Transcript Wolbachia Bacteria in the tiger beetles of Madre salva

Wolbachia Distribution in the Tiger Beetles of
Madre Selva, Peru
Claire Hirschberg
Results
Introduction
Question: Are the Tiger Beetles of the rainforest of Madre Selva Peru
infected with the Wolbachia Bacteria ?
Hypothesis: As previous research suggests that an estimate of 66% of all
insect species are infected with Wolbachia bacteria there
would be a good chance that the tiger beetles of Madre
Selva would have Wolbachia. Therefore I hypothesized
that there would be Wolbachia present in the Tiger
Beetles sampled.
Background
Wolbachia:
•
Wolbachia is a intercellular bacteria found widespread in
arthropods
•
Wolbachia is a reproductive parasite known for altering its
hosts in 4 main ways. It causes Parthenogenesis,
Feminization of males, Male killing and cytoplasmic
incompatibility.
•
The long term effect of these reproductive changes caused
by the Wolbachia bacteria is therefore a population with a
skewed sex ratio favoring females.
•
Effects of Wolbachia:
Part 2- Molecular
To look for the presence of Wolbachia we first extracted the DNA, then
we amplified segments of it that would indicate the presence of
Wolbachia through a PCR . Finally we loaded and ran a gel to analyze
the DNA.
1 – DNA extraction
To look for the presence of Wolbachia we first must extract DNA from
the beetle samples. (diagram to the left of text summarizes the steps)
1 - macerate the sample (originally we used the whole
beetle but then switched to just the end of its abdomen)
2 - add buffer ATL and proteinase K and buffer AL. the buffer
ATL helps break open the cells. The Proteinase K destroys
the DNase enzymes (which degrade the DNA) and the
buffer AL breaks open the cells.
3 - Vortex and incubate at 70˚C – this high temperature is
optimal for the lysis to occur
4 - Add ethanol – the ethanol precipitates the DNA
5 - Spin down the mixture
6 - pipette the liquid into the DNeasy Mini spin column –
centrifuge the mixture. This step means that the DNA
binds to the filter, and centrifuging it down removes debris
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7 - add buffer AW1 to the spin tube. Centrifuge. Discard flow
ures/qiaamp.jpg
through.
8 - Add buffer AW2 to the spin tube. Centrifuge. Discard flow through.
Buffer AW1 and AW2 are used to wash away contaminants from the DNA
that is attached to the column membrane
9 - add buffer AE which is a DNA elution buffer to remove the clean DNA
from the column and into the collection tube
2 – DNA Amplification (PCR)
Part 1: collection and
identification
Location:
http://www.uvm.edu/giee/ateliers/Camisea/Introduction2.htm
Madre Selva, Peru is located off a tributary on the Amazon River in the
north eastern rainforest of Peru. The forest of the area are
both terra Firma and Flood forests.
Collection
The tiger beetles of Madre Selva roost on the leaves of the under
growth at night.
To collect them we went on a night hike with jars of alcohol and brushed
the roosting beetles from the leaves into the alcohol
The collection was essentially random as I sampled from multiple sites
and did not discriminate between which beetles I
collected.
Collection was done on 2 occasions, off 2 separate trails. Both trails we
in the terra Firma but near the flood forests
The Samples were stored in 95% alcohol and in a freezer to prevent DNA
from degrading
The next step is to amplify the DNA. This reaction will amplify the
fragment of DNA that if present codes for the Wolbachia bacteria –
specifically the small subunit ribosomal RNA (16S rRNA). As a control l it
will also amplify the cytochrome oxidase 1 protein in animal
mitochondria. This means that if the PCR is successful when we run the
gels regardless of the presence of Wolbachia there should be a band for
the CO1.
The PCR reaction comes with readymade reaction tubes which have
already in them (tubes pictured below):
- Taq polymerase, MgCl₂, buffer and
dNTps
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we add water, extracted DNA, W-spec
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forward, primer W-spec reverse
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primer (these adds the nucleotides to
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the forward side and the reverse
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sides of the DNA when replicating the
fragment for the Wolbachia.), CO1
forward primer and CO1 reverse primer (these add the nucleotides to
the forward and reverse sides of the DNA when replicating the fragment
for the cytochrome oxidase 1 protein)
The PCR machine then goes through a thermal cycle set at optimal
temperatures for the reaction to occur.
3 – DNA analysis
(gel Electrophoresis)
•
•
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The figure below show a female (left) and male (right) tiger
beetle collected from Madre Selva. You can see the
differences in the shape of their mandibles, the males
being more squared to aid their reproductive success as
they are used to grip onto the female beetle during mating.
The male beetles are also significantly smaller than the
female.
