I must thank the entire BRET office for all the time and effort they put into enriching my time here, including Dr. I would also like to thank the members of my dissertation committee for all their support and helpful advice: Dr. Thanks to Sarah Bordenstein for being the unwavering pillar that holds the lab together through success and hardship, and to my fellow (former and current) labmates and friends: Drs.
Thanks also to my wonderful undergraduate mentee Ananya Sharma for all her hard work on the projects presented in this thesis; she will make a great doctor. To my parents and in-laws, I thank you for being the best listeners and supporters, especially when I needed both. Lastly, to my wife Laura, absolutely none of this would be possible without you by my side.
INTRODUCTION*
Note the morphological differences in wing size between species and sexes (drawing based on Loehlin et al. 2010). One of the most striking aspects of Wolbachia is undoubtedly its ability to manipulate host reproduction in various ways (Werren et al. 2008). These results indicate that the host genotype can determine the fate of paternal chromosomes in fertilized eggs independently of Wolbachia (Bordenstein et al. 2003).
For example, Wolbachia plays a crucial role in inducing premating isolation between hemispecies of the Drosophila paulistorum species complex (Miller et al. 2010). For example, species-specific cuticular hydrocarbons aid in mate discrimination in Nasonia (Buellesbach et al. 2013), but. Behavioral issues arising in hybrids (Clark et al. 2010) may have a microbial basis.
THE MATERNAL EFFECT GENE WDS CONTROLS WOLBACHIA TITER IN NASONIA ֒
Introgression of a specific Wolbachia strain (wVitA) from a Nasonia species (N. vitripennis) into a naïve, closely related species (N. giraulti) results in a major disturbance of the symbiosis in which the relative density of Wolbachia increases by two order of magnitude, and there is an associated reduction in fecundity in N. For each cross, the genotype (male × female) is followed by the cytotype in parentheses (V, N. vitripennis; G, N. giraulti; .. number, estimated percentage of the genotype ). Inset) Wolbachia density (mean ± SEM) of offspring based on maternal genotype at each QTL peak (V, N. vitripennis; G, N. giraulti).
Furthermore, we compared embryonic wVitA densities from mothers injected with dsRNA against the Nasonia gene LOC100679394 (Mucin-5AC), a gene significantly upregulated in N. vitripennis but immediately outside the chromosome 3 candidate region. vitripennis allele of Wds suppresses densities of vertically transmitted. To further validate the effect of Wdsv on Wolbachia densities, females from recombinant line R6-3 (homozygous N. vitripennis only for the 32-gene candidate region) were injected with. In contrast, the pI difference for the Mucin-5AC control is minimal (ΔpI = 0.04). vitripennis allele of Wds is under positive selection.
PHYLOSYMBIOSIS: RELATIONSHIPS AND FUNCTIONAL EFFECTS OF MICROBIAL COMMUNITIES ACROSS HOST EVOLUTIONARY HISTORY †
Indeed, diet is a stronger determinant of whole microbial community structure than genotype in laboratory-reared mice (Carmody et al. 2015). Sequence quality control and OTU analyzes were performed using QIIME version 1.8.0 (Lo et al. 2002). A phylogenetic tree of the representative sequences was built in QIIME (Lo et al. 2002) with the FastTree method and midpoint rooting (Russell et al. 2008).
Taxonomy was then assigned to the OTU representatives with the UCLUST method against the GreenGenes 13_5 database (Edgar et al. 2011). OTU tables were constructed in QIIME (Lo et al. 2002) and sorted by sample IDs alphabetically. The community profile (Figure III-2B) for the meta-analysis was generated using a custom python script and BIOM tools (Price et al. 2010).
Quantification of congruence between host phylogeny and microbiota dendrogram relationships (Figure III-3) was performed with a custom python script and the TreeCmp program (Guindon et al. 2010). These results are similar to previous observations that microbial community relationships parallel those in host phylogeny (Ochman et al. 201). In Peromyscus , we followed a previously established protocol ( Kohl et al. 2016 ) to transplant microbial communities from six rodent donor species into a single recipient species, P .
Hosts produce glycans and mucins on the intestinal mucosa that can serve as biomolecular regulators of microbial communities (Hooper and Gordon 2001; McLoughlin et al. 2016). For example, hosts have been compared to ecological islands where environmental selection of the microbiota through niche availability can occur (Costello et al. 2012). Later, the mouse gut microbiota dominated and outcompeted the human gut microbiota (Seedorf et al. 2014).
For example, sponges exhibit vertical transfer of a diverse set of microbes in embryos (Sharp et al. 2007).
AN OPTIMIZED APPROACH TO GERM-FREE REARING IN THE JEWEL WASP NASONIA ֡
Germ-free rearing of Nasonia involves two main components: (i) sterilization of Nasonia embryos and (ii) provision of larvae with sterilized food in an in vitro system. This study removes these three main components of the original NRM and optimizes the procedure by eliminating extraneous steps and using faster approaches. All plates were stored in a sterile Tupperware box at 25 ± 2 °C under constant light conditions for the duration of the experiment.
