Stable isotope analysis of the contribution of microalgal diets to the growth and survival of pacific oyster Crassostrea gigas (Thunberg, 1979) larvae.
KEY WORDS: oyster, Crassostrea gigas, larvae, Pavlova lutheri, Tahitian Isochrysis aff. galbana, Chaetoceros calcitrans, stable isotope analysis, hatchery
The Pacific oyster Crassostrea gigas (Thunberg, 1795) is the main bivalve species farmed globally with production in 2013 estimated at 560,000 tonnes, and a value of over USD 1.2 billion (FAO 2013). In Australia, C. gigas farming is a well-established industry that is worth around AUD 50 million (NSW Department of Primary Industries 2012). Commercial production of C. gigas in hatcheries is increasing, with most of the seed for the Australian and U.S. industries coming from hatcheries (Lavoie 2005, Bishop et al. 2010) and 70% in Spain (European Food Safety Authority 2010). Hatchery production of C. gigas in France is increasing due to ostreid herpesvirus and demand for triploid oysters; approximately 3 billion spat were produced in 2012 (Degremont & Benabdelmouna 2014).
Oyster hatcheries rely on the quantity and quality of microalgae to condition broodstock for spawning and to culture larvae and spat (Ponis et al. 2003, Ponis et al. 2006, Rico-Villa et al. 2006). To date, many studies have relied on biochemical analyses, growth and survival experiments to determine the nutritional value of microalgal species in oyster larvae diets (Brown et al. 1997, Knauer & Southgate 1999, Powell et al. 2002, Ponis et al. 2003, Rico-Villa et al. 2006). Success and profitability for hatcheries is fundamentally linked to increased outputs along with reduced operational time and rearing costs. Although plurispecific diets offer sufficient nutritional value for bivalve larvae, production of live microalgal cultures can account for between 15% and 85% of hatchery operational costs (Coutteau & Sorgeloos 1992, Knauer & Southgate 1999). In addition to reducing the rearing time and cost of larval production in hatcheries, it is important to identify optimal diets to attain high larval growth and survival, and to ensure that metamorphosis is achieved as soon as possible. It is also important to understand any changing larval nutritional requirements during ontogeny, that is, the stage at which larvae best assimilate nutrients from particular diets.
Larval rearing in hatcheries is particularly difficult. For example, hatcheries commonly experience problems when larval energy demands transition from reliance on endogenous egg reserves to exogenous nutrition from the water column (O'Connor & Dove 2009). Studies have suggested that dietary need may alter larval ontogeny for Saccostrea glomerate (O'Connor et al. 1992) and Pecten maximus (Tremblay et al. 2007).
Stable isotope analysis (SIA) is a technique that is widely used in ecology to determine the nutrient assimilation and relative dietary contribution of various food sources to organisms (Peterson & Fry 1987, Post 2002, Mazumder et al. 2011); its application is increasing in aquaculture research (Schlechtriem et al. 2004). Many nutrient assimilation studies on marine larvae have used indirect methods, such as using added markers (e.g., enriched isotopes) (Verschoor et al. 2005, Conceifao et al. 2007, Gamboa-Delgado & Le Vay 2009, Le Vay & GamboaDelgado 2011). Studies have also successfully obtained quantitative information on nutritional requirements, feed intake, relative digestion, and assimilation of nutrients in fish and crustacean larvae using radioisotopes (Schlechtriem et al. 2004, Conceifao et al. 2007, Gamboa-Delgado et al. 2008, Jomori et al. 2008, Gamboa-Delgado & Le Vay 2009). Stable isotope analysis was used in several studies on adult bivalve dietary requirements and physiology (Kang et al. 1999, Piola et al. 2006, Rubio 2007, 2008), and larval shell development (Waldbusser et al. 2013). In this study, carbon and nitrogen stable isotopes were used instead of stomach content analysis to increase accuracy, precision, and efficiency in defining dietary carbon and nitrogen assimilation for Crassostrea gigas larvae. Conventional stomach content analysis relies on the identification of ingested materials from the stomach content. Ingestion of materials, however, does not necessarily mean assimilation of nutrients necessary for tissue building. The underpinning theory is that the isotopic composition of an organism reflects the isotopic composition of its diet (DeNiro & Epstein 1978, Mazumder et al. 2016); in this context, SIA provides a direct measure of nutrient assimilation and utilization (Peterson & Fry 1987, Verschoor et al. 2005, Le Vay & Gamboa-Delgado 2011) that cannot be fully explained by commonly used growth performance measures. In general, the carbon to nitrogen ratio (C:N) is an indicator of diet quality (Fantle et al. 1999, Hill & McQuaid 2009). As nitrogen is present mostly in protein, diets with a C:N ratio higher than six represent a low proportion of protein (Yokoyama et al. 2005). The discrimination factor (A, the difference of isotopic signature between an organism and its diets) is an indication of dietary assimilation; the final equilibrium values are normally calculated by adding the discrimination factor to the diet's isotopic values (Fry & Arnold 1982). Discrimination factors have been observed to vary with species, type of tissue, age, and the growth rate of the organism (Roth & Hobson 2000, Pearson et al. 2003, Waddington & MacArthur 2008, Robbins et al. 2010). Kaufman et al. (2008) suggested using a model to obtain the nonarbitrary estimated equilibrium values.
