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INTRODUCTIONBivalve aquaculture almost entirely depends on the seed production from hatcheries due to the unreliability of wild seed collection (Wijsman et al., 2019). The use of hatchery‐produced seed is important not only for the growth of the bivalve aquaculture industry, but it also minimises pressure on natural recruitment and near‐shore ecosystems (Carranza & Zu Ermgassen, 2020). Bivalve hatcheries generally use live microalgae as the primary food source for all life stages, including larvae, juveniles and broodstock (Brown & Blackburn, 2013). Post‐set juveniles are still cultured inside a hatchery to maximise production output before transferring to nursery and grow‐out systems. A typical bivalve hatchery maintains millions of juveniles at any given time, generating a huge demand for microalgae.Live microalgae production is costly and labour intensive, representing 30%–50% of the operational cost for a bivalve hatchery (Oostlander et al., 2020). Therefore, identifying an optimal microalgae diet that improves productivity and cost‐effectiveness has crucial implications in a hatchery operation. The choice of microalgae species depends on several factors, including (a) appropriate cell size and morphology for filter feeding, (b) digestibility and nutritional profile that suit bivalve species at various life stages, (c) non‐toxicity to the consumer and (d) suitability of culture at large scale (Brown & Blackburn, 2013). The most widely used microalgae species for bivalves are Isochrysis galbana, Tisochrysis lutea (previously known as Isochrysis aff. galbana or T. Iso; Bendif et al., 2013), Tetraselmis spp., Pavlova lutheri, Chaetoceros spp., Thalassiosira spp. and Skeletonema spp. (Brown & Blackburn, 2013). A mixed microalgae diet generally offers better nutritional value than a mono‐species diet due to the complementary nutritional benefits needed for the physiological and nutritional processes of bivalve species (Cheng et al., 2020).The nutritional value of bivalve diets largely depends on the fatty acid profile of microalgae. Due to the inability of bivalves to ‘de novo’ synthesise long‐chain essential fatty acids from shorter chain precursors, the requirement of essential fatty acids must be met through diet. Microalgae species rich in n‐3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA; 20:5n‐3) and docosahexaenoic acid (DHA; 22:6n‐3) support better growth and survival at the early life stage (Liu et al., 2016; Reis Batista et al., 2014). The n‐6 PUFAs linoleic acid (18:2n‐6) and arachidonic acid (ARA; 20:4n‐6) act as metabolic precursors of EPA and DHA synthesis in bivalves; therefore, the ratio of n‐3/n‐6 also influences the quality of a bivalve diet (Soudant et al., 1999). Significant research efforts have been made to evaluate the dietary influence of fatty acids profile in many bivalve species, including oysters (Ronquillo et al., 2012; Wikfors et al., 1996), mussels (Nevejan et al., 2007), clams (Albentosa et al., 1996; Fernández‐Reiriz et al., 2006; Reis Batista et al., 2014) and scallops (Milke et al., 2004). Insufficient dietary fatty acids coupled with a low‐temperature environment triggered higher mortality rates in Mercenaria mercenaria juveniles (Portilla, 2016; Portilla et al., 2015), which demands a better understanding of a suitable microalgae diet. However, there have been limited studies on the effect of microalgae diet on the fatty acid profile of hard clam, M. mercenaria, juveniles.Hard clams are an important bivalve species for aquaculture on the East coast of the United States, with over 300 farms in 12 states generating over $65 million annually in sales (USDA, 2019). Farming of this species primarily depends on the hatchery and nursery for seed production. The techniques for hatchery and nursery production of hard clams have been described by Hadley and Whetstone (2007). They suggested a diet consisting of Isochrysis and Chaetoceros for the hard clam juvenile based on standard industry practice. Past research has evaluated the correlation between the gross biochemical composition of the diet and juvenile hard clam growth (Epifanio, 1979; Wikfors et al., 1992). To improve understanding of the dietary requirements for hard clam hatchery production, our lab has recently evaluated live microalgae and microalgae concentrate diets for larvae (Hassan et al., 2021a, 2021b) and the variations of filtration and ingestion rates of various microalgae species by larvae and juveniles (Hassan et al., 2022). This study aims to identify an optimal microalgae diet based on growth, survival and fatty acid profile for the culture of hard clam juveniles using combinations of T. lutea, P. lutheri, Chaetoceros gracilis and Cyclotella nana.MATERIALS AND METHODSJuvenile acquisition and rearing systemThis experiment was conducted at the Florida Atlantic University–Harbor Branch Oceanographic Institute (FAU‐HBOI) aquaculture facility. Seaventure Clam Co., a commercial hatchery, supplied juveniles for this study (size: 852.4 ± 225.1 µm [mean ± SD]; age: 33 days post‐fertilisation [DPF]). In the hatchery, larvae were fed a mono species diet of T. lutea up to 14 DPF, followed by a bi‐species diet of T. lutea and C. gracilis for post‐set juveniles at 15–33 DPF. During the 6‐week experiment, a complete water exchange with UV‐treated, 1‐µm‐filtered