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Effect of temperature on growth and survival of Crassostrea corteziensis spat during late-nursery culturing at the hatchery
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Aquaculture 272 (2007) 417 – 422 www.elsevier.com/locate/aqua-online
Effect of temperature on growth and survival of Crassostrea corteziensis spat during late-nursery culturing at the hatchery Jorge I. Cáceres-Puig, Fernando Abasolo-Pacheco, José M. Mazón-Suastegui, Alfonso N. Maeda-Martínez, Pedro E. Saucedo ⁎ Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S., 23090, Mexico Received 2 March 2007; received in revised form 20 June 2007; accepted 21 June 2007
Abstract Nine temperatures (16, 18, 20, 22, 24, 26, 28, 30, and 32 °C) within the natural range of distribution of the Cortez oyster Crassostrea corteziensis were tested in a first experiment to determine the optimal temperature for growth and survival. Based on these results, a second study assessed two temperatures above this range (34 and 36 °C) to determine upper median lethal temperature for the species. The species was thermo-tolerant between 16–32 °C, grew faster and larger at 24 to 30 °C, and had optimal growth at 28–30 °C. The lower tolerance of the species appears far from the lowest value tested (16 °C). In contrast, the upper tolerance temperature was near 32 °C, since 100% spat mortality occurred within 96 h at 34 and 36 °C. These results are being used to develop a protocol for large-scale hatchery culture of the species in Mexico. © 2007 Published by Elsevier B.V. Keywords: Mexico; Oyster; Spat; Temperature; Nursery culture; Hatchery
1. Introduction In marine bivalves, temperature is recognized as one of the fundamental exogenous factors influencing most aspects of the ecology and biology of the species (see reviews by Kinne, 1971 and Shumway, 1982). It limits the general distribution of a species within large geographic areas, as well as specific habitat occupation between diverse communities or populations. Temperature also regulates many physiological functions of organisms, such as cilia activity during filter-feeding, cardiac and growth rates, gonad development and reproductive ⁎ Corresponding author. Tel.: +52 612 123 8464; fax: +52 612 125 3625. E-mail address: [email protected] (P.E. Saucedo). 0044-8486/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.aquaculture.2007.06.030
output, energy budget, and metabolic activity of enzymes (Widdows, 1973; Bayne et al., 1976; Newell and Branch, 1980; Shpigel et al., 1992; Barber and Blake, 2006). Additionally, changes in temperature affect survival of larvae, juveniles, and adults of most marine bivalve species, not only in wild populations, but also in laboratory experiments (Holland, 1978). Among the diverse marine bivalve species inhabiting the Pacific Ocean, the Cortez oyster Crassostrea corteziensis (Hertlein, 1951), whose distribution covers most of the tropical and subtropical coasts from Mexico to Peru, is of particular interest (Stuardo and Martínez, 1975; Mazón-Suástegui, 1996; Chávez-Villalba et al., 2005). In the states of Sonora and Sinaloa in Mexico, the species has been overexploited and most natural beds show severe signs of depletion or even virtual extinction
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(Hoyos-Chairez and Robles-Mungaray, 1990; ChávezVillalba et al., 2005). Consequently, the oyster industry in the northwest coastal Mexico has concentrated its efforts on cultivating the Pacific oyster C. gigas (Thunberg, 1793). However, this species has a natural temperate distribution, and although it has been successfully introduced to Mexico decades ago, it has suffered large die-offs in recent years. Given this situation, C. corteziensis has been receiving great deal of attention because of its relatively low production costs, excellent taste, and high nutritional value. Currently, the incipient fishery developed with this species does not provide the numbers of oysters demanded by the local and national markets, and therefore, scientific improvement of cultivation methods is essential in the short future (Mazón-Suástegui et al., 2001; Chávez-Villalba et al., 2005). So far, there is some understanding of the ecology and biology of the species, including population dynamics (Stuardo and Martínez, 1975), changes in tissue chemical composition (Páez-Osuna et al., 1993), reproductive cycle (Frías-Espericueta et al., 1997), field cultivation (Chávez-Villalba et al., 2005), and more recently, determination of nutritional requirements of broodstock using artificial low-cost products (Leyva-Miranda, 2005). However, other aspects involving physiological regulation of the species still remain unclear. As part of a broad project aimed to develop a protocol for large-scale hatchery culture of C. corteziensis in Mexico, this paper studied the response of spat exposed to a wide range of temperatures during late-nursery culture at the hatchery. The goal was to find the optimum temperature for growth and survival and the upper median lethal temperature during the juvenile stage prior to transfer to the field for growth to commercial size. 2. Materials and methods The optimum temperature for growth and survival was determined in a first experiment by measuring growth rates of juveniles exposed to a wide range of temperatures occurring in the natural habitat where the species is distributed (Mazón-Suástegui et al., 2001; Chávez-Villalba et al., 2005; Leyva-Miranda, 2005). Upper median lethal temperature was estimated in a second experiment with survival data of juveniles exposed to high temperatures.
