Gene silencing reveals multiple functions of Na+/K+-ATPase in the salmon louse (Lepeophtheirus salmonis)

Abstract

Na⁺/K⁺-ATPase has a key function in a variety of physiological processes including membrane excitability, osmoregulation, regulation of cell volume, and transport of nutrients. While knowledge about Na⁺/K⁺-ATPase function in osmoregulation in crustaceans is extensive, the role of this enzyme in other physiological and developmental processes is scarce. Here, we report characterization, transcriptional distribution and likely functions of the newly identified L. salmonis Na⁺/K⁺-ATPase (LsalNa⁺/K⁺-ATPase) α subunit in various developmental stages. The complete mRNA sequence was identified, with 3003 bp open reading frame encoding a putative protein of 1001 amino acids. Putative protein sequence of LsalNa⁺/K⁺-ATPase revealed all typical features of Na⁺/K⁺-ATPase and demonstrated high sequence identity to other invertebrate and vertebrate species. Quantitative RT-PCR analysis revealed higher LsalNa⁺/K⁺-ATPase transcript level in free-living stages in comparison to parasitic stages. In situ hybridization analysis of copepodids and adult lice revealed LsalNa⁺/K⁺-ATPase transcript localization in a wide variety of tissues such as nervous system, intestine, reproductive system, and subcuticular and glandular tissue. RNAi mediated knock-down of LsalNa⁺/K⁺-ATPase caused locomotion impairment, and affected reproduction and feeding. Morphological analysis of dsRNA treated animals revealed muscle degeneration in larval stages, severe changes in the oocyte formation and maturation in females and abnormalities in tegmental glands. Thus, the study represents an important foundation for further functional investigation and identification of physiological pathways in which Na⁺/K⁺-ATPase is directly or indirectly involved.

Keywords: Feeding; Na(+)/K(+)-ATPase; RNA interference; Reproduction; Sea louse; α-subunit.