaum, A., Fale, P., 2008. Support for the Tongan pearl industry. SPC Pearl Oyster Inf. Bull. 18, 11–14. Wassnig, M., Southgate, P.C., 2012. Embryonic and larval development of Pteria penguin (Röding, 1798) (Bivalvia: Pteriidae). J Molluscan Stud 78 (1), 134–141. Yamamoto, T., Tanaka, H., 1997. Potential of commercial development of mabe pearl farming in Vava'u islands, Kingdom of Tonga (field document no. 5). South Pacific Aquaculture Development Project (Phase II), Food and Agriculture Organization of the United Nations (GCP/RAS/116/JPN), Suva, Fiji (22 pp.). Yu, X., Wang, M., Yie, F., 2000. Development and artificial propagation of Pteria (Magnavicula) penguin Roding. Natural Science Journal of Hainan University 3, 54–67.