salt well water was done at 48‐h intervals. Temperature, salinity and pH in the culture system were maintained at 25–28°C, 28–30 ppt and 8.0–8.3, respectively.A downwelling flow system was built to culture juveniles using 150‐µm bottom screen fitted between a PVC pipe and coupler. The downweller dimensions were 15.2 cm diameter and 25.4 cm height (Figure 1). The downwellers were placed inside a 60‐L tank and maintained 7.6 cm off the bottom with a plastic grid that allowed proper circulation of water inside the tank. An air‐stone was strung inside of a 2.5‐cm‐diameter pipe and the pipe was placed outside the wall of the downweller. Each downweller was stocked with 2388 ± 39 juveniles. Food and circulated water were supplied in the downweller with the airlifted waterflow, which was then passed through the bottom screen.1FIGUREThe downwelling water flow system used to culture hard clam, Mercenaria mercenaria, juveniles. Figure reproduced from Hassan et al. (2021a)Microalgae culture and weight standardisationThe four microalgae species used in this study were T. lutea, P. lutheri, C. gracilis and C. nana. All the microalgae inoculums were purchased from Algagen (https://www.algagen.com/, Lot # 201205) and batch cultured in flasks, 18‐L carboys and 200‐L fiberglass tanks using UV‐treated, 1‐µm‐filtered salt well water in the indoor microalgae culture facility at FAU‐HBOI. Temperature and pH of microalgae were maintained at 20°C and 8.0–8.2, respectively. The f/2 nutrient media was used for all species, and metasilicates were added to diatom cultures. All the microalgae used in this study were collected from 200‐L fiberglass tanks.Size variations among microalgae species were standardised based on the dry weight of each species, according to Abbas et al. (2018). Briefly, 100 ml microalgae from each species were filtered in triplicate using pre‐weighted 1.2‐µm glass microfibre filters (GF) and washed with 50 ml 0.5 M ammonium formate to remove salt residue. The filters were then dried in an oven at 70°C for 18 h and total weight of microalgae was recorded. Microalgae cell concentrations were determined using a hemacytometer and dry weight of algae cell was then calculated accordingly. The dry weight (pg/cell) of microalgae used in the study was as follows: T. lutea, 13.1 pg; P. lutheri, 13.4 pg; C. gracilis, 24.6 pg; and C. nana, 174.5 pg.Experimental dietsSix diets were used to feed juveniles using bi‐, tri‐ and tetra‐species combinations for 43 days, with three replicates for each dietary treatment. Juveniles were starved for 48 h prior to the start of the experiment. In the first week, juveniles were fed once daily with 1 × 106 T. lutea per day (or equivalent dry weight of other microalgae species), and feeding ration was increased 20% each of the following weeks. Dietary treatments used in this study are described in Table 1.1TABLEMicroalgae diets used to feed hard clam, Mercenaria mercenaria, juvenilesTreatmentsRation typeMicroalgae (inclusion level)T1Flagellate only (TL + PL)Tisochrysis lutea (50%) + Pavlova lutheri (50%)T2Diatom only (CG + CN)Chaetoceros gracilis (50%) + Cyclotella nana (50%)T3Equal proportion (1 flagellate + 1 diatom species, TL + CG)T. lutea (50%) + C. gracilis (50%)T4Equal proportion (2 flagellate + 2 diatom species, TL + PL + CG + CN)T. lutea (25%) + P. lutheri (25%) + C. gracilis (25%) + C. nana (25%)T5Flagellate dominant (TL + PL + CG)T. lutea (33.3%) + Pavlova lutheri (33.3%) + C. gracilis (33.3%)T6Diatom dominant (TL + CG + CN)T. lutea (33.3%) + C. gracilis (33.3%) + C. nana (33.3%)Assessment of growth parametersJuveniles were measured from the anterior–posterior margin of the shell using an Olympus SZX7 stereo microscope installed with cellSens Standard software at the beginning of the experiment. As post‐set juveniles grew, growth was measured with a vernier calliper fortnightly by taking 1.0–2.5 ml subsamples. A total of 20 post‐set clams were measured in each replicate, with three replicates per treatment.SurvivalSurvival was determined by taking subsamples of at least 50 post‐set juveniles from each replicate, with three replicates per treatment. A juvenile with an open valve or empty shell was considered dead.Fatty acid analysisRelative fatty acid content was determined by Fatty Acid Methyl Ester (FAME) profile both for microalgae and post‐set juveniles. Fatty acid analysis was performed by Microbial ID lab in Delaware, USA (https://microbialid.com/). Microalgae were filtered on a 2‐µm GF filter and scrapped off the filter paper into 15‐ml falcon tubes. Both algae and post‐set clam were freeze dried for 24–48 h and then pulverised using mortar and pestle. FAME profiles of freeze‐dried samples (∼20 mg microalgae, ∼50–70 mg post‐set clams) were identified according to Sasser (2006). FAME were separated using gas chromatography (GC) using hydrogen as the carrier gas and nitrogen as the makeup gas. The electric signal from GC detector was compared to stored database of the Sherlock pattern recognition software. Fatty acids from microalgae and juveniles are presented as the percentage of total fatty acids (Tables 2 and 4).2TABLEFatty acid profile (% of total fatty acid) of different microalgae species used to feed hard clam, Mercenaria mercenaria, juvenilesFatty acidTisochrysis luteaPavlova lutheriChaetoceros gracilisCyclotella nana12:00.10 ± 0.010.08 ± 0.010.14 ± 