Sarcophaga crassipalpis Heat Protection

We examined that certain hsp transcripts are expressed in response to desiccation stress. We also tested the ability of desiccation as a pretreatment of heat stress, to induce cross-tolerance to high (45°C) temperatures. The expression patterns of two heat shock proteins Hsp70 and Hsp23 were compared in response to heat stress (45oC for up to 60 minutes) and sub-lethal desiccation (0% relative humidity for up to 24 hours) with a control group for cross protection and expression. After desiccating the samples and analyzing them using qPCR and gel electrophoresis, the results showed that the expression of Hsp70 and Hsp23 was more profound in desiccated experimental groups than in control group. This may point out that desiccation response is linked to the expression of hsp70/23 after environmental insult.

Global warming best justifies the increasing rise in temperatures in the climate which significantly determines the distribution of Adult Male Flesh Flies Sarcophaga crassipalpis in the planet. Increased human activities and industrialization has resulted in destruction of the Ozone layer. The increased release of poisonous gases into the environment will then prevent the rays from the sun from being reflected back from the earth surface causing rise in temperatures (Chen, 1991). This global warming affects the population distribution of S. crassipalpis across the various geographical locations. They play a very crucial role in protein folding and protection from all sources of stress. Of all the members of the HSPs, Hsp70 shows the greatest expression upon high temperatures. It is conserved greatly among all prokaryotes and eukaryotes (Parsell et.

al, 1993). Additionally, they are the most dominant in terms of expression following exposure to environmental insults. Although the expression of heat shock proteins due to environmental stress such as high temperature has been well studied, their expression under conditions of dehydration has not been thoroughly investigated. Previous studies have shown that Heat shock proteins play a strong role in preventing aggregation of proteins under desiccation stress through increased representation. However, very little information is available to understand fully if the expression of Hsps cross protects against heat stress, and therefore it is imperative to understand how desiccation stress helps in inducing hsp transcripts (Chen, 1991). In this study we examined the expression of two heat shock mRNA (hsp70 and hsp 23) transcripts in adult male flesh fly Sarcophaga crassipalpis during desiccation stress.

The purpose of this study was to investigate if desiccation stress cross protects against high temperatures in the adult flies of Sarcophaga crassipalpis. H (Table 1). Table 1 Experimental groups displaying stress type, the temperature of stress, and duration of stress (Lab protocols – Environmental Physiology – spring 2015 – Hatle. Stress Temperature/Humidity Stress duration Desiccation 25°C/0%R. H 12, 18, 24 hours Desiccation with Heat shock 45°C/0%R. H 24 hours + 60 min heat shock Heat shock 45°C/N/A 60 minutes Control 25°C/50%R. Then the pellet was re-suspended in 50ul water and heated at 58 °C to 60 °C for 10 minutes. The RNA quality and concentration were then measured spectrophotometrically using the 260/280nm ratio. Control Heat Shock Pre-Desiccated Desiccated ↓ ↓ ↓ ↓ 0 1 Hour 12 Hours 0 - 8 - 12 - 24Hours Temp 25 Temp 45 Heat Shock 0% R. H ↓ Temp 45/ 0% R.

H ↑ RNA of Heat Shock and Desiccated flies Figure 1. Fluorescence was measured during at the end of each annealing step. To ascertain the amplification specificity, an analysis using an after-amplification curve of melting was conducted. Fluorescence data were recorded after every 0. 5C step during this period. All samples including experimental cDNA, control cDNA and an existing sample of control cDNA (from Env Phys lab) were examined for expression of heat shock protein 70 mRNA with RP49 as the reference gene. Thermal Stress of Cross-Tolerance To inquire if cross-tolerance to thermal stress was stimulated by desiccation, flies were pre-treated at 0% R. H for 24 h at 25°C. Six similar flies were used as controls for non-desiccated and pre-desiccated experimental samples. All flies from control samples and experimental samples were left at 45°C for 60 minutes.

The sample flies were extracted out of the heat shock and examined for Hsp70 and Hsp23 expression, as a proxy for cross tolerance. The expression of hsp70 was also increased dramatically as compared to the reference gene RP49. (Fig. However, when exposed to heat shock post desiccation, the general trend of Hsp70 expression decreased at the same temperature (45oC) compared to the heat shock sample (Fig. 2) Fig. Quantization of levels of hsp70 and RP 49 detected for A1 Control group, B1 heat stressed (at 45oC for 60 minutes), A2 desiccated (0% RH for 24hrs), and B2 heat stressed post desiccation flies (desiccated at 0% RH for 24h and heat shock at 45°C for 60 minutes). Figure 3B. Hsp70 and Hsp23 2 ΔΔCt expression using qPCR for group of flies exposed to heat shock at 45°C for 60 minutes, flies that were desiccated for 24hrs at 0% RH, and flies that were exposed to a 45°C heat shock post desiccation.

