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University of Waterloo
BIOL 370
Dinu Nesan

Results HIGH-TEMPERATURE SURVIVAL • General trend: Mortality rate increased as temperature increased • From 4°C - 25°C  larvae had a greater rate of survival • At 30°C  adult had a greater rate of survival • Conclusion: up-regulation of heat shock proteins at 30°C in adults, but not in larvae Median lethal time: The period of time required for 50% of a large group of organisms to die following a specific dose of an injurious agent, such as a drug or radiation. -The 3 experiment that was performed was a test of survival where they measured and compared the median lethal times at various temperatures for adult and larvae. -The median lethal time: period of time required for 50% of the organisms to die due to the heat stress -As you can see, the general trend was that mortality rate increased as the temperature increased for both larvae and adult midges -However, what they found was that from 4-25°C, larvae had a greater survival rate than adults, but once it reached 30°C, the adult midges had greater survival than the larvae -So they concluded that the reason why adults have a greater median lethal time compared to larvae at 30°C was most likely due to the induced expression and up-regulation of heat shock proteins in adults as a result of their inducible heat shock response -On the other hand, the reason why larvae began to die significantly at 30°C was because they weren’t able to up-regulate their heat shock proteins in response to the extreme stress like the adults did; and this is because the larvae only express their heat shock proteins constitutively rather than in response to extreme temperatures -these graphs are just showing the same general trend as the table from the previous slide -graph A is for the larvae and graph B is for the adults -as you can see, at 20 and 25°C, larvae maintained a greater percent survival for a longer period of time compared to adults, but at 30°C, adults survived longer than the larvae THERMOPROTECTION GENERATED BY PRIOR EXPOSURE TO LESS SEVERE HIGH TEMPERATURES Hypothesis: prior exposure to a less severe high temperature would generate thermoprotection at 30°C by first exposing the larvae for 2 hr to 15, 20, or 25°C and then exposing them to 30°C for 3 hr. 2 hr exposure to 15, 20, and 25°C did not impair or enhance survival of larvae placed at 30°C for 3 hr. The 2hr conditioning of larvae caused thermosensitivity and reduced survival at 30°C. Larvae Conclusion: Prior exposure to a less severe thermal stress failed to elicit protection from injury at either 25 or 30°C To test capacity for generation of thermoprotection in adults, first exposed them for 1 hr to 20 or 25°C and then transferred them to 30°C for 6 hr. Adult Conclusion: 1 hr at 25°C significantly increased survival at 30°C, thus indicating that adults are capable of generating thermoprotection Final Conclusion: results indicate that the adults, but not the larvae, are capable of generating thermoprotection EXPRESSION IN RESPONSE TO TEMPERATURE STRESS In this test, a northern blot was performed to detect hsp expression in B. Antarctica larvae and adults. Northern blot hybridization revealed that all 3 hsp transcripts (a small hsp, hsp70, hsp90) were expressed at high levels in larvae maintained at 4°C whereas adults held at 4°C exhibited little or no hsp expression. 4°C was used as the ambient temperature because that’s the typical summer air temperature in Antarctica. When exposed to both high and low temperatures, the amount of hsp transcripts did not change in larvae.  this shows that hsps are not up-regulated in larvae when exposed to extreme temperatures (instead, they are constitutively expressed at the same level at all times) At 2 hours of exposure to 30°C and at exposure to 35°C, the hsp signals disappeared, but it was most likely due to death of the insect at such a high temperature rather than down- regulation of the hsps. At 4°C, there is little/no expression of any of the 3 hsps in adults, but all 3 hsp transcripts responded to up-regulated in response to heat stress and were expressed when exposed to 30°C. Similar to the larvae, the reasons why no hsp expression was found in the northern blot hybridization of adults at 35°C was likely due to death of the insect at that high temperature rather than down-regulation of hsps. Conclusion: As seen in the northern blot hybridization, larvae constitutively express their hsps whereas adults only do so in response to temperature stress. PROTEIN AGGREGATION IN RESPONSE TO ENVIRONMENTAL STRESS • Low protein aggregation in larvae at ambient temperature (4°C), freezing temperatures (- 5°C), anoxia and mild heat shock (30°C/1h) • High protein aggregation at severe heat shock (30°C/2h) • Conclusion: although larvae lack the typical heat shock response of up-regulation due to environmental stressors, they are able to prevent protein aggregation in response to stress due to constitutive expression of heat shock proteins; however, once -the last experiment was a test of protein aggregation in response to different environmental stressors and it was only performed on the midge larvae -as we learned in class, proteins tend to denature at high temperatures, and as they denature, the hydrophobic regions of the proteins become exposed and start to interact with the hydrophobic regions of other proteins, resulting in aggregation and clumping of the denatured proteins -and this clumping of proteins will end up inhibiting normal metabolic processes of the cell -so since the heat shock response works to prevent the denaturation and aggregation of proteins, the protein aggregation levels were expected to be low when heat shock proteins are expressed -so what they found was that at ambient temperature (4°C), there was low protein aggregation -at freezing temperatures, anoxic conditions, and mild heat shock there was also low protein aggregation -from this, the results showed that the constitutive expression of heat shock proteins in larvae does a fairly good job maintaining survival in various stressful conditions -however, in severe heat shock conditions (where the larvae were held at 30°C for 2 hours instead of 1 hour), there was an increase in protein aggregation -this happened because the larvae don’t have an inducible heat shock response like adults do, so similar to what happened in the previous experiment, once they were exposed to severe heat shock, proteins started to accumulate and the larvae started to die because the temperature was just too extreme for the larvae to survive using just its constitutive expression of heat shock proteins -although this test was only done with larvae, it would be expected for adults to have low protein aggregation even in severe heat
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