There are no studies that have used stable isotopes to investigate bivalve larval diets, their assimilation, and subsequent contribution to growth and survival. Marine larval nutrition and physiology studies for other species have shown that SIA can yield relevant information to assess growth performance between treatments (Schlechtriem et al. 2004, Verschoor et al. 2005, Conceipao et al. 2007). Therefore, applying SIA to the selection of microalgal species to improved diet formulations could potentially reduce larval rearing time and operational costs for different developmental stages of hatchery-reared Crassostrea gigas.
This study focused on the first 15 days of the larval stage of Crassostrea gigas development before metamorphosis (Powell et al. 2002, Rico-Villa et al. 2006). The overall objective of this study was to assess the use of SIA as a tool to investigate the value of particular microalgal species to C. gigas larval nutrition. In this study, isotopic turnover rates, discrimination factors, and C:N ratios were used as indicators of diet performance and nutrient assimilation of larvae in relation to their diet (Roth & Hobson 2000. Mente et al. 2002, Kaufman et al. 2008, Le Vay & Gamboa-Delgado 2011). In addition, SIA could be used to develop optimal diets for other farmed bivalve species.
MATERIALS AND METHODS
Feeding Trial on Crassostrea gigas Larvae
D-veliger larvae were obtained at 24-h postfertilization from a commercial hatchery (Southern Cross Shellfish Hatchery, Shoal Bay, Port Stephens, NSW, Australia) and transferred to the Port Stephens Fisheries Institute's mollusc hatchery for experimentation. Approximately 1.5 million larvae were distributed into fifteen 10-1 tanks at a density of 10 larvae/ml. The mean temperature of the aquaria was 20.1 [+ or -] 0.9[degrees]C for the duration of the feeding trial. Larvae were reared in 1 [micro]m filtered seawater and the density was reduced to 5 larvae/ml at Day 7. Three commonly used species of microalgal feed for hatchery-reared bivalve molluscs were used: Pavlova lutheri (P), Tahitian Isochrysis aff. galbana (T), and Chaetoceros calcitrans forma pumilum (C) (Brown 2002). These species were reared as monospecific diets (P, T, and C separately) and compared with a trispecific diet [25P:25T:50C (PTC)]. Each treatment was carried out in triplicate and an unfed control was also included to quantify any potential contribution of nutrients from filtered seawater.
Water was completely changed every 2 days during the 15-day trial. At each water change, larvae were retained on a 45-pm nylon mesh screen and transferred to a new tank. To ensure the consistency of larval density, water volume for every replicate was adjusted after each water change according to larval survival. Larvae were fed each evening and food quantity was adjusted based on larval size following the method of O'Connor et al. (2008).
Larval Growth and Survival
Before every water change, 30 larvae were collected from each replicate to estimate mortality by counting empty larval shells (translucent shells) using light microscopy (100X magnifications). Growth measurements of larvae included total shell length (from anterior to posterior margin) using a calibrated eyepiece graticule and dry tissue weight (DTW) using a standard length-weight model for Crassostrea gigas larvae (Bochenek et al. 2001).
