1. Assessing the Prevalence of Wolbachia pipientis in Common Arthropods from Various NC Habitats Tamica Stubbs, Jennifer Telschow, Chanelle Whitehurst E.E. Waddell High School, Charlotte, NC 28217 ABSTRACT MATERIALS AND METHODS RESULTS CONCLUSIONS Collect various species of Arthropods, classify by class and order, and preserve. Collection sites – Figure 4 Isolate abdomen and macerate to lyse cells. Extract DNA from collected arthropods and known positive control. Analyze results to determine the presence of the known Wolbachia strand. Add samples to an agarose gel and run gel electrophoresis procedure. Amplify gene fragments mitochondrial Cytochrome Oxidase 1 (658bp) and Wolbachia 16S rDNA (438bp) using known primers with a PCR Process. Figure 2: Endosymbiosis involves two separate entities becoming one entity inside another. Wolbachia (represented in green) is believed to have once been it’s own entity. Figure 1: Cytoplasmic incompatibility exemplified in Wolbachia-infected arthropods. Affected males can only mate with affected females to successfully produce offspring. Wolbachia pipentis, a cytoplasmic gram-negative proteobacteria, is one of the world’s most prominent parasitic microbes. Wolbachia forms an intracellular infection within its host, affecting reproductive abilities by generating cytoplasmic incompatibility, or CI. CI (Figure 1) is the incompatibility between the sperm of infected males and eggs of uninfected females or females infected with a different Wolbachia strain. In other words, CI results from the modification of an infected male’s sperm that can only be undone by the same strain of Wolbachia in the female’s eggs. This modification causes the disrtiupon of mitotic events and eventual zygotic death. This characteristic effect, along with parthenogenesis, or development of an embryo without male fertilization, helps to ensure the perpetuation of the Wolbachia bacteria within the arthropod population by maintaining it within the female population. Wolbachia is believed to have obtained its ability to be located solely inside of a host through endosymbiosis. Endosymbiosis (Figure 2) is a symbiotic relationship in which the symbiont (Wolbachia) lives within the tissue of the host (arthropods). Once its own separate entity, the Wolbachia bacteria was engulfed into hosts, where it now thrives and is dependent on the host for survival. Wolbachia is extremely common, affecting approximately twenty percent of all arthropod species. Wolbachia is also common among noninsect intervertebrates, such as nematodes, crustaceans, and mites, which often serve as mediums between humans and infected species. For example, with this medium, Wolbachia-derived antigens within infected nematodes trigger innate immune responses, leading to such illnesses as River Blindness and Dengue Fever. Affected arthropod and noninsect organisms, though different in structure and physiological capabilities, represent closely related phylogenic clades evolved beyond that of Platyhelminthes. In the course of animal evolution, organisms that evolved after Playhelminthes were typically capable of adapting to a multitude of environments. The phyla of arthropoda and nematoda (Figure 3), representing all arthropod insects and nematodes, exhibit the strongest cases of habitat diversity within a single phylum. Nematodes are particularly apt to thrive in diverse, even extreme, habitats including arid, freezing, and anaerobic environments. Given the ability to undergo cryptobiosis, or resorting to an ametabolic state, nematodes can live indefinitely in inhospitable conditions, only returning with metabolic activity when conditions are optimal. Arthropods also have adapted to an array of habitats and living conditions. Various species of arthropods can be found in terrestrial habitats, ranging from arid deserts to humid forests. They are also found in a range of aquatic environments, including saltwater and freshwater sources. Both nematodes and a high percentage of arthropods prefer moist environments to progress reproductively. INTRODUCTION HYPOTHESIS Knowledge of the unique ability of arthropods and nematodes to exist in a variety of extreme habitats coupled with the data indicating Wolbachia’s preference to locate within these organisms leads us to hypothesize that it is, in fact, the habitat adaptation abilities of these organisms that has allowed for Wolbachia’s continued existence mainly within them. Thus, because Wolbachia’s high incidence within arthropods can be attributed to their adaptive abilities in diverse habitats, if we collect various arthropod specimens from a range of habitats, including aquatic and moist soil environments, then we will expect to find an equal incidence (~20%) of Wolbachia in arthropods from all studied habitats. Figure 3: Animal evolution phylogenetic tree. Arthropoda and Nematoda, outlined in red, are the phyla of interest. Figure 4: Specimens collected from sites in Mecklenburg County (left) and from Carolina Beach, Wilmington NC (right - highlighted in red). Mecklenburg County Water Quality levels are indicated. (Source: State of Environment, 