Progress 04/01/24 to 03/31/25
Outputs
Target Audience:Research was presented by M. Doremus to an audience ofresearchers at the annual conference of the Entomological Society of America (Phoenix, AZ)andat the University of Illinois Urbana-Champaign. The first manuscript detailing research funded by this award has also been submitted to an academic journal and is awaiting review. This study explored feminization of spiders infected with the symbiont Wolbachia. A preprint version of the manuscript is available on BioRxiv (https://doi.org/10.1101/2025.02.13.638118). A news article targeting a general audience covering symbiont-induced feminization of spiders is expected to be released in conjuction with this manuscript publication. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?M. Doremus has accepted an assistant professor position in the Department of Entomology at the University of Illinois Champaign-Urbana. This achievement was due in no small part to the USDA-NIFA postdoctoral award, which funded both the majority of his postdoctoral research and his attendence at multiple conferences. This project has also funded opportunities for threeundergraduate researchersinterested in agricultural research, withtwo conducting independent projects. They will be co-authors on several upcoming manuscript featuring their results,including the tempurature-feminization manuscript. Undergraduate researchers receive training in DNA extraction, PCR, gel electrophoresis, and DNA sequencing. As this award has been extended through 2026, it will continue to fund undergraduate research opportunities at the University of Illinois How have the results been disseminated to communities of interest?Results of the study confirming Wolbachia as the causitive agent of feminization in M. fradeorum, as well as the synergistic effect of symbiont coinfection on feminization efficacy, have been submitted to the journal Environmental Microbiology for review and publication. A preprint has been uploaded to BioRxiv for public access prior to the publishing of the reviewed manuscript. This work has also been presented at the 2024 conference of the Entomological Society of America. Results of experiments showing the sensitivity of Wolbachia feminization to temperature stress and the multi-generational consequences of temperature exposure have been presented atthe 2024 conference of the Entomological Society of America. A component of our symbiont survey study has been presented atthe 2024 conference of the Entomological Society of America. Presented work focused on the geographic variation in symbiont infection in M. fradeorum. What do you plan to do during the next reporting period to accomplish the goals?This project has been extended with a no-cost extension through March 31st, 2026. In this final year of the project, M. Doremus will conduct a final year of spider collections to further bolster the survey study. Collections will target midwestern/central locations (MO, AK, NE, MN, IL) and potentially sites closer to the east coast to better explore a possible east-west infection gradient. Collections may extend to monthly spider surveys over the 2025 growing season in IL and KY to track symbiont seasonal fluctuations. Additionally, we will focus on the final research goal: analyzing genomes of M. fradeorum symbionts. Specifically we will characterize the general features of these bacterial genomes, their biosynthetic potential, putative factors involved in host interaction and manipulation, and temperature stress responses. Preliminary analyses have already revealed potential candidate factors for Rickettsiella-induced CI, although these require more thorough analyses for full consideration as candidate factors. Additional temperature studies better characterizing the timepoints of Wolbachia feminization and/or Rickettsiella cytoplasmic incompatibility will, together will genome analyses, form the basis of future work exploring gene expression and bacterial localization during timepoints relevent to host manipulation.
Impacts
What was accomplished under these goals?
Initial experiments used lines of the dwarf spider, Mermessus fradeorum, that harbored different combinations of five heritable symbionts to determine which symbiont is responsible for feminization (i.e. genetic males develop as phenotypic females). This work found that a strain of Wolbachia is required for feminization but feminization rates are highest when this Wolbachia co-occurs in hosts with all five symbionts. In nature, this Wolbachia strain is most commonly found in quintuple infections; other infection types involving the feminizing Wolbachia are rare. This work suggests that the synergistic effect of coinfection improves Wolbachia-induced feminization of its spider host and may reinforce transmission of the full five member symbiont community. Additionally we found that the improved feminization rates in quintuply-infected spidersdo not correlate with an increase in the amount of Wolbachia in the spider, but the feminizing Wolbachia does consistently dominate the bacterial community when present. This work is available as a preprinthttps://www.biorxiv.org/content/10.1101/2025.02.13.638118v1 and we expect the peer-reviewed version will be published within the year. A seperate set of experiments then explored how temperature stress affectsWolbachia-induced feminization ofM. fradeorum. We treated spiderlings either withwarm temperatures (28C) or cool (20C) temperatures starting upon emergence from their egg mass until they mated. This initial warm temperature exposure did not affect feminization rate, which remained near perfect. However, the F1 offspring of heat-treated spiders, which themselves experienced only the benign 20C treatment,lost their feminization phenotype and exhibited a near 1:1 sex ratio.This indicates a delayed response to temperature stress and demonstratesthat maternalcondition can influence feminization induction in offspring. This phenotype failure was associated with reduced Wolbachia transmission rates;however,transmission rates still remained high (~80% at the warm temperature vs. 100% at the cool temperature) indicating that phenotype failure was not the result of transmission failure alone. The feminization phenotype recovered in the F2 offspring that retained their Wolbachia infection, shifting the sex ratio to be near-perfectly female. These shifts in phenotype are associated with shifts in symbiont titer. Wolbachia titer curiously increases during initial heat exposure, then its titer plummets in the F1 generation, followed by a stabilization to pre-treatment levels in the F2 generation. A replicate experiment using a temperature cycle of 27Cday/24Cnight as the warm treatment produced nearly identical results, indicating that a nighttime recovery period does not seem to improve symbiont temperature sensitivity. We are currently prepping this work for publication and expect it to be published this year. As a final temperature experiment, we conducted heat treatments during various specific M. fradeorum lifestages to characterize the timing of Wolbachia feminization. Lifestages tested included the mother, early embryogenesis, early instar (still in egg mass), and lateinstar (after emergence from egg mass). Each exposure period lasted for one week. Temperature only disrupted feminization when exposure occurred during themother stageand early embryogenesis, indicating that Wolbachia likely feminizes its host at a timepoint in early embryogenesis. This work has implications for the mechanism of Wolbachia feminization and will inform future work characterizing that mechanism. We conducted a two year multi-statesurvey ofsymbionts in spiders from alfalfa fields. From over 2000 specimens, we found five spider species, includingM. fradeorum, in enough abundance to examine their symbiont communities. Heritable symbionts uniformly dominated spider microbiomes, with species harboring between 3 and 5 symbiont species. Most species, including M. fradeorum, displayed striking geographic variation in symbiont infection on both a north-south and east-west gradient. Based on our prior work with M. fradeorum, we suspect that temperature is a major factor that shapes arthropod symbiont communities. We have successfully established lab cultures of two other spider species collected in this survey, Glenognatha foxi and Grammonota inornata, which will be the focus of future research efforts. PacBio long-read sequencing data and draft genomes have been generated for all five M. fradeorum heritable symbionts, as well as for a novel strain of the symbiont Rickettsiella that is more prevalent in southern spider populations. Preliminary tests suggest both Rickettsiella strains cause CI, however the southern strain causes a weak incompatible phenotype, killing only ~20% of uninfected offspring compared to ~80% of offspring by the northern strain's CI. Both CI strains are mutually compatible, suggesting that the southern strain retains full rescue potential to restore infected offspring survival. PacBio reads have also been generated for three symbionts of the spider Grammonota inornata for future study (Wolbachia and two strains of Cardinium).
