DISSECTION OF INSECT-PLANT-ENVIRONMENT INTERACTION VIA MOLECULAR AND GENOMIC APPROACHES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016725
• Project Start Date: Aug 23, 2018
• Project End Date: Aug 1, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Entomology

Non Technical Summary
Worldwide losses in stored grains and other agricultural products caused by insects have been estimated to be between 5-10%, and this number can reach 10-40% in some developing countries (Shaaya, Kostjukovski et al. 1997, Weaver and Petroff 2005). Direct consumption of kernels by the insects results in reduction in weight, nutritional value and germination rate of stored seeds. Insect infestation also causes sanitary problems due to accumulations of frass, exuviae, webbing and insect cadavers. Grain of such quality may completely lose its market value, as they are no longer acceptable for processing into food for human consumption.The most effective current method to control storage insects is through fumigation with chemical insecticides. Use of these chemicals, however, can result in excessive pesticide residues in treated grain or grain products that cause human health and environmental problems, as well as potential resistance development in insects. For example, methyl bromide has largely been phased out in developed countries due to its damage to the earth's ozone layer (Fields and White 2002). Another common fumigant, phosphine, has lost its effectiveness because of resistance development (Benhalima, Chaudhry et al. 2004, Collins, Daglish et al. 2005, Pimentel, Faroni et al. 2009).Modified atmospheres with low O2 (hypoxia) and/or high CO2 (hypercapnia) environments in airtight storage withhold the O2 required for insect development, thus preventing damage by insect pests in stored grain (Banks and Annis 1990, Fleurat-Lessard 1990, Sanon, Dabiré-Bins et al. 2011, Navarro, Timlick et al. 2012). Alteration of the composition of the storage atmosphere can be achieved biologically; the respiration of any insects present, together with the respiration of grain embryos, converts O2 into CO2, altering the internal atmosphere. A modern method modifies the atmosphere by purging the storage container with CO2 or N2 and then sealing it hermetically. Such hermetic storage is a cost-effective and environmentally friendly alternative measure to chemical fumigation, and has been used to control postharvest insect pests and various pests that affect a large number of raw and manufactured food products (Fleurat-Lessard 1990, van Epenhuijsen, Carpenter et al. 2002). Over the last several decades, considerable research has been carried out on the effectiveness of control via modified atmospheres using different gas compositions for various stored pests, especially well-known cosmopolitan pests such as Tribolium castaneum, T. confusum, Sitophilus zeamais, S. oryzae, Rhyzopertha dominica, Cryptolestes ferrugineus and Oryzaephilus surinamensis (Finkelman, Navarro et al. 2006, Chiappini, Molinari et al. 2009, Lord 2009, Noomhorm, Sirisoontaralak et al. 2009, Riudavets, Castane et al. 2009). However, knowledge gaps still remain. For example, although sensitivity of insects to low O2 is known to be dependent on the species and life stage (Hoback and Stanley 2001), little is understood of the underlying molecular mechanisms leading to injury or adaptation in stored insects, nor the association between O2 demand and hypoxic tolerance.The cowpea bruchid (Callosobruchus maculatus) is the most important storage pest of cowpea (Vigna unguiculata) and other legumes throughout the world (Giga and Smith 1987, Langyintuo, Lowenberg-DeBoer et al. 2003). Infestation of cowpea by bruchids starts in the field and proliferates in storage. The females lay their eggs (approximately 50 eggs per female) on the surface of cowpea seeds. Larvae feed inside the seeds, where they complete the entire four-instar larval development and pupate in a month. Adults start to lay eggs soon after they emerge, initiating another round of infestation. One hundred percent damage can occur to unprotected cowpea seeds after three to five months in storage (Zhu-Salzman and Murdock 2006). Using the cowpea bruchid as our model insect, we intend to obtain a more thorough understanding of developmental and survival parameters when insects are subjected to hypoxia treatment. We systematically characterized the response of this insect to hypoxia at different developmental stages. Our results revealed that late larval stages (4th instar) exhibited the strongest tolerance to low O2 stress and had the highest survival rate compared to other stages, although under normoxic atmospheric conditions the 4th instar larvae consumed the most O2, and ought to have been the most sensitive to low O2 (Cheng, Lei et al. 2012). Thus cowpea bruchid larvae may have evolved an adaptive strategy to handle hypoxia that is associated with their habitat. Unfortunately, very little is known about the molecular mechanisms mounted by storage pests in response to hypoxia.