In this study, the PCR products were tested for molecular size by gel electrophoresis to confirm the correct product was made. Both were differentially regulated. Fig. Cross-protection between desiccation and heat stress To test whether the elevated levels of the two hsp transcripts in response to desiccation conferred cross-tolerance to temperature extremes in flesh flies, flies were desiccated for 24 h, then subjected to heat shock (45°C) for 60 minutes. Non-desiccated control flies were also heat shocked at the same temperatures for the same durations. The data collected after the various treatments was analyzed at 95% confidence intervals for the observed average gene expression. The overall mean of gene expression was found to be 2. 33, the standard deviation equals 1. From the ANOVA it was deduced that there is significant difference between the expression of Hsp70 and Hsp23 transcripts expression at 60minutes within the groups.

(ANOVA; p=0. 001, and p=0. 03 and degree of freedom = 5). Discussion Previous studies have indicated that desiccation failed to enhance tolerance to heat stress, but rather caused a negative response to future stress in the pupae of Sarcophaga crassipalpis. This may point out that desiccation response is linked to the expression of hsps70/23 after environmental insult especially after heat shock. The study also showed that Hsp70 and Hsps23 expression levels followed expected trends that have been seen in previous studies. The trend of high Hsp70 at moderate temperatures that decreases with more severe temperature has also been seen in previous studies. Hsp70 has been characterized to have a maximum threshold level at 45oC that decreases after approximately 47°C. In terms of heat shock protein 23, the expression was found to be different than hsp70 which was highly expressed during heat shock and decreased during desiccation (Figure.

This observation in Hsp23 mRNA transcripts regulation results because Hsp23 protein expressed in the flies is small ATP-dependent protein mediating essential protein functions thus reducing protein structure alteration and thus degradation. Again, these proteins are continuously synthesized in the insect’s body when induced by environmental stressing factors. They function to maintain the insect’s cellular levels of homeostasis and thus stabilizing the insects physiological state. Another factor contributing to this behavior is the low energy production during the diapause stage of an insect whereby they undergo differential expression with minimal or no disruption of the mRNA transcript making them sequester. Hsps are mRNA transcripts found to be involved in dehydration responses of insects and sometimes speed up by other biotic and abiotic factors.

It is observed that Hsps play important role responding to dehydration stress without the interaction of other cellular processes. The influence of other physiological stress mechanisms on the expression of Hsps could help explain the dissimilarity in Hsp transcription expression between desiccated, heat shocked, diapausing and non-diapausing samples. Conclusion This study demonstrated that exposing adult male flesh flies Sarcophaga crassipalpis to desiccation at 0% R. H for 24hrs was non-lethal and reduced the cross tolerance to high temperature. The study also showed that Hsp70 and Hsp23 were up-regulated in the experimental groups of flies (desiccated, heat shocked and heat shocked post desiccation). Alternatively, tolerance to temperature extremes may require additional physiological adjustments not elicited by desiccation. Heat shock proteins are abundant group of proteins for cellular response to stress across many living organisms.

Understanding of the primary mRNA transcripts responsible for the regulation of these heat stress proteins to response to rehydration and desiccation is very important. It assists in identification of the distinct role of individual Hsps in responding in Sarcophaga crassipalpis where both Hsp23 and Hsp70 are upregulated by desiccation. Knowledge of Hsp proteins induction and regulation together with biotic and abiotic factors are very important factors for insects’ survival in response to global warming. "Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential auto-regulatory factor. " The Journal of Cell Biology 117, no. Bayley, M. and Holmstrup, M. Water vapor absorption in arthropods by accumulation of myoinositol and glucose. J. and Welch, W. J. Molecular chaperones and the centrosome.

J. Chen, H. , Xu, X. , Li, Y. , Wu, J. Characterization of heat shock protein 90, 70 and their transcriptional expression patterns on high temperature in adult of Grapholita molesta (Busck). P. Lima, and Alexandre A. Peixoto. "Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae). " Memórias do Instituto Oswaldo Cruz 100, no. (1991a). Developmental expression and hormonal regulation of a desiccation stress protein in Tenebrio molitor. Insect Biochem. Kroeker, E. M. Annual Review of Genetics 22, 631–677. Liang, P. and MacRae, T. H. The synthesis of a small heat shock/α-crystallin protein in Artemia and its relationship to stress tolerance during development. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins.