Variable food alters responses of larval crown-of-thorns starfish to ocean warming but not acidification

Marine ecosystems are facing a multitude of stressors, primarily ocean warming and acidification driven by anthropogenic CO2 emissions. These stressors are intensifying, with surface ocean temperatures already ~0.75 °C warmer and ~0.1 pH units more acidic than mid-20th-century levels. Predictions for the end of the 21st century suggest further increases of 1–4 °C in temperature and a pH drop of 0.1–0.3 units. These changes, alongside nutrient limitation, are causing a decline in phytoplankton abundance in tropical oceans, a trend expected to worsen with increased climate variability. Phytoplankton are crucial for marine invertebrate larvae, with at least 70% relying on them as a primary food source. Therefore, food availability significantly influences larval invertebrate responses to ocean change stressors. While high food availability can buffer against low pH or warming, low food supply hinders coping mechanisms. This study focuses on the crown-of-thorns starfish (*Acanthaster* sp.), a key coral predator whose outbreaks have devastated coral reefs. Outbreaks are often linked to increased nutrient influxes, boosting phytoplankton blooms and larval survival. While the relationship between food abundance and CoTS larval development is known, the impact of food variability on CoTS responses to ocean change stressors remains largely unexplored. This research investigates how low, high, and variable food availability affect CoTS larvae under present-day and future (warmed and acidified) ocean conditions, aiming to clarify the interplay between food supply and the species' resilience to climate change.

Existing research shows that CoTS fertilization and early development are generally unaffected by warming and acidification, although temperatures above 29 °C can negatively affect later larval stages. pH decreases of ≥0.2 units slow development and can increase mortality. Studies examining ocean change stressors on CoTS larvae have primarily focused on food levels supporting normal development. However, only one study has explored the influence of different food availabilities on resilience to these stressors, showing faster growth at 30 °C with abundant food. No studies had previously examined the effect of varying food availability on CoTS larvae under combined acidification and warming, or the effect of food variability on their responses.

The study assessed the impact of three food availability regimes (low, low-then-high, high) on CoTS larvae exposed to present-day (26 °C, pH 8.0) and future (30 °C, pH 7.6) ocean conditions. Adult *Acanthaster* sp. were collected from the Great Barrier Reef, and gametes were obtained using standard protocols. Fertilization occurred in experimental seawater (four treatments: temperature and pH combinations). Fertilized embryos were placed in flow-through downwellers at standardized density. Feeding commenced at ~72 hours post-fertilization using *Proteomonas sulcata* at rates corresponding to the assigned food treatment. Larval density, survival, size (length and width), developmental stage, and the proportion of abnormal larvae were assessed at 11 and 18 days post-fertilization (dpf). Competence to settle was evaluated from 14 dpf. Statistical analyses included three-way PERMANOVA for larval size, proportion of abnormal larvae, and proportion of early brachiolaria at 11 dpf, repeated-measures ANOVA for survival, and two-way ANOVA for the proportion of late brachiolaria at 18 dpf. Binary logistic regression was used to analyze larval competence to settle. The low and high food levels used reflected background levels and eutrophic conditions on the Great Barrier Reef, respectively. The low-then-high treatment simulated the variable conditions of phytoplankton abundance observed in contemporary oceans. The experiment was conducted in a purpose-built flow-through seawater system to maintain controlled environmental conditions.

Larvae fed a high food ration (5 × 10⁴ cells mL⁻¹) grew and developed faster than those fed a low ration (1 × 10³ cells mL⁻¹). A high food ration helped larvae cope with warming (30 °C), with some larvae fed variable or high food reaching the settling stage by 18 dpf, while larvae fed low food in warmed conditions experienced arrested development and death by week 6. The variable food treatment (initially low, then high) mitigated the negative effects of initial food limitation on development rate and abnormalities but resulted in larvae 16–17% smaller than those fed high food continuously. Acidification (pH 7.6) negatively affected growth, development, and increased abnormalities regardless of the food regime. Statistical analyses (PERMANOVA, ANOVA, binary logistic regression) supported these findings. Specifically, the binary logistic regression revealed a significant effect of temperature, pH, and food level on larval competence to settle, with warmer temperatures and lower pH significantly reducing the likelihood of competence. High food availability significantly increased the probability of larvae becoming competent to settle.

The results indicate that phytoplankton abundance significantly influences CoTS larval development and survival under warming conditions, with high food availability buffering against the negative effects of elevated temperatures. The ability of larvae to compensate for initial food limitation by exhibiting compensatory growth when food levels increase highlights the importance of food variability in shaping their resilience. However, the smaller size of these larvae at 18 dpf suggests potential carry-over effects impacting later life stages. The lack of a significant effect of food on the response to acidification suggests that other mechanisms are more important in mediating the effects of ocean acidification on CoTS larvae. These findings underscore the complex interplay between food availability and CoTS' response to climate change stressors, and that even brief periods of food limitation can have long-lasting impacts on larval development and ultimately, the success of the species.

This study demonstrates that food availability, particularly under warming conditions, is a crucial factor determining the success of crown-of-thorns starfish larvae. High food rations buffer against the detrimental effects of elevated temperatures, allowing some larvae to complete larval development, while low food rations lead to significantly reduced growth, development and mortality. The capacity of CoTS larvae to exhibit compensatory growth when food limitations are reversed, although not completely negating the impact of previous stress, highlights a degree of resilience. Future research should focus on examining the long-term consequences of this compensatory growth, examining the combined effects of additional stressors, and investigating the species' responses to different types of microalgae under these conditions.

The study focused on a single species of microalgae (*Proteomonas sulcata*), limiting the generalization to natural food conditions. The duration of the experiment might not fully capture the long-term impacts of early life-stage stress. While the flow-through system aimed for controlled conditions, subtle variations may have still occurred. Finally, the relatively high level of food considered 'high' might be infrequently encountered in the natural environment.