Isochrysis galbana and Chaetoceros muelleri at a 1:1 ratio (cell count) and a density of 80× 103 cells ml−1. After acclimation, they were divided into nine experimental treatments for exposure to the following temperatures held constant throughout the study: 16, 18, 20, 22, 24, 26, 28, 30, and 32 °C. Experimental treatments at each temperature consisted of triplicate 20-L plastic containers holding thirty specimens placed in plastic mesh bags. They were fed an equal mixture (cell count) of I. galbana and C. muelleri at 80 × 103 cells ml−1 (days 1 through 7) and 100 ×103 cells ml−1 (after day 8). Each container used 1-μm filtered and UV sterilized seawater (salinity of 37 ± 1). The containers were drained, washed, and refilled with clean seawater every three days. At the beginning of the experiment (t0), initial shell dimensions and wet weight of 100 juvenile oysters were measured. Shell and weight growth rates were estimated by the difference between final and initial values, divided by the experimental time (days). Subsequent measurements of both dimensions were made on days 7 (t1), 14 (t2), 21 (t3), and 28 (t4), using 10 specimens randomly selected from each container (30 specimens per thermal treatment). 2.2. Temperature tolerance In the second experiment, the upper temperature tolerance (LT50) of the species was determined by the median lethal dose method (Sprague, 1973); 200 hatchery-reared spat (4.1 ± 0.1 mm shell height) were exposed to constant temperatures of 34 and 36 °C, using the same acclimation procedures and experimental protocol as the first experiment. These temperatures were chosen because no deaths occurred in the range of 16–32 °C in the first experiment. Survival was estimated until all oysters were dead. Death was determined using the criteria of a gaping shell. The upper LT50 value and its 95% confidence limits were calculated for the first six days of temperature exposure with a computer program based on the method of Finney (1971), which transforms raw mortality data into probit mortality. The estimated probit line, together with results of a chi-square test for goodness-of-fit and a z-test to compare the two LT50 values (at 5% significance) were determined. 2.3. Statistical treatment of data
2.1. Optimal temperature for growth Spat used for this study was produced at the hatchery following the methods of Mazón-Suástegui et al. (2001). From this, 810 juveniles (5.0 ± 0.1 mm shell height) were acclimated for one week at a temperature of 26 ± 1 °C and a salinity of 37 ± 1. During acclimation, juveniles were fed
Group normality (in shell height and wet weight of spat) was initially evaluated with the Kolmogorov–Smirnov test and then with two-way, nested ANOVA for significant differences in growth of spat as a function of temperature (main factor with nine levels) and batch replication (nested factor with three levels) (Sokal and Rohlf, 1981). When
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3. Results 3.1. Absolute growth Absolute growth of spat at each temperature is shown in Fig. 1. Increase of shell height occurred in a linear pattern, while wet weight gain fitted an exponential model, with correlation coefficients N0.95 in both cases (Table 1). Maximum growth of spat (14.12 mm shell height and 0.532 mg wet weight) occurred at 28 °C; minimum growth (0.981 mm and 0.0045 mg) occurred at 16 °C. Changes in temperature did not reveal significant differences in absolute growth of spat between batch replicates (F = 0.739; P ≥ 0.05), but differences were significant when analyzed for shell height (F = 3.67; P ≤ 0.05) and wet weight (F = 2.39; P ≤ 0.05). According to Tukey's test (Table 1), spat raised at 28–30 °C (for shell height) and 26–28 °C (for wet weight) grew significantly faster and larger than at all other treatments, especially within the 16–20 °C range, where specimens were significantly smaller. Since growth rates of spat at the lower range (16– 20 °C) were insignificant compared to the higher thermalrange tested, they were excluded from further analyses. Shell and weight growth rates in relation to temperature were described by significant third degree polynomial equations as follows: Fig. 1. Growth in shell height (a) and wet weight (b) of Crassostrea corteziensis spat at different experimental temperatures.
necessary, post-hoc analyses with the Tukey's test were included. Results from growth rates were fit into polynomial equations to determine the optimum thermal range for spat growth. Significance level of analyses was set at P ≤ 0.05 for significant differences and at P ≤ 0.001 for highly significant differences.
Shell height ðhÞ ¼ 0:0049x3 þ 0:0427x2 0:0781x þ 0:456 ðr ¼ 0:99Þ Wet weight ðwÞ ¼ 0:1889x3 þ 1:3726x2 1:7956x þ 16:4 ðr ¼ 0:98Þ: 3.2. Growth rates For shell height, highest growth rate of 0.52 mm per day occurred in the 28–30 °C thermal range (Fig. 2a).
Table 1 Equation fitness and correlation coefficients for growth in shell height and wet weight of Crassostrea corteziensis spat at different experimental temperatures Temperature (°C)
Shell height
Wet weight
Total increment (mm)
Equation
Correlation
Total increment (mg)
Equation
Correlation
22 24 26 28 30 32 34 36
11.503a 11.492a 13.124ab 14.115b 14.098b 13.162ab
y = 0.417x + 4.83 y = 0.428x + 5.11 y = 0.476x + 5.57 y = 0.518x + 5.34 y = 0.518x + 5.42 y = 0.471x+ 4.46 No data No data
r = 0.99 r = 0.98 r = 0.98 r = 0.99 r = 0.99 r = 0.99
0.439ab 0.455ab 0.521b 0.532b 0.487ab 0.405a
y = 0.0159x – 0.031 y = 0.0164x – 0.027 y = 0.0187x – 0.023 y = 0.019x – 0.033 y = 0.018x + 0.027 y = 0.0143x – 0.028 No data No data
r = 0.96 r = 0.97 r = 0.98 r = 0.97 r = 0.97 r = 0.95
y = shell height and wet weight, respectively; x = intercept. Means within columns sharing superscripts are not significantly different (p N 0.05).
