HARNESSING CHEMICAL ECOLOGY TO ADDRESS AGRICULTURAL PEST AND POLLINATOR PRIORITIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1021581
• Project No.: MIS-311350
• Multistate No.: NE-1501
• Project Start Date: Jan 1, 2020
• Project End Date: Sep 30, 2020

Project Director
Ahn, SE, .

Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762

Performing Department
Biochemistry & Molecular Biology

Non Technical Summary
Plants defend themselves from insect herbivores with a variety of noxious chemical compounds, but many herbivorous insect species have evolved counter-adaptation mechanisms to overcome these defense compounds. Detoxification system in insects is, therefore, an important component of the adaptation and survivorship in many agricultural pests. Enzymes involved in the insect detox machinery include cytochrome P450s (P450s), glutathione S-transferases (GSTs), carboxylesterases (CCEs), and uridine-diphospate glycosyltransferases (UGTs). They are produced in various tissues such as salivary glands, gut, Malpighian tubules, and fat body taking part in different detoxification roles. They are also inducible by feeding on different food plants (therein toxic compounds) or by pesticide exposure.Corn earworm (Helicoverpa zea) is an economically important, nation-wide pest species of corn, cotton and soybean. But, its recent development of resistance to insecticides and Bt toxins might cause serious problems in agricultural practices and environmental issues. Therefore, it is demanding to develop alternative strategies to enhance its integrated pest management. Recently, the corn earworm genome sequencing has been completed and its rich bio-molecular information is ready to be utilized. One of the promising biotechnology-mediated novel strategies is RNA interference (RNAi), which is a sequence-specific gene silencing technology originally from the natural process for gene regulation and defense against pathogens. A starting point of this novel strategy is to find out potential target genes. Among others, the insect detox machinery can be a promising target, because it is highly responsible for the host adaptability and insecticide resistance particularly in herbivorous insects including the corn earworm. By disarming the insect detox machinery, we can make insects vulnerable to natural plant toxins or at least susceptible to lower dosages of insecticide applications. In this study, we aim to identify a complete repertoire of insect detox genes and to screen those associated with different diet challenges in various tissues of the corn earworm. To achieve this goal, four objectives will be pursued as described below:Collection of different tissue samples from the corn earworm and RNA isolation (0.25 yr): The corn earworm larvae will be dissected out into different tissues, after being challenged by different host plants including cotton, corn and soybean, and artificial diet. Total RNA will be isolated from these samples and purified for sequencing.RNA Sequencing and transcriptome expression profiling (0.25 yr): The purified RNA will be sent to a biotech company for the next-generation sequencing. The raw sequence reads generated will be assembled to create longer contiguous fragments with an expression value for each.Identification of detoxification gene families associated with tissue and diet challenge (0.25 yr): The complete repertoire of P450, GST, CCE, and UGT multigene families will be identified from the transcriptome and genome databases by BLAST searches. Genomic structure and transcriptomic variants will be analyzed together with their gene expression profiles in various tissues/diets.Validation of gene expression levels by quantitative real-time PCR analysis (0.25 yr): Selected detox genes showing significant expression levels will be PCR amplified and cloned for Sanger sequencing to confirm their sequences. Transcript variants, if any, will be further verified by PCR and the gene expression levels will be validated by quantitative real-time PCR. Phylogenetic analysis with other lepidopteran species will provide a gene tree reflecting their evolutionary pathways, leading to shortlist candidate genes for further analyses.

Animal Health Component

0%

Research Effort Categories

Basic

50%

Applied

30%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3110	1040	100%

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

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology;

Keywords

corn earworm

detoxification

transcriptome

insect-plant interaction

Goals / Objectives
Develop chemical ecology tools and information to support sustainable agriculture by reducing damage by pests in crops such as potatoes, brassicas, cucurbits, apples, blueberries, and sweet corn, while maintaining pollinator health in agricultural systems.

