Determining a microalgal cell concentration appropriate for the specific feeding regime within the novel RAS was an essential first step towards developing production-oriented D. antillarum culture protocols. Broadly, Hodin et al. (2018) state that most echinoderm larvae grow rapidly when fed between 5.0–10.0×103 cells ml− 1 of ~ 10-µm diameter live microalgae every 2–3 days. Optimal cell concentrations, however, can be species-specific and depend on the microalgae type and feeding regime used. Two separate small-scale D. antillarum culture attempts reported the most success when larvae were exposed to 5.0–10.0×103 cells ml− 1 mixtures of R. lens + T. lutea (Eckert, 1998), and different 10.0×103 cells ml− 1 combinations of T. luteau, C. gracilis, and R. lens (Leber et al., 2009). These feedings occurred every 3–7 days after 100% water exchange in standalone 1- 2.4- L vessels. Larger-scale culture attempts in standalone 50-L vessels were supplied between 30.0–50.0×103 cells ml− 1 combinations of R. lens + T. lutea + C. gracilis every 3–4 days after 100% water exchange (Moe, 2014). Production-oriented recirculating or flow-through culture systems require more frequent feeding of fewer cells than standalone tanks due to constant or periodic water exchange and active removal of unconsumed food. For example, Paracentrotus lividus larvae exhibit greater survival and similar growth when cultured in flow-through systems and fed daily with 1.0–6.0×103 cells ml− 1 compared to standalone tanks fed every 3 days with 3.0–18.0×103 cells ml− 1 (Carboni et al., 2012). In the present study, D. antillarum were fed every 24-hours after 8-hours of flow-through representing at least 100% water exchange. In experiment 1, growth and survival between the 10.0×103 and 40.0×103 cells ml− 1 treatment combinations of T. lutea + C. muelleri were similar. This suggested that larvae were not limited by the lower cell concentration over 21 DPF with this feeding regime and diet composition. An even lower initial concentration of 4.4×103 cells ml− 1 of R. lens + C. muelleri improved growth and survival compared to 10.0×103 cells ml− 1 of T. lutea + C. muelleri in experiment 2. Disregarding differences in diet quality between these treatments, this result suggests that particle encounter rates were likely sufficient down to 4.4–10.0×103 cells ml− 1 over a 16-hour feeding period. Low initial cell concentrations were preferred to limit the amount of unconsumed food and reduce the risk of fouling and disease. Despite the lack of statistical differences in growth between treatments in experiment 1, numerical divergence between treatments after 14 DPF suggested that moderately increasing the cell concentration at this timepoint could be beneficial. Regardless, similarly poor survival from both treatments averaging 17–26% indicated that some factor(s) other than food quantity, such as food quality and/or larval stocking density, impacted performance.
Comparisons between carbon-equivalent microalgae compositions in experiments 2–4 revealed the importance of diet quality on D. antillarum larval development. Larvae were stocked at equivalent densities across treatments within each experiment, unlikely to be limited by algal cell concentrations and had access to the same quantity of dietary carbon. Therefore, differences in larval performance likely resulted from different algal compositions. The T. lutea + C. gracilis reference diet used in experiment 1 also helped to improve the quality of diets over successive experiments through comparisons to a benchmark (Glencross et al., 2007). The incorporation of R. lens improved D. antillarum larval performance, corroborating prior recommendations (Eckert, 1998; Leber et al., 2009; Moe, 2014). Both diets containing R. lens in experiment 2 significantly improved growth and survival at 21 DPF compared to the reference diet without R. lens. A similar trend extended into late larval development, as both diets containing R. lens in experiment 3 significantly improved survival at 42 DPF compared to the reference diet. The apparent dietary benefit of this microalgae is not unique to D. antillarum. Rhodomonas spp. have been deemed a high-quality diet for numerous filter-feeding invertebrates including copepods (Dayras et al., 2021; Knuckey et al., 2005; Ohs et al., 2010), artemia (Seixas et al., 2009), rotifers (Coutinho et al., 2019), scallops (Tremblay et al., 2007), oysters (Brown et al., 1998), mussels (Jose Fernández-Reiriz et al., 2015) and sea urchins (Castilla-Gavillán et al., 2018; Gomes et al., 2021; Hinegardner, 1969). Nutritional factors including cell size and morphology, biochemical composition, and/or digestibility vary by microalgae species (Brown et al., 1997; Guedes and Malcata, 2012) and can help to explain improved D. antillarum larval performances from diets containing R. lens.
Filter feeding invertebrates can ingest a variety of food particles, however optimum size ranges exist (Fernandez, 2001; Lavens and Sorgeloos, 1996) and larger, yet still ingestible, microalgae are thought to improve growth (Cárcamo et al., 2005; Fernández-Reiriz et al., 2015; Seixas et al., 2009). Echinoderm larvae cannot actively select food prior to ingestion and instead consume particles that can be captured efficiently and passed through the esophagus into the gut (Strathmann, 1971). The ideal particle size range for larval D. antillarum is unknown, however smaller particles are thought to be less readily captured by urchin larvae than larger ingestible particles (Strathmann et al., 1972). Thus, it’s possible that fewer T. lutea and Chaetoceros sp. (3–5µm and 5–8µm, respectively [Brown et al., 1997]) cells were captured and ingested relative to larger R. lens cells (8–12µm [Brown et al., 1997]). Increased cell size and capture efficiency is unlikely to fully explain improved D. antillarum larval performance as a similarly sized and commonly used microalgae species, Dunaliella tertiolecta (10–12µm [Brown et al., 1997]), yielded poor results in other studies (Leber et al., 2009; Wijers et al., 2021 unpublished data). Rhodomonas spp. have favorable biochemical properties for marine aquaculture in general due to desirable fatty acid profiles and relatively high protein and carbohydrate contents (Brown et al., 1997; Castilla-Gavilán et al., 2018; Coutinho et al., 2019; Dunstan et al., 2005; Fernández-Reiriz et al., 2015; Seixas et al., 2009). These microalgae and other cryptophytes can notably synthesize a diversity of polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA; 22:6 n-3) (Peltomaa et al., 2018), which are both essential lipids for healthy marine larval development (Sorgeloos et al., 1998). The importance of individual or relative proportions of these long-chain PUFAs for D. antillarum development is unknown, as is the ability to create them from constituent fatty acids through elongation and desaturation which has been observed in other urchin species (Liu et al., 2007; Schiopu et al., 2006). Interestingly, Chaetoceros spp. and T. lutea also contain relatively high amounts of long-chain PUFAs, but neither are rich in both EPA and DHA (Brown et al., 1997), and in this study they underperformed in combination in the absence of R. lens. Castilla-Gavillán et al. (2018) demonstrated that Paracentrotus lividus larvae fed Rhodomonas sp. contained higher total lipid content than those fed other microalgae. A similar dynamic could have positive implications for larval D. antillarum development.
Dietary protein is an important nutritional source of nitrogen and amino acids and can directly influence growth rates of marine invertebrates (Enright et al., 1986; Kreeger and Langdon, 1993). Direct comparisons of protein content between all three microalgae species used in this study are unavailable and can vary depending on culture conditions. Despite this, R. lens has been shown to produce and contain higher levels of total protein than T. lutea (Fernández-Reiriz et al., 2015; Seixas et al., 2009) and T. lutea has also been shown to have higher proportional protein content than C. gracilis (Lora-Vilchis et al., 2004). Rhodomonas spp. produce a phycobiliprotein pigment called phycoerythrin that can account for up to 12% of total protein content (Seixas et al., 2009) and may have additional undescribed nutritional benefits. Nutrients within poorly digestible cells are unlikely to be assimilated effectively, regardless of cell size or biochemical composition. The digestive capabilities of larval D. antillarum are unknown, however, enzymes capable of hydrolyzing carbohydrates, lipids, and proteins have been found in other urchin species (Annunziata et al., 2014; Fenaux, 2020; Stumpp et al., 2013) and larvae are thought to prioritize defecating less digestible particles (Strathmann, 1971). Diet quality in this study could have been affected by differences in digestibility of microalgae cells and respective degrees of nutrient and energy assimilation. In separate studies, mussel and oyster species exhibited higher absorption rates and efficiencies when fed Rhodomonas sp. compared to T. lutea (Fernández-Reiriz et al., 2015; González-Araya et al., 2012). Larval sea urchins reportedly do not break down T. lutea within the gut as easily as other microalgae and, while diatoms are thought to be digestible (Strathmann, 1971), the silica-based frustules that comprise Chaetoceros spp. cell walls could be more recalcitrant than R. lens membranes.