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For total wet weight, greatest yield of 19 mg per day occurred at 26–28 °C (Fig. 2b). The relationship between shell height and length and shell height and wet weight when spat was grown at 28 °C was significant in both cases (r = 0.88 and 0.89 respectively) and fitted the equations: Shell height ðHÞ vs: shell length ðLÞ: L ¼ 0:78 þ 1:305H Shell height ðHÞ vs: shell weight ðW Þ: W ¼ ð0:0004ÞH 2:4 : 3.3. Survival No deaths of spat were registered during the first experiment. However, the upper temperature tolerance (LT50) of C. corteziensis spat determined during the second experiment showed different results (Fig. 3). No deaths were registered on day 1, but from days 2 to 5, LT50 declined gradually from 34. 9 °C on day 2 to
Fig. 2. Mean growth rates in shell height (a) and wet weight (b) of Crassostrea corteziensis spat at different experimental temperatures.
Fig. 3. Upper lethal temperatures (LT50) and 95% confidence limits for Crassostrea corteziensis spat (4.1 ± 0.1 mm shell height) acclimated at temperature of 26 ± 1 °C for various lengths of exposure.
33.8 °C on day 5. Total die-off of specimens occurred on day 6. 4. Discussion Our results confirm general findings that growth increases rapidly with increasing temperature up to a maximum, above which, it gradually decreases and then stops (Kinne, 1971). Subtropical C. corteziensis probed to be a eurythermic species within a wide range (16 to 32 °C), but was thermo-dependant between 24 and 30 °C. Within this narrower range, maximum (optimum) growth of spat occurred at 28 °C. The upper thermal limit of the species was observed near 32 °C. For the same species, Barraza-Guardado (1983) reported reduced growth rates of spat, juveniles, and adults subjected to temperatures from 32.7 to 33.3 °C. Flores-Vergara et al. (2004) also reported that temperatures above 32 °C inhibited growth and caused metabolic damage in C. gigas spat during hatchery culture. Despite similarities between species, comparisons of C. corteziensis and C. gigas should be made with caution because they inhabit different geographic provinces and exhibit different physiological strategies to deal with stress and regulate metabolism (Páez-Osuna et al., 1993; Mazón-Suástegui, 1996; Frías-Espericueta et al., 1997; Chávez-Villalba et al., 2005). C. corteziensis inhabits tropical and subtropical areas of the eastern Pacific Ocean, while C. gigas inhabits cool temperate areas of the northeastern Pacific (Stuardo and Martínez, 1975). Moreover, many C. gigas populations inhabit transitional ecotones between subtropical and temperate areas (e.g. Laguna Manuela in the middle part of the Baja California Peninsula; 27.58°N, 114.06°W), where it behaves as a thermo-tolerant species from 15 to 36 °C,
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but exhibits low optimum temperature for growth near 18 °C (Sicard et al., 2006). This value is at least ten degrees below the optimum temperature determined for C. corteziensis spat. Since the tolerance range of the species was not found between 16–20 °C, but at temperatures above 32 °C, growth and survival were affected much more by warm than cold temperatures. The low range tested caused no other damage that a significant reduction in growth of spat (shell height and wet weight), but was not sufficiently low to provoke death. Moreover, the response and physiological condition of spat was independent of temperature within the 16–20 °C range. Consequently, under this condition, there was very little information about growth rates and polynomial correlation fitness. During field cultivation, Chávez-Villalba et al. (2005) reported decreased growth in adult C. corteziensis when the temperature dropped below 18 °C, but at 34 to 36 °C, lethal conditions prevailed. From these results and previous studies of the Mytilidae and Pectinidae bivalve families, high temperatures cause severe physiological and metabolic stress, as well as denaturizing of proteins and enzymes, when energy reserves are preferentially reallocated for survival than for growth (see Kinne, 1971; Widdows, 1973; Bayne et al., 1976; Newell and Branch, 1980; Shpigel et al., 1992; Barber and Blake, 2006; Thompson and MacDonald, 2006). Regardless of the thermal treatment, growth of spat fit a linear model for shell height and an exponential model for wet weight, followed by a power law for the relationship between shell height and wet weight. Similarly, growth was isometric for the relationships shell height to shell length and shell height to wet weight. This suggests that sexually immature oysters allocate similar amounts of energy to all somatic body tissues, including the shell. These relationships agree with findings of Barraza-Guardado (1983) and ChávezVillalba et al. (2005) on C. corteziensis and S. palmula and Cáceres-Martínez and García-Bustamente (1990) on C. gigas. Similarly, an isometric relationship during growth occurs in young Pinctada mazatlanica and Pteria sterna (Saucedo et al., 1998) and Nodipecten subnodosus (Barrios-Ruíz et al., 2003). Conversely, an allometric growth relationship occurs in adult P. mazatlanica (Saucedo et al., 1998), S. palmula (Cabrera-Peña et al., 2001), and C. iridescens (Melchor-Aragón et al., 2002). Control and monitoring water temperature during cultivation of spat of this and other bivalve species is one of the key factors in promoting faster growth in less time and at lower cost. The results of this study are useful for increasing larval viability and spat yield in the