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
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

microarray

cowpea bruchid

responsive genes

adaptation

transcription factor

plant defense

Goals / Objectives
The goal of this research is to define the molecular bases of insect adaption to hypoxia. The working hypothesis of the proposed research is that insects activate diverse signaling pathways to induce sets of coordinately regulated hypoxia-responsive genes to mitigate the effect of low O2. Insight into activation and function of these hypoxia-regulated insect genes is important not only from a basic research perspective, but it is also critical to the success of environmentally friendly pest management strategies. I propose the following specific objectives using cowpea bruchids:1) Perform transcriptomic analysis for hypoxia-responsive genes2) Identify regulatory elements that control transcription of hypoxia-responsive genes

Project Methods
Objective 1. Perform transcriptomic analysis for hypoxia-responsive genesWe aim to define the molecular bases of insect adaption to hypoxia. Experimentally, age synchronized 4th instar larvae will be subjected to the following treatments of precision certified pre-mixed gases for 48 hrs: (1) 20% O2+80% N2 (normoxia control), (2) 2% O2+98% N2 (hypoxia). Such selections are based on our preliminary tests. Upon completion of treatment, total RNA will be extracted from the tissues, and the RNA quantity and quality will be verified using a NanoDrop spectrophotometer (NanoDrop Technologies) and by RNA 6000 Pico Chip on a 2100 Bioanalyzer (Agilent).RNA will be submitted for cDNA library production and Illumina sequencing, which will be performed at TAMU AgriLife Genomics & Bioinformatics Services, using the Illumina-HiSeq. The cDNA libraries will be constructed using the Illumina RNA-TruSeq kit. A total of three biological replicates (3 independent libraries) will be barcoded and sequenced. Data analysis will be performed at the AgriLife Genomics & Bioinformatics Services, TAMU. Obtained reads will be assembled using the CLC Bio software and Trinity running on the Brazos computing cluster. Since the genome sequence of the cowpea bruchid is not yet available, sequencing using a long (100 nucleotides) paired-end strategy is necessary to assemble the obtained reads. Unigenes will then be evaluated using BLASTx against the NCBI database (http://ncbi.nlm.nih.gov). Functional (Gene Ontology-GO) annotation will be obtained using Blast2go software with default parameters (http://www.blast2go.de). Generation of this differential gene set represents the first step toward understanding of insect resistance to low O2, and will shed light on how a representative insect species copes with low O2 environments.Objective 2. Identify regulatory elements that control transcription of hypoxia-responsive genesThe goal of this proposal is to define the specific molecular mechanisms that regulate activation of insects' hypoxia-responsive genes. Based on gene function and fold changes derived from follow-up qPCR analyses of a large number of genes, we will select 8-10 differentially regulated genes from several functional groups for further analysis. The promoters for these genes will be cloned and analyzed for promoter activity by transient expression assays in Drosophila S2 cells. Electrophoretic mobility shift assays will be used to identify specific sequences that regulate the response to hypoxia. Proteins that bind to the regulatory sequences identified above will be cloned. The regulatory genes will then be sequenced, analyzed, and transferred to over-expression vectors. The DNA binding activity of the trans-acting factors will be confirmed and characterized after protein expression and purification. Regulatory function of the trans-factors will be confirmed by transient expression assay.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Researchers, teachers, extension specialists and graduate/undergraduate students in the areas of entomology, plant protection, post-harvest products and pest management will benefit from this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The graduate student has learned various molecular techniques that benefit her career development. How have the results been disseminated to communities of interest?Publishing in scientific journals, presented in scientific meeting and guest lecture. What do you plan to do during the next reporting period to accomplish the goals?Continue to investigate the molecular mechanisms of insect tolerance.

