Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
(N/A)
Non Technical Summary
Arthropods play major roles in plant health and production by acting as both biological control agents andcrop pests. Arthropods also host heritable bacteria that are transmitted directly from mother to offspring. These bacteria are known as heritable endosymbionts and mediate essential aspects of arthropod biology, including providing their hsot with essential nutrients, protecting their hosts from imprant natural enemiesspecies interactions, and sabotaging host reproduction to favor symbiont maternal transmission. Endosymbionts cannot survive outside their host and this host-restricted lifecycle often decays endsymbiont gene functionality. Endosymbionts can have a limited ability to respond to environmental variation, with high temperatures destabilizing microbial symbioses by reducing transmission efficacy and phenotype penetrance. Variation in environmental variables like temperaturehas unclear consequences for heritable endosymbionts and their arthropod hosts. Linyphiid spiders are an important yet understudied guild of generalist biological control agents and emerging models for studying heritable symbioses. This proposal will use field surveys, laboratory-based experiments, and genome analyses to address three questions: 1) Do symbiont frequencies vary geographically across US spider populations? 2) Does temperature stress destabilize heritable symbioses? 3) Do symbiont genomes encode responses to temperature stress? Results will reveal how environmental variables influencelinyphiid symbiont communitiesand will establish an independent research system for studying these symbioses.This project will complete the work initiated in an AFRI EWD postdoctoral fellowship award addressing the program goal of cultivating future independent researchers that can solve emerging agricultural challenges by providing training in technical, pedagogical, and management skills. This project also meets the AFRI EWD program goals of advancing fundamental agricultural research by funding research on beneficial arthropod species associated with agricultural systems. Ultimately, this project will help the PD establish an independent research program studying symbioses of agriculturally-relevant arthropods at a tenure-track position in a research institution.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
This project has three goals:1) Complete a multi-state survey demonstrating how agriculturally relevant spider symbiont communities vary across an environmental gradient.2) Conduct laboratory assays comparing the thermal senesitivty of two geographically isolated strains of the common symbiont, Rickettsiella, in the spider Mermessus fradeorum.3) Analyze the genomes of the six member M. fradeorumsymbiont community to identify genes involved in symbiont responses to thermal stress.
Project Methods
Objective 1:Does temperature shape symbiont frequencies in US spider populations?We will collect spiders from alfalfa fields in northern (New York, South Dakota), eastern (Virginia), and western sites (Nebraska, Colorado, Oklahoma).I will locate spider webbing within quadrats and will collect ~ 100 spiders per site (n = 400 total) by hand or mouth aspirator. Spiders will be preserved in 95% ethanol in 1.5 mL tubes and stored at -20°C until DNA extraction. Spiders will be identified using morphology and cytochrome oxidase I (COI) barcoding with the Barcode of Life Database (boldsystems.org). I will identify symbionts of the five most common spider species using the variable V4 region of the bacterial 16S rRNA gene; microbiomes from ~700 specimens will be multiplexed into two Illumina MiSeq 500 sequencing runs using specimen-specific indexed primers. Raw sequencing reads will be filtered and quality checked using Basespace (Illumina), then analyzed using the Qiime2 microbiome analysis pipeline.Objective 2:Do elevated temperatures affect the stability of linyphiid symbioses and penetrance of symbiont-conferred phenotypes?I will test whether high temperatures affect the CI phenotype of a southern Rickettsiella strain, R2, by treating developing R2 spiderlings with either a control temperature of 20C or a warm treatment of 28C on a long day cycle (16:8h day:night). For all CI modification experiments, males will mate with either R2+ (Rescue compatible cross) or uninfected (CI) females one week after adult maturation. The rescue cross serves as a control for possible deleterious effects on male reproductive viability caused by temperature exposure. CI strength will be measured as the proportion of unhatched eggs. Males will be stored post-mating at -20C in 95% ethanol for DNA extraction and symbiont titer estimation using dPCR. Symbiont titer will be analyzed using ANOVA and offspring mortality will be analyzed using logistic regression in R.Objective 3:Do symbiont genomes encode responses to thermal stress?Draft genomes have been assembled for all five M. fradeorum symbionts using PacBio sequencing. These genomes are still undergoing assembly, with most having adequate read depth of >30X. I plan on performing PacBio sequencing on the most recently identified, southern Rickettsiella strain R2. Genomes, plasmids, and phages will be functionally annotated using the NCBI Prokaryotic Genome Annotation Pipeline, PLSDB, and PHASTER, respectively. I will next identify homologs of temperature stress proteins and chaperonins in these genomes using TBLASTN and HMMer v3.3.2 using search models based on bacterial temperature shock genes. I will compare the copy number of intact and psuedogenized temperature shock genes present in symbiont genomes with their temperature sensitivity as characterized in Q2 and in the previous version of this proposal.