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hatchery, which in turn enhances spat performance during field cultivation to commercial size. Increasing spat vigor through better hatchery conditions during late-nursery care is a very important task to prepare stocking of juveniles in the field under conditions of high density and low temperature without causing serious physiological damage, yet allowing rapid recovery and growth. Acknowledgements The authors are grateful to Gustavo Villavicencio, Mario Osuna, and Pablo Ormart at CIBNOR for technical support during hatchery cultivation of spat. We thank Carlos Cáceres-Martínez and Ira Fogel for valuable comments and editorial clarification. Funding was received from SAGARPA-CONACYT (Grant 2003-002061) and AVANCE-CONACYT (Grant C01-275 R&D). References Barber, B.J., Blake, N.J., 2006. Reproductive physiology, In: Shumway, S.E., Parsons, G.J. (Eds.), 2nd Ed. Scallops: Biology, Ecology, and Aquaculture. Elsevier, Amsterdam, pp. 357–416. Barraza-Guardado, R.H., 1983. Contribución al conocimiento sobre algunas especies comerciales de moluscos bivalvos Crassostrea corteziensis Hertlein, 1951, Saccostrea palmula Carpenter, 1857 y Atrina maura Sowerby, 1835 en el estero El Pozole, Sinaloa, México, 1982–1983. Bs. Thesis, Universidad Autónoma Sinaloa, Mazatlán, Mexico. Barrios-Ruiz, D., Chávez-Villalba, J., Cáceres-Martínez, C., 2003. Growth of Nodipecten subnodosus (Bivalvia: Pectinidae) in La Paz Bay, México. Aquac. Res. 34, 633–639. Bayne, B.L., Thompson, R.J., Widdows, J., 1976. Physiological integrations. In: Bayne, B.L. (Ed.), Marine Mussels: Their Ecology and Physiology. Cambridge Univ. Press, London, pp. 261–292. Cabrera-Peña, J.H., Protti-Quesada, M., Urriola-Hernández, M., Sáenz-Vargas, O., 2001. Growth of Nodipecten subnodosus (Bivalvia: Pectinidae) in La Paz Bay, México. Aquac. Res. 34, 633–639. Cáceres-Martínez, C., García-Bustamante, S., 1990. Cultivo piloto de ostión Crassostrea gigas en costales sobre estantes en la zona intermareal en Bahía Magdalena, B.C.S., influencia de la densidad sobre el crecimiento. In: De la Lanza-Espino, G., ArredondoFigueroa, J.L. (Eds.), La Acuicultura en México: de los Conceptos a la Producción. Ed. Limusa, Mexico, D.F, pp. 162–169. Chávez-Villalba, J., Mazón-Suástegui, J.M., Osuna-García, M., Robles-Mungaray, M., 2005. Growth of the oyster Crassostrea corteziensis (Hertlein, 1951) in Sonora, Mexico. Aquac. Res. 36, 1337–1344. Finney, D., 1971. Probit Analysis, 3rd. Ed. Cambridge University Press, London. (333 pp.). Flores-Vergara, C., Cordero-Esquivel, B., Ceron-Ortiz, A.N., Arredondo-Vega, B.O., 2004. Combined effects of temperature and diet on growth and biochemical composition of the Pacific oyster Crassostrea gigas (Thunberg) spat. Aquac. Res. 35, 1131–1140. Frías-Espericueta, M.G., Páez-Osuna, F., Osuna-López, I., 1997. Seasonal changes in the gonadal state of the oyster Crassostrea
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corteziensis (Filibranchia: Ostreidae) in the Northwest coast of México. Rev. Biol. Trop. 45, 1061–1065. Holland, D.L., 1978. Laid reserves and energy metabolism in the larvae of benthic marine invertebrates. In: Malins, D.C., Sargent, J.R. (Eds.), Biochemical and Biophysical Perspectives in Marine Biology. Academic Press, London, pp. 85–123. Hoyos-Chaires, F., Robles-Mungaray, M., 1990. Estudio sobre el cultivo piloto de ostión Crassostrea corteziensis en un criadero comercial. II) Crecimiento a talla comercial. Abstracts IV Cong. Nac. Acuic. AMAC-90. Hermosillo, Sonora, Mexico. Kinne, O., 1971. Temperature. 3.3. Animals. Invertebrates. In: Kinne, O. (Ed.), Marine Ecology, Environmental Factors, Part 1, vol. 1. Wiley-Interscience, London, pp. 407–514. Leyva-Miranda, G., 2005. Cultivo experimental de ostión de placer Crassostrea corteziensis (Hertlein, 1951) preengordado en laboratorio con tres dietas distintas para compensar su crecimiento en canastas en el Estero El Riíto, Huatabampo, Sonora. BS. Thesis, Centro de Estudios Superiores del Estado de Sonora, Navojoa, Sonora, Mexico. Mazón-Suástegui, J.M., 1996. Cultivo de ostión japonés Crassostrea gigas. In: Casas-Valdez, M., Ponce-Díaz, G. (Eds.), Estudio del Potencial Pesquero y Acuícola de Baja California Sur. FAOUABCS-CIBNOR-CICIMAR-CRIP, La Paz, B.C.S., Mexico, pp. 625–650. Mazón-Suástegui, J.M., Robles-Mungaray, M., Avilés-Quevedo, S., Flores-Higuera, F., Monsalvo-Spencer, P., Osuna-García, M., 2001. Avances en la producción y cultivo de semilla de ostión nativo Crassostrea corteziensis en Bahía de Ceuta, Sinaloa, Mexico. Mem. 1er Foro Estatal Cienc. y Tecn. Culiacán, Sinaloa, Mexico. Melchor-Aragón, J.M., Ruiz-Luna, A., Terrazas-Gaxiola, R., AcostaCastañeda, C., 2002. Mortality and growth of the rock oyster, Crassostrea iridescens (Hanley, 1854) in San Ignacio, Sinaloa, México. Cienc. Mar. 28, 125–132. Newell, R.C., Branch, G.M., 1980. The influence of temperature on the maintenance of metabolic energy balance in marine invertebrates. Adv. Mar. Biol. 17, 329–396.