Impacts
What was accomplished under these goals? To investigate transcriptional response of cowpea bruchids to hypoxia, we performed RNA-seq using midguts of hypoxia-treated and control 4th instar larvae, respectively. After quality trimming, approximately 277 million reads generated by Illumina sequencing were assembled into 121,227 unigenes covering a total length of 112,775,418 bp. Unigenes, ranging from 224 to 32,100 bp, have an average length of 930 bp and an N50 of 1,611 bp. All unigenes were annotated against the NCBI non-redundant protein (Nr) database using BLASTx with a cut-off E-value of 10-5. Low O2 stress caused significant expression changes of 602 genes, including 408 up- and 194 down-regulated genes, with a cutoff of log2(fold change) ≥ 1 and the adjusted P-value ≤ 0.05. To begin to understand transcriptional co-regulation in response to hypoxia, we attempted to obtain promoter cis-regulatory elements common to up- or down-regulated genes using Common TFs software and determined whether these cis-elements were involved in insect response to hypoxia using electrophoretic mobility shift assays. We also identified a transcription factor that interacted with promoters of hypoxia-responsive genes. Functional evaluation of the transcription factor is currently on-going.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Lei, J., J. Meng, I.W. Chen, W. Cheng, A.L. Beam, Md-Sajedul Islam, W.D. Bailey, S. Pillai and K. Zhu-Salzman (2020) Deleterious effects of electron beam irradiation on development and reproduction of tomato/potato psyllids, Bactericera cockerelli. Insect Sci. 27: 1311-1321.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Electron beam irradiation induces DNA endoreplication in holometabolous juvenile insects: a rapid flow cytometry-based diagnosis.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Wang, Q., E. Yuan, X. Ling, K. Zhu-Salzman, H. Guo, F. Ge, Y. Sun (2020) An aphid facultative symbiont suppresses plant defense by manipulating aphid gene expression in salivary glands. Plant, Cell & Environment 43:23112322
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang, M-Z, C-H. Sun, H-Q. Feng, H-W. Chang, S-N. Cao, H. Zhang, J. Hou, K. Zhu-Salzman, G-H Li and Q-M Qin (2020) Transcriptome analysis and functional validation reveal a novel gene BcCGF1 that enhances fungal virulence by promoting infection-related development and host penetration. Molecular Plant Pathology 21:834853
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Cheng, W., Y. Zhang, W. Liu, G. Li and K. Zhu-Salzman (2020) Molecular and functional characterization of three odorant-binding proteins from the wheat blossom midge, Sitodiplosis mosellana (G�hin) (Diptera: Cecidomyiidae). Insect Sci. 27: 721-734
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Meng, J., J. Lei, A. Davitt, J.R. Holt, J. Huang, R. Gold, E.L. Vargo, A.M. Tarone and K. Zhu-Salzman (2020) Suppressing tawny crazy ant (Nylanderia fulva) by RNAi technology. Insect Sci. 27:113-121
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Cheng, W., X. Li, J. Zhao and K. Zhu-Salzman (2020) Cloning and characterization of Methoprene-tolerant (Met) and Kr�ppel homolog1 (Kr-h1) genes in the wheat blossom midge, Sitodiplosis mosellana. Insect Sci. 27:292-303

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Researchers, teachers, extension specialists and graduate/undergraduate students in the areas of entomology, plant protection, post-harvest products and pest management will benefit from this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The postdoc and students learned techniques related to envrionmental control of pests, which will benefit their career development. How have the results been disseminated to communities of interest?publishing in scientific journals, presented in scientific meeting and guest lecture. What do you plan to do during the next reporting period to accomplish the goals?Continue to investigate the molecular mechanisms of insect tolerance.

Impacts
What was accomplished under these goals? Hermetic storage and electron beam (eBeam) irradiation are safe and effective technologies to protect stored products. Although hypoxic environment improves performance of some irradiated insects, whether hypoxia affects irradiation of storage insects and impacts pest control efficacy remains to be investigated. Using cowpea bruchid (Callosobruchus maculatus) larvae, we showed that, relative to eBeam irradiation under normoxia, the adult emergence rate increased substantially if they were exposed to hypoxia prior to and during eBeam treatment. Conversely, exposure to hypoxia only after eBeam irradiation did not have this protective effect. eBeam irradiation caused an increase in reactive oxygen species (ROS) in normoxic larvae but not in hypoxic larvae. The activity of citrate synthase, a pace-making enzyme in the citric acid cycle, was suppressed under hypoxia but resumed normal function within hours of reoxygenation, suggesting that reduced mitochondrial activity, and thus less ROS production under hypoxia increased insect tolerance to irradiation. Furthermore, reoxygenation accelerated eBeam-induced glutathione S-transferase activation and potentiated eBeam-enhanced catalase activities. Faster and stronger detoxification capacity in eBeam-irradiated, hypoxic larvae may have protected them from oxidative damage. Taken together, hypoxic environment enhanced radiotolerance of bruchid larvae, presumably due to limited ROS production and elevated antioxidant enzymatic activities after reoxygenation.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Sang, W., R. Ji, C.L. Lei and K. Zhu-Salzman (2019) Parental hypoxic exposure influences performance of offspring in Callosobruchus maculatus. Pest Manag. Sci. 75:2810-2819
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, R-L., K. Zhu-Salzman M. E. A. Elzaki, Q-Q. Huang, S. Chen, Z-H. Ma, S-W Liu and J-E. Zhang (2019) Mikania micrantha wilt virus alters insect vectors host preference to enhance its own spread. Viruses 11: pii: E336. doi: 10.3390/v11040336
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Chang, Y., X. Zhang, M. Lu, Y. Du and K. Zhu-Salzman (2019) Molecular cloning and characterization of small heat shock protein genes in the invasive leafminer fly, Liriomyza trifolii. Genes 10, 775; doi:10.3390/genes10100775
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, L., W. Cheng, J. Meng, M. Speakmon, J. Qiu, S.D. Pillai and K. Zhu-Salzman (2019) Hypoxic environment protects cowpea bruchid (Callosobruchus maculatus) from electron beam irradiation damage. Pest Manag. Sci. 75: 726-735