Sampling and SIA [alpha]
Larval samples were collected for SIA on Day 1,7, and 15 of the experiment. These sampling days were selected to cover the transition from the larvae's dependency on internal energy reserves to exogenous feeding (Waldbusser et al. 2013). The larval samples were retained on a 30-[micro]m mesh screen, rinsed with fresh water, and immediately placed in a -80[degrees]C freezer (Thermo Scientific Cold Storage 907). Before sampling, half of the larvae were transferred into clean tanks with seawater that contained no added microalgae for 48 h to ensure larvae had empty stomachs (verified by light microscopy) for accurate isotopic reading. Isolating specific tissues of an individual larva is problematic, therefore pooled samples were used. All samples were rinsed with Milli-Q water, transferred into a glass petri dish, oven-dried at 60[degrees]C overnight, and homogenized (Mazumder et al. 2011). Before analysis, all larvae were acidified by adding five to six drops of 1 mol HCl to eliminate carbonates from the shell that would interfere with the isotopic measurements (Mazumder et al. 2010). Because the acid washing process enriches [delta][sup.15]N (Pinnegar & Polunin 1999), untreated larvae subsamples were analyzed for [delta][sup.15]N and acid-treated samples were used for the [delta][sup.13]C analyses. Microalgae cultures were vacuum filtered through 0.7-[micro]m glass microfiber filter papers (Filtech Grade 453) and oven-dried. Samples were analyzed at the Australian Nuclear and Technology Organisation. Samples were loaded in tin capsules and analyzed with a continuous flow isotope ratio mass spectrometer, model Delta V Plus (Thermo Scientific Corporation), interfaced with an elemental analyzer (Thermo Fisher Flash 2000 HT EA, Thermo Electron Corporation). Data were reported relative to the International Atomic Energy Agency secondary standards that have been certified relative to Vienna PeeDee Belemnite for [delta][sup.13]C and air for [delta][sup.15]N. A two-point calibration was used to normalize the data based on standards that bracket the samples being analyzed. Two quality control references were also included in each run. Results were accurate to 1% for both C% and N% and [+ or -] 0.3 [per thousand] for [delta][sup.13]C and [delta][sup.15]N. Stable isotope values were reported in delta (8) units in parts per thousand ([per thousand]) relative to the international standard and determined as follows:
X ([per thousand]) = ([P.sub.sample] / [R.sub.standard] - 1) x 1000 (1)
where X = [delta][sup.13]C or [delta][sup.15]N, and R = [sup.13]C/[sup.12]C or [sup.15]N/[sup.14]N, respectively. Replicates (n = 2-3) from each treatment were analyzed.
Estimation of Nutrient Assimilation and Isotopic Turnover Rate
To estimate whether larvae have assimilated their diets or not. the isotopic turnover rate was calculated by fitting to an exponential model proposed by Tieszen et al. (1983) (Eq. 2):
[C.sub.larvae] = [C.sub.n] + ([C.sub.0] - [C.sub.n]) x [e.sub.-ct] (2)
where [C.sub.larvae] is the larval tissue isotope value at time t, that is, 1-15 days, [C.sub.0] = initial isotope value of the larvae, [C.sub.n] = final isotope value reached when larvae are in equilibrium with the diet (approached asymptotically), t = time (days), and c = turnover rate constant (per day). Both [C.sub.n] and c were derived from a least squares optimization for the fit of the predicted values (using Eq. 2) to the measured data. Constant c represents both growth and metabolic factors. To understand which factor was responsible for isotopic turnover, growth and metabolic turnover rate were estimated using the models proposed by Hesslein et al. (1993) (Eqs. 3 and 4):
[C.sub.larvae] = [C.sub.n] + ([C.sub.0] - [C.sub.n]) x [e.sup.-(k+m)t] (3)
where m = metabolic turnover rate (per day) and k = estimated growth rate (per day) obtained from an exponential growth equation (Eq. 4) (Hesslein et al. 1993):
W = [W.sub.0-] x [e.sup.kt] (4)
where W = final DTW, [W.sub.0] = initial larval weight, t = time, and k can be derived from rearranging the equation. Estimated days for isotopic values of larvae to equilibrate with its diet values were calculated when the deviation between predicted values (fitted to Eq. 3) and observed values were similar (e.g., 0.1). The diet-tissue discrimination factor (A) was calculated for Crassostrea gigas, as it is an indication of the diet performance (DeNiro & Epstein 1978, 1981):
[DELTA]X = [delta] [X.sub.issue] - [delta][X.sub.diet] (5)
where X = [sup.13]C and/or [sup.15]N at the end of the diet exposure period, that is, Day 15.