0.02ND14:025.44 ± 0.25a12.39 ± 0.01c20.94 ± 0.67b12.96 ± 0.72c15:00.64 ± 0.08b1.07 ± 0.19ab1.04 ± 0.23ab1.42 ± 0.18a16:010.60 ± 1.29b21.23 ± 0.93a18.51 ± 0.69a18.56 ± 2.11a17:03.10 ± 1.202.12 ± 0.112.48 ± 3.132.62 ± 2.9819:0ND0.11 ± 0.010.33 ± 0.010.10 ± 0.0120:00.15 ± 0.01c2.19 ± 0.06a0.94 ± 0.01b2.63 ± 0.14aTotal SFA40.02 ± 0.09ab39.17 ± 1.04ab44.36 ± 1.57a38.28 ± 0.17b14:1n‐50.42 ± 0.01a0.06 ± 0.01b0.42 ± 0.02a0.32 ± 0.02ab14:1n‐70.08 ± 0.010.11 ± 0.010.13 ± 0.01ND15:1n‐60.07 ± 0.01b0.09 ± 0.00b0.23 ± 0.01ab0.46 ± 0.04a16:1n‐3NDND0.59 ± 0.031.65 ± 0.2016:1n‐50.13 ± 0.01b0.16 ± 0.01b0.60 ± 0.02ab0.87 ± 0.07a16:1n‐60.99 ± 0.04c0.69 ± 0.01c3.21 ± 0.12b11.87 ± 0.02a16:1n‐76.34 ± 0.12c21.16 ± 0.40bc38.13 ± 0.91a27.67 ± 2.05b17:1n‐80.55 ± 0.01ND0.15 ± 0.030.13 ± 0.0218:1n‐71.38 ± 0.01c8.20 ± 0.46a2.36 ± 0.02b2.21 ± 0.59b18:1n‐89.71 ± 0.30NDNDND18:1n‐912.64 ± 1.13aND0.78 ± 0.02b0.88 ± 0.13bTotal MUFA32.29 ± 1.05b30.46 ± 0.88b46.57 ± 1.07a40.11 ± 5.28ab18:2n‐64.75 ± 0.05a0.47 ± 0.02b0.89 ± 0.02b0.54 ± 0.06b18:3n‐60.33 ± 0.02c0.18 ± 0.01c0.92 ± 0.05b3.48 ± 0.03a20:4n‐6 (ARA)0.20 ± 0.05b0.23 ± 0.02b0.71 ± 0.01aND22:5n‐60.82 ± 0.01b0.27 ± 0.03cND1.20 ± 0.20aTotal PUFA (n‐6)6.09 ± 0.01a1.14 ± 0.08b2.51 ± 0.08b5.22 ± 0.09a18:4n‐313.44 ± 0.81a5.18 ± 0.05b0.48 ± 0.05c0.80 ± 0.11c20:5n‐3 (EPA)0.65 ± 0.01c17.85 ± 0.16a5.48 ± 0.21b14.99 ± 1.69a22:6n‐3 (DHA)6.81 ± 0.20a5.05 ± 0.14a0.16 ± 0.01c1.25 ± 0.21bTotal HUFA8.48 ± 0.27c23.4 ± 0.35a6.35 ± 0.23c17.44 ± 0.21bTotal PUFA (n‐3)20.90 ± 0.99b28.08 ± 0.30a6.12 ± 0.18c17.04 ± 2.02bn‐3/n‐63.43 ± 0.16c24.78 ± 1.43a2.44 ± 0.01c6.71 ± 4.66bTotal FAs99.29 ± 0.0498.84 ± 0.2299.56 ± 0.2398.90 ± 0.38Note: Different letters in the same column indicate significant differences among the treatments (one‐way ANOVA, α = 0.05, a > b). Microalgae were filtered on 2‐µm GF filter and freeze dried for 24 h prior to fatty acid analysis.Abbreviations: ARA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FAs, fatty acids; HUFA, highly unsaturated fatty acid; MUFA, monounsaturated fatty acid; ND, not detected; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid.Statistical analysisAll data were tested for assumptions of normality and homogeneity of variances using Shapiro–Wilk test and Levene's test statistics. All the proportional data were arcsine transformed prior to analysis. The differences in growth, survival and fatty acid content among treatments were analysed with one‐way analysis of variance (ANOVA). When significant differences were observed between treatments, Tukey post hoc test was used. The Kruskal–Wallis test along with Dunn's nonparametric post hoc analysis was performed when assumptions on normality or homogeneity of variances were violated. The data were considered statistically significant at p < 0.05. Data were analysed using SPSS v.27 (IBM, Armonk, NY).RESULTSGrowthSignificant differences were found in final size of juveniles fed different microalgae diets (F = 12.5, df = 5, p < 0.05). Juveniles fed the tetra‐species diet T. lutea, P. lutheri, C. gracilis and C. nana (T4) numerically grew larger than all other treatments (Table 3), but growth rate was statistically similar in T. lutea and P. lutheri (T1), T. lutea and C. gracilis (T3) and T. lutea, C. gracilis and C. nana (T6) diets. Juveniles fed a diatom dominant diet (T. lutea, C. gracilis and C. nana; T6) numerically grew larger than those fed a flagellate dominant diet (T. lutea, P. lutheri and C. gracilis; T5), but no statistical difference was found. Juveniles fed the flagellate‐only diet (T. lutea and P. lutheri; T1) numerically grew larger than those fed the diatom‐only diet (C. gracilis and C. nana; T2), but no statistical difference was found.3TABLESize (mm, mean length ± SD) of hard clam, Mercenaria mercenaria, juveniles fed live microalgae dietsDiet treatmentDuration of feeding2 weeks (Age: 49 DPF)4 weeks (Age: 63 DPF)6 weeks (Age: 77 DPF)T12.3 ± 0.9ab3.0 ± 0.7ab3.6 ± 1.1abT21.9 ± 0.7b2.6 ± 0.8b3.2 ± 1.4bT32.5 ± 0.7ab3.0 ± 0.8ab3.8 ± 1.1abT42.9 ± 0.6a3.3 ± 0.6a4.0 ± 1.2aT52.7 ± 0.9ab3.0 ± 0.6ab3.3 ± 0.9bT62.6 ± 0.7ab3.1 ± 0.7ab3.7 ± 1.1abNote: Different letters in the same column indicate significant differences among the treatments (one‐way ANOVA, α = 0.05, a > b).Abbreviation: DPF, days post‐fertilisation.Significant differences were found in the daily growth rates of juvenile clams fed different microalgae diets (F = 3.8, df = 5, p < 0.05). Juveniles fed the tetra‐species diet of T. lutea, P. lutheri, C. gracilis and C. nana (T4) attained a higher daily growth rate (74.9 µm/day), but the daily growth rate was statistically similar among T1, T3, T4 and T6 (Figure 2). Juveniles fed the bi‐species diet of C. gracilis and C. nana (T2) attained lower daily growth rate than other treatments (57.1 µm/day). Among clams fed equal proportions of flagellates and diatoms, the daily growth rate was numerically higher with the tetra‐species diet T. lutea, P. lutheri, C. gracilis and C. nana (T4) than the bi‐species diet T. lutea and C. gracilis (T3), but no statistical difference was found.2FIGUREDaily growth rate (µm/day) of hard clam, Mercenaria mercenaria, juveniles fed live microalgae diets. Different letters indicate significant differences among the treatments (one‐way ANOVA, α = 0.05, a > b). Each bar represents mean ± SD of three replicates.SurvivalSignificant differences were found in final survival of juveniles