MOLECULAR ANALYSIS OF INSECTICIDE RESISTANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1003738
• Project Start Date: Oct 1, 2014
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Entomology

Non Technical Summary
Colorado potato beetle (CPB) is a notorious pest. High fecundity, diverse and flexible life history and an ability to detoxify insecticides make this insect difficult to manage. The coevolution of this beetle with its host plants that contain toxic chemicals may have led to its remarkable insecticide resistance. This beetle has developed resistance to all classes of synthetic chemicals introduced for its control. We will employ RNA sequencing to identify detoxification enzymes induced by plant chemicals. The top 100 xenobiotic enzymes identified will be confirmed qRT-PCR. We will employ RNAi to study the function and mechanisms of plant chemical induction of 20 selected enzymes. Two selected enzymes will be tested in greenhouse experiments to develop methods for fighting insecticide resistance.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	1310	1130	100%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1310 - Potato;

Field Of Science
1130 - Entomology and acarology;

Keywords

pest

insecticide

resistance

rnai

insect

Goals / Objectives
1. Identification of genes coding for detoxification enzymes and their regulators that contribute to insecticide resistance. We will employ RNA sequencing and quantitative real-time PCR approaches to identify genes that are differentially expressed in resistant and susceptible populations.2. Test identified genes for their use in RNAi-aided suppression of insecticide resistance. We will assay the efficacy of neonicotionoid insecticides in resistant CPB that had RNAi-aided knock-down in expression of genes identified as those contributing to insecticide resistance. We will also test the utility of feeding RNAi in insecticide resistance management.

