Tricks Not Treats: Wolbachia’s Manipulation of Sex in Infected D. subquinaria Offspring

Madeline Sheppard, a student at Eckerd College, worked in the lab of Dr. Kelly Dyer

Abstract Wolbachia is a bacteria that is found in up to 60% of all insects, which is transmitted exclusively from mother to offspring through the egg. In many host species Wolbachia infection does not Wolbachia are gram-negative maternally transmitted bacterial endosymbionts that are found in upwards of 60% of arthropods. Here, we aimed to determine the phenotype expressed in D. subquinaria when they are infected with a strain of Wolbachia originally found in a closely related species, D. recens. Wolbachia infection is expressed as one of two phenotypes: Cytoplasmic incompatibility (CI), which causes the deaths of most infected offspring, and Male Killing (MK), which causes the death of the sons of infected males. Additionally, we tested how Wolbachia interacts with the varied genetic backgrounds of D. subquinaria populations, as well as how that would influence which phenotype is displayed in infected offspring. We tested a total of 15 lines of D. subquinaria by taking 2 to 3 infected females and crossing them with 1 uninfected male. The F1 offspring from those crosses were collected, and the sex distribution per line’s offspring was determined. Ten lines produced a significant number of flies (n>10). We tested for Wolbachia infection by using PCR, and determined the F1 generation was positive for Wolbachia. All lines expressed evidence of the MK phenotype, and four lines showed nearly complete MK (98-100% female). The remaining six expressed partial MK (60-84% female). In the future, we aim to determine whether there is a genetic suppressor of the MK phenotype in D. subquinaria, as well as to generalize the results of this project to further understand the population dynamics of Wolbachia-infected flies.


Wolbachia and its effects on mating preference in two Drosophila species

Kareena Collins, a students at the University of Maryland Eastern Shore, worked with Paul Ginsberg and Dr. Kelly Dyer.

Abstract: Wolbachia is a maternally inherited intracellular endosymbiont that can manipulate reproduction in many different species of arthropod hosts, enabling its invasion into novel host populations. The most common types of reproductive manipulation is cytoplasmic incompatibility (CI), where mating between an uninfected females and infected males results in embryonic mortality.  Two Drosophila species, D. recens and D. subquinaria, were used to investigate whether Wolbachia can affect mating preference in a native versus non-native host species. D. recens is the infected species of Wolbachia with a frequency ~ 98%, while D. subquinaria is the uninfected host of Wolbachia. In the geographic region where both species overlap there is gene flow between species. We introgressed Wolbachia from D. recens into D. subquinaria in the laboratory. Both species show the CI phenotype in the lab when there is a cross with an uninfected female and an infected male. We conducted no choice mate trials for all crosses among infected and uninfected individuals for each species (all intraspecific crosses), and watched for mating for a three hour observation period. We found that Wolbachia had no effect on mating preference in the native host, D. recens. However, in the non-native host, D. subquinaria, Wolbachia had a huge effect on mating preference, with a significant reduction of mating rate in the cross between an uninfected female and an infected male ( the “incompatible” cross). Because Wolbachia had such a significant effect on mating preference only in the non-native host of D. subquinaria, it has potential implications for Wolbachia’s inability to become established as a native host in the population and/or species.


Identifying bacterial pathogens in natural Drosophila populations

Kailene Richbow Dozier, a junior at Virginia Union University, worked with Dr. Kelly Dyer to examine the pathogens present in wild populations of Drosophila.

Abstract: An estimated 70% of the world’s insects are infected with the gram-negative bacteria, Wolbachia pipientis. Wolbachia is known to protect organisms against viral pathogens, however little is known about how it interacts with bacterial pathogens. The aim of this study was to isolate and identify bacterial pathogens within wild, mushroom feeding Drosophila. With this knowledge, we hope that we can aid future research studies trying to understand how Wolbachia increases host resistance to bacterial pathogens as compared to viral pathogens. Two species of flies, Drosophila Putrida and Drosophila Tripunctata were captured from Oconee State Park located in Athens, GA. The captured flies were separated by gender. Female flies were put aside to generate Iso-Female lines, while male flies were homogenized and streaked onto LB agar plates for bacteria growth. Once colonies began to grow after incubation, they were isolated and morphologically identified. 35 bacterial isolates were obtained from the 18 D. Tripunctata flies streaked. 20 bacterial isolates were obtained from the 20 D. Putrida flies streaked. A total of nine bacterial species were identified morphologically, seven of which were shared between the two species of wild flies. Similarities between the abundance of Micrococcus luteus and Enterococcus within both species of flies were observed. D. Tripunctata was seen to slightly differ from D. Putrida by carrying the bacterium Bacillus subtilis and Bacillus megaterium. For future exploration, 16S gene sequencing is needed to accurately confirm the identification of the bacterial pathogens discovered. Afterwards, testing for pathogenicity of the bacterial pathogens needs to be conducted as well as testing for Wolbachia’s interaction with the pathogens.