Progress 08/23/18 to 09/30/18

Outputs
Target Audience:Researchers, teachers, extension specialists and graduate/undergraduate students in the areas of entomology, plant protection, post-harvest products and pest management will benefit from this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The students and postdoc leanred related techniques, which will benefit their career development. How have the results been disseminated to communities of interest?Manuscripts What do you plan to do during the next reporting period to accomplish the goals?We will continue to investigate the combined effect of electron beam and hypoxia.

Impacts
What was accomplished under these goals? Modi?ed atmosphere based on lack of O2 can prevent stored grains from insect pest damage. Previous research revealed that although population expansion of cowpea bruchid (Callosobruchus maculatus) could be temporarily arrested when exposed to 2% O2, this insect could survive extended periods of hypoxia and continue its normal development if normoxic conditions resume. It is not clear, however, whether parental hypoxic treatment has any effects on offspring's performance and response to hypoxia.Developmental time, body weight, sex ratio and reproduction of insects with hypoxia experience themselves as well as their progenies either challenged with hypoxia again or reared under normoxia were documented. Hypoxia postponed development of treated parental bruchids. Its negative effects on oviposition and hatch rate of these eggs were significant only when hypoxia was administered at the parental 4th instar larval stage or later. When F1 generation was exposed to hypoxia at the 4th instar larval stage, they exhibited comparable developmental delay, and reduction in adult emergence and fecundity whether the parents experienced hypoxia or not. Interestingly, eggs laid by hypoxia-treated F1s had increased hatch rates if their parents had also been exposed to hypoxia at the late larval stage. Stronger suppression of the digestive protease gene CatL and elevated basal expression of the stress responsive gene Hsp27 were observed in F1 larvae with parental hypoxic experience. Although hypoxia impaired the performance of both parental and F1 generations of bruchids, the parental hypoxic experience appeared to better prepare the F1 progenies for further hypoxic challenge. The tawny crazy ant (Nylanderia fulva) is a new invasive pest in the United States. At present, its management mainly relies on the use of synthetic insecticides, which are generally ineffective at producing lasting control of the pest, necessitating alternative environmentally friendly measures. In this study, we evaluated the feasibility of gene silencing to control this ant species. Six housekeeping genes encoding actin (NfActin), coatomer subunit β (NfCOPβ), arginine kinase (NfArgK), and V-type proton ATPase subunits A (NfvATPaseA), B (NfvATPaseB) and E (NfvATPaseE) were cloned. Phylogenetic analysis revealed high sequence similarity to homologs from other ant species, particularly the Florida carpenter ant (Camponotus floridanus). To silence these genes, vector L4440 was used to generate 6 specific RNAi constructs for bacterial expression. Heat-inactivated, dsRNA-expressing Escherichia coli were incorporated into artificial diet. Worker ants exhibited reduced endogenous gene expression after feeding on such diet for 9 days. However, only ingestion of dsRNAs of NfCOPβ (a gene involved in protein trafficking) and NfArgK (a cellular energy reserve regulatory gene in invertebrates) caused modest but significantly higher ant mortality than the control. These results suggest that bacterially expressed dsRNA can be orally delivered to ant cells as a mean to target its vulnerabilities. Improved efficacy is necessary for the RNAi-based approach to be useful in tawny crazy ant management.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhao, J.J., X.J. Li, Z.Z. Liu, W. Cheng and K. Zhu-Salzman (2018) Cloning of heat shock protein gene SmHsp40 and its expression during diapause and under extreme temperature stress in Sitodiplosis mosellana (Diptera: Cedidomyiidae). Acta Entomol. Sinica. 61: 1253-1262
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: . Meng, J., J. Lei, A. Davitt, J.R. Holt, J. Huang, R. Gold, E.L. Vargo, A.M. Tarone and K. Zhu-Salzman (2018) Suppressing tawny crazy ant (Nylanderia fulva) by RNAi technology. Insect Sci. (in press)