One-way analysis of variance was performed using SPSS software (IBM SPSS Statistics Version 22) to determine the influence of diets on larval growth and survival, whereas significant differences (P < 0.05) between diets were compared using the post hoc Scheffe's test. A Student-Newman-Keuls post hoc test was used to determine the differences in isotopic values between sampling times.
Isotopic Values in Diets
The four different diets fed to Crassostrea gigas larvae showed significant differences for [delta][sup.13]C (df = 3, F = 7,511, P < 0.001) but not for [delta][sup.15]N and C:N ratios. The mean [delta][sup.13]C value of Pavlova lutheri (-46.45 [per thousand]) was significantly lower and Chaetoceros calcitrans was significantly higher (~34.55 [per thousand]) compared with other microalgal diets (Table 1).
Growth and Survival
At the end of the feeding trial (Day 15), Crassostrea gigas larvae increased in shell length and DTW, and ranged from 82.97 to 125.94 [micro]m and 54.83 to 162.54 ng, respectively (Table 2). Significant differences (df = 4, F = 51.71, P < 0.001) in larval growth between dietary treatments were found and the ranking was C = PTC>P = T (Table 2). Larvae fed on the Chaetoceros calcitrans and PTC diets had the highest growth, 162.54 and 151.86 ng, respectively, whereas unfed larvae had the lowest growth, 54.83 ng. For survival, a significant difference (P < 0.001) was only detected on Day 15 using analysis of variance. All dietary treatments that had microalgae added had similar survival (77.78% 88.89%, Table 2); however, larvae in the unfed treatment had significantly lower survival (60%) compared with other treatments (Table 2).
The carbon and nitrogen stable isotope values of Crassostrea gigas depleted toward diets from Day 1 to Day 15 showing assimilation of diet isotope values over time (Figs. 1 and 2). Among the four diets, the [delta][sup.13]C of Chaetoceros calcitrans and PTC were assimilated faster by C. gigas and were observed to be closer to the isotopic values of their diets on Day 15, and estimated to reach equilibrium by Day 20 (Fig. 1C, D). Compared with [delta][sup.13]C (0.327 [+ or -] 0.20/day), C. gigas larvae showed slower assimilation of [delta][sup.15]N (0.075 [+ or -] 0.02/day). For [delta][sup.15]N, C. gigas were observed to be closer to the diet values by Day 15 for C. calcitrans and PTC diets. The estimated time to reach equilibrium with diet values will be 60-70 days for C. calcitrans and 20-25 days (Fig. 2C) for PTC diet (Fig. 2D).
Isotopic turnover in tissue is governed by growth and metabolic factors (Eqs. 3 and 4). Based on 15 days of observed measurements, the contribution of growth to the [delta][sup.13]C turnover was 20% and 23%, whereas for [delta][sup.15]N, it was 54% and 100% for Chaetoceros calcitrans and PTC diets, respectively. These two diets were assimilated better than the other two diets. It is likely that the prediction growth model (Eq. 4) overestimated the growth contribution because larvae did not equilibrate with their diets by the end of the 15-day feeding trial. Nevertheless, the results indicate that a major portion of dietary carbon is used for metabolic turnover and dietary nitrogen for growth in Crassostrea gigas larvae.
Despite variable assimilation time for [delta][sup.13]C and [delta][sup.15]N, at the end of the 15-day feeding trial, the diet-tissue discrimination values (Eq. 5) for [delta][sup.13]C ranged from 5.82 [per thousand] to 16.64 [per thousand] with Chaetoceros calcitrans (5.82 [per thousand]) and PTC (7.20 [per thousand]) closer to the common range in aquatic larvae ([DELTA][sup.13]C: 0.4[per thousand]-4.1 [per thousand], Le Vay & Gamboa-Delgado 2011). The 815N discrimination values ([DELTA][sup.15]N: 2.21 [per thousand]-4.37 [per thousand]) for all dietary treatments were within the typical range ([DELTA][sup.15]N: 0.1 [per thousand]-5.3 [per thousand], Le Vay & Gamboa-Delgado 2011).