Páez-Osuna, F., Zazueta-Padilla, H.M., Osuna-López, J.I., 1993. Biochemical composition of the oysters Crassostrea iridescens Hanley and Crassostrea corteziensis Hertlein in the Northwest coast of Mexico: seasonal changes. J. Exp. Mar. Biol. Ecol. 170, 1–9. Saucedo, P.E., Monteforte, M., Blanc, F., 1998. Changes in shell dimensions of pearl oysters, Pinctada mazatlanica (Hanley 1856) and Pteria sterna (Gould 1851) during growth as criteria for Mabe pearl implants. Aquac. Res. 29, 801–814. Shpigel, M., Barber, B.J., Mann, R., 1992. Effects of elevated temperature on growth, gametogenesis, physiology, and biochemical composition in diploid and triploid Pacific oysters, Crassostrea gigas Thunberg. J. Exp. Mar. Biol. Ecol. 161, 15–25. Shumway, S.E., 1982. Oxygen consumption in oysters: an overview. Mar. Biol. Lett. 3, 1–23. Sicard, M.T., Maeda-Martínez, A.N., Lluch-Cota, S.E., LodeirosSeijo, C., Roldán-Carrillo, L.M., Mendoza-Alfaro, R., 2006. Frequent monitoring of temperature: an essential requirement for site selection in bivalve aquaculture in tropical-temperate transition zones. Aquac. Res. 37, 1040–1049. Sokal, R.R., Rohlf, F.J., 1981. Biometry, 2nd ed. W.H. Freeman, San Francisco. (859 pp.). Sprague, J.B., 1973. The ABC's of pollutant bioassay using fish. In: Cairns, J., Dickson, K.L (Eds.), Biological Methods for the Assessment of Water Quality, 528. ASTM STP American Society for Testing and Materials, pp. 6–30. Stuardo, J., Martínez, A.,1975. Relaciones entre algunos factores ecológicos y la biología de poblaciones de Crassostrea corteziensis Hertlein 1951, de San Blas, Nayarit, México. Anales Centro Cienc. Mar y Limnol. UNAM 2, 89–130. Thompson, R., MacDonald, B., 2006. Physiological integrations and energy partitioning, In: Shumway, S.E., Parsons, G.J. (Eds.), 2nd Ed. Scallops: Biology, Ecology, and Aquaculture. Elsevier, Amsterdam, pp. 493–520. Widdows, J., 1973. Effect of temperature and food on the heart beat, ventilation rate and oxygen uptake of Mytilus edulis. Mar. Biol. 20, 276–279.
Aquaculture 272 (2007) 417 – 422 www.elsevier.com/locate/aqua-online
Effect of temperature on growth and survival of Crassostrea corteziensis spat during late-nursery culturing at the hatchery Jorge I. Cáceres-Puig, Fernando Abasolo-Pacheco, José M. Mazón-Suastegui, Alfonso N. Maeda-Martínez, Pedro E. Saucedo ⁎ Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S., 23090, Mexico Received 2 March 2007; received in revised form 20 June 2007; accepted 21 June 2007
Abstract Nine temperatures (16, 18, 20, 22, 24, 26, 28, 30, and 32 °C) within the natural range of distribution of the Cortez oyster Crassostrea corteziensis were tested in a first experiment to determine the optimal temperature for growth and survival. Based on these results, a second study assessed two temperatures above this range (34 and 36 °C) to determine upper median lethal temperature for the species. The species was thermo-tolerant between 16–32 °C, grew faster and larger at 24 to 30 °C, and had optimal growth at 28–30 °C. The lower tolerance of the species appears far from the lowest value tested (16 °C). In contrast, the upper tolerance temperature was near 32 °C, since 100% spat mortality occurred within 96 h at 34 and 36 °C. These results are being used to develop a protocol for large-scale hatchery culture of the species in Mexico. © 2007 Published by Elsevier B.V. Keywords: Mexico; Oyster; Spat; Temperature; Nursery culture; Hatchery
1. Introduction In marine bivalves, temperature is recognized as one of the fundamental exogenous factors influencing most aspects of the ecology and biology of the species (see reviews by Kinne, 1971 and Shumway, 1982). It limits the general distribution of a species within large geographic areas, as well as specific habitat occupation between diverse communities or populations. Temperature also regulates many physiological functions of organisms, such as cilia activity during filter-feeding, cardiac and growth rates, gonad development and reproductive ⁎ Corresponding author. Tel.: +52 612 123 8464; fax: +52 612 125 3625. E-mail address: [email protected] (P.E. Saucedo). 0044-8486/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.aquaculture.2007.06.030
output, energy budget, and metabolic activity of enzymes (Widdows, 1973; Bayne et al., 1976; Newell and Branch, 1980; Shpigel et al., 1992; Barber and Blake, 2006). Additionally, changes in temperature affect survival of larvae, juveniles, and adults of most marine bivalve species, not only in wild populations, but also in laboratory experiments (Holland, 1978). Among the diverse marine bivalve species inhabiting the Pacific Ocean, the Cortez oyster Crassostrea corteziensis (Hertlein, 1951), whose distribution covers most of the tropical and subtropical coasts from Mexico to Peru, is of particular interest (Stuardo and Martínez, 1975; Mazón-Suástegui, 1996; Chávez-Villalba et al., 2005). In the states of Sonora and Sinaloa in Mexico, the species has been overexploited and most natural beds show severe signs of depletion or even virtual extinction
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(Hoyos-Chairez and Robles-Mungaray, 1990; ChávezVillalba et al., 2005). Consequently, the oyster industry in the northwest coastal Mexico has concentrated its efforts on cultivating the Pacific oyster C. gigas (Thunberg, 1793). However, this species has a natural temperate distribution, and although it has been successfully introduced to Mexico decades ago, it has suffered large die-offs in recent years. Given this situation, C. corteziensis has been receiving great deal of attention because of its relatively low production costs, excellent taste, and high nutritional value. Currently, the incipient fishery developed with this species does not provide the numbers of oysters demanded by the local and national markets, and therefore, scientific improvement of cultivation methods is essential in the short future (Mazón-Suástegui et al., 2001; Chávez-Villalba et al., 2005). So far, there is some understanding