Source: OHIO STATE UNIVERSITY submitted to
MOLECULAR CHARACTERIZATION OF INDIRECT SELECTION FOR INCREASED SUSCEPTIBILITY TO INSECTICIDES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0219507
Grant No.
2009-65104-05740
Project No.
OHO01063-SS
Proposal No.
2009-02874
Multistate No.
(N/A)
Program Code
91111
Project Start Date
Sep 1, 2009
Project End Date
Aug 31, 2013
Grant Year
2009
Project Director
Mittapalli, O.
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Entomology
Non Technical Summary
Understanding the genetic correlation between insect behavior and their physiology in pest adaptation is an important and understudied aspect of pest management and insect plant interactions. We have been exploring the behavioral responses of insect pests to insecticides and their genetic correlation with physiological tolerance to these toxins. The practical significance of a genetic correlation between these traits is that selection on one trait can cause a change in both simultaneously. Previous studies have shown that selection for behavioral avoidance of permethrin resulted in a simultaneous shift to greater physiological susceptibility in a population of diamond-back moth (Plutella xylostella). This suggests that we could simultaneously prevent resistance development while using the insecticide with lower concentrations required over time. Resistance is very difficult to cure, so an additional method of reducing resistance levels in a population would be extremely helpful in many situations where it has occurred. For genetically engineered crops, alternatives to the high dose-refuge strategy would be valuable, because a high dose relative to the tolerance of target pests is not always feasible and the logistics of implementing the strategy can be difficult even if the high dose can be achieved. The benefits to agriculture are the sustainable use of insecticides and insect resistance traits, and at lower concentrations of toxins. A sustainable strategy that results in shifting pest damage away from the harvestable, economically important parts of a crop could be preferable to a non-sustainable strategy that kills the pests. Stakeholders would benefit by reduced costs of insect resistance to insecticides or plant traits, reduced costs of pest control, better integration of controls in an IPM framework, and improved protection of the environment.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110104080%
2113110113010%
2163110113010%
Goals / Objectives
It has been shown that an increase in susceptibility within a population can be achieved even during continued insecticide use through a targeted but heterogeneous, low-dose application. Understanding the genetic correlation between behavior and physiology in pest adaptation is an important and understudied aspect of pest management and insect plant interactions. We propose experiments to unlock the genetic mechanisms leading to this genetic correlation using laboratory and field-based population genomic analyses of diamond-back moth (Plutella xylostella). We aim to create new lines under the same selection regimes to 1) identify and map the genomic region(s) responsible for the correlation, 2) link QTL uncovered in laboratory strains to field populations in areas where agricultural production results in different selection pressures and 3) compare and contrast gene expression patterns at the population level in candidate genes for both traits. The work we propose is integral to our understanding of how pests respond to plant defenses and insecticides. During the first year, we will perform our microsatellite screen to test for amplification and polymorphism among 147 available loci. We will also collect individuals from our field populations and construct F1 populations. Towards the end of the first year, we will begin our linkage map construction from 15 individual families from our 5th generation, as well as begin the genome scan of field samples. Functional analysis for the 10 candidate genes will be performed during the second year on the F1 populations using qRT-PCR. The third year will entail mapping of QTL.. The following are the expected outcomes: 1) The phenotypic effects and strong heritabilities of our traits strongly suggest the presence of genetic control at the same loci. We anticipate that traits will share QTL as well as have unique loci, given the complexity of the traits. 2) The genetic variation in field populations will mimic our laboratory lines. While environmental conditions and selection pressures may differ substantially, our primary interest is in loci implicated through our QTL study. However, using a genomic scan approach will allow us to look at not only the level of selection with the QTL, but other genomic areas as well that may be contributing to these traits. Therefore, we predict that loci exhibiting genetic differentiation in our laboratory lines will also demonstrate differentiation in the field. 3) We expect to find an up-regulation of transcripts for some of the candidate genes specific to each of the trait under investigation (behavioral and physiological) while the others show a more buffered profile.
Project Methods
Lines for tolerance and avoidance will be generated from previously described methods. For the F5, we will perform individual family crosses to generate linkage maps, using previously characterized microsatellite loci. As recombination does not occur in females in DBM, establishing linkage groups is straightforward. We will use MAPMAKER/EXP, construct a composite map of all families. Once differences in physiological tolerance have been reached (estimated at generation 25 to ensure shifts in the behaviorally selected line) we will set up 2 mass crosses among the physiology line and the behavioral avoidance line; F1A will use 20 physiology males and 20 behavioral females where as F1B will use 20 physiology females and 20 behavior males. Cross cages will be held in separate rearing rooms to prohibit contamination among crosses. A fraction of this F1 progeny will be used in 4 single-pair backcrosses to either the physiology line or the behavior line. For backcrosses BC-A and BC-B, we will use 10 F1A males crossed with 10 physiology-line females and 10 behavior-line females, respectively. For BC-C and BC-D we will use 10 F1B males crossed with 10 physiology-line females and 10 behavior-line females, respectively For QTL associations, ≥30 offspring from each of 4 largest BC-A-D crosses will be genotyped with all microsatellite loci available from our screen. Linkage group consistency and map distances among these BC crosses will be checked against our linkage map using MAPMAKER/EXP. For QTL associations, we will use a maximum-likelihood interval analysis provided in MAPMAKER/QTL 1.0. Our significance threshold will be determined by the equation LOD = log10(1/α/M), where M is the number of independent markers. We will collect 100 larvae from 5 populations in the Southern US and 5 populations in the Northern US. We will use the program fDist to compare simulated neutral divergence from observed divergence at putative selected loci. In total 10 candidate genes will be profiled for mRNA levels across all 10 field populations. The mRNA levels will be determined in adults, whole larvae as well as larval tissues. Four general odor binding proteins (GOBP) belonging to two subfamilies (GOBP1 and GOBP2) and six cytochrome P450 genes encoded by the DBM genome will be analyzed. The selection criteria for the P450s are based on their putative role in pyrethroid resistance and their amenability to oral RNAi. Quantitative PCR will be used to assess transcript levels of all 10 candidate genes in adults, whole larvae and larval tissues. For calculations of significance, the logs of the REVs for each gene will be analyzed by ANOVA (Analysis of Variance) using the PROC MIXED procedure of SAS. The different populations will represent different biological replicates and therefore will be included as a random effect in the analysis model.