fed different microalgae diets (F = 7.2, df = 5, p < 0.05, Figure 3). Juvenile clams fed the tetra‐species diet of T. lutea, P. lutheri, C. gracilis and C. nana (T4) numerically attained the highest survival rate (87.4%) and juveniles fed the tri‐species diet of T. lutea, C. gracilis and C. nana (T6) numerically attained the lowest survival rate (75.3%). There was no statistical difference in survival among juveniles fed T1, T3, T4 and T5 diets.3FIGUREFinal survival rate of hard clam, Mercenaria mercenaria, juveniles fed live microalgae diets. Different letters indicate significant differences among the treatments (one‐way ANOVA, α = 0.05, a > b > c). Each bar represents mean ± SD of three replicates.Fatty acid profile of juvenilesThe fatty acid content of juveniles fed different microalgae diets is presented in Table 4. No statistical difference was found in the percentage of saturated fatty acid (SFA) content (F = 2.8, df = 5, p = 0.06), but significant differences were found in the percentage of monounsaturated fatty acid (MUFA) content (F = 69.3, df = 5, p < 0.05) of juvenile clams in various treatments. Among the long‐chain PUFAs, significant differences were found in the percentage of ARA (F = 93.8, df = 5, p < 0.05), EPA (F = 168.2, df = 5, p < 0.05), DHA (F = 116.3, df = 5, p < 0.05), n‐3 PUFA (F = 9.4, df = 5, p < 0.05) and n‐6 PUFA (F = 16.8, df = 5, p < 0.05) contents. Juveniles fed the flagellate‐only diet (T1) had a higher percentage of MUFA and DHA but a lower percentage of SFA and PUFA n‐6. In contrast, juveniles fed a diatom‐only diet (T2) had a higher percentage of ARA and EPA but a lower percentage of DHA. Juveniles fed the tetra‐species diet of T. lutea, P. lutheri, C. gracilis and C. nana (T4) had neither very high nor very low percentages of ARA, EPA, DHA and total PUFA content but maintained moderate levels of all the essential fatty acids compared to those in other treatments.4TABLEFatty acid profile (% of total fatty acid) of hard clam, Mercenaria mercenaria, juveniles fed microalgae dietsFatty acidTreatment 1Treatment 2Treatment 3Treatment 4Treatment 5Treatment 614:06.93 ± 0.68a1.43 ± 0.02c3.60 ± 0.23b4.13 ± 0.38b4.64 ± 0.53b2.32 ± 0.12c15:00.66 ± 0.040.54 ± 0.160.58 ± 0.020.67 ± 0.050.59 ± 0.060.56 ± 0.0216:024.49 ± 0.34b27.1 ± 0.06ab28.63 ± 0.51ab30.27 ± 1.02a27.93 ± 0.52ab28.15 ± 0.21ab17:05.66 ± 0.41ab7.76 ± 0.10a4.63 ± 2.61b2.66 ± 0.43c7.57 ± 1.25a6.44 ± 0.46a20:00.94 ± 0.03c3.71 ± 0.08a2.17 ± 0.12ab1.56 ± 0.12b1.29 ± 0.05b2.33 ± 0.20ab21:01.06 ± 0.22abND0.64 ± 0.11c0.85 ± 0.07b1.18 ± 0.41a0.60 ± 0.05cTotal SFA39.74 ± 0.3240.57 ± 0.0940.26 ± 2.4340.15 ± 1.6243.20 ± 1.0140.39 ± 0.2716:1n‐60.22 ± 0.020.32 ± 0.030.26 ± 0.040.29 ± 0.02ND0.22 ± 0.0216:1n‐72.83 ± 0.17c6.66 ± 0.10a4.44 ± 0.14b3.92 ± 0.39b3.38 ± 0.40bc4.11 ± 0.13b18:1n‐60.24 ± 0.03e1.21 ± 0.02a0.42 ± 0.05 cd0.53 ± 0.09bc0.32 ± 0.05de0.65 ± 0.01b18:1n‐76.46 ± 0.23a5.92 ± 0.16abc6.04 ± 0.15ab5.70 ± 0.21bc5.55 ± 0.52b5.40 ± 0.02b18:1n‐98.11 ± 0.13a1.51 ± 0.07d3.21 ± 0.10c5.75 ± 0.16b6.22 ± 0.29b4.05 ± 0.07bc19:1n‐7ND0.36 ± 0.020.34 ± 0.04NDND0.36 ± 0.0320:1n‐61.16 ± 0.06c3.86 ± 0.08a2.13 ± 0.01ab1.99 ± 0.25ab1.61 ± 0.04b2.38 ± 0.07ab20:1n‐95.63 ± 0.19aND2.87 ± 0.69b3.94 ± 0.44ab4.73 ± 0.47abND22:1n‐30.30 ± 0.02ND0.33 ± 0.060.35 ± 0.05ND0.25 ± 0.0422:1n‐9ND0.77 ± 0.01ND1.28 ± 0.16ND1.10 ± 0.03Total MUFA24.94 ± 0.36a20.61 ± 0.21bc22.03 ± 0.24ab23.75 ± 0.69a21.82 ± 0.89b18.51 ± 0.21c18:2n‐62.14 ± 0.12a0.53 ± 0.01c1.23 ± 0.06b1.30 ± 0.14b1.45 ± 0.17b0.78 ± 0.01c20:2n‐67.05 ± 0.34c6.99 ± 0.13c9.91 ± 1.85b7.21 ± 1.02c7.71 ± 0.36c11.06 ± 0.34a20:3n‐60.78 ± 0.020.88 ± 0.010.56 ± 0.140.57 ± 0.140.46 ± 0.100.53 ± 0.1020:4n‐6 ARA3.04 ± 0.21d6.17 ± 0.12a5.25 ± 0.27b4.33 ± 0.27c4.23 ± 0.07c4.99 ± 0.03b22:4n‐60.34 ± 0.01d2.24 ± 0.05a0.93 ± 0.12b0.61 ± 0.01c0.59 ± 0.07c1.05 ± 0.08b22:5n‐63.87 ± 0.18b3.89 ± 0.01b3.26 ± 0.14c4.26 ± 0.28a3.60 ± 0.06bc4.31 ± 0.02aTotal PUFA n‐617.20 ± 0.60c20.70 ± 0.19ab21.13 ± 1.77a18.27 ± 1.03bc18.04 ± 0.58bc22.72 ± 0.34a15:4n‐30.20 ± 0.02e1.75 ± 0.04a0.61 ± 0.02c0.57 ± 0.06c0.36 ± 0.06d0.87 ± 0.02b18:4n‐31.90 ± 0.21aND1.16 ± 0.05b1.08 ± 0.13b1.26 ± 0.14b0.64 ± 0.02c20:5n‐3 (EPA)1.75 ± 0.04e9.09 ± 0.51a5.20 ± 0.35bc5.09 ± 0.64c3.50 ± 0.13d6.11 ± 0.15c22:5n‐3ND2.28 ± 0.06a0.78 ± 0.07bc0.69 ± 0.10c0.54 ± 0.08d1.00 ± 0.09b22:6n‐3 (DHA)11.18 ± 0.38a3.59 ± 0.22d9.60 ± 0.37bc9.37 ± 1.00bc10.24 ± 0.37ab8.25 ± 0.17cTotal HUFA28.01 ± 0.8935.13 ± 1.3235.49 ± 0.9332.13 ± 1.1830.87 ± 0.9737.3 ± 0.67Total PUFA n‐315.04 ± 0.22c16.72 ± 0.65ab17.34 ± 0.76a16.79 ± 0.50ab15.90 ± 0.18bc16.87 ± 0.21abn‐3/n‐60.87 ± 0.03a0.81 ± 0.04ab0.82 ± 0.07ab0.92 ± 0.05a0.88 ± 0.04a0.74 ± 0.02bTotal EPA+DHA12.93 ± 0.41ab12.68 ± 0.62ab14.79 ± 0.72a14.46 ± 0.50a13.74 ± 0.27a14.36 ± 0.11aTotal EPA+DHA+ARA15.97 ± 0.62b18.85 ± 0.52ab20.04 ± 0.98a18.78 ± 0.49ab17.97 ± 0.24ab19.35 ± 0.13aEPA/ARA0.58 ± 0.03c1.48 ± 0.11a0.99 ± 0.02b1.18 ± 0.20ab0.83 ± 0.03bc1.22 ± 0.03abDHA/ARA3.69 ± 0.13a0.58 ± 0.04d1.83 ± 0.02bc2.17 ± 0.24b2.42 ± 0.11b1.65 ± 0.03c(EPA+DHA)/ARA4.27 ± 0.16a2.06 ± 0.14c2.82 ± 0.01bc3.35 ± 0.26b3.25 ± 0.11b2.88 ± 0.01bcTotal FAs98.76 ± 2.0498.60 ± 0.4398.10 ± 0.9398.96 ± 1.0198.95 ± 0.3698.50 ± 0.26Note: Juveniles were freeze dried for 48 h and pulverised prior to fatty acid analysis. Different letters within a row indicate significant differences in fatty acid profile.Abbreviations: ARA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FAs, fatty