Publications
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Progress 04/01/23 to 03/31/24
Outputs
Target Audience:Presented research talks to a national and international audience of symbiosis and entomology researchers at three conferences. Conferences included the 4th ISF Research Workshop on Symbiosis hosted by the Hebrew University of Jerusalem, the 2023 Wolbachia meeting in Chania, Crete, and the Annual Entomological Society of Entomology conference in National Harbor, MD. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? PD Doremus has received experience mentoring a research team of undergraduates, as well as networking opportunites via his attendence at three research conferences. PD Doremus has also begun interviewing for faculty positions. Two undergraduate researchers have received training in molecular research techniques. One of these students performed her own study (a portion of the experiments testing how temperature affects feminization) and presented her work at the 2023 Entomology Society of America (ESA) conference. Her talk was awarded 2nd place in her section of undergraduate research talks. How have the results been disseminated to communities of interest?Research on the effects of temperature on M. fradeorum feminization, as well as the role of Wolbachia in this manipulation have been presented as research talks at ESA in National Harbor, Maryland, as well as at the 2023 Wolbachia conference in Chania, Crete. The Wolbachia conference is a well-attended meeting focusing on heritable symbioses that draws research from across the world. What do you plan to do during the next reporting period to accomplish the goals? Complete the first run of 16S rRNA sequencing and analysis ofmicrobiomes of linyphiids collectedin 2023. Based on the results of this initial run, we will target several sites to collect additional samples of certain species. Samples collected in 2024 will undergo processing, species identification, and microbiome sequencing. This second collection period will also yield additional spider species to start lab cultures. Perform experiments estimating effect of temperature and symbiont coinfection on symbiont titer Write and make publically available a manuscript of the effects of temperature on symbiont-induced feminization in M. fradeorum Write and make publically available a manuscript detailing the role of Wolbachia and coinfection in causing feminization. Extract high molecular weight symbiont DNA for PacBio long-read sequencing and begin construction and analysis of the M. fradeorum symbiont genomes Present research at the 2024 ESA conference in Phoenix, AZ
Impacts
What was accomplished under these goals?
Using several lab-based experiments exposing M. fradeorum to different temperature regimens, we found that warm temperatures representative of current summertime high temperatures in Kentucky reduce the effectiveness of two symbiont-induced forms of reproductive manipulation. Warm temperatures may influence the symbiont infection dynamics and host biology of the M. fradeorum system. One of these manipulations is the symbiont-induced feminization of male hosts, which resultsin a phenotypic female capable of vertically transmitting the symbiont to their offspring. Spider development at warm temperatures caused a delayed, trans-generational failure of feminization in their offspring resulting in a restoration of male production. This feminization failure was associated with a shift in the spider symbiont community caused by the vertical transmission failure of several symbionts from mother to offspring. This indicatesthat one or more of the affected bacteria are responsible for feminization. The feminization phenotype is restored following additional spider generations raised at cooler temperatures, suggesting that this phenotype may have variable efficacy throughout the growing season. This variable efficacy, combined with inefficient transmission rates, may be responsible for the lower infection rates for the feminizing symbionts. These temperature studies also revealed that the five symbionts infectingM. fradeorum vary in their thermal tolerance. Rickettsiella appears to be the most thermally robust symbiont, with stable transmission across multiple generations at warm temperatures. The three Wolbachia strains show intermediate tolerance and are variably lost following multiple generations at warm temperatures. Tisiphia is extremely temperature sensitive, exhibiting total transmission failure following initial exposure to warm temperatures. Using crossing assays with a number of differentially infected spider lines, we have identified one strain of Wolbachia as the causitive agent of feminization, although coinfection with multiple symbionts increases feminization efficacy. This synergystic effect may promote the persistance ofquintuple infections in host populations. To begin characterizing how climate shapes heritable symbiont communities of linyphiids, we have collected ~1000 spiders from 17 sites across 13 states representing a latitudinal gradient. Spiders are currently undergoing intial processing to identify species using a combination of morphology and CO1 sequencing. We are currently preparing a high-throughput sequencing run of the 16S rRNA gene to characterize the microbiome of a subset of these samples.
Publications
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