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.
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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)
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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
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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
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