2008) _____________________________________________________________________________________________________________________________________________________ _____________ Table 1. Distribution of Wolbachia by orders (Presence of Wolbachia (W) is indicated by +. Taxonomic position of tested samples refer to kingdom, phylum, class, order, & common name. For each category the number positive for Wolbachia is indicated in parentheses. The geographical origin of samples is shown as Carolina Beach, Wilmington (W), Lake Wylie, SC (LW), McDowell Nature Preserve Main Stream (M), Lake Mist Creek (C), and Mount Island Lake (L). _______________________________________________________________________________________________________________________________ Taxon W loc _______________________________________________________________________________________________________________________________ Animalia Arthropoda Insecta Coleoptera Japanese Beetle + C Flea Beetle LW Ground BeetleLW Japanese Beetle L Dermaptera EarwigLW Diptera MosquitoC Hemiptera Toad BugLW Stink BugL Hymenoptera Wasp + M Wasp LW Carpenter AntM Velvet AntC Odonata Water NymphC Malcostraca Decapoda CrayfishLW Sand CrabW Isopoda Pill BugLW Pill Bug C Arachnida Araneae Black-tailed Red SheetweaverLW Opiliones HarvestmenM Diplipoda Julida Giant MillipedeL ACKNOWLEDGEMENTS -Dr. Julie Goodliffe, Research Mentor, University of North Carolina, Charlotte -Funded By: Marine Biological Laboratory, Woods Hole, Massachusetts Figure 5: Graph displaying the Incidence of Wolbachia in North Carolina habitats, expressed in percent of total collected samples from each individual collection site. Figure 6: Above: Gel electrophoresis results testing for the presence of Wolbachia. Lanes 1 and 2 of each gel, top and bottom, contain the kilobase ladder and 123 Base Pair ladder, respectively. Ovals indicate bands positive for Wolbachia. Arrows point to bands produced from the mitochondrial Cytochrome Oxidase 1 primer. 33.3 % 16.6% Dobson..ca.uky.edu M.J. Farabee, 2001 www.hbwbiology.net Wolbachia pipentis, a cytoplasmic gram-negative proteobacteria, is one of the world’s most prominent parasitic microbes, affecting approximately twenty percent of all arthropod species and a large percentage of noninsect invertebrates, such as nematodes and crustaceans. The phyla that represent all arthropods and nematodes, arthropoda and nematoda, exhibit strong characteristic cases of habitat diversity. Nematodes are apt to thrive in diverse, even extreme, habitats while arthropods can adapt to an array of terrestrial and aquatic habitats. Knowledge of their ability to exist in a variety of extreme habitats coupled with the data indicating Wolbachia’s preference to locate within these organisms leads us to hypothesize that it is the habitat adaptation abilities of these organisms that has allowed for Wolbachia’s continued existence within them. Thus, we would expect to find equal incidence of Wolbachia in arthropods from a variety of habitats. Arthropods were collected from five collection sites, including conduits, lakes, and saltwater environments. DNA was extracted and primers were used to isolate regions characteristic of both common arthropods and Wolbachia; these sequences were amplified using Polymerase Chain Reaction and illuminated using the gel electrophoresis procedure. Of the five collection sites, one site was shown to have a 33.3% incidence of Wolbachia, another exhibited a 16.6% incidence. Both sites are similar to conduit habitats, indicating a preference for Wolbachia to locate within hosts of similar habitats. This does not fully support our hypothesis. A larger sample pool would be needed to yield more accurate results. Knowing the ability of arthropods and nematodes to adapt to a wide range of extreme habitats as well as research showing their high incidence of Wolbachia, we hypothesized that samples collected from a variety of habitats would yield an equal incidence of Wolbachia. Data collected shows a trend inconsistent with this hypothesis, as only two of the five collection sites yielded samples that tested positive for Wolbachia. These two sites were similar in that they both contained characteristics similar to that of conduits, containing small channels that link larger bodies of water. Our research would lead one to believe that Wolbachia thrives best in hosts within a conduit habitat as opposed to saltwater or mainly terrestrial environments. However, because prior research has demonstrated Wolbachia’s ability to thrive in saltwater and terrestrial habitats, we cannot fully support this conclusion. A significantly larger amount of samples would be needed to fully explore this hypothesis and produce sound results. Larger amount of samples and a wider range of habitats would yield more accurate percentages, and could provide for a better understanding of habitat preference. Future implications include exploring the environmental conditions, including salinity and pH, of habitats and any possible correlation to Wolbachia preference. Future studies may also include exploring the incidence of Wolbachia among the same species of arthropods in various habits to illuminate any trends and correlations.