WWW.WAS.ORG • WORLD AQUACULTURE • MARCH 2025 57 (Cal), serine/threonine-protein phosphatases (ST), leucinerich (Leu), ADP-ribosylation factor (ADP), pancreatic lipase (PL), carboxypeptidase (Car), von Willebrand factor (Von), and Mucin-5A (MU)] and two bacterial toxic genes (LuxR and ToxR). We normalized the expression level of each targeted gene according to the expression of the elongation factor (EL) gene (housekeeping gene) for each biological and technical sample. We converted the transcript levels of each targeted gene to fold changes by the relative quantification method and compared them between the control and treatment groups (Livak & Schmittgen, 2001; Schmittgen & Livak, 2008). We evaluated the relative fold changes for each gene between the control and infected groups for statistical significance using a t-test in SPSS v.25 (p-value < 0.05). We conducted ANOVA using SPSS v.25 (Liu et al., 2013) to assess the significance of gene expression levels at different salinities following infection by V. harveyi. We also performed absolute quantification of bacterial genes using the established absolute standard curve (Fukui & Sawabe, 2008). We analyzed Pearson correlation between targeted immune-related genes and bacterial toxin genes using SPSS v.25 prior to visualization using RStudio software v.3.3.3 (p-value < 0.05). Results and Discussion We discovered that all experimented shrimps were in a moribund state after 24-hour challenge test. All immune genes were up-regulated in L. vannamei cultivated at all three distinct salinity levels following Vibrio harveyi infection (Figure 4). This indicated that c-t, Per, Cal, ST, Leu, ADP, PL, Car, Von, and MU gene act as the first-line defense mechanism for penaeid shrimp in detecting V. harveyi, which serve a crucial role in the innate immunological defense of shrimp (Bi et al., 2020). Our qPCR analysis demonstrated that immune-related genes MU (1152.06fold), ST (17.712-fold), c-t (14.123-fold), PL (59.991-fold), and Per (16.795-fold) exhibited significantly elevated expression in shrimp cultivated at 5 ppt relative to those cultured at 20 ppt and 30 ppt (Figure 4). This demonstrated that shrimp cultivated at low salinity (5 ppt) were more susceptible to bacterial infections, such as V. harveyi, in contrast to those at greater salinity. Interestingly, our overall result showed that shrimp cultured at 20 ppt had lowered (CONTINUED ON PAGE 58) expression of immune-related genes compared to 5 and 30 ppt after V. harveyi infection, suggesting that shrimp cultured at 20 ppt are more resistance toward the V. harveyi infection. The fold-change analysis has demonstrated that the expression of LuxR gene (2.26fold change) was significantly highest in shrimp cultured at 30 ppt compared to 5 and 20 ppt (Figure 5). Our study indicates that shrimp cultured at 5 and 30 ppt are more susceptible to V. harveyi infection (Figure 5). The high expression of the LuxR gene has been previously reported to be elevated in shrimp infected with V. harveyi and V. parahaemolyticus (Muthukrishnam et al., 2022; Peeralil et al., 2020). Given the observed correlation between the expression of the LuxR and ToxR genes, we hypothesized that the expression of LuxR gene may be initiated by the presence of the ToxR gene in infected shrimp (Figure 6) following infection with V. harveyi. ToxR is a suitable candidate gene for the exclusive identification of all Vibrio species. Given that shrimp cultivated at 20 ppt had the lowest expression of the LuxR gene, this demonstrated that 20 ppt effectively reduces the infection rate in shrimp, as mentioned in previous studies, where the expression of ToxR gene were observed to be lower at higher or excessively low salinity level of 3 and 5 ppt (Boas et al., 2023; Brown, 2023). In conditions of excessively low and high salinity, shrimp may prioritize the regulation of proteins involved in osmotic pressure management over immune functions. Additional energy is required to modulate and stimulate ion transportation in the shrimp body in adapting to the changes of the salinity level (Farhadi et al., 2022; Nguyen et al., 2022; Li et al., 2022; Rahi et al., 2018). Moreover, both 5 and 30 ppt induce a substantial reduction in immune activity factors, such as c-type lectin, resulting in the inhibition of innate immune responses such as phagocytosis and stress regulation processes (Deris et al., 2024). Consistent with previous studies, our results highlighted that salinity changes impacted haemolymph osmosis, induced degradation of plasma membrane, inhibited cytoskeleton modification and intracellular ion transport which further suppress cell viability and phagocytic activities in L. vannamei (Zhao et al., 2016; Joseph & Philip, 2020). FIGURE 3. Dissection of shrimp. We normalized the expression level of each targeted gene according to the expression of the elongation factor (EL) gene (housekeeping gene) for each biological and technical sample. We converted the transcript levels of each targeted gene to fold changes by the relative quantification method and compared them between the control and treatment groups.
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