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The Battle of the Sperm: Observations of Sperm Competition in a Wolbachia Infected Species

LaTrice Montgomery, a student from Hampton University, worked with Paul Ginsberg in the lab of Dr. Kelly Dyer to study sperm competition in an infected fly species.

Abstract:  Wolbachia, a maternally inherited bacterium, is broadly distributed among arthropod hosts. It is also capable of reducing viral load in its host, preventing the transmission of human pathogens and thus raising the possibility of its use as a biocontrol agent. In this study, we ask whether Wolbachia has any effects on the mating system of its host, specifically whether it affects patterns of male fertility and/or sperm competition. We conducted mating trials using the fly, Drosophila recens, which is naturally infected with Wolbachia. We utilized an infected, dark eye recessive mutant D. recens and uninfected, red eye wild type D. recens to assess paternity of infected and uninfected males. We found that there are no advantages or disadvantages of Wolbachia infection for male fertility or sperm competition.

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Geographic variation of Wolbachia-induced cytoplasmic incompatibility in the fly Drosophila recens

Sydney Keane, a Biology and Chemistry major from East Texas Baptist University, worked with Dr. Kelly Dyer examining the effects of infection on reproduction in Drosophila.

Abstract:  are bacterial parasites that commonly infect arthropods and nematodes. These parasites have damaging effects on the progeny of those they infect, including cytoplasmic incompatibility (CI). CI occurs when an infected male and an uninfected female mate, resulting in fewer eggs that successfully hatch into larvae than normal. In this study, infected virgin males from Drosophila recens were collected from multiple strains across three locations, and coupled with uninfected virgin females from the same species. After allowing the females to lay eggs for 72 hours, I recorded the numbers of eggs that hatched and that did not hatch. Males were tested for Wolbachia infection using PCR. After analyzing the data, I found that the overall hatch rate in each location was low, the amount of CI in each location did not vary significantly, the amount of CI in the experimental group compared to the control was significantly high, and that the number of total eggs produced varied significantly between the locations. The overall percentage of CI found within all of the locations examined was approximately 72%. These results show that the presence of Wolbachia is similarly effecting various populations of the fly throughout North America and that the level of CI occurring within this species may cause a drastic decrease in the population size over time.


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Is the Transmission Rate of The Wolbachia Parasite Lower in Hybrids Compared To Pure Species?

Jasmine Gipson, from Kennesaw State University, worked with Dr. Kelly Dyer in the UGA Genetics department to study the transmission of Wolbachia, a parasite of insects.

Abstract: Wolbachia is an endosymbiont parasite that lives in the reproductive system 70% of all insects. It is passed down vertically to its offspring from the mother. Wolbachia occurs naturally in D. recens, but not D. subquinaria. In the wild, D. recens and D. subquinaria hybridize and about 2-3% of those hybrid offsprings contain a D. recenâ’s mitochondria, but not the wolbachia. This is a strange situation because the mitochondria is only passed down through the mother’s eggs, just like wolbachia. So how is it possible to have a D. recenâ’s mitochondria, but not the wolbachia as well? This peculiar scenario led to the question, is the transmission rate of wolbachia lower in hybrids compared to pure species? To answer this question, a D. recens female was crossed with a D. subquinaria. The F1 hybrid female was then backcrossed to a D. subquinaria male. The F1 and F2 generations were both test for wolbachia using PCR. The transmission rate for the F1 generation had a transmission rate of 100% and the F2 generation had a transmission rate of 95.7%. This shows that the transmission rate of wolbachia is lowered in hybrid species compared to pure species. Possible explanations for this decrease in transmission rate is because of the genetic variation between D. recens and D. subquinaria or the parasite could have been randomly loss due to oogenesis.


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