Progress 09/01/09 to 08/31/13

Outputs
Target Audience: The target audience has been small & large farmers, academia including universities and colleges and some industry audience. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The post-doctoral researcher working on this project was able to present data in several meetings and conferences and ultimately allowed her to establish as an independent researcher with solid skills relevant to this project. On the other hand, the technician (0.5FTE) who had worked on this project is currently enrolled as a graduate student i.e. into doctoral program. How have the results been disseminated to communities of interest? The primary means to disseminate the findings have been through informal gatherings with small and large farmers, and academic meetings and conferences at the national and international levels. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This report documents research employing functional and population genomics approaches to characterize the genes involved in behavioral responsiveness (e.g., avoidance) and physiological tolerance of the diamondback moth, Plutella xylostella, to a pyrethroid insecticide (i.e., permethrin). In 2010, we started the laboratory selection of diamondback moth lines for avoidance and physiological tolerance using a population from Celeryville, Ohio. No selection lines were also included. Avoidance lines are reared on cabbage sprayed on the upper and inner half of the plants with LC5 of permethrin ( low dose, heterogeneous application mode) , whereas the physiological tolerance lines are reared on cabbage plants sprayed on both upper and leaf surfaces (to runoff) with LC50 of permethrin ( uniform high dose application). The lines with no selection, on the other hand, are reared on cabbage plants sprayed with a spreading agent solution. The LC5 and LC50 values of permethrin used are from leaf-dip bioassay results obtained from the preceding generation. After 20, 25 and 30 generations, larvae from the physiological tolerance lines had 349-, 752-, and 591.6-fold levels of resistance, respectively, compared with the control lines. Relative to larvae from the avoidance line, larvae from physiological tolerance lines had 87-, 251- and 101.1- fold levels of resistance after 20, 25 and 30 generations, respectively. Gene expression analyses revealed that at least 2 detoxification genes encoding cytochrome P450s (CYP4M14 and CYP305B1) might be governing the high resistance level (up to 752-fold) observed in larvae from physiological tolerance lines, as well as the moderate resistance level (up to 5.0-fold) observed in larvae from the avoidance lines.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Ma. Bautista, Casey Hoy, Andrew Michel, Omprakash Mittapalli .2012. Gene responses to pyrethroids in the diamond back moth. Presented in the sympoisum "Function, Regulation and Evolution of Environmental Response Genes in Post-Genomic Era" at the 2012 Annual Entomological Society of America Meeting. November 12-14, 2012. Knoxville, TN.


Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: This report documents research employing functional and population genomics approaches to characterize the genes involved in behavioral responsiveness (e.g., avoidance) and physiological tolerance of the diamondback moth, Plutella xylostella, to a pyrethroid insecticide (i.e., permethrin). In 2010, we started the laboratory selection of diamondback moth lines for avoidance and physiological tolerance using a population from Celeryville, Ohio. No selection lines were also included. Avoidance lines are reared on cabbage sprayed on the upper and inner half of the plants with LC5 of permethrin ( low dose, heterogeneous application mode) , whereas the physiological tolerance lines are reared on cabbage plants sprayed on both upper and leaf surfaces (to runoff) with LC50 of permethrin ( uniform high dose application). The lines with no selection, on the other hand, are reared on cabbage plants sprayed with a spreading agent solution. The LC5 and LC50 values of permethrin used are from leaf-dip bioassay results obtained from the preceding generation. After 20, 25 and 30 generations, larvae from the physiological tolerance lines had 349-, 752-, and 591.6-fold levels of resistance, respectively, compared with the control lines. Relative to larvae from the avoidance line, larvae from physiological tolerance lines had 87-, 251- and 101.1- fold levels of resistance after 20, 25 and 30 generations, respectively. Gene expression analyses revealed that at least 2 detoxification genes encoding cytochrome P450s (CYP4M14 and CYP305B1) might be governing the high resistance level (up to 752-fold) observed in larvae from physiological tolerance lines, as well as the moderate resistance level (up to 5.0-fold) observed in larvae from the avoidance lines. The primary means to disseminate the findings have been through informal gathers with small and large farmers, and academic meetings and conferences at the national and international levels. PARTICIPANTS: PI: Omprakash Mittapalli Co-PIs: Andy Michel & Casey Hoy Post-doctoral researcher: Marianne Bautista Research Scholar: Marcelo Goyzueta Training/professional development: This project has provided adequate experience for the research scholar to currently apply for graduate school and the post-doctoral researcher to apply for potential research associate/faculty positions in her country. In addition, the post-doctoral researcher was able to participate in academic conferences and meetings. TARGET AUDIENCES: The target audience has been small & large farmers, academia including universities and colleges and some industry audience. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Specific Outcomes/Impacts: 1. Functional analysis by RNAi can be performed on genes possibly involved in avoidance strategy of DBM ( i.e. OBP13 and CSPs). 2. The SNPs we have developed can be used to determine migration patterns and SNP allele frequencies between resistant and susceptible DBM from field and laboratory populations. 3. Dataset from RNA-seq will provide complementary genomic resources for studies on mechanisms involved in physiological and behavioral resistanceof DBM to insecticides. Current changes in knowledge: For this year (2012), we have also validated single nucleotide polymorphism (SNP) loci that were predicted from the DBM 454 transcriptome dataset we reported earlier. An oligo pool assay (OPA) was used to genotype several individuals of P. xylostella populations collected from the 5 North American states: Texas, Ohio, New York. Michigan and Florida. Overall, 89 SNPs were converted into working assays (93% success rate) on 66 individuals from Texas, Ohio and Florida (30 individuals were not included in the population genetics study); 7 failed in the assay. However, of the 89 SNP loci, only 74 loci were polymorphic and neutral. Using these 74 loci, a lack of genetic differentiation was found among the 3 DBM populations, a pattern that is consistent with several earlier works on population genetics of DBM. Finally, a RNA-seq dataset was also generated by Illumina sequencing of DBM samples from all permethrin-selected and control lines. A total of 938,116,086 reads were obtained and assembled into 294,858 contigs. Of these assembled contigs, 57,977 unigenes were annotated. Global profiling of differentially expressed unigenes as well as validation is underway. This dataset is set to provide complementary genomic resources for studies on mechanisms involved in physiological and behavioral resistance of DBM to insecticides. The resources at the OARDC and activities pertaining to the dissemination of the results were crucial in laying out the final objective of this project. The informal discussion and one-on-one meetings at academic conferences were especially important in fine tuning the current outcomes. For example, we now have some very strong metabolic targets to pursue potential RNAi experiments, which were the result of the functional genomics experiments.