All microalgal diets used in this experiment exhibited similar quality (C:N ratio -8.5), however, variable growth was observed across different dietary treatments. The differences in growth in this study could be explained by the larval digestive physiology and feeding behavior (Rico-Villa ct al. 2006, Mazumder et al. 2016). The growth and survival of Crassostrea gigas larvae fed with a monospecific diet of Chaetoceros calcitrans and the PTC trispecific diet (25P:25T:50C) were lower to what is reported by Rico-Villa et al. (2006). Nevertheless, both C. calcitrans and PTC diets were shown to provide better growth and survival than the other diets tested in this study.
The results of this study showed that growth and survival in larvae were associated with assimilation of their diets. Larvae that were fed with Chaetoceros calcitrans and PTC diets showed faster isotopic turnover rates and time to equilibrate with their diets. The discrimination factor, an indication of dietary assimilation, further supports good growth performance of larvae reared on C. calcitrans and PTC diets. The discrimination values for both carbon (5.82 [per thousand] and 7.20 [per thousand], respectively) and nitrogen (2.97 [per thousand] and 3.79 [per thousand] respectively) obtained in this experiment were closer to the typical range for aquatic invertebrates ([DELTA][sup.13]C: 0.4[per thousand]-4.1[per thousand], [DELTA][sup.15]N: 0.1[per thousand]-5.3[per thousand], Le Vay & Gamboa-Delgado 2011) and suggest better assimilation of nutrients from these two diets (DeNiro & Epstein 1978, 1981) compared with other diets in this experiment. Furthermore, the results indicate that dietary carbon was mostly used for metabolism and dietary nitrogen was used for growth. Other studies have shown that dietary carbon (lipids and carbohydrates) is important for shell growth and development, whereas dietary nitrogen (protein) is important for tissue growth (His & Maurer 1988, Powell et al. 2002, Ponis et al. 2003, Rico-Villa et al. 2006).
The relatively poor growth performance from Pavlova lutheri and Tahitian Isochrysis aff. Galhanci treatments may have been related to the inability of larvae to completely assimilate specific nutrients from these diets (Metcalfe & Monaghan 2001, Martinez del Rio & Wolf 2005). Consequently, inadequate assimilation of nutrients by the larvae due to poor digestion, assimilation, and/ or preferential feeding can negatively affect growth (Rico-Villa et al. 2006, Rico-Villa et al. 2009).The species P. lutheri has been shown to produce good growth and survival for other species of bivalve larvae such as Pecten maximus (Delaunay et al. 1993), Mimachlamys asperrima (O'Connor & Heasman 1997), and Ostrea angasi (O'Connor et al. 2012).
The findings of this study indicate that both Chaetoceros calcitrans and PTC were the most suitable diets for larval growth and survival compared with the other tested microalgal diets. The increased growth in larvae from PTC could be due to the presence of C. calcitrans. Future studies could use mathematical mixing models (e.g., IsoSource and SIAR) to convert isotopic values of plurispecific diets into estimates of relative dietary contribution (Fry 2006, Phillips 2012). Further work could investigate dietary effects at metamorphosis and through the postlarval stages using SIA. This would facilitate the decision on appropriate diets and help to improve hatchery yields of Crassostrea gigas.
The results of this study showed that both Chaetoceros calcitrans and PTC induced the best larval growth and survival making them the most appropriate of the microalgal diets tested for the growth and survival of hatchery-reared Crassostrea gigas larvae. The good growth performance was supported by the fast isotopic turnover rates. In addition, the discrimination factors were closer to the common range for [delta][sup.13]C and [delta][sup.15]N. Stable carbon and nitrogen isotopes showed that C. calcitrans and PTC diets were better assimilated by the larvae compared with Pavlova lutheri and T. Iso diets. Because all dietary treatments exhibit similar nutritional quality, based on C:N ratios, the differences in assimilation and isotopic turnover rates in this study indicate that growth is strongly associated with digestive physiology and feeding behavior. The study demonstrated that SIA techniques can indeed be applied to nutritional studies for oyster larvae. Further research could also focus on the stages at which larvae are better at assimilating nutrients from specific microalgal species to optimize the larval feeding regime, which could potentially reduce the rearing time and cost of larval production in hatcheries. Stable isotope analysis would also be effective to fine tune diets for other commercial bivalve species and evaluate new and lower cost diets.