Project Methods
PROCEDURE/METHODS/APPROACH:Objective 1 Identification of genes coding for detoxification enzymes There are recent reports on development of resistance by the Colorado potato beetles to neonicotinoid insecticides [39, 44, 53]. The main goal is to determine if feeding RNAi could be used to knock-down the expression of genes that contribute to neonicotinoid resistance in these beetles so that a combination of feeding RNAi and insecticides could be used for managing the insecticide resistance in these beetles. This is similar to approaches that used synergists such as piperonyl butoxide used to overcome resistance to a few insecticides such as fenvalerate and azinphomethyl in this species [43, 55]. However, the chemical inhibitor approaches were not highly successful for many reasons including safety issues at high use rate, efficacy, and the fact that the inhibitors were developed for only a few detoxification enzymes. Feeding RNAi approach could overcome some of the problems such as unavailability of inhibitors for most enzymes.Previous efforts identified 9302 sequences from CPB. Blast searches of identified 76 genes coding for P450s in these sequences. The maximum number of genes coding for P450s identified in any one insect species so far is 166. It is remarkable that almost 50% of P450s identified in other insect species are present in 2240 contigs formed by CPB sequences. These studies suggest that either the P450 enzyme genes are highly expressed in this insect or this insect codes for an unusually large number of P450 enzymes. To distinguish these two possibilities and to identify metabolic enzymes and other genes differentially expressed between susceptible and insecticide resistant populations, we will perform sequencing of RNA isolated from insecticide susceptible and insecticide resistant colonies. Two colonies are currently available in our laboratory; a colony maintained in the laboratory for more than 10 years by New Jersey department of Agriculture (susceptible) and imidacloprid resistant colony collected in a commercial potato field in Long Island, New York. CPBs in that location are characterized by extremely high levels of resistance to a wide variety of chemicals, at least in part due to increased monooxygenase activity [44]. Sequencing of RNA isolated from susceptible and resistant populations will allow us to identify genes coding for metabolic enzymes and their regulators over expressed in resistant populations. The results obtained by RNA sequencing will be confirmed by quantitative reverse transcriptase real time PCR. The detoxification genes coding for enzymes and their regulators identified as overexpressed in resistant populations will be tested for their ability to suppress neonicotionoid and synthetic pyrethroid resistance.RNA sequencing and data analysisRNA will be isolated from whole body larvae, pupae, adult male and female beetles. Total RNA will be isolated using TRIZOL reagent following manufacturer's protocol (Invitrogen Three biological replicates will be used for each strain. Illumina TruSeq kit will be used for preparation of cDNA libraries and the libraries will be sequenced on HiSeq 2000 (Illumina). RSEM (RNA-Seq by Expectation Maximization, [59]) will be used to quantify the gene expression in resistant and susceptible populations. Up to 100 genes identified as differentially expressed between resistant and susceptible populations based on several parameters including GO term enrichment, annotation, levels of expression, previous reports on role of homologs in insecticide resistance in other insects will be verified by qRT-PCR using gene specific primers designed based on sequence of each selected gene, RNA isolated from two strains and qRT-PCR. The genes that are confirmed as those that are expressed at higher levels in resistant insects will be subjected to further characterization. KEGG automatic annotation server [60] will be used for Pathway mapping of genes. BLASTX search of KEGG database will be performed to assign unique enzyme commission (EC) numbers to each gene identified as differentially expressed in resistant strain. Top ten genes that are confirmed as differentially expressed between resistant and susceptible populations will be selected and the role of these identified genes in insecticide resistance will be determined using RNAi as described below.Evaluation of identified genes for their use in RNAi-aided suppression of insecticide resistance. We anticipate identifying a few detoxification enzymes and their regulators contributing to imidacloprid or deltamethrin resistance in CPB. In a similar analysis in the deltamethrin resistance strain of the red flour beetle we identified one P450 that plays a major role and eight other P450s that play minor role in deltamethrin resistance [61]. We will determine the effect of knockdown in expression of top ten identified insecticide resistance genes selected on the basis of their differential expression between susceptible and resistant populations and their function inferred from literature and sequence similarity searches. Genomic DNA will be isolated from CPB adults and this DNA and PCR primers designed based on EST sequences of select genes to amplify 200-400 bp regions of genes will be used in a PCR reaction to obtain gene specific fragments. The PCR fragments will contain 200-400 bp coding regions and intervening sequences that separate the exons. The PCR products (200-400 bp) will be cloned into T-tailed L4440 vector. This vector contains two convergent T7 polymerase promoters in opposite orientation separated by a multiple cloning sites and routinely used for feeding RNA experiments in nematodes [62]. For preparing dsRNA in bacteria, the L4440 vector containing PCR products will be transformed into HT115 (DE3), an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity. Double-stranded RNA will be prepared in bacteria and fed to adult beetles. Bioassays will be conducted using RNAi beetles that had ingested dsRNA for insecticide resistance genes or control malE. Comparison of LC50 values between beetles that had ingested dsRNA for detoxification enzymes and control malE dsRNA ingested beetles will show whether knock-down in expression of genes coding for insecticide resistance genes on the efficacy of imidacloprid or deltamethrin. These studies will identify genes detoxification enzymes and their regulators that could be used to increase the efficacy of insecticides.Objective 2: Test identified genes for their use in RNAi The genes coding for detoxification enzymes and their regulators identified will be used to test the hypothesis that RNAi could be used to improve the efficacy of insecticide by knocking-down the expression of genes coding for detoxification enzymes and other proteins that are responsible for insecticide resistance. Double-stranded RNA prepared using identified insecticide resistance gene or malE gene fragments as templates in bacteria will be fed to the insecticide resistant beetles either by incorporation into diet or spraying on potato leaves. The RNAi insects will be used to conduct diet incorporation or topical bioassays for testing the efficacy of deltamethrin and imidacloprid. Based on the results, we will select two dsRNAs that improved the efficacy of imidacloprid and two dsRNAs that improved the efficacy of deltamethrin and perform green house experiments.Green house experiments will be performed by spraying heat killed bacteria expressing insecticide resistance gene dsRNA or control malE dsRNA along with foliar label rates of imidacloprid or deltamethrin onto potted potato plants growing in green house. The plants will be caged and 20 second instar larvae from susceptible and resistant populations will be released into each cage. Mortality of larvae and plant defoliation will be monitored every other day until all survivors complete their development to adulthood.