While R. lens appeared to be a crucial dietary component, D. antillarum nonetheless benefited from mixed microalgae diets. A comparison of mono-algal R. lens and mixed R. lens + C. gracilis diets in experiment 4 resulted in significantly higher survival from the mixed diet. Similarly, the tripartite diet in experiment 3 resulted in significantly higher survival compared to the R. lens + C. gracilis diet, which indicated a possible positive correlation between overall diet diversity and performance. This is unsurprising, given that mixed diets are more likely to provide nutritional balance (Brown et al., 1997; Ohs et al., 2010) and have been shown to improve survival of other sea urchin larvae (Gomes et al., 2021). The comparative importance of T. lutea or Chaetoceros sp. as supplements to R. lens is unclear given statistically similar growth and survival between mixed diets in experiment 2. However, R. lens + Chaetoceros sp. diets performed well and produced the numerically largest larvae in experiments 2 and 3 and the statistically highest survival in experiment 4. Despite improved survival from higher diversity diets and potential benefits from the inclusion of a diatom, the mono-algal R. lens diet in experiment 4 produced significantly larger larvae than the mixed diet and the highest growth overall in this study. This suggests that like other urchin species (Castilla-Gavillán et al., 2018; Hinegardner, 1969), Rhodomonas sp. can viably be used as a mono-algal diet for D. antillarum.
A density-dependent relationship between larval survival and growth was observed and should be considered concurrently to diet quantity and quality. Higher survival from the mixed-algal diet treatment in experiment 4 corresponded with lower growth, indicating an inverse relationship between larval density and growth. Similarly, the tripartite diet in experiment 3 resulted in the highest survival and lowest growth. Within the same experiment, the production of unexpectedly large larvae from the reference diet treatment may have resulted from a drastic increase in algal cells per larvae due to extremely low survival of ~ 1%. In these instances, reduced survival and lower larval densities potentially lessened competition for physical space and/or resources, including food, leading to higher growth. This dynamic has been observed in the larval culture of other urchin species (Azad et al., 2012; Buitrago et al., 2005; Suckling et al., 2018) and warranted investigation in D. antillarum. Experiment 5 was conducted to test the hypothesis that, other factors being equal, D. antillarum growth and survival improves at lower larval densities. Indeed, a similar trend revealed that the lowest initial stocking density resulted in significantly larger larvae than the higher density treatments. While survival was statistically similar between all three larval density treatments, proportionally fewer larvae remained in the higher density cultures. Statistical differences could have resulted from extending the experiment past 35 DPF or from stocking the 1.5 and 2.25 larvae ml− 1 treatments at higher initial densities. Regardless of initial stocking densities and diet compositions, experiments extending past 30 DPF (experiments 3 & 5) into late larval development concluded at average densities less than 1 larvae ml− 1. This observation, in conjunction with the highest growth observed from the 0.75 larvae ml− 1 treatment in experiment 5, supports culturing D. antillarum at initial densities ≤ 1 larvae ml− 1.
This study represents a series of investigations intended to develop D. antillarum larval culture protocols within a novel RAS capable of scaled production for restoration. Variables of interest included, 1) microalgae diet quantity, 2) microalgae diet composition, and 3) larval stocking density, which improved outcomes over multiple culture attempts. In summary, daily fed microalgae concentrations down to 4.4–10.0×103 cells ml− 1 were adequate for rearing D. antillarum over 21 DPF with at least 100% daily water exchange. Gradually increasing cell concentrations as larvae grow and presumably increase consumption rates past this point was likely beneficial, but this was not empirically confirmed. The microalgae R. lens was a critical dietary component and other Rhodomonas species with similar nutritional profiles are likely to perform equally well. Increasingly diverse mixed diets containing R. lens improved larval survival and supplementing with a diatom may have been beneficial. Lastly, density-dependent growth dynamics were observed whereby reduced larval densities resulted in higher growth. Production-oriented D. antillarum larval cultures should be conducted at densities ≤ 1 larvae ml− 1. The experimentally derived protocols outlined here eventually resulted in complete development of this species within the novel RAS (Pilnick et al., 2021) and have since been used to produce over 1,000 juveniles. Further culture optimizations aimed at improving yields are necessary. Suggestions for additional research include determining prey capture and consumption rates, digestion efficiencies of different microalgae, and types and ratios of supplementary microalgae to best support R. lens diets. Subsequent understanding of how larval nutrition affects settlement and post-settlement success will further improve production viability. Concurrent investigations into juvenile growout (Hassan et al., 2022, in revision, Aquaculture Reports) and restocking methods can further advance D. antillarum restoration objectives.