A baseline for the number of larvae present in a well was determined by counting the number of larvae present in transwell photographs three days after embryo deposition on the transwell membranes (day 3). The proportion of adults produced by a transwell was determined as follows: (the number of larvae on day 3 − the number of dead larvae and pupae remaining on day 20) ÷ the number of larvae on day 3. To investigate whether the use of NRMv2 reduces the larvae improves adult survival, both the number of transwells producing adults and the average number of adults produced per transwell were compared between NRMv1 and NRMv2.
The previously established Nasonia in vitro germ-free growth protocol (Brucker and Bordenstein 2012b), which included sterilization of embryos and feeding of NRMv1 larvae, was crucial for conducting experiments on Nasonia-microbiota interactions (Brucker and Bordenstein 2013). However, this initial version of the germ-free growth system contained highly artificial elements such as washing with bleach, FBS, and antibiotics (Figure IV-1; NRMv2). For example, antibiotics are a confounding variable with unknown consequences for the biology of Nasonia, and they may inhibit the inoculation capabilities of the system by causing rapid changes in the composition of bacterial communities.
This new system allows the introduction of both autochthonous and allochthonous microbial communities, enabling investigations into the functional relevance of host specificity. This in vitro rearing system allows for the exploration of the interaction of microbes with host signals to test what role these complex interactions may have in adult behavior, insect communication and reproductive isolation. Parasitoid wasps are also difficult to study developmentally because the fly host's puparium hinders visualization of the Nasonia larvae and pupae, which occur over time multiple measures of a single individual.
The NRMv2 method allows investigation of the life cycle of Nasonia under gnotobiotic conditions, opening the door to multidisciplinary studies of host-microbiota interactions and adding to the utility of Nasonia as a model system.
A FUNCTIONAL ASSOCIATION OF PHYLOSYMBIOSIS WITHIN THE NASONIA WASP CLADE
In this study, the effects of allochthonous microbiota transplantation between three of the four Nasonia spp. This procedure was performed independently for each of the donor microbial communities one day before transplantation. On days 2-8 of transplantation, the transwell insert was drained on a sterile Kimwipe before adding 20 µL of microbiota suspension.
If any bacterial or fungal contamination occurred in the transwell during Nasonia development, the transwell and its data were removed from the experiment. Normalized larval growth per sample well was calculated as the mean length of Nasonia larvae per well divided by the mean length of larvae of the indigenous microbiota treatment group for each treatment day. Normalized pup reproduction per transwell sample was calculated as the percentage of Nasonia pups per transwell divided by the mean percentage of pups of the indigenous microbiota treatment group.
Normalized survival of adults per transwell sample was calculated as the percentage survival of Nasonia from 3 days to 20 days after hatching, divided by the mean percentage survival of the indigenous microbiota-treated group. To determine the effect of microbiota transplantation on the growth of Nasonia in the larval stage, I took pictures of the treated larvae during each day of pre-pupation development and measured their length. During this stage of development, larval length increased by 1.14 mm, especially between days 2 and 3 of development, which is equivalent to 75% of total larval growth before pupation.
To determine whether these larval length differences persist beyond peak larval growth, the effects of the microbiota transplants were observed on the 5th day of larval development. Considering a typical conventional Nasonia female/male sex ratio of ~0.9, there was no effect of the different microbiota transplants on male fertility (Figure V-4A). Here, we report that the adverse impact of the allochthonous microbiota transplants begins early in larval development.
Due to the restrictive nature of the heat-inactivated transplantation model, it remains possible that other mechanisms play a role in adaptation of the wasp host to its microbiota.
CONCLUSIONS AND FUTURE DIRECTIONS
As the Nasonia larvae undergo pupation and adult eclosion, we can also observe the changes in the presence and localization of the GFP-expressing bacteria during metamorphosis. Inter- and intraspecific comparison of the bacterial assemblages in the large intestine of humivorous dung beetle larvae (Pachnoda spp.). Phylogeny of the Nasonia species complex (Hymenoptera: Pteromalidae) inferred from an internal transcribed spacer (ITS2) and 28S rDNA sequences.
Bacterial infections associated with the syn-killer trait in the parasitoid wasp Nasonia (=Mormoniella) vitripennis (Hymenoptera: . Pteromalidae). Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the small intestinal mucosa. Interspecies interactions determine the influence of gut microbiota on nutrient allocation in Drosophila melanogaster.
Wolbachia induces reactive oxygen species (ROS)-dependent activation of the Toll pathway to control dengue virus in the mosquito Aedes aegypti. Single and double infections with Wolbachia in the parasitic wasp Nasonia vitripennis: effects on compatibility. Community heritability (H2C) emphasizes that the host is part of the ecosystem and measures the extent to which the phenotype variation of the entire community is due to genetic variation in the host.
H2C emphasizes that the host is part of an ecosystem and measures the extent to which variation in "whole-community" phenotype is due to genetic variation in the community's foundational (ie, host) species.