Progress 10/01/14 to 09/30/19

Outputs
Target Audience:Scientists working in universities, government and industry involved in the development of novel pest management method Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A technician and two graduate students have been trained in molecular methods How have the results been disseminated to communities of interest?Results have been published in refereed journals and presented at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Genes responsible for deltamethrin resistance in the red flour beetle and imidacloprid resistance in Colorado potato beetlewere identified. P450 genes and the regulators of these genes CnCc and Maf play important roles in resistance to bothinsecticides in these two pest insects. RNA interference-mediated knockdown of P450s, as well as the transcription factor,CnCc, reduced resistance levels to these insecticides. We also showed that knockdown of the gene coding for c-enzymeP450 reductase also could be used as a method to reduce resistance levels against the insecticides. These data could be used to develop novel methods using double-stranded RNA as a supplement to reduce insecticide resistance and prolong the life of commercial insecticides.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhu GH, Jiao Y, Chereddy SCRR, Noh MY, Palli SR. Knockout of juvenile hormone receptor, Methoprene-tolerant,induces black larval phenotype in the yellow fever mosquito, Aedes aegypti. Proc Natl Acad Sci U S A. 2019 Oct22;116(43):21501-21507. doi: 10.1073/pnas.1905729116. Epub 2019 Sep 30. PubMed PMID: 31570611; PubMed CentralPMCID: PMC6815201
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhu KY, Palli SR. Mechanisms, Applications, and Challenges of Insect RNA Interference. Annu Rev Entomol. 2019 Oct14;. doi: 10.1146/annurev-ento-011019-025224. [Epub ahead of print] PubMed PMID: 3161013
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Roy A, Palli SR. Epigenetic modifications acetylation and deacetylation play important roles in juvenile hormone action.BMC Genomics. 2018 Dec 14;19(1):934. doi: 10.1186/s12864-018-5323-4. PubMed PMID: 30547764; PubMed CentralPMCID: PMC6295036.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Dhandapani RK, Gurusamy D, Howell JL, Palli SR. Development of CS-TPP-dsRNA nanoparticles to enhance RNAiefficiency in the yellow fever mosquito, Aedes aegypti. Sci Rep. 2019 Jun 19;9(1):8775. doi: 10.1038/s41598-019-45019-z.PubMed PMID: 31217512; PubMed Central PMCID: PMC658473

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Scientists working on insecticide resistance Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Trained Technician and four graduate students. How have the results been disseminated to communities of interest?Results were published in peer-reviewedjournals and presented at conferences. What do you plan to do during the next reporting period to accomplish the goals?We will work on molecular mechanismsthat govern the regulation of detoxificationenzymes mediating insecticide resistance.

Impacts
What was accomplished under these goals? The Colorado potato beetle (CPB), Leptinotarsa decemlineata developed resistance to imidacloprid after exposure to this insecticide for multiple generations. Our previous studies showed that xenobiotic transcription factor, cap 'n' collar isoform C (CncC) regulates the expression of multiple cytochrome P450 genes, which play essential roles in resistance to plant allelochemicals and insecticides. In this study, we sought to obtain a comprehensive picture of the genes regulated by CncC in imidacloprid-resistant CPB. We performed sequencing of RNA isolated from imidacloprid-resistant CPB treated with dsRNA targeting CncC or gene coding for green fluorescent protein (control). Comparative transcriptome analysis showed that CncC regulated the expression of 1798 genes, out of which 1499 genes were downregulated in CncC knockdown beetles. Interestingly, expression of 79% of imidacloprid induced P450 genes requires CncC. We performed quantitative real-time PCR to verify the reduction in the expression of 20 genes including those coding for detoxification enzymes (P450s, glutathione S-transferases, and esterases) and ABC transporters. The genes coding for ABC transporters are induced in insecticide resistant CPB and require CncC for their expression. Knockdown of genes coding for ABC transporters simultaneously or individually caused an increase in imidacloprid-induced mortality in resistant beetles confirming their contribution to insecticide resistance. These studies identified CncC as a transcription factor involved in the regulation of genes responsible for imidacloprid resistance. Small molecule inhibitors of CncC or suppression of CncC by RNAi could provide effective synergists for pest control or management of insecticide resistance.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Gaddelapati SC, Kalsi M, Roy A, Palli SR. Cap 'n' collar C regulates genes responsible for imidacloprid resistance in the Colorado potato beetle, Leptinotarsa decemlineata. Insect Biochem Mol Biol. 2018 Aug;99:54-62.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Roy A, Palli SR. Epigenetic modifications acetylation and deacetylation play important roles in juvenile hormone action. BMC Genomics. 2018 Dec 14;19(1):934. doi: 10.1186/s12864-018-5323-4.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Yoon JS, Sahoo DK, Maiti IB, Palli SR. Identification of target genes for RNAi-mediated control of the Twospotted Spider Mite. Sci Rep. 2018 Oct 2;8(1):14687. doi: 10.1038/s41598-018-32742-2.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Yoon JS, Mogilicherla K, Gurusamy D, Chen X, Chereddy SCRR, Palli SR. Double-stranded RNA binding protein, Staufen, is required for the initiation of RNAi in coleopteran insects. Proc Natl Acad Sci U S A. 2018 Aug 14;115(33):8334-8339. doi: 10.1073/pnas.1809381115.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Scientists working on insecticide resistance Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and two post-doctoral fellows were trained. How have the results been disseminated to communities of interest?Published in journals What do you plan to do during the next reporting period to accomplish the goals?Work on identification CnCC target genes in Colorado Potato Beetle.

