Thermal Stress Biomarkers Research Articles

Metabolic energy sensors (AMPK and SIRT1), protein carbonylation, and cardiac failure as biomarkers of thermal stress in an intertidal limpet: linking energetic allocation with environmental temperature during aerial emersion

The effects of heat stress on organisms are manifested at the levels of organ function, metabolic activity, protein stability and gene expression. Here, we examined effects of high temperature on the intertidal limpet Cellana toreuma to determine how the temperatures at which (1) organ failure (cardiac function), (2) irreversible protein damage (carbonylation) and (3) expression of genes encoding proteins involved in molecular chaperoning (hsp70 and hsp90) and metabolic regulation (ampk and sirt1) occur compare with field temperatures, which commonly exceed 30°C and can reach 46°C. Heart failure, indexed by the Arrhenius break temperature, occurred at 34.3°C. Protein carbonylation rose significantly at 38°C. Genes for heat shock proteins HSP70 (hsp70) and HSP90 (hsp90), for two subunits of AMP-activated protein kinase (AMPK) (ampkα and ampkβ) and for histone/protein deacetylase SIRT1 (sirt1) all showed increased expression at 30°C. Temperatures of maximal expression differed among genes, as did temperatures at which upregulation ceased. Expression patterns for ampk and sirt1 indicate that heat stress influenced cellular energy homeostasis; above ~30°C, upregulation of ATP-generating pathways is suggested by elevated expression of genes for ampk; an altered balance between reliance on carbohydrate and lipid fuels is indicated by changes in expression of sirt1. These results show that C. toreuma commonly experiences temperatures that induce expression of genes associated with the stress response (hsp70 and hsp90) and regulation of energy metabolism (ampk and sirt1). At high temperatures, there is likely to be a shift away from anabolic processes such as growth to catabolic processes, to provide energy for coping with stress-induced damage, notably to proteins.

Temperature-Mediated Differential Expression of Immune and Stress-Related Genes in Aedes aegypti Larvae

The mechanisms by which natural populations of vector mosquitoes cope with daily and seasonal fluctuations in temperature are poorly understood. We examined the effect of water temperature on expression of stress and immune-related genes in Aedes aegypti larvae. Aedes aegypti 3rd instars were exposed for 24 h to one of 7 constant temperatures (10 degrees C, 15 degrees C, 20 degrees C, 25 degrees C [control], 32 degrees C, 36 degrees C, or 40 degrees C) and expression of antimicrobial peptides (cecropin, defensin), transferrin, and heat shock proteins (HSP70 and HSP83) quantified by real-time reverse-transcriptase polymerase chain reaction. Cecropin, defensin, and transferrin were overexpressed at 36 degrees C and underexpressed at 15 degrees C and 32 degrees C. HSP83 was overexpressed at 10 degrees C and 40 degrees C and underexpressed at 20 degrees C, while HSP70 was underexpressed at 15 degrees C, 32 degrees C, and 36 degrees C. These findings suggest that antimicrobial peptides can serve as biomarkers of thermal stress and that HSP83 may buffer mosquito larvae against extreme temperatures.

Strict thermal threshold identified by quantitative PCR in the sponge Rhopaloeides odorabile

In light of increasing sea surface temperatures, quantifying the expression of stress- inducible genes in coastal organisms is imperative to identify early biomarkers of thermal stress. In the present study we developed a quantitative PCR (qPCR) assay to test the molecular response to heat stress in the Great Barrier Reef sponge Rhopaloeides odorabile. Suitable reference genes (cod- ing for α-tubulin, 28S rRNA and ubiquitin) were identified among 5 candidates and then used to nor- malise expression of target genes (actin-related protein, calmodulin, ferritin, ubiquitin-conjugating enzyme, heat shock protein 90 (Hsp90) and heat shock protein 40 (Hsp40)) in samples exposed to high temperatures (31 and 32°C) for 1, 3, 14 and 15 d. A rapid down-regulation of most genes (actin- related protein, ferritin, calmodulin and Hsp90) was observed at both temperatures within 24 h, indi- cating an initial shut-down of the sponge's molecular systems in response to thermal stress. The increased expression of Hsp40 and Hsp90 in sponges at 32°C after 1 and 3 d respectively indicates an activation of the heat shock response system and is consistent with their role as chaperones for direct- ing degraded proteins to proteolysis, this last process being sustained by an induction of the ubiqui- tin-conjugating enzyme gene at this temperature. While sponges kept at 32°C only survived for the first 3 d, none of the genes in sponges kept at 31°C were significantly different from those in the 27°C controls after 14 d. This indicates a very strict thermal threshold for R. odorabile between 31 and 32°C and is consistent with previous findings based on sponge necrosis and symbiotic disruptions in this species.

Immediate early response of the marine sponge Suberites domuncula to heat stress: reduction of trehalose and glutathione concentrations and glutathione S-transferase activity

The marine sponge Suberites domuncula was used to identify early markers for thermal stress. Cubes from sponges have been kept for 30 min at 31°C (10 °C higher than the ambient temperature). After this treatment the sponge cubes were kept again at 21°C. To demonstrate that the animals reacted to the elevated temperature, the expression of heat shock protein (HSP) was determined. Using an antibody raised against HSP70, it was found by Western blotting that the animals specifically express a 45 kDa polypeptide after heat treatment. It was shown that even after 10 min of heat treatment the steady-state concentration of trehalose drops by 40% from a base level of 13 nmol/mg protein. The activity of the trehalose-degrading enzyme, trehalase, remained unchanged. Additional early biomarkers for thermal stress include the enzyme activity of glutathione S-transferase (GST) and the concentration of glutathione (GSH). After 5-min the activity of GST decreased by 40%. Similarly, the concentration of GSH dropped by 50% after 15 to 20 min exposure. The orginal levels of the biomarkers, trehalose, GSH and GST, were reached again after a recovery period of about 180 min. By contrast, the steady-state concentration of polyphosphates did not change during heat treatment. These data show that in S. domuncula the concentrations of trehalose, GSH, and the activity of GST, are biomarkers for immediate early response towards heat stress.