Lab Write-up #5: Cold Tolerance

Introduction:
Fruit flies are very common insects that are found all over the world. Because
they live is such a variety of climates, including ones that have a large range of
temperatures throughout the year, we question how they have evolved to tolerate the cold.
In attempt to investigate this question, we studied physiological adaptation to cold in
several Drosophila species from different climate locations. If cold tolerance is an
adaptation to climate we would expect to see a correlation between level of cold tolerance
and latitude from which the Drosophila came from. However, there is a problem with
phylogenetic pseudoreplication. We must consider that such a correlation could be due to
phylogeny. The species we observed are closely related, so their observed phenotypes
could have something to do with inheritance rather than adaptation to climate. This
would mean that our data points would not be phylogenetically independent. We
accounted for this by using independent contrasts to disentangle phylogeny from
adaptation. Our null hypothesis was that there would be no correlation between midpoint
latitude and mean wakeup time and our hypothesis was that there would be a correlation
due to adaptation.

Methods:
We were given five species of Drosophila, in test tubes, which had been placed in
an ice-bath for three hours. The species were placed in the ice in fifteen-minute intervals,
so we removed one species every fifteen minutes, in the same order that they were placed
in so that each species was in the ice-bath for the same amount of time. We first started
with Drosophila melanogaster and began the timer as we removed them from the ice. We
poured the dormant flies onto a sheet of white paper, labeled with the species, and
carefully flipped them onto their backs with a paintbrush. We then observed the flies
until they awoke. In order to be consistent, we considered when the flies rolled over onto
their feet to be the time of awakening. We recorded the time for each fly to awaken, and
repeated the same procedure with the four other species: mauritiana, testacea,
neotestacea, and falleni. We did not restart the clock for each species, but rather

subtracted the time in-between for each consecutive species later on. Once all of the data
were collected, we entered how many seconds it took each fly to awaken, into a
spreadsheet in order to find mean wake-up time versus midpoint latitude for each species.
We then plotted our raw data and took into account the possible pseudoreplication
problem by performing an independent contrast correction.
Results:

Figure 1: This figure shows the

phylogeny of the different Drosophila
species studied. It is important to
consider in order to account for the
possibility of a pseudoreplication
problem.

Figure 2: This graph shows the

correlation between midpoint latitude
(x axis) and average wake-up time (y
axis). It shows us that flies from
colder temperatures woke up faster.
It also displays the raw correlation
without independent contrast
correction (black) as well as the
correlation with independent contrast
(red). These two lines are identical.

Table one shows the mean wake-up time for the different species of Drosophila
based on midpoint latitude. Although we only observed five species, table 1 shows the
results for 16 species, some of which were studied in previous experiments. Drosophila
sechellia had the highest mean wake-up time (1544.92seconds) and one of the lowest
midpoint latitudes. Drosophila subquinaria had the lowest mean wake-up time
(67.65seconds) and one of the highest midpoint latitudes. The data points from this table
were used to create the raw correlation in figure 2.
Figure 1 displays the phylogenetic tree of the different Drosophila species that
were studied. The tree shows us that many of the species were closely related to one
another. Because this could cause a phylogenetic pseudoreplication problem, we used an
independent contrast correction (as seen in figure 2) to see if the phylogeny had an
impact.
As seen in figure 2, we plotted the raw correlation between midpoint latitude and
wake-up time and observed the solid black line, indicating a negative correlation between
the two variables. In order to account for the possible pseudoreplication problem, we then
plotted the line with the independent contrast and saw that the two lines were identical.
This indicates that there is no association between the phenotype and phylogeny because
there is no difference when an independent contrast correction is or is not taken into
account. Because latitude and phylogeny are independent of one another, a regression that
doesnt consider phylogeny was used. The p value was found to be 0.028, and the R2
value was found to be 0.3.
Discussion:
Our results allow us to reject our null hypothesis because they support our
hypothesis; there is a correlation, independent of phylogeny, between midpoint latitude
and wake-up time. As seen in figure 2, the p value was found to be 0.028, which is
significant and supports our results because it is such a low value. Looking at the R2
value, we can also see that there is a significant correlation between midpoint latitude and
wake-up time. An R2 value equal to one means you can perfectly predict one term from
the value of the other. With an R2 value equal to zero, we cannot predict the other term at

all. Our value was 0.3, indicating that we can predict wake-up time based on midpoint
latitude with some accuracy, though a higher value would have been more supportive.
Considering that phylogeny has no impact on our study, as determined by the
independent contrast correction, our results show that cold tolerance is likely adaptive
because as midpoint latitude of species increases, mean wake-up time decreases. The
Drosophila species that live in higher latitude and therefore colder environments
generally woke up more quickly after being exposed to cold than those that lived at lower
latitudes. These species have likely adapted to their environments via natural selection,
so those that live harsher climates are more able to withstand colder temperatures,
whereas those that live in warmer environments, which have no need for such
adaptations, can not tolerate the cold as well.
Improvements:
If we were to repeat this experiment, we could be more careful in watching the
flies to be sure we recorded the accurate wake-up time. It would also be beneficial if we
could test more species to get more data points for analysis. It would be interesting to
also test other insects and see if this correlation proves true for them as well, and to see
whether phylogeny plays an impact on other types of insects.