Project Methods
1. Collection of various tissue samples from the corn earworm and RNA isolationCorn earworm larvae will be obtained from the laboratory colony that has been maintained in the MSU Entomology insectary. After challenged by feeding on different host plants including cotton, corn and soybean, as well as on artificial diet as a control, the 5th instar larvae will be dissected out into different tissues such as salivary glands, gut, Malpighian tubules, fat body and integument. The samples will be collected in separate tubes on dry ice with three biological replicates, from which total RNA will be extracted using PureLink RNA Mini Kit (Thermo Fisher Scientific) and treated with Turbo DNase (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA will be further purified by using the RNeasy MinElute Cleanup Kit (Qiagen) and stored at - 80 ?. The RNA quantity and quality will be assessed using a NanoDrop Spectrophotometer ND-2000 (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies) based on RNA Integrity Number (RIN).2. RNA Sequencing and transcriptome expression profilingThe purified RNA will be sent to Psomagen, Inc (Rockville, MD) for the next-generation sequencing, where cDNA libraries will be prepared using TruSeq Stranded Total RNA Library Prep Kit (Illumina). The raw sequence reads generated by NovaSeq 6000 Sequencing System (Illumina) will be checked by FastQC for quality control and trimmed by Trimmomatic tool (ver. 0.32). Overlapping high-quality reads will be assembled to create longer contiguous fragments (contigs). Transcript abundance will be estimated using RSEM (ver. 1.2.15) to generate FPKM (fragments per kilobase per million mapped reads).3. Identification of detoxification gene families associated with tissue and diet challengeWe have already identified a list of detoxification multigene families from other lepidopteran species. By BLAST searches, the complete repertoire of P450, GST, CCE, and UGT multigene families will be identified from H. zea transcriptome and genome databases, and then annotated in the genomic scaffolds to understand their physical structure and orientation. Furthermore, each gene family will be compared with other lepidopteran species (moths and butterflies) including a closely-related congeneric species (Helicoverpa armigera) and a model insect silkworm (Bombyx mori) by constructing phylogenetic trees. Their deduced amino acid sequences will be aligned together and grouped according to their sequence similarity. Phylogenetic analysis of these amino acid sequences will result in a gene tree reflecting their evolutionary pathways, such as gene duplications, lineage-specific gene diversifications, gene loss/gain, etc. Taken together with gene expression profiles and genomic structure, the molecular evolutionary analysis will lead to screen candidate genes for further analyses.4. Validation of gene expression levels by quantitative real-time PCR analysisSelected genes showing significant expression levels will be PCR amplified and cloned for Sanger sequencing to confirm their sequences. Transcript variants, if any, will be further verified by PCR with various experimental samples. Gene expression levels will be validated by quantitative real-time PCR (qRT-PCR) using gene specific primers with SYBR Green RT-PCR Master Mix. By doing this, tissue- and diet-associated detoxicative enzymes will be screened. The gene structure and phylogenetic position of the selected candidate genes will provide more information for future studies.