We collected a total of 52 beetles with 11males and 41
females
From a Ztest of proportion I have convincing evidence that
the true proportion of male in the population is less than
.05
This sex ratio being significantly different from the
predicted 1:1 appears to be initial support for the presence
of Wolbachia
Below are pictures of our gels.
All of the gels roughly looked the same. With a ladder, a positive
control (a sample we knew to have the Wolbachia bacteria) and a
negative control (a sample we knew to not have the Wolbachia
bacteria) and then the samples. All of our PCRs were success as we
always had a band for the CO1 show up on our gels, meaning that our
results of finding of no Wolbachia are not due to error in the PCR or
extraction steps.
Discussion
My results lead me to another question, if it isn't the Wolbachia
The most likely hypothesis I have is that there are differences in
the life cycle of the 2 beetles. One paper found that in one species of
tiger beetles found in the flood plains of a part of the Peruvian
Amazon the life cycles for males and females differed, and the males
had a shorter lifespan than the females. (Amorim et al, 1997) Though
I don’t know yet if the tiger beetles I studied were the same
Pentacomia egregia that had this different life cycle but it is possible
that a life cycle like this could explain why there are more females
than males in my sample. The beetles I collected were not found in
forests that typically flooded seasonally but they were very close to
the flood forest meaning they inhabit a similar habitat to the
Pentacomia egregia. This supports the idea that my sample may be
this species, or that it would be closely related and therefore have a
similar life cycle.
References
Hilgenboecker, K., Hammerstein, P., Schlattmann, P., Telschow, A. and
Werren, J. H. (2008), How many species are infected with Wolbachia? – a statistical
analysis of current data. FEMS Microbiology Letters, 281: 215–220. Published 29
FEB 2008.
“Wolbachia Biology”, Warren Lab department of Biology at University of
Rochester. Accessed Sunday, May 22, 2011.
http://www.rochester.edu/college/bio/labs/WerrenLab/WerrenLabWolbachiaBiology.html
Amorim, M., Adis, J., Paarmann, W., “Ecology and adaptations of the Tiger
Beetle Pentacomia egregia (chaudior) (Cicindelinea: Carabidea) to Central
Amazonian Floodplains.” Ecotropica. Vol.3 No.2, 1997.
Bordenstein, S., Brothers, C., wolfe, G., Bahr, M., Minckley, R., Clark, M.,
Wernegreen, J., Bordenstein, S. R., Reznikoff, W., Werren, J. “Using the Wolbachia
Vacterial Symbiont to teach Inquiry-Based Science.” The American Biology Teacher,
Vol. 72, No. 8, October 2010.
Identification
•
According to Hilgenboecker et al when a species is infected with
Wolbachia the infection is either in 10% or the population or 90% of
the population. With these 2 values as my expected proportion a 1
proportion Z test shows that at both the expect p of .10 and .90 that
I have significant evidence to reject my hypothesis, meaning that it is
highly unlikely that the tiger beetles of Madre Selva are hosts to the
Wolbachia bacteria.
than what is causing the strange sex ratio?
http://www.rochester.edu/college/bio/labs/WerrenLab/WerrenLab-WolbachiaBiology.html
Tiger beetles:
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In the order Coleoptera, family Cicindelinae
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There are a estimated 2300 species of tiger beetles, found
in all types of environments from the rainforest to the
desert and on almost all surfaces of the earth.
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Despite being so widespread the different species within
the tiger beetle family are remarkably similar in their
adapted traits
In our 52 samples we found 0 samples that had the Wolbachia
bacteria present.
The final step was to take the PCR reaction
and load it into a 2% agarose gel.
(pictured above) The gel works as the
negatively charged DNA moves with the
electricity through the gel. Different
fragments of DNA have lengths that differ
enough that they move at different rates
through the gel.
The DNA is loaded with a dye so that you can see it moving in the gel
(above right picture) . Once the dye approaches the end of the gel the
gel has run and it is taken and stained so that the DNA and Ladder can be
seen.
Perching Heights and Nocturnal Communal Roosts of Some Tiger Beetles
(Coleoptera: Cicindelidae) in Southeastern Peru
David L. Pearson and Joseph J. Anderson, Biotropica, Vol. 17, No. 2 (Jun., 1985), pp.
126-129
Werren, J., Windsor, D., Guo, L., “Distribution of Wolbachia amung
Neotropical Arthropods” the Royal Society, 1995.
Discover the Microbes Within! The Wolbachia project. Accessed May 22,
2011. http://discover.mbl.edu/class_resources.htm
Acknowledgements
Many thanks to: Elizabeth Fox, Jim Serach, Leah Domb, The
Lawrenceville school, Project Amazonas and all my friends on
the Amazon trip who helped me catch beetles!