Introduction
Sea urchin populations have dramatically declined due to continuous alterations in ocean conditions caused by global climate change. Elevated temperatures increase the energetic cost of maintaining essential physiological functions and consequently disrupt defense mechanisms against reactive oxygen species (ROS) in marine invertebrates. This leads to oxidative stress and lipid peroxidation, ultimately reducing their fitness and increasing their susceptibility to mortality (Leiva et al., 2022). Recent research highlights that sea urchins can alter their physiological responses through phenotypic plasticity after acclimation . Dietary carotenoids can help counteract oxidative damage , protecting cellular components and potentially improving the efficiency of the immune response and reproductive success. Major yolk protein (MYP) is the nutritional reserve in oocytes, and it is essential for embryonic development. Temperature-induced reproductive disruptions inevitably affect the availability and function of MYP. Conversely, heat shock proteins (HSPs) are activated under thermal stress, act as molecular chaperones to protect and refold denatured proteins, and play an important role in thermal adaptation. Understanding the interplay between these factors is crucial for developing effective strategies for the sustainable production and conservation of sea urchins in a changing environment. The present study aimed at evaluating the impact of high and low carotenoid diets on the reproductive performance, oxidative stress , and gene expression of myp and heat shock proteins (hsp70, hsp90, hsp60 , and hsp56 ) in the sea urchin Paracentrotus lividus (Lamarck, 1816) under high temperature.
Materials and Methods
P. lividus broodstock was acclimated in recirculating aquaculture systems (RAS) and starved for one month before the experimental conditions. S ea urchins (N=168) were reared for four months on two temperature scenarios: local environmental temperature (18°C) and warming temperature (+3.5°C) , following IPCC 2023 predictions for 2100. During conditioning, the individuals were fed with two isoproteic a nd isolipidic jellified diets: a high carotenoid diet (HC) and a low carotenoid diet (LC). The microalgae Nannochloropsis sp. was used as a source of carotenoids since it enhances gonad development and immune function (Tsushima et al., 1991) . During acclimation, a 12L:12D photoperiod was used to mimic natural light conditions. The temperature was digitally controlled, simulating the two scenarios. By the end of the trial, several physiological parameters were assessed, including maturity level, gonadosomatic index (GSI) , and oxidative stress markers such as superoxide dismutase (SOD) and catalase (CAT) activity, as well as lipid peroxidation (LPO). Subsequently, total RNA was extracted from gonad tissue and used to perform RT-qPCR for gene expression analysis of myp and the heat shock protein genes hsp70, hsp90, hsp60 , and hsp56.
Results
Conditioning temperature significantly influenced gonad development. Sea urchins maintained at +3.5ºC and fed with both diets presented a higher proportion of advanced stages of gonad development (mature; partly spawned; spent). GSI was significantly affected by temperature, with lower values observed in sea urchins exposed to +3.5°C and fed the HC diet (7 %). The activities of antioxidant enzymes (SOD and CAT) and the level of LPO were affected by temperature and dietary treatments. SOD activity showed a decrease of over 10% when sea urchins were exposed to +3.5°C and fed with both diets, with no significant differences observed between the HC and LC diets at this temperature. In contrast, CAT activity significantly increased (39 Umg/protein) in sea urchins fed the LC diet and exposed to +3.5°C, compared to those kept at 18°C (11 Umg/protein ). When the sea urchins were fed by the HC diet, no significant differences in CAT activity were observed between the two temperature treatments, with activity levels remaining relatively stable (between 10-16 Umg/protein). A similar trend was observed for LPO. Significantly higher LPO levels (0.05 nmol/g wet weight) were detected in sea urchins exposed to +3.5°C and fed the LC diet. The expression of myp was significantly up regulated in sea urchins exposed to the +3.5°C temperature, regardless of the dietary treatment. Conversely, the expression of heat shock protein genes (hsp70 , hsp90 , hsp60 , and hsp56) increased in sea urchins exposed to +3.5°C, particularly in those fed the LC diet.
Discussion
Sea urchins fed the HC diet demonstrated the strongest antioxidant defense when exposed to a higher temperature. These individuals exhibited hig h SOD concentration and significantly reduced CAT activity, resulting in low LPO values. Tsushima et al. (1991 ) reported that the carotenoid supplementation in a diet improves the immune function of sea urchins. Although CAT activity was higher when sea urchins were exposed to a higher temperature and fed with LC diet, it was insufficient to prevent lipid peroxidation, resulting in the highest LPO values. The expression of the heat shock protein genes was up regulated when the individuals were exposed to higher temperatures and fed with an LC diet. The combination of high temperature and a low carotenoid diet creates a synergistic stress effect. The high temperature directly induces protein misfolding and ROS production, while the low carotenoid diet weakens the sea urchins’ ability to counteract the resulting oxidative stress. The up regulation of myp gene expression indicates that sea urchins were responding to higher temperatures by enhancing their reproductive processes, specifically egg development. The increase of nutrient accumulation in the gonads could be a mechanism to increase the offspring’s resilience and maximize the reproductive success in a changing environment.
References
Leiva, L., Tremblay , N., Torres, G., Boersma , M., Krone , R., & Giménez, L. (2022). European Lobster Larval Development and Fitness Under a Temperature Gradient and Ocean Acidification. Frontiers in Physiology , 13 . https://doi.org/10.3389/fphys.2022.809929
Tsushima, M., Matsuno, T., & Hirano, R. (1991). Effect of β -carotene on gonad growth and carotenoid content in the sea urchin Pseudocentrotus depressus . Nippon Suisan Gakkaishi , 57(11), 2053-2057.