Introduction
Whiteleg shrimp Litopenaeus vannamei is one of the most economically significant species in global aquaculture. It is primarily farmed in ponds which are increasingly threatened by extreme weather induced by climate change (Galappaththi et al., 2020). Whilst the effects of individual stressors have been documented (Zheng et al., 2017; Wang et al., 2020), the physiological impact of combined environmental shifts, such as rapid changes in temperature and salinity caused by heatwaves and heavy rainfall, remains poorly understood. These stressors may induce physiological stress, leading to disrupted immune regulation and microbiome dysbiosis. As a result, this study hypothesises that the synergistic effect of these stressors would induce acute oxidative stress and compromise mucosal barrier integrity, leading to systemic physiological instability and extensive lipid peroxidation within haemocytes and immune-related tissues. Therefore, this study aimed to investigate the effects of extreme environmental shifts on the mucosal immunity of L. vannamei.
Materials and Methods
Early juvenile Litopenaeus vannamei (mean weight: 0.20 ± 0.10 g) were acclimatised in a recirculating aquaculture system (27°C, 25 ppt) for two weeks before being randomly allocated into three groups in quadruplicate: 1] high temperature and high salinity group (HTHS: 33°C, 40 ppt), 2] low temperature and low salinity group (LTLS: 20°C, 10 ppt), and 3] a control group (27°C, 25 ppt). Environmental parameters were adjusted during a three day ramping phase at a rate of change of 3°C and 5 ppt per day. The sampling regime comprised nine shrimp per group, sampled across the four replicate tanks with 2-3 shrimp per tank at each sampling point. Sampling points were on the day before the ramping phase (T0), and at 24 hours (T1) and 5 days (T2) after achieving the target temperature and salinity. Haemolymph was extracted using a syringe pre-loaded with anticoagulant, and haemocytes were subsequently collected via centrifugation at 800 x g for 30 minutes at 4°C. The cell pellets were then lysed in 1/3 PBS to quantify the activities of lysozyme (LYZ) and superoxide dismutase (SOD), and the levels of malondialdehyde (MDA), using commercial assay kits (Abcam, Cambridge, UK) according to the manufacturer's instructions. Statistical significance was determined using the Kruskal-Wallis test followed by Dunn's post-hoc test with Bonferroni correction for multiple comparisons (P < 0.05), analysed using the R software environment. Furthermore, gills and intestines were preserved in ethanol for microbiome analysis while gills, intestines, and hepatopancreas were excised for immune gene expression analysis via RT-qPCR.
Results
At the initial sampling points, no significant differences in the biochemical markers were observed between the control and the stress treatment groups (P > 0.05). In contrast, at T2, the lysozyme activity (Figure 1A) in the HTHS group (0.87 ± 0.19 mU mg-1) was significantly higher than that of the control group (0.35 ± 0.06 mU mg-1) (P = 0.03). Similarly, SOD activity (Figure 1B) was significantly higher in the LTLS group (566.70 ± 133.03 U mg-1) compared to the control group (282.08 ± 35.24 U mg-1) (P = 0.01). Most notably, MDA levels (Figure 1C) in both the HTHS (2.40 ± 0.70 nmol mg-1) and LTLS (3.45 ± 1.31 nmol mg-1) groups were significantly higher than the control group (0.38 ± 0.04 nmol mg-1) (P = 0.0018 & 0.0001, respectively).
Discussion
These findings indicate that combined temperature and salinity shifts pose a severe physiological burden on early juvenile L. vannamei. Particularly, the significant elevation in MDA levels within the LTLS group indicates lipid peroxidation, suggesting that oxidative stress has exceeded the host's antioxidant capacity (Li et al., 2017). Whilst significant increases in SOD and lysozyme activities reflect an active mobilisation of the antioxidant defence and innate immunity, such a prolonged response may impose a substantial metabolic cost. This may confer a metabolic trade-off between immune tolerance and physiological state. These results show the vulnerability of early-stage shrimp to simulated extreme weather events. Ongoing microbiome profiling, integrated with host immune gene expression analyses, will clarify whether these biochemical alterations are associated with intestinal dysbiosis and concomitant modulation of specific immunometabolic pathways. This integrative approach will provide mechanistic insight into host-microbe-immune interactions, thereby informing the development of targeted intervention strategies to enhance immune competence and resilience in commercial aquaculture systems. />
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Figure 1. Biochemical markers in the haemocytes of L. vannamei under control (27°C, 25 ppt), HTHS (33°C, 40 ppt), and LTLS (20°C, 10 ppt) regimes: (A) Lysozyme (LYZ), (B) Superoxide dismutase (SOD), and (C) Malondialdehyde (MDA). Data are mean ± S.E.M. (n = 9) sampled at T0 (baseline), T1 (24 h), and T2 (5 d) post ramping. Different superscript letters indicate significant differences between control group and experimental groups at each specific time point (P < 0.05), distinct letter sets have been applied to T1 and T2 respectively.
Acknowledgment
This work was funded by Centre of Research excellence in Intelligent and Sustainable Productive Systems (CRISPS), University of Plymouth and Lallemand SAS, Blagnac, France.
References
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