Introduction:

Wolbachia is an endosymbiont found mainly in insects, which causes many

reproductive alterations in their hosts. Wolbachia is present in many insects and is one of
the most widespread infections on Earth. In this lab, we screened three insects for the
presence of Wolbachia by extracting DNA and selectively amplifying sequences using
PCR. PCR (polymerase chain reaction) is a technique used to amplify a few copies of a
DNA sequence to get orders of great magnitude, which can then be used for a variety of
experiments. The sample is first heated to denature the DNA into two separate strands.
Then, the temperature is lowered so that the primers can attach to the template during the
annealing phase. Next, an enzyme called taq polymerase synthesizes two new strands of
DNA. Taq is from the prokaryote Thermus aquaticus and is a good polymerase for PCR
because it can handle the drastic temperature changes without denaturing. We also used
Gel Electrophoresis in this lab, which is a method of separating substances in an
electrical field. During electrophoresis, a gel is submerged in a vessel that contains a
buffer solution and a positive and negative electrode. The DNA then moves according to
various factors, and the different bands formed can be interpreted. We can look at the
results to see if our insects were infected with Wolbachia, and use phylogenies to help us
distinguish that Wolbachia is spread both vertically and horizontally. According to
Moran et al (1993), which investigated endosymbionts in aphids, monophyletic groups
with a phylogeny completely concordant with that of their hosts implies long-term cospeciation. Endosymbionts with phylogenies that are mirror images to their hosts
phylogenies are subject to vertical transmission, not horizontal. Endosymbionts with
horizontal transmission will have phylogenies that are patchy looking. In this
experiment, our evidence points to the idea that Wolbachia is transmitted both vertically
and horizontally.

Methods:
We were given three insects, two of which we identified as Hymenoptera and one
of which we identified as Coleoptera based upon certain phenotypic traits. The insects
used were preserved in EtOH at the time they were collected to prevent the DNA from
being destroyed. The first step in this experiment was DNA extraction and isolation. To
isolate the Wolbachia DNA, we used primers that only bind to Wolbachia DNA, not
insect DNA. We began by isolating the insects abdomens for use and then rinsing them
with distilled water to remove any excess EtOH. We then immersed the insects in
distilled water in a tube. After allowing them to sit for a minute, we removed the water
from the tube using a pipet. We then added Buffer ATL and Proteinase K to the tubes.
The Buffer ATL induces lysis of cell tissues in order to release and expose the insects
DNA. However, this also exposes the insects DNA to hydrolytic DNases, which cleave
bonds via hydrolysis. Therefore, we also added Proteinase K. This enzyme denatures

hydrolytic enzymes so that the DNA stays intact. Once that was complete, we macerated
the insects in the tubes using pestles. We then incubated the tubes in a heat bath for over
an hour and afterwards mixed them by vortexing. This essentially denatured the
hydrolytic enzymes so that we could continue with the DNA isolation.
At this point we added buffer AL and ethanol to the tubes. The buffer AL breaks
open cells and their nuclei to extract DNA for our analysis. The ethanol precipitates the
DNA because DNA is insoluble to alcohol, so it aggregates together upon centrifugation.
We then pipetted the solution into flow-through tubes, leaving behind large insect chunks.
We then centrifuged the tubes for one minute at 8,000rpm. We discarded the flowthrough, added Buffer AW1 and centrifuged again for 1 minute at 6,000rpm. We again
discarded the flow-through and then added AW2 and centrifuged for 1 minute at
6,000rpm. We discarded the flow-through once again. These two additions of buffer
were a wash-through process. We then repeated the centrifugation for 3 minutes at
14,000rpm. We then transferred the spin columns into new tubes and added buffer AE
onto the membrane. Buffer AE is an elution buffer that is pulled through the matrix
during the final centrifugation, bringing with it the DNA. We allowed the tubes to
incubate at room temperature for a few minutes and then centrifuged a final time for 1
minute at 14,000rpm. We now had our DNA.
The next step was DNA amplification via PCR. We used a technique called
multiplexing, which is running multiple primer pairs in a PCR reaction. We did this by
using a primer specific for the WSP gene in Wolbachia, which is not present in
eukaryotes, and also using a primer specific for a mitochondrial gene, CO1, which is not
present in Wolbachia because Wolbachia do not have mitochondria. The purpose behind
doing this was to make sure we got a sample that was good enough for PCR. If the CO1
was not present, we would know that our DNA didnt have good enough quality and our
results regarding Wolbachia would be invalid, but if the CO1 was present, and WSP was
not, we could say that our samples were actually negative for Wolbachia.
We had 6 PCR tubes labeled 1 through 6. We added PCR mix containing DNA
polymerase, MgCl2, dNTPs, primers and buffers to all of the tubes. Then, in each tube
we added a different substance: The first tube contained the positive control (insect DNA
known to carry Wolbachia), the second tube contained the negative control (insect DNA
known not to carry Wolbachia), the third tube contained the DNA sample from our first
insect (hymenoptera 1), the fourth tube contained the DNA sample from our second
insect (hymenoptera 2), the fifth tube contained the DNA sample from our third insect
(coleoptera), and the sixth tube contained water, as a control, which should not create any
bands in the gel electrophoresis. If it does, we know there was some contamination. The
positive control allowed us to have a band to compare our specimens to and see if
Wolbachia is present. The negative control allowed us to make sure insect DNA was
present, which indicates whether our DNA samples were good enough for the
experiment.