acids; HUFA, highly unsaturated fatty acid; MUFA, monounsaturated fatty acid; ND, not detected; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid.DISCUSSIONThe requirements of microalgae diets for juvenile culture have been evaluated for many commercially important bivalve species, but this is the first study that determined an optimal diet for M. mercenaria juveniles. In this study, juvenile clams attained numerically higher growth and survival when fed a tetra‐species diet composed of T. lutea, P. lutheri, C. gracilis and C. nana, but production parameters of juveniles were statistically similar in diets composed of T. lutea and P. lutheri; T. lutea and C. gracilis; or T. lutea, C. gracilis and C. nana. While better growth and survival of juveniles was achieved by feeding a tetra‐species diet, the cost for multiple microalgae species production should also be taken into consideration for hatchery/nursery operations. Microalgae diets composed of diatoms only (C. gracilis and C. nana) resulted in decreased production; therefore, a mix of flagellate and diatom microalgae should be included in juvenile diets.The determination of microalgae species used to feed bivalves at different life stages depends on the nutritional quality and the ability of that bivalve to ingest and digest microalgae. Ingestion is influenced by the microalgae cell size and presence or absence of spines, whereas digestion is influenced by the thickness and nature of the microalgae cell wall and the presence or absence of toxins (Martínez‐Fernández et al., 2004). Microalgae species used in this study (T lutea, P. lutheri and C. gracilis) are widely used for bivalve hatchery production. The diatom Cyclotella is not as commonly used globally, but it has been found suitable for triangle sail mussel Hyriopsis cumingii culture (Chen et al., 2021) and has been used to successfully culture other species at the FAU‐HBOI aquaculture facility (unreported data). The average size range of the microalgae used in this study was 4.5–10.6 µm (Hassan et al., 2022). In this study, hard clam juveniles had an average initial shell length of 852.4 µm; therefore, they were of sufficient size to ingest and digest the microalgae species. Consequently, the observed differences in growth and survival are linked to the nutritional quality of microalgae. Since bivalves have a limited capacity to synthesise PUFAs from their fatty acid precursors, the concentration of PUFAs (especially EPA and DHA) in the microalgae diets was the contributing factor for the observed differences in juvenile production parameters.A juvenile bivalve diet composed of multiple microalgae species has been found to result in better production than single microalgae species in several studies (Liu et al., 2009, 2016; Milke et al., 2004, 2006; Ren et al., 2015). Mixed microalgae diets provide complementary nutritional benefits leading to higher growth and survival of juveniles. The choice of a mixed microalgae diet depends on the suitability of microalgae for each bivalve species. Thereby, various microalgae species have been reported as a suitable diet for different bivalve species, including P. lutheri and C. calcitrans for the sea scallop Placopecten magellanicus (Milke et al., 2004) and bay scallop Argopecten irradians (Milke et al., 2006); Nannochloropsis oculata and P. lutheri for the European flat oyster Ostrea edulis (Ronquillo et al., 2012); and T. lutea and Chaetoceros muelleri for the Pacific geoduck clam, Panopea generosa (Ren et al., 2015). However, the inclusion of unsuitable microalgae in the mixed diet can lower the growth and survival of bivalves. The triangle sail mussel Hyriopsis cumingii grew slowly due to the inclusion of Monoraphidium contortum in a mixed microalgae diet (Chen et al., 2021). Bivalve hatcheries typically use a combination of flagellate and diatom microalgae for juvenile culture. In this study, higher growth and survival of juvenile clams were achieved with mixed microalgae diets compared to diatom (CG + CN) only diets. When mixed microalgae diets with equal proportions of flagellates and diatoms were compared, diets composed of two flagellate and two diatom species attained similar growth rate as the diet composed of one flagellate and one diatom species. Although the production parameters in the tetra‐species diet were numerically higher than that in the bi‐species diet, the additional cost and labour required for production of multiple microalgae species need careful financial consideration of hatchery/nursery operators.Among the dietary nutritional components, PUFAs play essential roles in supporting nutritional requirements. The requirements of PUFAs for the maintenance of cellular membrane integrity and physiological functions are primarily met through exogenous food supplies (Soudant et al., 2000; Tan and Zheng, 2022). Among the microalgae species used in this study, P. lutheri and C. nana had a high EPA content and T. lutea and P. lutheri had a high DHA content. A similar trend in the variations of EPA and DHA content among flagellate and diatom microalgae species has also been reported in other studies (Brown et al., 1997; Rivero‐Rodríguez et al., 2007; Volkman et al., 1989). The juvenile clams fed a flagellate‐only diet in this study had the highest DHA but lowest EPA content. In contrast, juveniles fed a diatom‐only