Publications

  • Bautista,M.A.M., Mittapalli, O., Hoy, C., Michel, A. 2012. Single nucleotide polymorphism discovery in the diamondback moth, Plutella xylostella (L.). Molecular Ecology Resources. (in press)


Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: To characterize the genes involved in behavioral responsiveness (e.g., avoidance) and physiological tolerance of the diamondback moth, Plutella xylostella, to a pyrethroid insecticide (i.e., permethrin), population and functional genomics approaches are employed in this project. Diamondback moth lines selected for increased susceptibility (avoidance line) and physiological tolerance were started in July 2010 using a population collected from Celeryville, OH and since then have been maintained in the laboratory. Non-selected lines were also included. The lines with no selection are reared on cabbage plants sprayed with a spreading agent solution. Avoidance lines are reared on cabbage sprayed on the upper and inner half of the plants with LC5 of permethrin, whereas the selection for physiological tolerance lines are reared on cabbage plants sprayed on both upper and leaf surfaces (to runoff) with LC50 of permethrin. The LC5 and LC50 values of permethrin used are from leaf-dip bioassay results obtained on preceding generation. All lines are in their 22nd generation. Each generation began with 200 pupae harvested from the previous generation. Generations are maintained in a discrete and synchronized manner. The physiologically tolerant lines have developed ca. 185-fold levels of resistance within 22 generations compared with the original colony. In the absence of selection pressure, the LC50 values and resistance ratios of the control lines decrease ca. 2-fold. For generations 1-22, lines selected with LC5 remained slightly higher in LC50 values compared with unselected control line and has 2 to 3-fold levels of resistance compared with the original colony. Apparently, there is a delay in response to indirect selection for physiological susceptibility, which may be due to the low frequency of alleles related to the behavioral response in the founder population. In addition, results of a behavioral response assay from the 20th generation showed that about 60% of larvae (n=15) from both control and avoidance lines and 50% of larvae from physiologically tolerant were behaviorally responsive. 454 pyrosequencing of 3rd instar larvae and adult heads was employed to hasten the search for potential genes conferring for both resistance traits. Candidate sequences of genes mined include cytochrome P450s (P450s), glutathione S-transferases (GSTs), odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs) and ionotropic glutamine receptors (iGluR). In recent qRT-PCR experiments we found that the expressions of some detoxification enzymes and chemosensation genes in 3rd instars differ among the three population lines in two generations (13th and 15th). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: pesticide and insecticide companies; farmers (large- and small-scale); research scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our results for the second year will have no immediate impact on DBM resistance management. Nonetheless, the 454 sequencing effort allowed the identification of candidate genes encoding cytochrome P450s (P450s), glutathione S-transferases (GSTs), odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs) and ionotropic glutamine receptors (iGluR). Expression profiling of these candidate detoxification and chemosensation genes among lines in earlier generations provide insights into the likely involvement/interaction of both traits and gene(s) that actively participate in conferring an individual trait (behavioral avoidance or physiological tolerance). Some of these genes could also be potential RNAi targets to further decipher gene function. Genome scans using Single Nucleotide Polymorphisms (SNPs) on 5 field populations are currently being investigated. Scans are to be extended later on individuals from additional field populations and from the 3 laboratory selected and non-selected lines. Results obtained suggest further monitoring of susceptibility and resistance levels, as well as behavioral responsiveness, in succeeding generations. We predict that the traits will become more pronounced at or after the 25th generation. The overarching impact(s) of the results obtained will provide a scenario of how genes are regulated in a population that is receiving different or no selection pressure in a time-course manner.

