Wolbachia Distribution in the Tiger Beetles of Madre Selva, Peru

Claire Hirschberg

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.

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:

Tiger beetles:• In the order Coleoptera, family Cicindelinae• 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.

• Despite being so widespread the different species within the tiger beetle family are remarkably similar in their adapted traits

Location:

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.

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 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

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.

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 7 - add buffer AW1 to the spin tube. Centrifuge. Discard flow 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 membrane9 - add buffer AE which is a DNA elution buffer to remove the clean DNA from the column and into the collection tube

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 we add water, extracted DNA, W-spec forward, primer W-spec reverse primer (these adds the nucleotides to the forward side and the reverse 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.

The final step was to take the PCR reactionand 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.

In our 52 samples we found 0 samples that had the Wolbachia bacteria present.

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. 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.

My results lead me to another question, if it isn't the Wolbachia than what is causing the strange sex ratio?

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.

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/WerrenLab-WolbachiaBiology.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.

Perching Heights and Nocturnal Communal Roosts of Some Tiger Beetles (Coleoptera: Cicindelidae) in Southeastern PeruDavid 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

Introduction

Background

Part 1: collection and identification

Part 2- Molecular

1 – DNA extraction

Identification

Collection

2 – DNA Amplification

(PCR)

3 – DNA analysis

(gel Electrophoresi

s)

Results

Discussion

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!

References

http://research.foxyresearch.com/pictures/qiaamp.jpg

http://upload.wikimedia.org/wikipedia/commons/thumb/8/81/PCR_tubes.png/300px-PCR_tubes.png

http://www.uvm.edu/giee/ateliers/Camisea/Introduction2.htm

http://www.rochester.edu/college/bio/labs/WerrenLab/WerrenLab-WolbachiaBiology.html