PCR is conducted by first raising the temperature to around 95C. This causes the
DNA to denature and separate into single strands. Then the temperature is lowered to
between 50 and 60C for the annealing phase. During this phase, primers attach to the
DNA and the polymerase begins to replicate a few base pairs. Then the temperature is
raised to 72C, which is the optimum temperature for taq polymerase to function. Then
elongation begins and the polymerase replicates the two strands into two new strands of
DNA. This process is then repeated several times and the amount of DNA increases
exponentially.
The next step was Gel Electrophoresis. We began by dissolving agarose in TAE
buffer and heating it until the solution became clear. We added SYBR safe, which is a
stain that will fluoresce under certain conditions, and then let it cool to an extent before
pouring it into the casting tray. We put the comb into place on the tray and then allowed
the solution to cool and solidify for about 10 minutes. We removed the comb, which
created wells in the gel. We then placed the gel into the electrophoresis chamber
containing TAE buffer that covered the gel. We added loading buffer to each of the six
PCR tubes. The loading buffer contains a dye that assesses how fast our gel runs and a
reagent to make the samples denser than the running buffer so that the samples sink into
the wells. We then loaded 10L of each sample into wells 2 through 6 in the gel and
ladder, which is a solution of DNA molecules of different lengths used to reference the
size of unknown DNA molecules, into well 1. We connected the electrodes and turned it
on to 100 volts, and let it run for about 45 minutes. The samples moved towards the
positive end because DNA is negatively charged and opposites attract. We then turned it
off, removed the electrodes, and removed our gel. We then photographed the results for
analysis.
The next part of the lab involved phylogenies. We downloaded a file containing
CO1 sequences from insects infected with Wolbachia, and we also downloaded a file
containing the sequence of the fstz gene from Wolbachia extracted from these insects.
We used online computer programs to interpret these files and create phylogenetic trees.
We changed the format of the tree to a cladogram so that we could see the branches
clearly. We could then compare these trees.

Results:

Figure
1:

Figure
2:

The first lane contains the positive control (insect DNA known
to carry Wolbachia), the second lane contains the negative
control (insect DNA known not to carry Wolbachia), the third
lane contains the DNA sample from our first insect
(hymenoptera 1), the fourth lane contains the DNA sample
from our second insect (hymenoptera 2), the fifth lane contains
L 1
2
3
4
the DNA sample from our third insect (coleoptera), and the
5 water,
6 as a control,
These
phylogenetic
treeswhich
showshould
insect phylogeny
ThisWolbachia
is an idealphylogeny
gel. It eachonlane
the contains
right. These
the same
two entities as
sixth lane contains
not and on the left and
whileinsomewhat
similar,
not that
mirror
arefigure
more 1,
patchy
howeverlooking,
in this gel,
which
all suggests
three insects
that are positive for
does not createtrees,
any bands
the gel. This
gel are
shows
all images. They in
Figure
Wolbachia
is transferred
horizontally rather than vertically. Wolbachia.
three of our insects
were negative
for Wolbachia.