diet had the highest EPA but lowest DHA content. However, juveniles fed tetra‐species diet had moderate levels of both EPA and DHA. Based on these results, the current study argues that the tetra‐species microalgae diet provided balance of PUFAs such as EPA and DHA which supported enhanced growth and survival. Pacific oyster juveniles had slower growth when fed a microalgae species (Dunaliella tertiolecta) with a very low EPA and DHA content (McCausland et al., 1999). The Pacific geoduck clam, P. generosa, achieved higher production when fed a diet containing microalgae species rich in EPA (Tetraselmis suecica, Phaeodactylum tricornutum, Thalassiosira pseudonana) and DHA (T. lutea) (Ren et al., 2015). Studies conducted with other bivalve species also show superior production outcomes with diets balanced in EPA and DHA content (Caers et al., 1998; Milke et al., 2004, 2006; Pernet and Tremblay, 2004).In addition to EPA and DHA, ARA is considered one of the important PUFAs known to play a role in forming eicosanoids that are crucial in various physiological and pathological processes in marine bivalves (Delaporte et al., 2006; Hurtado et al., 2009). The ARA content was relatively low in the microalgae diets (less than 1%) used in this study compared to juvenile clam tissues (3%–6.2%) in all the dietary treatments, yet this lower ARA content did not seem to affect production parameters. Results in this study indicated that a diet low in ARA content did not limit the growth and survival of juveniles when EPA and DHA content in the diet was balanced. In the common cockle, Cerastoderma edule, relatively higher ARA content was found in the tissues of juveniles fed microalgae diets with low ARA content, and ARA content did not limit juvenile growth (Reis Batista et al., 2014). Hurtado et al. (2009) underscored the importance of ARA in maturation and immune response but did not find an effect on the growth of the mangrove oyster, Crassostrea corteziensis. Although the limited capacity of bivalves to synthesise PUFAs from the fatty acid precursors is well accepted, results of this and other studies evoke the possibility of de novo ARA synthesis (Waldock & Holland, 1984). A potential limitation for the claim in the present study is that the absolute ARA content in juvenile tissue at the beginning of the experiment is unknown. An elevated level of PUFA seen in Pacific oyster, Crassostrea gigas, broodstock tissue compared to levels in the diets also suggested the possibility of elongation and desaturation of EPA and DHA from fatty acid precursors (Rato et al., 2019). Waldock and Holland (1984) suggested that Pacific oyster, C. gigas, juveniles were able to incorporate dietary 14C label into long‐chain fatty acids, but the level of synthesis was insufficient to sustain optimal growth. Further research on the ability of hard calm juveniles to synthesise ARA from the fatty acid precursors would be intriguing.In addition to PUFAs, other nutritional dietary components such as protein, lipids, carbohydrates, vitamins and minerals may affect juvenile production performance. When the essential nutritional components (such as PUFAs) are present adequately, the differences in proximate composition among diets can affect production. Microalgae diets with adequate PUFAs and high carbohydrate content resulted in higher growth of juvenile European oysters, Ostrea edulis (Enright et al., 1986), but higher protein content resulted in higher growth in juvenile mussels, Mytilus trossulus (Kreeger & Langdon, 1993). Growth of juvenile scallops Pecten maximus was positively correlated to the carbohydrate but not the lipid content of the diet (Reitan, 2011). Unfortunately, due to the amount of clam tissue required for proximate composition determination, this analysis was not done in the present study.CONCLUSIONThis study concludes that a microalgae diet that consists of flagellates and diatoms is a better option for hard clam juveniles than diatom only diets due to nutritionally balanced profile. While the tetra‐species diet was numerically more suitable for enhanced growth and survival, the cost of production of multiple microalgae species is likely to be higher. The benefit–cost analysis of multiple microalgae species production versus enhanced production parameters of juveniles should be undertaken by commercial hatchery/nursery operations to determine whether an economic benefit exists. The dietary fatty acids EPA and DHA appeared to be necessary; therefore, a mixed microalgae diet containing high levels of both EPA and DHA are preferable for growth and survival. Results of this study could directly be applied to improve productivity and profitability in hard clam hatcheries. Hard clam juveniles fed different microalgae diet exposed to different environmental conditions could contribute to understanding the role of nutrition in immunity and environmental adaptability.AUTHOR CONTRIBUTIONSMd Mahbubul Hassan designed experiment, collected and analysed data and prepared the manuscript. Edward Perri designed the juvenile culture system and collected data. Victoria Parks facilitated logistic supply. Susan Laramore involved in funding acquisition, project administration and manuscript revision. All authors read and approved the final manuscript for publication.ACKNOWLEDGEMENTSWe acknowledge the support from Seaventure Clam Co. for