We only have one month at Palmer Station this year (2005), but our research is scheduled for similar trips in 2006 and 2007. In science, a month is very little time to do both field and lab work on any subject. As a result, our work is sort of "commando science", working fast and shifting our focus as we realize what areas of the investigation are giving us the most interesting results. In addition, many months of lab work will follow upon our return.
In the big picture, our work focuses on the ways terrestrial animals survive the dramatic, rapid environmental changes that are typical of this part of Antarctica. Specifically, we are investigating the survival mechanisms of the wingless midge Belgica antarctica, which is an interesting animal for many reasons:
- Belgica is the world’s southernmost true insect.
- It is Antarctica's largest completely terrestrial animal (penguins and seals are considered marine animals).
- It tolerates a wide variety of harsh conditions, including freezing, dehydration, lack of oxygen and wide fluctuations in pH and salinity.
- It spends two years as a larva, and less than two weeks as an adult, mating, laying eggs and dying in about 10 days.
I. Field Work
In order to study Belgica, we have to find it! Field collecting trips are made in a Zodiac boat to different islands, where we look in the soil under rocks for both larvae and adults. When we find a good site for larvae, we take some of the soil they're in, put it in a labeled Ziploc bag, and take it back to our research lab at the station. When we’re lucky enough to find adults, we use a device called an aspirator to suck the adults into a plastic jar. Also, we are interested in the environmental conditions Belgica actually experiences, so we sometimes leave devices called HOBO temperature loggers in the places we find Belgica — when we collect the HOBOs later, we can download the temperature data for the period of time they were in the field.
II. Isolation of Specimens
Back in the lab, we need to get the insects out of the mud and vegetation they're in. To do this we use a modification of a device called a Berlese funnel. Basically we spread the soil and vegetation out on a big screen, with a tray of ice water underneath. We aim lights at the soil from above. As the soil slowly dries and warms, the larvae move downward, away from the heat and light, and eventually drop into the ice water, in which they survive very well. From there we can pick them out of the ice water with plastic transfer pipets.
III. Experimental Methods
A. Heat Shock and Cold Shock
1. Survival curves —
It is important to find out exactly what temperature extremes Belgica is able to survive, and so, using small tubes of larvae in several different temperatures of water, we see how well the larvae survive in these temperatures for different lengths of time.
2. Thermoprotection and rapid cold hardening —
Insects often show more tolerance to extreme temperatures if they are first exposed to a less extreme temperature for a short time. For example, insects that normally die at 30°C (86°F) might survive that temperature if they first spend an hour at 25°C (77°F). That's thermoprotection — rapid cold hardening is the same thing at low temperatures. Belgica seems a likely candidate for both of these mechanisms.
As in the temperature experiments, we put larvae at different humidity levels for different periods of time to see how well they survive, and whether they are able to control their water loss. We also want to know if Belgica can undergo cryoprotective dehydration — loss of water that protects an organism from very low temperatures.
C. HSP-70 and Northern Blots
1. HSP-70 —
is a gene that codes for a protein that almost all animals make, and which helps protect cells from damage caused by heating, chilling, dehydration, and many other kinds of stress — it’s called a chaperone protein, meaning it actually wraps around important cellular molecules, protecting them from the denaturing (unraveling, which makes them nonfunctional) that would normally occur. We want to know if Belgica increases its production of HSP-70 (upregulating the HSP-70 gene) in response to any of the environmental stresses it experiences.
2. Northern blots —
are one way to tell if a gene has been upregulated. This is a type of gel electrophoresis (in which you use electricity to move molecules through a gel filter, sorting them by size and weight) that allows us to determine what kinds of mRNA an organism is making. mRNA is the messenger molecule that brings DNA's protein-building instructions to the ribosome (protein factory of the cell) so that a particular protein can be built — so knowing what mRNA a critter is making tells us which genes it is actively expressing, and thus which proteins it is producing. Specifically, we are looking for the RNA from the gene that codes for HSP-70 — if we find it, it means that Belgica was actively producing HSP-70, and that's a starting point for testing which conditions cause upregulation of HSP-70 expression.
Finally, we are using an instrument called an osmometer to measure the vapor pressure of larvae and the soil in which they live. Vapor pressure, essentially, is the tendency of something to give off moisture (as water vapor) to its surroundings. It is a function of both the amount of water in a sample and the concentration of solutes in that water. Knowing the vapor pressure of Belgica in different situations will help us understand things about how it dehydrates, and whether the dehydration is a cryoprotective mechanism (something that helps it survive the cold.)
Our research has gone exceptionally well, and we have discovered many things about Belgica's stress tolerance. Here are just a few of them, the ones that will probably go into the first publications of our work.
1. Heat stress survival —
Adult Belgica are able to tolerate high temperature stress better than larvae. This agrees with our field temperature measurements. Adults, crawling on the surface of relatively warm (20°C/ 68°F) moss and rocks, are exposed to much higher temperatures than larvae buried in the mud.
2. Cold stress survival —
Larval Belgica are able to tolerate low temperature stress better than adults. Again, this makes sense. Unlike adults, larvae must survive two Antarctic winters, and so must be cold-tolerant.
3. Northern blot on heat stressed Belgica larvae —
Larvae show a constant expression of HSP-70, which is not upregulated when heat stressed. This is an interesting result because most animals do not produce HSP-70 when they aren't stressed — in fact HSP-70 is harmful to most animals in normal conditions, interfering with growth and other vital cell processes.
4. Northern blot on heat stressed Belgica adults —
Adults do not normally express HSP-70, but will begin to produce it in response to heat stress.
5. Thermoprotection —
Adults show thermoprotection. When exposed to 25°C (77°F) for one hour, adults are better able to survive at 30°C (86°F).
6. Dehydration of larvae —
Larvae are able to survive extensive dehydration, losing up to 70% of their body water. After a week of slow dehydration at high humidity, the larvae actually begin to reabsorb water from the air. The reabsorption is probably made possible by an active increase in Belgica's solute concentration — we are in the process of measuring this with the osmometer.
- Luke Sandro