Publications

  • No publications reported this period


Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: A decrease in tolerance to an insecticide (i.e., permethrin) in the diamondback moth (DBM) population while using the insecticide has been shown to result from the negative correlation between behavioral responsiveness and physiological tolerance to the insecticide. This implies that selection for avoidance may select indirectly for increased susceptibility to the insecticide. The next logical step is to characterize the genes involved in each trait or genes playing dual roles in behavior and tolerance. To characterize the genes involved in each or both traits both population and functional genomics approaches are being employed in this project. The first few months of the project focused on establishing the laboratory selection lines for avoidance and tolerance, and control (no selection) lines. A DBM population (from Celeryville, OH) with which we had conducted experiments in the past to exhibit a negative correlation between behavioral and physiological responses to permethrin. Therefore, large variation in tolerance was used to establish our laboratory lines in July 2010. The selection experiments are being conducted on 8 cabbage plants using 0.8 x 0.6 x 0.3 m home-made cages. Two hundred pupae were used to start each selection line in three replicates. The no selection lines are reared on cabbage plants sprayed with a spreading agent solution. On the other hand, the selection for avoidance lines are reared on cabbage sprayed on the upper and inner half of the plants with LC5 of permethrin, whereas the selection for physiological tolerance lines are reared on cabbage plants sprayed on both upper and leaf surfaces (to runoff) with LC50 of permethrin. The LC5 and LC50 values of permethrin used are from assays on previous generation. Microsatellite screening has been done as part of our plan to use them in detecting genomic regions under selection and/or adaptation to the insecticide. The microsatellite markers redesigned from the already available 147 loci for DBM were tested in a set of 20 to 24 individuals from four North American populations (Ohio, Georgia and 2 of Hawaii). Of the loci screened, 17 consistently seemed to be polymorphic. For genome scans, we intend to use Single Nucleotide Polymorphism (SNPs) instead of the polymorphic markers. We started collecting populations of DBM from cabbage fields in the Northern states (OH, NY and MI) of which F1 populations were obtained for future genome scans and current assessment of resistance to permethrin based on LC50 analysis. The other field populations will be collected in spring. 454-based pyrosequencing of 3rd instar larvae and adult heads was employed to search for potential genes conferring for both the resistance traits. The data is being currently analyzed. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Target audiences include: companies, farmers, and consumers. Efforts to disseminate the learnt knowledge include presentations at professional conferences/meetings, extension/outreach programs, training of interns (research aides) in laboratory techniques and insect rearing. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The 17 polymorphic microsatellites that were genotyped will be used to obtain knowledge of population structure of the DBM in North America, which in turn can help estimate migration between and among DBM populations and support more effective approaches to control them in the future. The 454 pyrosequencing data yielded a total of 198,753 reads which were assembled into 5,159 isotigs, 5,587 contigs, and 55,471 singletons). Functional annotations of sequences with similarities in nucleotide and protein databases have been obtained using Gene Ontology (GO) classification. Candidate sequences of genes likely involved in conferring physiological resistance and/or behavioral avoidance (detoxification and olfactory) to permethrin have been mined from the database and await validation via qPCR. This sequencing effort will: i) increase the pest's genetic and genomic resources necessary for more mechanistic studies; ii) allow us to identify and functionally characterize the genes likely involved in behavioral avoidance and physiological tolerance to permethrin; and iii) provides an excellent platform for mining gene-associated SNPs, which will be used for future genome scans.

Publications

  • No publications reported this period