A newly developed ELISA showing the effect of environmental stress on levels of hsp86 in Cherax quadricarinatus and Penaeus monodon.
The induction of hsps by stress in Cherax quadricarinatus and Penaeus monodon was investigated using SDS-PAGE, Western blotting and ELISA techniques. Western blotting showed the presence of an immuno-reactive protein to mouse alpha-human hsp70 IgG1 monoclonal antibody at a mass of 86 kDa (hsp86) in pleopod samples but was not sensitive enough to detect differences in response to stress. An enzyme-linked immunosorbent assay (ELISA) was developed using this antibody for the detection of hsp86 in the pleopods of C. quadricarinatus and P. monodon. Using this assay, significantly higher levels of hsp86 were detected in hyperthermally stressed C. quadricarinatus (21 to 70 g) and P. monodon (14 to 32 g) and hypoosmotically stressed P. monodon (14 to 32 g). Male C. quadricarinatus and P. monodon were thermally stressed with an increase in temperature from 24 to 33 degrees C for a period of 2 h then a recovery period of 6 h. SDS-PAGE gels of thermally stressed C. quadricarinatus and P. monodon samples revealed an increase in protein band intensity at 97 kDa (C. quadricarinatus) and 43 and 35 kDa in P. monodon. A 25 kDa mass protein was induced in C. quadricarinatus when thermally stressed. P. monodon were osmotically stressed with a decrease from 31 to 15 ppt for 2 h with a recovery of 6 h. SDS-PAGE gels revealed increased intensity of bands at 35 and 43 kDa and a 100 kDa band was induced demonstrating a wide range response of protein profile to stress in these species. SDS-PAGE gels of both species investigated also revealed an apparent reduction in band intensity of the haemocyanin subunits in stressed samples. The ELISA described here constitutes the first quantitative assay for the detection of a hsp in crustaceans and the following investigations are believed to be the first to describe the response of hsps to stress in C. quadricarinatus and P. monodon. In doing so, they provide a sound basis for future studies of the role of hsps in physiological functions in commercially cultured crustaceans.
Cimino
Elisabeth J
EJ
Microbiology and Immunology, James Cook University, Townsville, Australia.
Owens
Leigh
L
Bromage
Erin
E
Anderson
Trevor A
TA
D000222:Adaptation, Physiological; D000818:Animals; D003400:Astacoidea / Q000737:chemistry*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018841:HSP90 Heat-Shock Proteins / Q000032:analysis*; D006361:Heating; D009995:Osmosis; D033561:Penaeidae / Q000737:chemistry*; D013312:Stress, Physiological / Q000378:metabolism*
D018841:HSP90 Heat-Shock Proteins; C107835:Hspca protein, mouse