E. E. Waddell High School Charlotte, nc 28217




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Assessing the Prevalence of Wolbachia pipientis in Common Arthropods from Various North Carolina Habitats

Marine Biological Laboratory

Summer Envisionship

2009


Tamica Stubbs

Jennifer Telschow

Chanelle Whitehurst

E.E. Waddell High School

Charlotte, NC 28217
ABSTRACT
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 Wolbachia thrive within hosts of similar habitats. This does not fully support our hypothesis. A larger sample pool would be needed to yield more accurate results.

INTRODUCTION

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 strand of Wolbachia in the female’s eggs. This modification causes the disruption 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.



Figure 1: Cytoplasmic incompatibility exemplified in Wolbachia-infected arthropods. Affected males can only mate with affected females to successfully produce offspring

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.

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 separate entity.

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.



Figure 3: Animal evolution phylogenetic tree.

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, 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.

REVIEW OF LITERATURE

Bouchon, DiDier, et al. Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization. (1998) Proceedings of the Royal Society. 265, 1081-1090

Casiraghi, M. et al. Phylogeny of Wolbachia pipientis (2005) Microbiology 151, 4015- 4022

Dobson, S. & Tanouye, M. The Paternal sex ratio chromosome induces chromosome loss independently of Wolbachia. (1996) Development Genes and Evolution 206, 207- 217

Kyung-Tai, M. & Benzer, S. (1997) Proc. Natl. Acad. Sci. USA. 64, 10792-10796

Weeks, A.R. & Breeuwer, J.A. Wolbachia-induced parthenogenesis in a genus of phytophagous mites. (2001) Proc. Biol. Sci. 268, 2245-2251

Werren, J. H. Genetic Invasion of the Insect Body Snatchers (1997) Proc. Natl. Acad. Sci. USA. 94, 11154-11155

Werren, J. H. (1994) Natural History – Evolution. 36-38



RESULTS

_____________________________________________________________________________________________________________________


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 Beetle LW

Japanese Beetle L

Dermaptera

Earwig LW

Diptera

Mosquito C

Mosquito C

Hemiptera

Toad Bug LW

Stink Bug L

Hymenoptera

Wasp LW

Wasp + M

Carpenter Ant M

Velvet Ant C

Odonata

Water Nymph C

Malcostraca

Decapoda

Crayfish LW

Sand Crab W

Sand Crab W

Sand Crab W

Sand Crab W

Isopoda


Pill Bug LW

Pill Bug C
Arachnida

Araneae


Black-tailed Red Sheetweaver LW

Black-tailed Red Sheetweaver LW

Opiliones



Harvestmen M

Diplipoda

Julida

Giant Millipede L



RESULTS (CONTINUED)





RESULTS (CONTINUED)


Well # Sample Well # Sample

8 __ Empty 8 __ Empty

7 __ 4 –W4 7 __ 8 – C1

6 __ 3 -W3 6 __ 7 – L3

5 __ 2- W2 5 __ 6 – L2

4 __ 1- W1 4 __ 5 – L1

3 __ Positive Control 3 __ Positive Control

2 __ 123 Ladder 2 __ 123 Ladder

1 __ Kilobase Ladder 1 __ Kilobase Ladder

Left Top Left Bottom






RESULTS (CONTINUED)


Well # Sample Well # Sample

8 __ Empty 8 __ Empty

7 __ 12 – C5 7 __ 16 – M3

6 __ 11 – C4 6 __ 15 – M2

5 __ 10 – C3 5 __ 14 – M1

4 __ 9 – C2 4 __ 13 – C6

3 __ Positive Control 3 __ Positive Control

2 __ 123 Ladder 2 __ 123 Ladder

1 __ Kilobase Ladder 1 __ Kilobase Ladder

Left Top Left Bottom






RESULTS (CONTINUED)


Well # Sample Well # Sample

8 __ Empty 8 __ 25 – LW9

7 __ 20 – LW4 7 __ 24 – LW8

6 __ 19 – LW3 6 __ 23 – LW7

5 __ 18 – LW2 5 __ 22 – LW6

4 __ 17 - LW1 4 __ 21 – LW5

3 __ Positive Control 3 __ Positive Control

2 __ 123 Ladder 2 __ 123 Ladder

1 __ Kilobase Ladder 1 __ Kilobase Ladder

Left Top Left Bottom






RESULTS (CONTINUED)

Organism

Common Name

Taxonomic Classification

Wolbachia Present

Collection Site:

Lake Mist Creek

Land








C-1

Velvet Ant (Actually a Wasp)

Class: Insecta

Order: Hymenoptera







C-2

Japanese Beetle

Class: Insecta

Order: Coleoptera



Positive


C-3

Rolly polly


Class: Malacostraca

Order: Isopoda







C-4

Mosquito Larvae

Class: Insecta

Order: Diptera

































Water








C-5

Water Nymph


Class: Insecta

Order: Odonata














































C-6

Mosquito Larvae

Class: Insecta

Order: Diptera






























Collection Site: McDowell Park & Reserve and Streams / Mecklenburg County

Stream pH: 6.5












M-1



Daddy Long Legs
Captured in the stream.

Class: Arachnid

Order: Opiliones (harvestmen)







M-2


Wasp
Captured +/- 6 feet from stream.

Class: Insecta

Order: Hymoneptera




Positive



M-3

Carpenter Ant
Captured 1 foot from stream.

Class: Insecta

Order: Hymoneptera






Collection Site: Lake Wyle, South Carolina


Lake Sample pH: 6











LW-1

Wasp
Captured +/- 20 feet from the shoreline.

Class: Insecta

Order: Hymenoptera








LW-2

Toad bug
Captured +/- 3 feet from shoreline.

Class: Insecta

Order: Hemiptera








LW-3

Flea Beetle
Captured +/- 3 feet from shoreline.

Class: Insecta

Order: Coleoptera








LW-4

Ground Beetle
Captured +/- 6 feet from shoreline.

Class: Insecta

Order: Coleoptera








LW-5

Pill Bug
Captured +/- 10 feet from shoreline.

Class: Crustacea

Order: Isopoda








LW-6


Black-tailed Red Sheetweaver
Captured +/- 6 feet from shoreline.

Class: Arachnida

Order: Araneae







LW-7

Black-tailed Red Sheetweaver
Captured 1.5 feet from shoreline.

Class: Arachnida

Order: Araneae








LW-8

Crayfish
Captured in Lake.

Class: Malacostraca

Order: Decapoda










LW-9


Earwig
Captured +/- 10 feet from shoreline.


Class: Insecta

Order: Dermaptera






Collection Site:

Carolina Beach, NC / Wilmington















W-1

Sand Crab

Class: Malcostraca

Order: Decapoda








W-2

Sand Crab

Class: Malcostraca

Order: Decapoda








W-3

Sand Crab

Class: Malcostraca

Order: Decapoda







Collection Site:

Mount Island Lake
















L-1

Giant Millipede

Class: Diplipoda

Order: Julida







L-2

Stink Bug

Class: Insecta

Order: Hemiptera







L-3

Japenese Beetle

Class: Insecta

Order: Coleoptera








CONCLUSION

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.



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