FEATURED ARTICLE

Investigating the presence of Wolbachia pipientis in various mosquito species

Yuan Kang and Brian Dempsey*
Department of Science Acton Boxborough Regional High School, 36 Charter Road, Acton, MA 01720 *Correspondence: bdempsey@mail.ab.mec.edu

Wolbachia pipientis are endosymbionts that infect many insects, arachnids, and nematodes. By comparing the Wolbachia DNA sequences contained in different infected insect species, one can investigate the evolutionary history of arthropods and the modes of transmission of this reproductive parasite. We collected and extracted DNA from several mosquito species to test for the presence of Wolbachia. Multiple positive sequences were generated by PCR and DNA sequencing of 16S DNA from Culex restuans samples. When these sequences were analyzed using the NCBI BLAST database, we confirmed the occurrence of transmission of Wolbachia DNA in insects and arachnids. However, due to contamination of multiple samples, we were unable to conclusively compare the Wolbachia sequences between different mosquito species.

Abstract

Introduction

Wolbachia pipientis is an -proteobacterial reproductive parasite that infects a wide variety of arthropods, including 17-75% of all insects (Huigens 2003). Since it is vertically transmitted (passed on to offspring) exclusively in the cytoplasm of egg cells, Wolbachia triggers phenotypic changes in hosts to increase transmission through females. Host manipulations include parthenogenesis, feminization of males, killing of male embryos, and cytoplasmic incompatibility between uninfected females and infected males. These effects skew insect populations in favor of infected females, thus enhancing the parasites survival and reproduction (Doran 2001). In addition, reproductive barriers induced by Wolbachia infection can promote sympatric speciation (evolution of species living in the same environment) within the host population. Because Wolbachia is a widespread endosymbiont, it can be used to study the evolutionary history of arthropods (Bordenstein 2006). Comparisons of the 16S rRNA gene give insight to the relatedness of various infected species. Close Wolbachia gene

alignments also indicate the occurrence of lateral transmission between distantly related insects (Huigens 2003). Further study in identifying a wide range of host species and analyzing their 16S sequences can make Wolbachia a very useful tool for evolutionary biology and bioinformatics. Wolbachia infects an estimated 28% of mosquito species (Xi 2006). There are several potential applications of Wolbachia as a solution to global health issues such as lymphatic filariasis, malaria, and dengue. Entomologist Steven Dobson hopes to eliminate mosquito vectors of filariasis in the South Pacific by introducing infected males to the island populations. Dobson hypothesizes that cytoplasmic incompatibility-induced sterility will effectively reduce, and even eliminate, the harmful mosquito populations after many generations of reproduction. This principle is applicable to other mosquito-transmitted diseases as well. More detailed research regarding specific mosquito species and Wolbachia will aid in the development of these applications.

Materials and Methods

Mosquito larvae were collected from tree holes and discarded tires around the Worcester area. Several different species were identified in the laboratory using a light microscope and field guide: Culex restuans, Ochlerotatus japonicus, and Ochlerotatus triseriatus. Toxoryhnchites rutilus septentrionalis and Orthopodomyia signifera collected in Princeton, New Jersey, were also used to provide a more diverse range of mosquito species. Larvae were kept in plastic containers filled with water from their respective collection sites. Adults were raised in breeding cages at 80% humidity with a 10% sucrose solution food source. Insects for DNA extraction were preserved in 100% ethanol and stored at -20C. The Qiagen DNeasy Kit was used to extract DNA from the collected mosquitoes and the positive and negative Nasonia control insects. We initially followed the Wolbachia Project extraction protocol outlined by George Wolfe (Academy of Science, Loudon City, VA) but later found that the Qiagen protocol worked more

Yuan Kang and Brian Dempsey effectively. In both procedures, Proteinase K and lysis buffers were added to the macerated insect tissue. After incubation, ethanol was added to precipitate the DNA. Next, a series of wash steps using DNeasy spin columns cleaned the DNA. Finally, an elution buffer removed the clean DNA from the spin column filter. DNA samples were stored at -20C until PCR. PuReTaq Ready-To-Go PCR beads were used in the polymerase chain reaction. In addition to the premixed PCR pellets, each reaction tube also contained 2ul each of wspec forward primer, wspec reverse primer, and extracted DNA. These primers were used to test for 16S DNA. Positive Wolbachia DNA previously extracted from a Nasonia specimen served as the control for this step of the experiment. The following pcr conditions were used: 2 min @ 95 C for 1 cycle; 30 sec. @ 94 C, 45 sec @ 55 C, 90 sec @ 72 C for 38 cycles; and 10 min. @72 C for the last cycle. After PCR, samples were kept at 4C until gel electrophoresis. 5ul of each DNA sample was mixed with 1.5ul of 6X loading dye and pipetted into a prepared agarose gel containing ethidium bromide. 5ul of a DNA ladder solution was used in one lane to calculate the size of the unknown fragments. The gel was run at 80V for 30 minutes and then viewed under an ultraviolet light. Unknown samples that tested positive for Wolbachia and had good control results were cleaned with ethanol, resuspended in distilled water, and sent in to be sequenced. The DNA sequences were uploaded onto the NCBI BLAST to generate phylogenetic trees based on other sequences in the database.

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Figure 1: (A) Positive result for sample Tr5, but absence of signal from positive control and faint signal in negative control. (B) Strong PCR results from multiple test samples, but also strong signal in negative control and weak signal in positive control.