Figure 1 shows my gel results. The first lane contains the positive control (insect
DNA known to carry Wolbachia), the second lane contains the negative control (insect
DNA known not to carry Wolbachia), the third lane contains the DNA sample from our
first insect (hymenoptera 1), the fourth lane contains the DNA sample from our second
insect (hymenoptera 2), the fifth lane contains the DNA sample from our third insect
(coleoptera), and the sixth lane contains water, as a control, which should not and does
not create any bands in the gel. Our results show that all three of our insects are negative
for Wolbachia. The top row of bands on the gel shows insect DNA. It is present in lanes
1 through 5, and not in lane 6. The second row down shows Wolbachia DNA. A band
appears in the first lane containing the positive control, as was expected, however a band
does not appear in any of the other lanes. The fact that insect DNA showed up in all of
our three samples and that Wolbachia was shown to be present in the positive control,
allows us to conclude that our three specimens were free of Wolbachia. Our gel matches
the ideal gel in figure 2 in all except the fact that all three of the insects in the ideal gel
were positive for Wolbachia.
Looking at the phylogenetic trees, the evolutionary history of the insects and the
Wolbachia do not look the same, rather they look more patchy and separate. If the trees
were to be close mirror images of each other, it would suggest that Wolbachia is
transferred vertically in insects (from parent to offspring) because they would have coevolved and speciated at the same points. A more patchy appearance between the two
phylogenies, as is apparent in figure 3, suggests horizontal transfer of Wolbachia.
Horizontal transfer is transferring from one organism to another, not based on parent to
offspring inheritance, but by some kind of infection. As can be seen in the orange box on
the Wolbachia phylogeny in figure 3, one clade contains Wolbachia found in three
different orders of insects. This would suggest that the Wolbachia is closely related
among different orders of insects. However, in the blue box on the same phylogeny we
can see that the Wolbachia is more closely related within separate orders. The top clade
is mainly hymenoptera and the bottom clade is mainly diptera. As shown in the green
boxes, Wolbachia within the same order can be more distantly related than Wolbachia
between two different orders. However, because it varies throughout the entire tree, we
can conclude that the Wolbachia is not necessarily more similar within an order of insect
and is likely transferred horizontally between insects, without specification to order.
Because of the high variance among Wolbachia strains found in different orders of
insects, it is difficult to say whether the Wolbachia found in each of the three test subjects
of the ideal gel were the same strain.

Discussion:

Because insect DNA is present in lanes 1 through 5, and not in lane 6, our samples
were adequate because insect DNA showed up where it was supposed to and did not in
the water, which was a negative control that contained no DNA template. If DNA were
to show up in lane 6, we would have known there was a contamination and wouldnt have
been able to interpret our results. Our positive and negative control also worked, as
insect DNA appeared in both of them and Wolbachia DNA appeared in the positive
control but not the negative control. The fact that all three of our controls worked
indicates that we can assume our results are valid and that the lack of Wolbachia in our
specimens is not due to some error in the experiment. When comparing our gel (figure
1) to the ideal gel (figure 2), they are identical except for the presence of Wolbachia in
our specimens. This difference is likely just due to chance.
As described in the results on page 5, the comparison of the phylogenies of
insects and Wolbachia suggests horizontal transfer rather than vertical. However, we
know from previous research that there is also vertical maternal transfer of Wolbachia in
insects. Whether Wolbachia is transferred vertically or horizontally is biologically
relevant because it allows us to observe how it infects hosts and to gain insight into its
evolution, whether that be in concordance with their insect hosts or separate from them.
According to Heath et al (1999), although Wolbachia normally undergoes vertical
transmission through the maternal line of its host population, there is compelling
evidence from molecular phylogenies that extensive horizontal (intertaxon) transmission
must have occurred. One possible explanation that Heath et al investigates is
transmission between insect parisitoids and host insects. Insect parisitoids inject their
eggs into the haemocoel of their hosts, where the eggs undergo metamorphosis if the
host is a carrier of Wolbachia, this potentially exposes the developing parasitoid's stem
cells and somatic cells to infection (Heath et al). The exact method of horizontal
transfer between insects is still unknown, but this is one possible explanation. Wolbachia
transfer is not a black and white process. It has shown to be vertically transferred
maternally, but our phylogenetic evidence also suggests that it is transferred horizontally.
This means that there are many possible means of transmission, which will need further
research to uncover.
Improvements:
There does not seem to be any problems with our data so there are not many
improvements we could do methodically. However, it would be beneficial if we could
see the other students data to compare so that we could see how many of the insects that
were tested in our class were actually infected. This would more effectively demonstrate
to us how widespread Wolbachia is, being as none of our specimens were infected. Also,
it would be more beneficial if we could have discussed more as a class about the
possibilities of horizontal gene transfer and its implications.
Works Cited

Heath, Benjamin. Butcher, Robert. Whitfield, William. Hubbard, Stephen.

Horizontal transfer of Wolbachia between phylogenetically distant insect species by a
naturally occurring mechanism. Current Biology. 9: 313-316 (1999).
Morgan, Nancy. Munson, ark. Paul, Baumann. Ishikawa, Hajime. A molecular
clock in endosymbiotic bacteria is calibrated using the insect hosts. The Royal Society.
253: 167-171 (1993).
Nuigens, M. de Almeida, R. Boons, P. Luck, R. Stouthamer, R. Natural
interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia
in Trichogramma wasps. The Royal Society. 271: 509-515 (2004)