Immunomodulatory effect of squid ink extract on lipoxygenase pathways and MIH gene in Litopenaeus vannamei under different salinity condition

Lipoxygenase (LOX)-derived eicosanoids (12-/15-HETE) and Molt-Inhibiting Hormone (MIH) are key mediators of inflammation and stress physiology in Litopenaeus vannamei during bacterial challenge. Squid ink (Loligo sp.) contains bioactives (betaine, choline, cinnamic acid) with potential immunomodulatory activity. This study aim to evaluate whether dietary squid ink extract modulates MIH responses, LOX-related metabolites, and survival of L. vannamei challenged with Vibrio harveyi under different salinities. Squid ink was methanol-macerated and evaporated to paste; 8 g paste was dissolved in 1 L water and spray-coated onto per kg commercial feed (fed at 5% biomass/day, 4 times/day). Shrimp were reared in 12 aquaria at 24, 27, and 30 ppt, with an infected positive control at 33 ppt, then challenged by immersion with V. harveyi (10^6 CFU/mL). MIH was identified by PCR, 12-HETE/15-HETE were quantified by LC–MS, and ligand–protein interactions were explored by interaction in silico analysis. Squid ink-related treatments reduced 12-HETE from 2.0 ± 0.3 to 0.2–0.5 nmol/mg (ANOVA F = 6.929; p = 0.006), whereas 15-HETE remained near the observed range (5.5–6.0 nmol/mg). MIH amplicons (250–283 bp) showed the strongest band intensity at 30 ppt. Survival improved in supplemented groups (80–90%) relative to the positive control (73.33 ± 5.77%), with the highest survival at 30 ppt (90 ± 10%) (post hoc, p < 0.05). Dietary squid ink extract attenuated the pro-inflammatory 12-LOX pathway and improved post-challenge survival, with responses depending on salinity, supporting squid ink as a natural immunostimulant candidate for shrimp aquaculture.