of the ecology and biology of the species, including population dynamics (Stuardo and Martínez, 1975), changes in tissue chemical composition (Páez-Osuna et al., 1993), reproductive cycle (Frías-Espericueta et al., 1997), field cultivation (Chávez-Villalba et al., 2005), and more recently, determination of nutritional requirements of broodstock using artificial low-cost products (Leyva-Miranda, 2005). However, other aspects involving physiological regulation of the species still remain unclear. As part of a broad project aimed to develop a protocol for large-scale hatchery culture of C. corteziensis in Mexico, this paper studied the response of spat exposed to a wide range of temperatures during late-nursery culture at the hatchery. The goal was to find the optimum temperature for growth and survival and the upper median lethal temperature during the juvenile stage prior to transfer to the field for growth to commercial size. 2. Materials and methods The optimum temperature for growth and survival was determined in a first experiment by measuring growth rates of juveniles exposed to a wide range of temperatures occurring in the natural habitat where the species is distributed (Mazón-Suástegui et al., 2001; Chávez-Villalba et al., 2005; Leyva-Miranda, 2005). Upper median lethal temperature was estimated in a second experiment with survival data of juveniles exposed to high temperatures.
Isochrysis galbana and Chaetoceros muelleri at a 1:1 ratio (cell count) and a density of 80× 103 cells ml−1. After acclimation, they were divided into nine experimental treatments for exposure to the following temperatures held constant throughout the study: 16, 18, 20, 22, 24, 26, 28, 30, and 32 °C. Experimental treatments at each temperature consisted of triplicate 20-L plastic containers holding thirty specimens placed in plastic mesh bags. They were fed an equal mixture (cell count) of I. galbana and C. muelleri at 80 × 103 cells ml−1 (days 1 through 7) and 100 ×103 cells ml−1 (after day 8). Each container used 1-μm filtered and UV sterilized seawater (salinity of 37 ± 1). The containers were drained, washed, and refilled with clean seawater every three days. At the beginning of the experiment (t0), initial shell dimensions and wet weight of 100 juvenile oysters were measured. Shell and weight growth rates were estimated by the difference between final and initial values, divided by the experimental time (days). Subsequent measurements of both dimensions were made on days 7 (t1), 14 (t2), 21 (t3), and 28 (t4), using 10 specimens randomly selected from each container (30 specimens per thermal treatment). 2.2. Temperature tolerance In the second experiment, the upper temperature tolerance (LT50) of the species was determined by the median lethal dose method (Sprague, 1973); 200 hatchery-reared spat (4.1 ± 0.1 mm shell height) were exposed to constant temperatures of 34 and 36 °C, using the same acclimation procedures and experimental protocol as the first experiment. These temperatures were chosen because no deaths occurred in the range of 16–32 °C in the first experiment. Survival was estimated until all oysters were dead. Death was determined using the criteria of a gaping shell. The upper LT50 value and its 95% confidence limits were calculated for the first six days of temperature exposure with a computer program based on the method of Finney (1971), which transforms raw mortality data into probit mortality. The estimated probit line, together with results of a chi-square test for goodness-of-fit and a z-test to compare the two LT50 values (at 5% significance) were determined. 2.3. Statistical treatment of data
2.1. Optimal temperature for growth Spat used for this study was produced at the hatchery following the methods of Mazón-Suástegui et al. (2001). From this, 810 juveniles (5.0 ± 0.1 mm shell height) were acclimated for one week at a temperature of 26 ± 1 °C and a salinity of 37 ± 1. During acclimation, juveniles were fed
Group normality (in shell height and wet weight of spat) was initially evaluated with the Kolmogorov–Smirnov test and then with two-way, nested ANOVA for significant differences in growth of spat as a function of temperature (main factor with nine levels) and batch replication (nested factor with three levels) (Sokal and Rohlf, 1981). When
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3. Results 3.1. Absolute growth Absolute growth of spat at each temperature is shown in Fig. 1. Increase of shell height occurred in a linear pattern, while wet weight gain fitted an exponential model, with correlation coefficients N0.95 in both cases (Table 1). Maximum growth of spat (14.12 mm shell height and 0.532 mg wet weight) occurred at 28 °C; minimum growth (0.981 mm and 0.0045 mg) occurred at 16 °C. Changes in temperature did not reveal significant differences in absolute growth of spat between batch replicates (F = 0.739; P ≥ 0.05), but differences were significant when analyzed for shell height (F = 3.67; P ≤ 0.05) and wet weight (F = 2.39; P ≤ 0.05). According to Tukey's test (Table 1), spat raised at 28–30 °C (for shell height) and 26–28 °C (for wet weight) grew significantly faster and larger than at all other treatments, especially within the 16–20 °C range, where specimens were significantly smaller. Since growth rates of spat at the lower range (16– 20 °C) were insignificant compared to the higher thermalrange tested, they were excluded from further analyses. Shell and weight growth rates in relation to temperature were described by significant third degree polynomial equations as follows: Fig. 1. Growth in shell height (a) and wet weight (b) of Crassostrea corteziensis spat at different experimental temperatures.
necessary, post-hoc analyses with the Tukey's test were included. Results from growth rates were fit into polynomial equations to determine the optimum thermal range for spat growth. Significance level of analyses was set at P ≤ 0.05 for significant differences and at P ≤ 0.001 for highly significant differences.