Impacts
What was accomplished under these goals? Colorado potato beetle (CPB), Leptinotarsa decemlineata is a notorious pest of potato. Co-evolution with Solanaceae plants containing high levels of toxins (glycoalkaloids) helped this insect to develop an efficient detoxification system and resist almost every chemical insecticide introduced for its control. Even though the cross-resistance between plant allelochemicals and insecticides is well acknowledged, the underlying molecular mechanisms are not understood. Here, we investigated the molecular mechanisms involved in detoxification of potato plant allelochemicals and imidacloprid resistance in the field-collected CPB. Our results showed that the imidacloprid-resistant beetles employ metabolic detoxification of both potato plant allelochemicals and imidacloprid by upregulation of common cytochrome P450 genes. RNAi aided knockdown identified four cytochromes P450 genes (CYP6BJa/b, CYP6BJ1v1, CYP9Z25, and CYP9Z29) that are required for defense against both natural and synthetic chemicals. These P450 genes are regulated by the xenobiotic transcription factors Cap n Collar C, CncC and muscle aponeurosis fibromatosis, Maf. Studies on the CYP9Z25 promoter using the luciferase reporter assay identified two binding sites (i.e. GCAGAAT and GTACTGA) for CncC and Maf. Overall, these data showed that CPB employs the metabolic resistance mediated through xenobiotic transcription factors CncC and Maf to regulate multiple P450 genes and detoxify both imidacloprid and potato plant allelochemicals. In invertebrates, a heterodimer of xenobiotic transcription factors, cap n collar C isoform (CncC) and muscle aponeurosis fibromatosis (Maf) mediate cellular defense. In insects, these proteins regulate expression of genes involved in insecticide detoxification. In the current study, we performed sequencing of cDNA copied from RNA isolated from Tribolium castaneum pyrethroid resistant strain (QTC279) beetles injected with CncC or green fluorescence protein (GFP, control) dsRNA. Differential expression analysis of sequences identified 662 genes that showed a decrease and 91 genes that showed an increase in expression (p value≤0.01 and log2 fold change of ≥ 1.5) in CncC knockdown insects when compared to their expression in control insects. We selected a subset of 27 downregulated genes and verified their differential expression using qRT-PCR. This subset of 27 genes included 21 genes with a predicted function in xenobiotic detoxification. RNAi and insecticide bioassays were employed to study the function of six of these genes coding for CYP4G7, CYP4G14, GST-1 and four ABC transporters, ABCA-UB, ABCA-A1 and ABCA-A1L and ABCA-9B involved in all three phases of insecticide detoxification. These data suggest that CncC regulates genes coding for proteins involved in detoxification of insecticides.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kalsi M, Palli SR. Transcription factor cap n collar C regulates multiple cytochrome P450 genes conferring adaptation to potato plant allelochemicals and resistance to imidacloprid in Leptinotarsa decemlineata (Say). Insect Biochem Mol Biol. 2017 Apr;83:1-12. doi: 10.1016/j.ibmb.2017.02.002. Epub 2017 Feb 9. PubMed PMID: 28189748.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kalsi M, Palli SR. Cap n collar transcription factor regulates multiple genes coding for proteins involved in insecticide detoxification in the red flour beetle, Tribolium castaneum. Insect Biochem Mol Biol. 2017 Nov;90:43-52. doi: 10.1016/j.ibmb.2017.09.009. Epub 2017 Sep 23. PubMed PMID: 28951207.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Palli SR. New roles for old actors, ROS and PRMT1. Proc Natl Acad Sci U S A. 2017 Oct 10;114(41):10810-10812. doi: 10.1073/pnas.1715062114. Epub 2017 Oct 2. PubMed PMID: 28973953; PubMed Central PMCID: PMC5642735.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientists working in Academia, Industry and Government to develop methods for fighting insecticide resistance. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Trained a graduate student How have the results been disseminated to communities of interest?A paper has been published in Scientific Reports, an open access journal What do you plan to do during the next reporting period to accomplish the goals?We plan to identify transcription factors responsible for up-regulation of P450 genes in insecticide and plant chemical treated insects. We will perform bioassays to determine the effect of RNA interference-mediated silencing of P450 and transcription factor genes on insecticide resistance.