supplying microalgae and juvenile clams for this study. Special thanks to Richard Baptiste for helping with the juvenile culture system design. This research was supported by a sponsored research agreement between Florida Atlantic University and Seaventure Clam Co. (FAU‐SRA #19–284). This is FAU contribution #2314.CONFLICT OF INTERESTThe authors declare no conflict of interest.DATA AVAILABILITY STATEMENTData generated from this study will be made available upon reasonable request from the corresponding author.PEER REVIEWThe peer review history for this article is available at: https://publons.com/publon/10.1002/aff2.80ETHICS STATEMENTEthical approval was not required for the research conducted in association with this manuscript.REFERENCESAbbas, A.S., El‐Wazzan, E., Khafage, A.R., El‐Sayed, A.F.M. & Razek, F.A.A. (2018) Influence of different microalgal diets on gonadal development of the carpet shell clam Ruditapes decussatus broodstock. Aquaculture International, 26, 1297–1309.Albentosa, M., Labarta, U., Fernández‐Reiriz, M.J. & Pérez‐Camacho, A. (1996) Fatty acid composition of Ruditapes decussatus spat fed on different microalgae diets. Comparative Biochemistry and Physiology, 113, 113–119.Bendif, E.M., Probert, I., Schroeder, D.C. & de Vargas, C. (2013) On the description of Tisochrysis lutea gen. nov. sp. nov. and Isochrysis nuda sp. nov. in the Isochrysidales, and the transfer of Dicrateria to the Prymnesiales (Haptophyta). Journal of Applied Phycology, 25, 1763–1776.Brown, M.R. & Blackburn, S.I. (2013) Live microalgae as feeds in aquaculture hatcheries. In Allan, G., Burnell, G. (Eds.) Advances in aquaculture hatchery technology 117–158. Oxford: Woodhead Publishing.Brown, M.R., Jeffrey, S.W., Volkman, J.K. & Dunstan, G.A. (1997) Nutritional properties of microalgae for mariculture. Aquaculture, 151, 315–331.Caers, M., Coutteau, P., Lombeida, P. & Sorgeloos, P. (1998) The effect of lipid supplementation on growth and fatty acid composition of Tapes philippinarum spat. Aquaculture, 162, 287–299.Carranza, A. & Zu Ermgassen, P.S. (2020) A global overview of restorative shellfish mariculture. Frontiers in Marine Science, 7, 722.Chen, Q., Jiang, X., Han, Q., Sheng, P., Chai, Y., Peng, R. et al. (2021) Growth, calcium content, proximate composition, and fatty acid composition of triangle sail mussel (Hyriopsis cumingii) fed five different microalgal diets. Aquaculture, 530, 735719.Cheng, P., Zhou, C., Chu, R., Chang, T., Xu, J., Ruan, R., Chen, P. & Yan, X. (2020) Effect of microalgae diet and culture system on the rearing of bivalve mollusks: nutritional properties and potential cost improvements. Algal Research, 51, 102076.Delaporte, M., Soudant, P., Moal, J., Giudicelli, E., Lambert, C., Séguineau, C. et al. (2006) Impact of 20∶ 4n‐6 supplementation on the fatty acid composition and hemocyte parameters of the Pacific oyster Crassostrea gigas. Lipids, 41, 567–576.Enright, C.T., Newkirk, G.F., Craigie, J.S. & Castell, J.D. (1986) Evaluation of phytoplankton as diets for juvenile Ostrea edulis L. Journal of Experimental Marine Biology and Ecology, 96, 1–13.Epifanio, C.E. (1979) Growth in bivalve molluscs: nutritional effects of two or more species of algae in diets fed to the American oyster Crassostrea virginica (Gmelin) and the hard clam Mercenaria mercenaria (L.). Aquaculture, 18, 1–12.Fernández‐Reiriz, M.J., Labarta, U., Albentosa, M. & Pérez‐Camacho, A. (2006) Lipid composition of Ruditapes philippinarum spat: effect of ration and diet quality. Comparative Biochemistry and Physiology Part B: Biochemistry & Molecular Biology, 144, 229–237.Hadley, N.H. & Whetstone, J.M. (2007) Hard clam hatchery and nursery production. Stoneville, MS: Southern Regional Aquaculture Center.Hassan, M.M., Parks, V. & Laramore, S. (2021a) Optimizing microalgae diets for hard clam, Mercenaria mercenaria, larvae culture. Aquac. Reports, 20, 100716.Hassan, M.M., Parks, V. & Laramore, S. (2021b) Assessment of microalgae concentrate as diet for hard clam, Mercenaria mercenaria, larvae. Aquaculture Nutrition, 27, 1871–1879.Hassan, M.M., Parks, V. & Laramore, S. (2022) Variation in filtration and ingestion rates among different microalgae species by hard clam, Mercenaria mercenaria, larvae and post‐set juveniles. Aquaculture Research, 53, 684–688.Hurtado, M.A., Reza, M., Ibarra, A.M., Wille, M., Sorgeloos, P., Soudant, P. et al. 2009. Arachidonic acid (20: 4n‐6) effect on reproduction, immunology, and prostaglandin E2 levels in Crassostrea corteziensis (Hertlein, 1951). Aquaculture, 294, 300–305.Kreeger, D.A., Langdon, C.J. (1993) Effect of dietary protein content on growth of juvenile mussels, Mytilus trossulus (Gould 1850). The Biological Bulletin, 185, 123–139.Liu, W., Pearce, C.M., Alabi, A.O., Gurney‐Smith, H. (2009) Effects of microalgal diets on the growth and survival of larvae and post‐larvae of the basket cockle, Clinocardium nuttallii. Aquaculture, 293, 248–254.Liu, W., Pearce, C.M., McKinley, R.S. & Forster, I.P. (2016) Nutritional value of selected species of microalgae for larvae and early post‐set juveniles of the Pacific geoduck clam, Panopea generosa. Aquaculture, 452, 326–341.Martínez‐Fernández, E., Acosta‐Salmón, H. & Rangel‐Dávalos, C. (2004) Ingestion and digestion of 10 species of microalgae by winged pearl oyster Pteria sterna (Gould, 1851) larvae. Aquaculture, 230, 