Figure 2: Species showing positive results for Wolbachia. Lane 1: DNA ladder, lane 2: positive control, lane 3: negative control, lane 4: Culex sample 1, lane 5: Culex sample 2, lane 6: Culex sample 3, lane 7: Culex sample 4

Results

Most of our trials yielded unreliable control data and could not be sent in for sequencing (Table 1). In many cases, the negative Nasonia control displayed a positive band after gel electrophoresis, a sign of contaminated samples(Figure 1A). Several other species tested positive for Wolbachia, but their data is questionable due to poor Nasonia control results (Figure 1B). However, three Culex restuans samples showed solid bands around 400 bp long with good controls (Figure 2). We were unable to produce a phylogenetic tree.

Discussion

Several Culex restuans specimens tested positive for Wolbachia with reliable Nasonia control bands. BLAST results showed close alignment with other Diptera organisms, as expected, as well as some Hymenoptera, Lepidoptera, Coleoptera, and Prostigmata (arachnids). The latter supports the hypothesis that Wolbachia was transmitted both vertically and horizontally throughout evolutionary history, since insects and arachnids are fairly distant relatives. The DNA sequences from other mosquito species showed very little variation in BLAST results. This suggests that the

Yuan Kang and Brian Dempsey

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Table 1: Alignment of sequences of samples C2-C4

Genus ID Order Species

Genus species aligned with

Orders aligned with

C2

C3

C4

16S gene is not significantly variable among the mosquito species sampled. However, due to faulty controls in these later trials, the DNA sequences generated may be contaminated and unreliable. The extraction phase of this lab has the most potential for error. The Qiagen protocol proved to be more reliable at extracting DNA than the procedure provided by the Wolbachia Project website. Contamination of the negative Nasonia control was also a major problem during the extraction step. Since the controls were very inconsistent, another possible error is unreliable Nasonia specimens. Some of our positive controls lacked Wolbachia DNA, and some negative controls displayed false positive results. In both cases, the unknown mosquito DNA results could not be used because the entire trial was not reliable. In the future, it would be beneficial to amplify a portion of insect DNA in addition to the Wolbachia sequence to determine whether or not the DNA extraction step was successful. The bioinformatics analysis of our Wolbachia sequences was very vague, and there were no close matches to any mosquito species for the Culex samples or other species. In fact, many of the closest matches were to Nasonia and Trichogramma, suggesting possible contamination between the wasp controls and the unknown mosquito specimens. Because the Culex DNA sequences were not completely clean, editing for ambiguous base pairs was difficult. Only the C3 sample yielded decent forward and reverse sequences which could be manipulated and combined to form one clear sequence. The other two samples, C2 and C4, showed contamination on either the forward or reverse sequences, so they could not be edited. While one sequence for these two samples was still usable for BLAST, we did not have the opportunity to correct misalignments based on the complementary strand. This experiment would be more conclusive with a larger sampling pool, more reliable results, and cleaner DNA sequences. It is important to keep in mind that the phylogenetic tree generated by BLAST depends on the breadth of sequences in the NCBI database. Our DNA may not have generated matches to mosquito Wolbachia because those sequences have not been studied and uploaded yet. In the future, as Wolbachia research expands to cover more species, the database will be able to produce better DNA sequence matches and more complete phylogenies.

Yuan Kang and Brian Dempsey

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References

Bordenstein, S., Marshall, M., Fry, A., Kim, U. & Wernegreen J. 2006. The tripartite associations between bacteriophage, Wolbachia, and arthropods. PLoS Pathlog vol. 2, no. 5: e43. Charlat, S., Hurst, G. D. & Mercot, H. Evolutionary consequences of Wolbachia infections. TRENDS in Genetics vol. 19, no. 4. April 2003: 217-223. Doran, T. & Moore, R. 2001. Application of the reproductive parasite Wolbachia to the biological control of flystrike. Proc. FLICS Conference, 241244. Huigens, M. E., de Almeida, R. P., Boons, P. A. H., Luck, R. F. & Stouthamer, R. 2004. Natural interspecific and intraspecific horizontal transfer of parthenogenesis-inducing Wolbachia in Trichogramma wasps. Proc. R. Soc. Lond. B 271, 509-515. Rokas, A. Wolbachia as a speciation agent. TREE vol. 15, no. 2. February 2000: 44-45. Worley, Jeff. Mosquito Birth Control. Odyssey 2006. 15 August 2008. Xi, Z., Khoo, C. & Dobson, S. L. 2006. Interspecific transfer of Wolbachia into the mosquito disease vector Aedes albopictus. Proc. R. Soc. B 273, 13171322.

MBL and now at Vanderbilt University), Dr. William Reznikoff and Ms. Michele Bahr of the MBL. The Wolbachia Project which they created has inspired numerous students and teachers to explore cuttingedge science.

Acknowledgements

The authors are very grateful to the researchers at Clark University for their assistance with the Wolbachia Project. Dr. Todd Livdahl provided space in his lab and hours of time answering questions. Mr. Camilo Khatchikian and Ms. Jamie Fitzgerald helped troubleshoot laboratory problems and offered useful guidance. Dr. David Hibbett initiated the authors to molecular systematics and hosted students from the Wolbachia class to his lab. The authors are especially thankful for the leadership and vision of Dr. Seth Bordenstein (formally of the