Characterization and functional analysis of Litopenaeus vannamei Na+/K+/2Cl− cotransporter 1 under nitrite stress

Abstract

The function of Litopenaeus vannamei Na⁺/K⁺/2Cl⁻ cotransporter 1 (NKCC1) under nitrite stress was investigated. The full-length cDNA sequence of the L. vannamei NKCC1 gene was cloned using the rapid amplification of cDNA ends (RACE) technique, and the sequence was analysed using bioinformatics tools. Expression and localisation of NKCC1 in tissues were assessed using real-time quantitative PCR and in situ hybridisation, respectively. The impact of nitrite stress on the survival, physiology, biochemistry and tissue structure of L. vannamei was investigated following silencing of NKCC1 by RNA interference. The 3143 bp cDNA sequence of L. vannamei NKCC1 encodes a polypeptide of 918 amino acids. It is evolutionarily conserved. NKCC1 expression was highest in gill tissue, particularly within cuticle and gill epithelial cells. After silencing NKCC1, an increase in shrimp survival was observed, accompanied by a significant reduction in nitrite entry into the body (P < 0.05). Moreover, the oxidative stress enzyme system remained unaffected and damage to gill tissue was alleviated. The results suggest that NKCC1 is involved in regulating nitrite uptake, and plays a crucial role in facilitating nitrite entry into the organism through gill tissue. The findings provide a vital experimental basis for addressing concerns related to nitrite toxicity.