Non-Lethal Heat Shock Increased Hsp70 and Immune Protein Transcripts but Not Vibrio Tolerance in the White-Leg Shrimp

Abstract

Non-lethal heat shock boosts bacterial and viral disease tolerance in shrimp, possibly due to increases in endogenous heat shock protein 70 (Hsp70) and/or immune proteins. To further understand the mechanisms protecting shrimp against infection, Hsp70 and the mRNAs encoding the immune-related proteins prophenoloxidase (proPO), peroxinectin, penaeidin, crustin and hemocyanin were studied in post-larvae of the white-leg shrimp Litopenaeus vannamei, following a non-lethal heat shock. As indicated by RT-qPCR, a 30 min abrupt heat shock increased Hsp70 mRNA in comparison to non-heated animals. Immunoprobing of western blots and quantification by ELISA revealed that Hsp70 production after heat shock was correlated with enhanced Hsp70 mRNA. proPO and hemocyanin mRNA levels were augmented, whereas peroxinectin and crustin mRNA levels were unchanged following non-lethal heat shock. Penaeidin mRNA was decreased by all heat shock treatments. Thirty min abrupt heat shock failed to improve survival of post-larvae in a standardized challenge test with Vibrio harveyi, indicating that under the conditions of this study, L. vannamei tolerance to Vibrio infection was influenced neither by Hsp70 accumulation nor the changes in the immune-related proteins, observations dissimilar to other shrimp species examined.

Antimicrobial proteins: From old proteins, new tricks

“…Indeed, the use of heat shock has been argued by some authors (reviewed by Sung and MacRae, 2011) to be a useful non-traumatic method for enhancing humoral and cellular innate immunity in aquaculture species. Contrary to this belief, the study on L. vannamei by Loc et al (2013) further showed that heat shock confers no overall improvement in survival of post-larvae after challenge with V. harveyi, with expression of crustin and peroxinectin found to remain unchanged and mRNA levels of penaeidin actually reduced. Heat shocking therefore remains an unproven and controversial immune-stimulating strategy.…”

“…It could be via proteases liberated from haemocytes, as suggested originally by Destoumieux-Garzon et al (2001), but it could occur non-proteolytically as a recent paper has found that in the porcelain crab, Petrolisthes cinctipes, haemocyanin fragmentation occurs after non-lethal heat-shock (Garland et al, 2015). Interestingly, heatshock enhances haemocyanin mRNA expression in L. vannamei (Loc et al, 2013) possibly to replenish the haemocyanin pool depleted by fragmentation. This is an intriguing finding because non-lethal heat shocking has been documented to improve resistance of shrimp against bacterial and viral infections (de la Vega et al, 2006;Sung et al, 2007), contrary to the widely held expectation that stress impairs immune function in most animals.…”

Hsp70 expression in shrimp Litopenaeus vannamei in response to IHHNV and WSSV infection

“…In aquatic animals, Hsp70 has been shown to play an important role in relation to the host response to environmental pollutants, food toxins, and bacterial or viral infections [18]. Elucidating associations between Hsps, the immune system and pathogen resistance in shrimp represents an intriguing challenge [12]. Shrimps may express or suppress proteins in response to the viral infection.…”

Ingestion of food pellets containing Escherichia coli overexpressing the heat‐shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection

“…Vibrio harveyi , previously isolated from diseased shrimps (Loc et al . ), was grown at 28 °C on Marine Agar 2216 (Difco Laboratories). Colonies were transferred individually to Marine Broth 2216 (Difco Laboratories) and grown to stationary phase by incubation overnight with constant shaking at 28 °C.…”

“…The relationship between Hsps and immunity in aquatic organisms is important to aquaculture, and there is evidence that Hsp70/DnaK plays a role in the protection of fish, shrimp and molluscs against disease (Basu et al 2002;Roberts et al 2010;. Methods using Hsps to enhance the protection of aquatic organisms against pathogens include accumulation of endogenous Hsp70 upon heat shock, administration of stimulants which boost the synthesis of Hsp70, and provision of exogenous Hsps (Sung et al 2007(Sung et al , 2009aRoberts et al 2010;Ryckaert et al 2010;Baruah et al 2011;Loc et al 2013;Hu et al 2014). Injection represents an effective method to deliver Hsps to organisms, and an intracoelomal injection with 1 mg GroEL and DnaK reduces the mortality of platyfish by 20-25% during Yersinia ruckeri infection (Ryckaert et al 2010).…”