Shell height ðhÞ ¼ 0:0049x3 þ 0:0427x2 0:0781x þ 0:456 ðr ¼ 0:99Þ Wet weight ðwÞ ¼ 0:1889x3 þ 1:3726x2 1:7956x þ 16:4 ðr ¼ 0:98Þ: 3.2. Growth rates For shell height, highest growth rate of 0.52 mm per day occurred in the 28–30 °C thermal range (Fig. 2a).
Table 1 Equation fitness and correlation coefficients for growth in shell height and wet weight of Crassostrea corteziensis spat at different experimental temperatures Temperature (°C)
Shell height
Wet weight
Total increment (mm)
Equation
Correlation
Total increment (mg)
Equation
Correlation
22 24 26 28 30 32 34 36
11.503a 11.492a 13.124ab 14.115b 14.098b 13.162ab
y = 0.417x + 4.83 y = 0.428x + 5.11 y = 0.476x + 5.57 y = 0.518x + 5.34 y = 0.518x + 5.42 y = 0.471x+ 4.46 No data No data
r = 0.99 r = 0.98 r = 0.98 r = 0.99 r = 0.99 r = 0.99
0.439ab 0.455ab 0.521b 0.532b 0.487ab 0.405a
y = 0.0159x – 0.031 y = 0.0164x – 0.027 y = 0.0187x – 0.023 y = 0.019x – 0.033 y = 0.018x + 0.027 y = 0.0143x – 0.028 No data No data
r = 0.96 r = 0.97 r = 0.98 r = 0.97 r = 0.97 r = 0.95
y = shell height and wet weight, respectively; x = intercept. Means within columns sharing superscripts are not significantly different (p N 0.05).
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For total wet weight, greatest yield of 19 mg per day occurred at 26–28 °C (Fig. 2b). The relationship between shell height and length and shell height and wet weight when spat was grown at 28 °C was significant in both cases (r = 0.88 and 0.89 respectively) and fitted the equations: Shell height ðHÞ vs: shell length ðLÞ: L ¼ 0:78 þ 1:305H Shell height ðHÞ vs: shell weight ðW Þ: W ¼ ð0:0004ÞH 2:4 : 3.3. Survival No deaths of spat were registered during the first experiment. However, the upper temperature tolerance (LT50) of C. corteziensis spat determined during the second experiment showed different results (Fig. 3). No deaths were registered on day 1, but from days 2 to 5, LT50 declined gradually from 34. 9 °C on day 2 to
Fig. 2. Mean growth rates in shell height (a) and wet weight (b) of Crassostrea corteziensis spat at different experimental temperatures.
Fig. 3. Upper lethal temperatures (LT50) and 95% confidence limits for Crassostrea corteziensis spat (4.1 ± 0.1 mm shell height) acclimated at temperature of 26 ± 1 °C for various lengths of exposure.
33.8 °C on day 5. Total die-off of specimens occurred on day 6. 4. Discussion Our results confirm general findings that growth increases rapidly with increasing temperature up to a maximum, above which, it gradually decreases and then stops (Kinne, 1971). Subtropical C. corteziensis probed to be a eurythermic species within a wide range (16 to 32 °C), but was thermo-dependant between 24 and 30 °C. Within this narrower range, maximum (optimum) growth of spat occurred at 28 °C. The upper thermal limit of the species was observed near 32 °C. For the same species, Barraza-Guardado (1983) reported reduced growth rates of spat, juveniles, and adults subjected to temperatures from 32.7 to 33.3 °C. Flores-Vergara et al. (2004) also reported that temperatures above 32 °C inhibited growth and caused metabolic damage in C. gigas spat during hatchery culture. Despite similarities between species, comparisons of C. corteziensis and C. gigas should be made with caution because they inhabit different geographic provinces and exhibit different physiological strategies to deal with stress and regulate metabolism (Páez-Osuna et al., 1993; Mazón-Suástegui, 1996; Frías-Espericueta et al., 1997; Chávez-Villalba et al., 2005). C. corteziensis inhabits tropical and subtropical areas of the eastern Pacific Ocean, while C. gigas inhabits cool temperate areas of the northeastern Pacific (Stuardo and Martínez, 1975). Moreover, many C. gigas populations inhabit transitional ecotones between subtropical and temperate areas (e.g. Laguna Manuela in the middle part of the Baja California Peninsula; 27.58°N, 114.06°W), where it behaves as a thermo-tolerant species from 15 to 36 °C,
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but exhibits low optimum temperature for growth near 18 °C (Sicard et al., 2006). This value is at least ten degrees below the optimum temperature determined for C. corteziensis spat. Since the tolerance range of the species was not found between 16–20 °C, but at temperatures above 32 °C, growth and survival were affected much more by warm than cold temperatures. The low range tested caused no other damage that a significant reduction in growth of spat (shell height and wet weight), but was not sufficiently low to provoke death. Moreover, the response and physiological condition of spat was independent of temperature within the 16–20 °C range. Consequently, under this condition, there was very little information about growth rates and polynomial correlation fitness. During field cultivation, Chávez-Villalba et al. (2005) reported