Impacts
What was accomplished under these goals? We identified the molecular basis of xenobiotic adaptation in a specialist herbivore, Colorado Potato Beetle. This insect develops resistance to chemicals introduced for its control within a short time. The research conducted in this project showed that up-regulation of multiple P450s that are involved in detoxification of both insecticides and plant allelochemicals. The identified genes could be used for fighting insecticide resistance in this as well as other major pests.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhu F, Moural TW, Nelson DR, Palli SR. A specialist herbivore pest adaptation to xenobiotics through up-regulation of multiple Cytochrome P450s. Sci Rep. 2016 Feb 10;6:20421. doi: 10.1038/srep20421. PubMed PMID: 26861263; PubMed Central PMCID: PMC4748221.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:US Scientists and farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Trained PDF and a graduate student How have the results been disseminated to communities of interest?Published in journals and presneted at the meetings What do you plan to do during the next reporting period to accomplish the goals?We will work on identification of P450s and their regulators in Cololrado potato beetle.

Impacts
What was accomplished under these goals? Insecticide resistance is a global problem that presents an ongoing challenge in pest managemnt. Insects acquire resistance by increasing the levels of their detoxification enzymes includingthe cytochrome P450 monooxygenases (P450's). We recentlyshowed that the pyrethroid resistance in QTC279 strain of Tribolium castaneum is mediatedthrough constitutive overexpression of the P450 gene CYP6BQ9 by 200-fold higher in the resistant strain as compared to that in the susceptible strain. RNAi-aided knockdown of CYP6BQ9 reduced deltamethrin resistance levles in QTC279. We employed RNAi to identifygenes that regulate P450 gene expression in QTC279.Cap 'n' collar C (CncC) and muscle aponeurosis fibromatosis (Maf) family transcription factors were identified as the key regulator of these genes, CncC and Maf regulate expression of multiple genes in the CYP6BQ cluster. Studies on the promoters of these genes using reporter assays identified binding sites that mediate CncC and Maf regulation of CYP6BQ gene expression.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Kalsi, M. & Palli, S. R. (2015) Transcription factors, CncC and Maf, regulate expression of CYP6BQ genes responsible for deltamethrin resistance in Tribolium castaneum, Insect Biochem Molec. 65, 47-56.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Palli, S. R. (2014) RNA interference in Colorado potato beetle: steps toward development of dsRNA as a commercial insecticide. , Curr Opin Insect Sci 6, 1-8.