Progress 01/01/20 to 09/30/20

Outputs
Target Audience:Target audiences of my research include undergraduate students, graduate students, research scientists and other professionals in academia, and personnel in government and industry sectors who areworking on insect-plant interactions and agricultural pest management. My efforts for the duration of this project have been delivered by publications, seminars, laboratory workshops, and classroom instructions. Changes/Problems:COVID-19 pandemic hasaffected this project during and after the period of Mar. 15 till Sep. 30, 2020. It has sloweddown the laboratory activities and hindered the potential interactions among multistate collaborators. What opportunities for training and professional development has the project provided?During the project period, Jan. 1, 2020 to Sep. 30, 2020, this projecthastrained two undergraduate students and two graduate students for basic molecular biology tools used in the study of chemical ecology, such as nsect rearing, molecular cloning, insect cell culture, and microscopy. For the graduate students, this projectprovided a couple of opportunities to design experiments, develop short-term and long-term projects, and give a presentation in the regular lab meetings. How have the results been disseminated to communities of interest?I have disseminated the project results to communities of interest in the form of journal articles and conference presentations, as listed below: 1. Journal articleonAntennal transcriptome of Spotted Wing Drosophila (SWD): Ahn, S.-J., Oh, H.W. Corcoran, J., Kim, J.A., Park, K.C., Park, C.G., Choi, M.-Y. 2020. Sex-biased gene expression in antennae of Drosophila suzukii. Archives of Insect Biochemistry and Physiology, e21660. https://doi.org/10.1002/arch.21660 2. Journal article on Neuropeptides and their receptors of Brown Marmorated Stink Bug (BMSB): Ahn, S.-J., Corcoran, J., Vander Meer, R.K., Choi, M.-Y. 2020. Identification and Characterization of GPCRs for Pyrokinin and CAPA Peptides in the Brown Marmorated Stink Bug, Halyomorpha halys (Hemiptera: Pentatomidae). Frontiers in Physiology, 11: 559. https://doi.org/10.3389/fphys.2020.00559 3. Conference presentation onFirst molluscan PRXamide neuropeptides and their receptors in the slug: Ahn S.-J., Choi M.-Y. 2020. First molluscan PRXamide neuropeptides and their receptors in the gray garden slug. Eighty-fourth annual meeting of Mississippi Academy of Sciences, February 20-21, 2020, Mississippi Gulf Coast and Convention Center, Biloxi, MS 4. Conference presentation on UDP-glycosyltransferase (UGT) multigene family in arthropods: Ahn, S.-J. 2020. Molecular evolution of UDP-glycosyltransferase (UGT) multigene family in arthropods. 13th Annual Arthropod Genomics Symposium. July 21-23. (virtual) What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Revealing insect detox machinery by transcriptome analysis of the corn earworm: The corn earworm (Helicoverpa zea) is one of the serious insect pests found throughout the United States. Its polyphagous host usage has enabled the voracious caterpillars to feed a wide range of plants including several important agricultural crops, such as corn, cotton, soybean, sorghum and tomato. It has been postulated that generalist herbivores like the corn earworm can avoid the toxicity of the host plant's defensive chemicals by excreting noxious xenobiotics out of the body faster than other specialist insects. In fact, our previous work revealed that capsaicin, an alkaloid compound that is responsible for pungency in Capsicum fruits, is more readily excreted in the corn earworm than the congeneric specialist, the oriental tobacco budworm (H. assulta), while another generalist, H. armigera, also releases it as fast as the corn earworm. To get rid of the toxic compounds, insects utilize an enzymatic system that conjugates the xenobiotics with a sugar molecule(s), which increases their water solubility, making easier to excrete after all. The enzyme responsible for the sugar conjugation is called uridine diphosphate glycosyltransferase (UGT), which is composed of multiple genes in insect genomes, and it was revealed from the previous year research that the corn earworm has 45 UGT genes in total. I was able to identify a complete set of UGT gene family from the corn earworm transcriptome. Differential gene expression analysis showed that several UGT genes were expressed in a tissue-specific manner. For example, UGT42b, UGT41a, UGT40a, UGT40g, UGT40h, UGT33e, UGT33h, UGT33s and UGT33u were highly expressed in midgut; UGT40e, UGT33b, UGT33c, UGT33j and UGT33t were in fat body; UGT33o and UGT33t were in Malpighian tubules. These tissues are generally known as "battle fields" of digestion and detoxification in insects. Interestingly, UGT34 was exclusively highly expressed in silk gland and UGT40d in central nervous system, suggesting a novel function in relation to nondigestive/detox activities in the corn earworm. In addition, a different set of UGT genes also showed induced expression in different feeding treatments. For example, UGT41a, UGT41c, and UGT40a were induced by corn leaf feeding; UGT40a, UGT40c, UGT40d and UGT33u were induced by Bt corn feeding; and UGT41c and UGT33t were higher in the