Performing Department
(N/A)
Non Technical Summary
Arthropods play major roles in agricultural production by acting as biological control agents or crop pests. Most arthropods also host maternally inherited, intracellular bacterial symbionts that can provide their host with required nutrients, protect hosts from important natural enemies, or manipulate the outcomes of host reproduction. High temperatures can destabilize these symbioses by reducing transmission efficacy and preventing the symbiont from modifying their host. The predicted increase in global temperatures has unclear consequences for heritable symbionts and their arthropod hosts. Spiders are an important but understudied group of generalist predators that often harbor complex heritable symbiont communities and are emerging models for studying heritable symbionts. This project will use field surveys, laboratory-based experiments exposing spiders to current and predicted high temperatures, and genomic analyses to address three questions: 1) Does climate shape symbiont frequency in US spider populations? 2) Does temperature stress destabilize heritable symbioses? 3) Do symbiont genomes encode responses to temperature stress? Results will reveal how climate influences spider symbionts and will establish a new spider model system for studying heritable symbioses. This research will further characterize the role of symbionts in arthropod biology and how climate change might affect these widespread symbioses. Data from this project will be made publicly available for potential use in other research, including developing models predicting the impact of climate change on arthropod populations and their heritable symbionts. This project will also generate a highly trained and competitive workforce that are well prepared to address emerging agricultural challenges, including agricultural climate adaptation, by providing postdoctoral and undergraduate research, mentorship, teaching, and project management training. It will also increase scientific literacy in the general public by developing an educational module on DNA sequencing for undergraduate and high school students. This module will include hands on experience extracting DNA and analyzing DNA sequencing, common technique employed by biology labs.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The major goals for this project are to:Research how climate and temperature stress affect heritable symbioses between arthropods (linyphiid spiders) and their intracellular, bacterial symbionts. It will use the emerging spider model Mermessus fradeorum and establish lab cultures ofa second spider system to serve as a new model for spider-microbesymbiosis research.Determine how temperature stress affects the stability of linyphiid heritable symbiont communities, including how high temperatures affect symbiont-induced manipulation of arthropod reproduction.Determine whether climate shapes heritable symbiontcommunities in natural populations of linyphiid spiders in US alfalfa fieldsSequence and analyze the genomes of the M. fradeorum symbiont community and the community of a second novel spider model. Genomes will be used to identify canidate symbiont effectors involved in manipulating host reproduction and the symbiont thermal stress response.Establish an interactive educational module for undergraduate and high school students on DNA sequencing and COI barcoding.Provide training in project management, mentorship, bioinformatics techniques, and networking opportunities for PD Doremus to facilitate his transition to a research-focused faculty position.Provide training in molecular research techniques for two undergraduate researchers
Project Methods
We will survey natural linyphiid populations from alfalfa fields in 8 states across two climatic zones representing a "cool" and "warm" climate. Spider collection metadata will be recorded and spiders will undergo DNA extraction and COI barcoding to identify spider species. We will characterize the microbiome of the 4-5 most prevalent spider species using 16S rRNA sequencing to identify bacterial symbionts, focusing primarily on heritable symbionts. I will analyze symbiont occurrance for each spider species using generalized linear models incorporating vegetation status, locality, and climatic variables. We will establish a lab culture for one of these spider species for use in downstream experiments testing the effect of temperature stress on symbiont transmission.We will next use laboratory based experiments with the linyphiid M. fradeorum to measure the effect of temperature stress on symbiont-induced cytoplasmic incompatibility (CI, a form of lethal reproductive sabotage of uninfected offspring) and feminization. Spiders will develop at either a cool control temperature at which the symbiosis is stable or an elevated temperature representing current high temperatures experienced by M. fradeorum in Kentucky. Following temperature exposure, spiders will be used in experimental crosses to measure the symbiont-induced phenotypes(sex ratio for feminization experiments, hatch rate for