An Integrated System for the Automated Recording and Analysis of Insect Behavior in T-maze Arrays.

Host-plant resistance to insects like thrips and aphids is a complex trait that is difficult to phenotype quickly and reliably. Here, we introduce novel hardware and software to facilitate insect choice assays and automate the acquisition and analysis of movement tracks. The hardware consists of an array of individual T-mazes allowing simultaneous release of up to 90 insect individuals from their individual cage below each T-maze with choice of two leaf disks under a video camera. Insect movement tracks are acquired with computer vision software (EthoVision) and analyzed with EthoAnalysis, a novel software package that allows for automated reporting of highly detailed behavior parameters and statistical analysis. To validate the benefits of the system we contrasted two Arabidopsis accessions that were previously analyzed for differential resistance to western flower thrips. Results of two trials with 40 T-mazes are reported and we show how we arrived at optimized settings for the different filters and statistics. The statistics are reported in terms of frequency, duration, distance and speed of behavior events, both as sum totals and event averages, and both for the total trial period and in time bins of 1 h. Also included are higher level analyses with subcategories like short-medium-long events and slow-medium-fast events. The time bins showed how some behavior elements are more descriptive of differences between the genotypes during the first hours, whereas others are constant or become more relevant at the end of an 8 h recording. The three overarching behavior categories, i.e., choice, movement, and halting, were automatically corrected for the percentage of time thrips were detected and 24 out of 38 statistics of behavior parameters differed by a factor 2-6 between the accessions. The analysis resulted in much larger contrasts in behavior traits than reported previously. Compared to leaf damage assays on whole plants or detached leaves that take a week or more to complete, results were obtained in 8 h, with more detail, fewer individuals and higher significance. The potential value of the new integrated system, named EntoLab, for discovery of genetic traits in plants and insects by high throughput screening of large populations is discussed.

Genome-wide association studies on the phyllosphere microbiome: Embracing complexity in host-microbe interactions.

Environmental sequencing shows that plants harbor complex communities of microbes that vary across environments. However, many approaches for mapping plant genetic variation to microbe-related traits were developed in the relatively simple context of binary host-microbe interactions under controlled conditions. Recent advances in sequencing and statistics make genome-wide association studies (GWAS) an increasingly promising approach for identifying the plant genetic variation associated with microbes in a community context. This review discusses early efforts on GWAS of the plant phyllosphere microbiome and the outlook for future studies based on human microbiome GWAS. A workflow for GWAS of the phyllosphere microbiome is then presented, with particular attention to how perspectives on the mechanisms, evolution and environmental dependence of plant-microbe interactions will influence the choice of traits to be mapped.

Natural variation in life history strategy of Arabidopsis thaliana determines stress responses to drought and insects of different feeding guilds.

Plants are sessile organisms and, consequently, are exposed to a plethora of stresses in their local habitat. As a result, different populations of a species are subject to different selection pressures leading to adaptation to local conditions and intraspecific divergence. The annual brassicaceous plant Arabidopsis thaliana is an attractive model for ecologists and evolutionary biologists due to the availability of a large collection of resequenced natural accessions. Accessions of A. thaliana display one of two different life cycle strategies: summer and winter annuals. We exposed a collection of 308 European Arabidopsis accessions, that have been genotyped for 250K SNPs, to a range of stresses: one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips and Myzus persicae aphids) and two combined stresses (drought plus P. rapae and Botrytis cinerea fungus plus P. rapae). We identified heritable genetic variation for responses to the different stresses, estimated by narrow-sense heritability. We found that accessions displaying different life cycle strategies differ in their response to stresses. Winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars. Summer annuals are also more resistant to the combined stresses of drought plus P. rapae and infection by the fungus Botryris cinerea plus herbivory by P. rapae. Adaptation to drought displayed a longitudinal gradient. Finally, trade-offs were recorded between the response to drought and responses to herbivory by caterpillars of the specialist herbivore P. rapae.

Genetic architecture of plant stress resistance: multi-trait genome-wide association mapping.

Plants are exposed to combinations of various biotic and abiotic stresses, but stress responses are usually investigated for single stresses only. Here, we investigated the genetic architecture underlying plant responses to 11 single stresses and several of their combinations by phenotyping 350 Arabidopsis thaliana accessions. A set of 214 000 single nucleotide polymorphisms (SNPs) was screened for marker-trait associations in genome-wide association (GWA) analyses using tailored multi-trait mixed models. Stress responses that share phytohormonal signaling pathways also share genetic architecture underlying these responses. After removing the effects of general robustness, for the 30 most significant SNPs, average quantitative trait locus (QTL) effect sizes were larger for dual stresses than for single stresses. Plants appear to deploy broad-spectrum defensive mechanisms influencing multiple traits in response to combined stresses. Association analyses identified QTLs with contrasting and with similar responses to biotic vs abiotic stresses, and below-ground vs above-ground stresses. Our approach allowed for an unprecedented comprehensive genetic analysis of how plants deal with a wide spectrum of stress conditions.

Automated video tracking of thrips behavior to assess host-plant resistance in multiple parallel two-choice setups.

BACKGROUND: Piercing-sucking insects cause severe damage in crops. Breeding for host-plant resistance can significantly reduce the yield losses caused by these insects, but host-plant resistance is a complex trait that is difficult to phenotype quickly and reliably. Current phenotyping methods mainly focus on labor-intensive and time-consuming end-point measurements of plant fitness. Characterizing insect behavior as a proxy for host-plant resistance could be a promising time-saving alternative to end-point measurements. RESULTS: We present a phenotyping platform that allows screening for host-plant resistance against Western flower thrips (WFT, Frankliniella occidentalis (Pergande)) in a parallel two-choice setup using automated video tracking of thrips behavior. The platform was used to establish host-plant preference of WFT with a large plant population of 345 wild Arabidopsis accessions and the method was optimized with two extreme accessions from this population that differed in resistance towards WFT. To this end, the behavior of 88 WFT individuals was simultaneously tracked in 88 parallel two-choice arenas during 8 h. Host-plant preference of WFT was established both by the time thrips spent on either accession and various behavioral parameters related to movement (searching) and non-movement (feeding) events. CONCLUSION: In comparison to 6-day end-point choice assays with whole plants or detached leaves, the automated video-tracking choice assay developed here delivered similar results, but with higher time- and resource efficiency. This method can therefore be a reliable and effective high throughput phenotyping tool to assess host-plant resistance to thrips in large plant populations.

Association mapping of plant resistance to insects.

Association mapping is rapidly becoming an important method to explore the genetic architecture of complex traits in plants and offers unique opportunities for studying resistance to insect herbivores. Recent studies indicate that there is a trade-off between resistance against generalist and specialist insects. Most studies, however, use a targeted approach that will easily miss important components of insect resistance. Genome-wide association mapping provides a comprehensive approach to explore the whole array of plant defense mechanisms in the context of the generalist-specialist paradigm. As association mapping involves the screening of large numbers of plant lines, specific and accurate high-throughput phenotyping (HTP) methods are needed. Here, we discuss the prospects of association mapping for insect resistance and HTP requirements.