tomato fruit-fed larvae. It is noteworthy that many genes of the UGT40 subfamily in particular showed a significant association with different feeding regimes, indicating that UGT40 subfamily enzymes might be major players in detox machinery. Antennal transcriptome of Spotted Wing Drosophila (SWD): SWD is a native from Southeast Asia, and has successfully spread to all other continents, becoming a serious pest of the small and stone fruits including blueberries in North America. SWD differs from other members of the genus Drosophila in its host preference and oviposition behavior. The flies are attracted to ripening fruits, and females have a serrated ovipositor enabling eggs to be laid inside the fruit. In addition to its huge economic impact, its unique chemoecological, morphological, and physiological characteristics have garnered considerable research interests. I analyzed D. suzukii antennal transcriptomes to identify sex?biased genes by comparison of differential gene expressions between male antennae (MA) and female antennae (FA). Among 13,583 total genes of the fly genome, 11,787 genes were expressed in either MA or FA. There are only 132 genes (9 in MA, 7 in FA, and 116 in both, FPKM >1) were expressed in antennae exclusively, and 2,570 genes (9 in MA, 0 in FA, and 2,561 in both) were enriched in antennae containing 185 and 113 sex?biased genes in MA and FA, respectively. Interestingly, many immune?related genes were highly expressed in MA, whereas several chemosensory genes were at high rank in FA. I identified 27 sex-biased chemosensory genes including odorant and gustatory receptors, odorant?binding proteins, chemosensory proteins, ionotropic receptors, and cytochrome P450s, and validated the gene expressions using quantitative real?time PCR. The highly expressed sex?biased genes in antennae are likely involved in the fly specific mating, host?finding behaviors, or sex?specific functions. The molecular results obtained from this project will facilitate to find the unique chemoreception of D. suzukii, as well as on the development of new management strategies for this pest. Neuropeptides and their receptors of Brown Marmorated Stink Bug (BMSB): BMSB is an invasive true bug that causes significant economic losses to various agricultural products around the world. I studied the pheromone biosynthesis-activating neuropeptide (PBAN) genes and its receptors from BMSB, identifying the pyrokinin and capa genes from BMSB. In this year, I further identified their receptors, six pyrokinin and capa G protein-coupled receptors (GPCRs) in total and evaluated their (a) ability to respond to neuropeptides in cell-based assays, and (b) expression levels by RT-PCR. Functional studies revealed that the BMSB pyrokinin receptor-1 (HalhaPK-R1a & b) responded to the pyrokinin 2 (PK2) type peptide. RT-PCR results revealed that these receptors had little or no expression in the tissues tested, including the whole body, central nervous system, midgut, Malpighian tubules, and reproductive organs of males and females. HalhaPK-R2 showed the strongest response to PK2 peptides and a moderate response to pyrokinin 1 (PK1) type peptides (= DH, diapause hormone), and was expressed in all tissues tested. HalhaPK-R3a & b responded to both PK1 and PK2 peptides. Their gene expression was restricted mostly to the central nervous system and Malpighian tubules. All PK receptors were dominantly expressed in the fifth nymph. HalhaCAPA-R responded specifically to CAPA-PVK peptides (PVK1 and PVK2), and was highly expressed in the Malpighian tubules with low to moderate expression in other tissues, and life stages. Of the six GPCRs, HalhaPK-R3b showed the strongest response to PK1. Our experiments associated the following peptide ligands to the six GPCRs: HalhaPK-R1a & b and HalhaPK-R2 are activated by PK2 peptides, HalhaPK-R3a & b are activated by PK1 (= DH) peptides, and HalhaCAPA-R is activated by PVK peptides. These results pave the way for investigations into the biological functions of BMSB PK and CAPA peptides, and possible species-specific management of BMSB.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ahn, S.-J., Oh, H.W. Corcoran, J., Kim, J.A., Park, K.C., Park, C.G., Choi, M.-Y. 2020. Sex-biased gene expression in antennae of Drosophila suzukii. Archives of Insect Biochemistry and Physiology, e21660. https://doi.org/10.1002/arch.21660
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ahn, S.-J., Corcoran, J., Vander Meer, R.K., Choi, M.-Y. 2020. Identification and Characterization of GPCRs for Pyrokinin and CAPA Peptides in the Brown Marmorated Stink Bug, Halyomorpha halys (Hemiptera: Pentatomidae). Frontiers in Physiology, 11: 559. https://doi.org/10.3389/fphys.2020.00559
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ahn S.-J., Choi M.-Y. 2020. First molluscan PRXamide neuropeptides and their receptors in the gray garden slug. Eighty-fourth annual meeting of Mississippi Academy of Sciences, February 20-21, 2020, Mississippi Gulf Coast and Convention Center, Biloxi, MS
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ahn, S.-J. 2020. Molecular evolution of UDP-glycosyltransferase (UGT) multigene family in arthropods. 13th Annual Arthropod Genomics Symposium. July 21-23. (virtual)