RNA interference of Hsp70 in Artemia franciscana nauplii and its effect on morphology, growth, survival and immune response

Abstract

Brine shrimp Artemia franciscana release either encysted embryos via oviparous reproduction or motile nauplii via ovoviviparous reproduction. Artemia cysts contain an abundant amount of ArHsp21, ArHsp22, artemin, and p26, small heat shock proteins which regulate diapause and stress tolerance of this branchiopod crustacean. However, not much is known of the role of molecular chaperone, Hsp70 in this animal. In this study, the immunostimulatory role of Hsp70 in A. franciscana was investigated, work which included examining the function of this protein in embryo development, growth, survival and stress tolerance. Knockdown of Hsp70 was successfully performed by RNA interference (RNAi), with specific Hsp70 dsRNA construct delivered to female broods by microinjection to knockdown the protein in nauplii. The loss of Hsp70 neither affected the growth nor morphology as Artemia nauplii hatched normally and thereafter grew to adult within 28 days, observations similar to the control animals, the latter injected with GFP dsRNA. The survival of nauplii lacking Hsp70 appeared lower upon 28 days culture but the differences were insignificant when comparisons were made with the controls (P > .05). On other aspect, knockdown of Hsp70 reduced approximately 31% survival upon Vibrio campbellii challenge, indicating that Hsp70 assisted in the tolerance of Artemia nauplii during pathogen infection, perhaps through immune stimulation. Initial findings revealed that Hsp70 knockdown did not affect the expression of prophenoloxidase (proPO), a crucial immune protein of the shrimp innate immunity. By examining the morphology, growth, viability and molecular changes upon Hsp70 elimination, this study contributes to our understanding of the important roles of Hsp70 in A. franciscana, an important live food species used in aquaculture.