decreased growth in adult C. corteziensis when the temperature dropped below 18 °C, but at 34 to 36 °C, lethal conditions prevailed. From these results and previous studies of the Mytilidae and Pectinidae bivalve families, high temperatures cause severe physiological and metabolic stress, as well as denaturizing of proteins and enzymes, when energy reserves are preferentially reallocated for survival than for growth (see Kinne, 1971; Widdows, 1973; Bayne et al., 1976; Newell and Branch, 1980; Shpigel et al., 1992; Barber and Blake, 2006; Thompson and MacDonald, 2006). Regardless of the thermal treatment, growth of spat fit a linear model for shell height and an exponential model for wet weight, followed by a power law for the relationship between shell height and wet weight. Similarly, growth was isometric for the relationships shell height to shell length and shell height to wet weight. This suggests that sexually immature oysters allocate similar amounts of energy to all somatic body tissues, including the shell. These relationships agree with findings of Barraza-Guardado (1983) and ChávezVillalba et al. (2005) on C. corteziensis and S. palmula and Cáceres-Martínez and García-Bustamente (1990) on C. gigas. Similarly, an isometric relationship during growth occurs in young Pinctada mazatlanica and Pteria sterna (Saucedo et al., 1998) and Nodipecten subnodosus (Barrios-Ruíz et al., 2003). Conversely, an allometric growth relationship occurs in adult P. mazatlanica (Saucedo et al., 1998), S. palmula (Cabrera-Peña et al., 2001), and C. iridescens (Melchor-Aragón et al., 2002). Control and monitoring water temperature during cultivation of spat of this and other bivalve species is one of the key factors in promoting faster growth in less time and at lower cost. The results of this study are useful for increasing larval viability and spat yield in the
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hatchery, which in turn enhances spat performance during field cultivation to commercial size. Increasing spat vigor through better hatchery conditions during late-nursery care is a very important task to prepare stocking of juveniles in the field under conditions of high density and low temperature without causing serious physiological damage, yet allowing rapid recovery and growth. Acknowledgements The authors are grateful to Gustavo Villavicencio, Mario Osuna, and Pablo Ormart at CIBNOR for technical support during hatchery cultivation of spat. We thank Carlos Cáceres-Martínez and Ira Fogel for valuable comments and editorial clarification. Funding was received from SAGARPA-CONACYT (Grant 2003-002061) and AVANCE-CONACYT (Grant C01-275 R&D). References Barber, B.J., Blake, N.J., 2006. Reproductive physiology, In: Shumway, S.E., Parsons, G.J. (Eds.), 2nd Ed. Scallops: Biology, Ecology, and Aquaculture. Elsevier, Amsterdam, pp. 357–416. Barraza-Guardado, R.H., 1983. Contribución al conocimiento sobre algunas especies comerciales de moluscos bivalvos Crassostrea corteziensis Hertlein, 1951, Saccostrea palmula Carpenter, 1857 y Atrina maura Sowerby, 1835 en el estero El Pozole, Sinaloa, México, 1982–1983. Bs. Thesis, Universidad Autónoma Sinaloa, Mazatlán, Mexico. Barrios-Ruiz, D., Chávez-Villalba, J., Cáceres-Martínez, C., 2003. Growth of Nodipecten subnodosus (Bivalvia: Pectinidae) in La Paz Bay, México. Aquac. Res. 34, 633–639. Bayne, B.L., Thompson, R.J., Widdows, J., 1976. Physiological integrations. In: Bayne, B.L. (Ed.), Marine Mussels: Their Ecology and Physiology. Cambridge Univ. Press, London, pp. 261–292. Cabrera-Peña, J.H., Protti-Quesada, M., Urriola-Hernández, M., Sáenz-Vargas, O., 2001. Growth of Nodipecten subnodosus (Bivalvia: Pectinidae) in La Paz Bay, México. Aquac. Res. 34, 633–639. Cáceres-Martínez, C., García-Bustamante, S., 1990. Cultivo piloto de ostión Crassostrea gigas en costales sobre estantes en la zona intermareal en Bahía Magdalena, B.C.S., influencia de la densidad sobre el crecimiento. In: De la Lanza-Espino, G., ArredondoFigueroa, J.L. (Eds.), La Acuicultura en México: de los Conceptos a la Producción. Ed. Limusa, Mexico, D.F, pp. 162–169. Chávez-Villalba, J., Mazón-Suástegui, J.M., Osuna-García, M., Robles-Mungaray, M., 2005. Growth of the oyster Crassostrea corteziensis (Hertlein, 1951) in Sonora, Mexico. Aquac. Res. 36, 1337–1344. Finney, D., 1971. Probit Analysis, 3rd. Ed. Cambridge University Press, London. (333 pp.). Flores-Vergara, C., Cordero-Esquivel, B., Ceron-Ortiz, A.N., Arredondo-Vega, B.O., 2004. Combined effects of temperature and diet on growth and biochemical composition of the Pacific oyster Crassostrea gigas (Thunberg) spat. Aquac. Res. 35, 1131–1140. Frías-Espericueta, M.G., Páez-Osuna, F., Osuna-López, I., 1997. Seasonal changes in the gonadal state of the oyster Crassostrea
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