417–423.McCausland, M.A., Brown, M.R., Barrett, S.M., Diemar, J.A. & Heasman, M.P. (1999). Evaluation of live microalgae and microalgal pastes as supplementary food for juvenile Pacific oysters (Crassostrea gigas). Aquaculture, 174, 323–342.Milke, L.M., Bricelj, V.M. & Parrish, C.C. (2004) Growth of postlarval sea scallops, Placopecten magellanicus, on microalgal diets, with emphasis on the nutritional role of lipids and fatty acids. Aquaculture, 234, 293–317.Milke, L.M., Bricelj, V.M. & Parrish, C.C. (2006) Comparison of early life history stages of the bay scallop, Argopecten irradians: effects of microalgal diets on growth and biochemical composition. Aquaculture, 260, 272–289.Nevejan, N., Davis, J., Little, K. & Kiliona, A. (2007) Use of a formulated diet for mussel spat Mytilus galloprovincialis (Lamarck 1819) in a commercial hatchery. Journal of Shellfish Research, 26, 357–363.Oostlander, P.C., van Houcke, J., Wijffels, R.H. & Barbosa, M.J. (2020) Microalgae production cost in aquaculture hatcheries. Aquaculture, 525, 735310.Pernet, F. & Tremblay, R. (2004). Effect of varying levels of dietary essential fatty acid during early ontogeny of the sea scallop Placopecten magellanicus. Journal of Experimental Marine Biology and Ecology, 310, 73–86.Portilla, S.E., Branco, B.F. & Tanacredi, J.T. (2015) Preliminary investigation into the effects of two dietary fatty acids, 20: 5n‐3 and 22: 6n‐3, on mortality of juvenile Mercenaria mercenaria during the approach to winter. Aquaculture International, 23, 1357–1376.Portilla, S.E. (2016) Mortality of first‐year cultured northern quahogs, Mercenaria mercenaria, through thermal decline: impacts of low temperature, the rate of temperature decrease and dietary 20: 5n‐3 and 22: 6n‐3. Aquaculture, 454, 130–139.Rato, A., Pereira, L.F., Joaquim, S., Gomes, R., Afonso, C., Cardoso, C. et al. (2019) Fatty acid profile of Pacific oyster, Crassostrea gigas, fed different ratios of dietary seaweed and microalgae during broodstock conditioning. Lipids, 54, 531–542.Reis Batista, I., Kamermans, P., Verdegem, M.C.J. & Smaal, A.C. (2014) Growth and fatty acid composition of juvenile Cerastoderma edule (L.) fed live microalgae diets with different fatty acid profiles. Aquaculture Nutrition, 20, 132–142.Reitan, K.I. (2011) Digestion of lipids and carbohydrates from microalgae (Chaetoceros muelleri Lemmermann and Isochrysis aff. galbana clone T‐ISO) in juvenile scallops (Pecten maximus L.). Aquaculture Research, 42, 1530–1538.Ren, Y., Liu, W., Pearce, C.M., Forster, I. & McKinley, R.S. (2015) Effects of selected mixed‐algal diets on growth and survival of early postset juveniles of the Pacific geoduck clam, Panopea generosa (Gould, 1850). Aquaculture Nutrition, 21, 152–161.Rivero‐Rodríguez, S., Beaumont, A.R. & Lora‐Vilchis, M.C. (2007) The effect of microalgal diets on growth, biochemical composition, and fatty acid profile of Crassostrea corteziensis (Hertlein) juveniles. Aquaculture, 263, 199–210.Ronquillo, J.D., Fraser, J. & McConkey, A.J. (2012) Effect of mixed microalgal diets on growth and polyunsaturated fatty acid profile of European oyster (Ostrea edulis) juveniles. Aquaculture, 360, 64–68.Sasser, M. (2006) Bacterial identification by gas chromatographic analysis of fatty acid methyl esters (GC‐FAME). Newark, NY: MIDI‐Inc.Soudant, P., Chu, F.L.E. & Samain, J.F. (2000) Lipids requirements in some economically important marine bivalves. Journal of Shellfish Research, 19, 605.Soudant, P., Van Ryckeghem, K., Marty, Y., Moal, J., Samain, J.F. & Sorgeloos, P. (1999) Comparison of the lipid class and fatty acid composition between a reproductive cycle in nature and a standard hatchery conditioning of the Pacific oyster Crassostrea gigas. Comparative Biochemistry and Physiology Part B: Biochemistry & Molecular Biology, 123, 209–222.Tan, K. & Zheng, H. (2022) Endogenous LC‐PUFA biosynthesis capability in commercially important mollusks. Critical Reviews in Food Science and Nutrition, 62, 2836–2844.United States Department of Agriculture (USDA). (2019) 2017 Census of Agriculture: Census of Aquaculture 2018. https://www.nass.usda.gov/Publications/AgCensus/2017/Online_Resources/Aquaculture/Aqua.pdfVolkman, J.K., Jeffrey, S.W., Nichols, P.D., Rogers, G.I. & Garland, C.D. (1989) Fatty acid and lipid composition of 10 species of microalgae used in mariculture. Journal of Experimental Marine Biology and Ecology, 128, 219–240.Waldock, M.J. & Holland, D.L. (1984) Fatty acid metabolism in young oysters, Crassostrea gigas: polyunsaturated fatty acids. Lipids, 19, 332–336.Wijsman, J.W.M., Troost, K., Fang, J. & Roncarati, A. (2019) Global production of marine bivalves. Trends and challenges. In: Smaal, A., Ferreira, J., Grant, J., Petersen, J., Strand, Ø. (eds), Goods and services of marine bivalves, 7–26. Cham: Springer.Wikfors, G.H., Ferris, G.E. & Smith, B.C. (1992) The relationship between gross biochemical composition of cultured algal foods and growth of the hard clam, Mercenaria mercenaria (L.). Aquaculture, 108, 135–154.Wikfors, G.H., Patterson, G.W., Ghosh, P., Lewin, R.A., Smith, B.C. & Alix, J.H. (1996) Growth of post‐set oysters, Crassostrea virginica, on high‐lipid strains of algal flagellates Tetraselmis spp. Aquaculture, 143, 411–419.
Aquaculture Fish and Fisheries – Wiley
Published: Feb 1, 2023
Keywords: bivalve; diet; hard clam; Mercenaria mercenaria; microalgae
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