CI). All spiders will be stored in 95% EtOH for DNA extraction and symbiont infection confirmation using PCR and symbiont density estimation following temperature exposure using quanititative PCR. Experiments will be replicated using high temperatures reflecting predicted high temperatures experienced by spiders by the end of the century. Symbiont transmission rates (from mother to offspring) will be recorded following temperature exposure for M. fradeorum and thesecond linyphiid spider. Sex ratio and hatch rates will be analyzed using logistic regression, while symbiont density will be analyzed using ANOVAs, and transmission rate will be analyzed using Fisher's exact tests.Genomes for M. fradeorum symbionts and the symbionts of the newly established spider culture will be sequenced using a combination of Illumina short reads and PacBio long reads to resolved repetitive regions in the genome. Genomes will be assembled, circularized, quality checked, and rotated to the origin of replication using UniCycler. Genomes will be annotated using the NCBI Prokaryote Genome Annotation Pipeline, PLSDB (for plasmid annotation), and PHASTER (phage annotation). I will identify homologs of classical temperature stress proteins and chaperonins using TBLASTN and HMMer and will compare the number of intact or psuedogenized temperature shock genes with the symbiont's temperature sensitivity as determined by the previous lab experiments. I will next test if putativetemperature stress response genes are expressed following temperature shock. Spiders exposed to either high temperatures or cool control temperatures will be flash frozen in liquid nitrogen prior to having their RNA extracted. Using reverse transcriptase qPCR, I will estimate the expression of putative symbiont temperature shock genes relative to a housekeeping gene. Gene expression will be analyzed using ANOVAs. I will use similar methods to also identify putative effectors involved in symbiont-induced CI and feminization, using TBLASTN and HMMer to search for effectors homologous to other known manipulative effectors, as well as genes encoding predicted eukaryotic interaction domains like ankyrins.Beyond research, this project will fund the development of two educational courses/modules. The first is a graduate level course on insect-microbe symbioses co-taught by PD Doremus and Mentor White. PD Doremus will be responsible for course design and leading discussions. The second unit is an educational module on DNA sequencing and COI barcoding using local spiders. This module will initially be presented as a portion of Mentor White's General Entomology course. Students will collect and stored a local specimen of spider in 95% EtOH. During a class and lab period, PD Doremus will lead a discussion on the underlying principles of DNA sequencing, COI Baracoding, and their usage in biology. During this period, students will extract spider DNA. In a subsequent lab period, students will amplify the COI sequence from their spider DNA using PCR. PD Doremus will perform PCR clean ups and send samples to an offsite sequencing facility. Students will then analyze sequence chromatograms, then use popular platforms like NCBI or the Barcode of Life Database to identify their spider species. This unit can potentially be adapted for high school students. The ultimate goal for this module to provide students hands on experience with common biological research techniques and to build a database of local spider species for downstream use in research.We will primarily use publication of results in peer reviewed journals as the final evaluation for research aims. We will also preesnt research results at conferences for feedback from peers prior to publication. During the project, yearly updates will be provided to anadvisory committee composed of the co-mentors White and Dunning Hotopp, and well as our collaborator Yuval Gottlieb (Israel) on project progress. This committee will offer feedback on the progress towards major goals. PD Doremus will additionally meet with Mentor White on a weekly basis and mentor Dunning Hotopp on a monthly basis for project udpates and feedback.For mentorship goals, Mentor White will evaluate mentorship ofundergraduate students supervised by PD Doremus, meeting with them once in the midpoint of them first year and hosting a final evaluation at the end of their second year. Their feedback will be provided to PD Doremus so he can adjust mentorship accordly. Undergraduate reserach will also be evaluated via presentations at the Undergraduate Research Showcase at the University of Kentucky (UKY) each spring.For teaching goals, PD Doremus will incorporate pedagogical guidance and feedback from the Center for Enhancement of Learning and Teaching (CELT) at UKY during module and course development. Mentor White will also provide feedback on course design and teaching. Students will be provided with an opportunity to evaluate the course/module and PD Doremus' teaching at the end of the educational course. This feedback will be incorporated in subsequent iterations of the educational course.