We acknowledge and thank the Port Stephens Fisheries Institute and Australian Nuclear and Technology Organisation staff for their assistance with equipment set up and sample analyses. A special thanks to Southern Cross Shellfish Hatchery for providing the oyster larvae, and Stephen O'Connor and Dr. Wayne O'Connor for their helpful comments.
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ANGELA LIU, (1) DEBASHISH MAZUMDER, (2) MICHAEL C. DOVE, (3) TATT SHENG LAI, (1) JAGODA CRAWFORD (2) AND JESMOND SAMMUT (1) *
(1) School of Biological, Earth and Environmental Sciences, The University of New South Wales, Sydney, NSW 2052, Australia; (2) Australian Nuclear Science and Technology Organisation, Locked Bag 2001 Kirrawee DC, NS W 2232, Australia;3Department of Primary Industries, Port Stephens Fisheries Institute, Taylors Beach, NSW 2316, Australia
* Corresponding author. E-mail: email@example.com
TABLE 1. Mean of stable carbon and nitrogen isotope values and elemental ratios (C:N) of Crassostrea gigas diets. Diet [delta][sup.13]C [delta][sup.15]N C:N P -46.45 (0.06) (a) -1.88 (l.OO) (a) 8.28 (0.21) (a) T -36.40 (0) (b) -2.39 (0.31) (a) 8.43 (0.20) (a) C -34.55 (0.12) (c) -1.53 (1.00) (a) 8.49 (0.2l) (a) PTC -36.32 (0.17) (b) -2.50 (0.66) (a) 8.52 (0.70) (a) Mean [+ or -] SD; n = 3. Column values with different superscripts indicate significant differences at P < 0.05. TABLE 2. Mean shell length, DTW, and survival of unfed (control) Crassostrea gigas larvae and larvae fed on monospecific and trispecific diets on Days 1 (D1), 7 (D7), and 15 (D15). Length ([micro]m) Diet D1 D7 D15 Unfed 72.25 (1.37) (a) 82.50 (5.33) (a) 82.97 (5.15) (a) (control) P 72.25 (1.37) (a) 92.81 (6.03) (b) 92.24 (16.49) (b) T 72.25 (1.37) (a) 90.33 (5.96) (b) 90.86 (12.37) (ab) C 72.25 (1.37) (a) 98.81 (8.68) (c) 125.94 (16.43) (c) PTC 72.25 (1.37) (a) 97.82 (10.61) (c) 115.78 (34.97) (c) DTW (ng) Diet Dl D7 D15 Unfed 42.55 (4.98) (a) 54.07 (9.24) (a) 54.83 (9.02) (a) (control) P 42.55 (4.98) (a) 73.07 (12.48) (b) 80.53 (17.76) (a) T 42.55 (4.98) (a) 68.21 (11,75) (b) 71.30 (31.76) (a) C 42.55 (4.98) (a) 86.34 (19.20) (c) 162.54 (48.84) (b) PTC 42.55 (4.98) (a) 84.83 (24.55) (c) 151.86 (135.59) (b) Survival (%) Diet D1 D7 D15 Unfed 100 (0) (a) 87.78 (11.7) (a) 60.0 (6.67) (a) (control) P 100 (0) (a) 97.78 (1.92) (a) 88.89 (1.92) (b) T 100 (0) (a) 98.89 (1.92) (a) 77.78 (3.85) (b) C 100 (0) (a) 97.78 (1.92) (a) 86.67 (0) (b) PTC 100 (0) (a) 96.67 (5.77) (a) 88.89 (5.09) (b) Mean [+ or -] SD; n = 3. Column values with different superscripts indicate significant differences at P < 0.05.
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|Author:||Liu, Angela; Mazumder, Debashish; Dove, Michael C.; Lai, Tatt Sheng; Crawford, Jagoda; Sammut, Jesmo|
|Publication:||Journal of Shellfish Research|
|Date:||Apr 1, 2016|
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