• 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
-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
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
EXPRESSION IN RESPONSE TO TEMPERATURE STRESS
In this test, a northern blot was performed to detect hsp expression in B. Antarctica larvae and
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
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
-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
-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
-although this test was only done with larvae, it would be expected for adults to have low protein
aggregation even in severe heat