Injection of an extract of fresh cacao pod husks into Litopenaeus vannamei upregulates immune responses via innate immune signaling pathways.

Immunostimulation is a novel method and a promising development in aquaculture. Products derived from plants exhibit various biological activities. In this study, the hot-water extract isolated from fresh cacao (Theobroma cacao) pod husks (CPHs) was administered by injection to evaluate cellular signaling pathways of innate immunity, the immunostimulating potential, disease resistance, and hypothermal tolerance of white shrimp, Litopenaeus vannamei. Results showed significant increases in the total hemocyte count, semigranular cells, granular cells, phenoloxidase activity, and respiratory bursts (RBs) of hemocytes per unit of hemolymph at 1 day, and in phagocytic activity toward and the clearance efficiency of Vibrio alginolyticus at 1-3 days after shrimp were injected with fresh CPH extract at 40 μg shrimp. However, only RBs per hemocyte had significantly decreased at 1 day after the injection. All immune parameters had returned to control levels by 3 days after receiving fresh CPH extract except for RBs, phagocytic activity, and the clearance efficiency, which had returned to control values by 7 days. Furthermore, at 1 day after the injection, the peroxinectin, prophenoloxidase (proPO) II, toll-like receptors (TLR) 3, signal transducer and activator of transcription (STAT), and crustin in shrimp receiving fresh CPH extract at 20 μg shrimp, and the lipopolysaccharide and β-1,3-glucan-binding protein, proPO II, TLR1, and STAT in shrimp receiving fresh CPH extract at 40 μg shrimp were significantly higher than those of shrimp receiving saline. After injecting fresh CPH extract at 10-40 μg shrimp and 40 μg shrimp, the mortality of shrimp challenged with V. alginolyticus and exposed to 14 °C for 96 h significantly decreased, respectively. These results suggest that fresh CPH extract can be used as an immunostimulant and a physiological regulator for shrimp through injection administration to enhance immunological and physiological responses, which can elevate the resistance against V. alginolyticus and tolerance against hypothermal stress in L. vannamei.