Progress 09/01/11 to 08/31/16

Outputs
Target Audience:Regulatory agencies and government Scientists from both academia and industry Stakeholders including farmers, farm communities, and private sectors General public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This proposal represents a collaborative effort among scientists from the University of Kentucky (PI Zhou) and the University of Nebraska (Co-PI Siegfried). Richard Hellmich (USDA-ARS) and Ann L. Rypstra (Miami University) kindly provided monarch butterfly eggs and Sinella curviseta cultures for the development of their respective RNAi toxicity assays. Funding from this proposal supported the training for two post-doctoral researchers at the University of Kentucky and at the University of Nebraska, a graduate student, and two temporary laboratory technicians (exchange graduate students from the China Agricultural University and Hunan Agricultural University, respectively) at the University of Kentucky. How have the results been disseminated to communities of interest? Participated in a class entitled "Introduction to Genetically Engineered Crops, Risks and Benefits II". This course (PPA-631) was offered by Dr. Paul Vincelli, an Extension Professor at the Department of Plant Pathology. Invited by Paul, I lectured on Feb. 10, 2016 about the current status of the risk assessment of RNAi transgenic crops, and interacted with students and county agents through radio. Peer-reviewed publications (11 published, 4 submitted, and 1 in preparation). Results from the proposed research have been presented at the national meetings (2), international conferences (4), and universities and research institutions (>10)in U.S. and China. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. Clone and sequence vacuolar ATPase (V-ATPase) subunit-a, the target sequence for control of the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), with transgenic RNAi maize from selected surrogate species of non-target arthropods (NTAs) representing different habitats and diverse ecological functions. (Year 1; Completion: 100%) Outcomes: We have successfully obtained the complete open reading frames (ORFs) of V-ATPase subunit-A in target pest, D. virgifera virgifera, and selected surrogate species using standardized molecular cloning techniques. Non-target arthropods tested in this study included honeybee (incidental pollinator), monarch butterfly (eco-indicator), four species of lady beetles (biological control agents) and two species of springtails (soil decomposers). A 400bp region which has the highest sequence similarity among target pest and nontarget surrogate species was selected as the template to synthesize dsRNAs for the subsequent in vivo dietary RNAi toxicity assay. Objective 2. Develop a standard in vivo RNAi-toxicity test using a worst case scenario. (Year 2-3; Completion: 100%) Outcomes: Sequence similarity, dose, and exposure are the key factors that define the worst case scenario for the RNAi toxicity assay.Sequence identity, especially identical sequences of at least 21 nucleotides (21mers) between target and non-target arthropods of arthropod-active genes, specificity of transgenic RNAi maize. Interestingly, different surrogate species, even within the same taxonomic group, respond to ingested dsRNAs differently. Our preliminary results showed that ladybugs are more sensitive toward dietary RNAi than honeybees. However, among the two ladybug species, H. convergens was significantly more responsive toward ingested dsRNAs than H. axyridis, displayed strong cross-reaction with the Western corn rootworm dsRNA. In addition to mortality, we also documented life history traits, including body weight, development time, and fecundity, as the potential endpoints for the measuring ecological risks associated with the RNAi crops, especially under the sublethal exposure. To validate newly developed dietary RNAi toxicity assays, we have established standardized qRT-PCR analysis protocols for both target and non-target insect species following the MIQE (Minimum Information for publication of Quantitative real-time PCR Experiments) guideline. qRT-PCR is a rapid and reliable method for the detection and quantification of gene expression during different biological processes. To ensure the accuracy, a critical component in qRT-PCR analysis is to normalize data by measuring in parallel the expression of a reference gene from the same samples. Housekeeping genes, involving in basic cellular functions, typically maintain stable and constitutive expression in all cells, regardless of physiological conditions. We have concluded reference gene selection studies on the majority of the tested surrogate NTA species, as well as several species of sap-sucking insects. The inclusion of aphids and spider mites, the prey of bio-control agents (e.g., lady beetles) and the vehicle of insect-active dsRNAs, is for the development of dietary RNAi toxicity assays using live preys instead of synthesized dsRNAs. Objective 3. Carry out the early-tier risk assessment of arthropod-resistant transgenic RNAi maize on selected non-target arthropods using an RNAi-toxicity test developed in Objective 2. (Year 4-5; Completion: 100%) Outcomes: Assessed the potential risks associated with dietary RNAi for the incidental pollen feeder, honeybee, A. mellifera, eco-indicator, monarch butterfly, D. plexippus, biological control agents, convergent lady beetle, H. convergens, multicolored Asian lady beetle, H. axyridis, pink spotted lady beetle, Coleomegilla maculata, and the seven-spotted ladybeetle, Coccinella septempunctata, and soil decomposers S. curviseta, and F. candida, respectively. Combined results from our dietary RNAi toxicity assays and validation studies suggested that ingested dsRNAs have negligible biological impacts on honeybee, monarch butterfly, and soil decomposers. However, predatory lady beetles exhibited apparent differential responses to the ingested vATPase dsRNAs, derived either from themselves or the target insect pest Western corn rootworm, D. virgifera virgifera. Specifically, the convergent, pink spotted, and seven spotted lady beetles were sensitive to the dietary RNAi, with H. convergens showing the highest sensitivity (>80% mortality), while the multicolored Asian lady beetle was not responsive (<10% mortality). Surprisingly, in comparison to H. convergens, H. axyridis vATPase has greater number of 21mer matches with D. virgifera virgifera vATPase. This result is not consistent with the notion that the amount of 21mer matches (the number of the exact same 21 nucleotide sequences between the target and nontarget species) dictates the sensitivity of the surrogate species to dietary RNAi. The genetic basis of differential responses toward ingested dsRNAs, even within the same taxon, is a focus of our current research. Impacts: Summary The development of dietary RNAi toxicity assays for relevant non-target arthropods within the RNAi crop agroecosystem is the critical first step to establish a standardized ERA protocol to evaluate potential risks associated with this novel biotechnology. Results from this project reflect the complexity in the formulation of ERA for RNAi transgenic crops. Although dietary RNAi has negligible impacts on the majority of tested surrogate species, we did observe some effects on their life history traits, which warrants further investigation. The differential responses to ingested dsRNAs in lady beetles indicates that 1) arthropod-active dsRNAs can negatively impact NTAs under the worst case scenarios; and 2) sequence identity (e.g., 21mer match) between the target and non-target arthropods, in some cases, is not informative of the potential impacts on NTAs. Therefore, results from the dietary RNAi toxicity assay, not bioinformatics analysis, should be the key permissible evidences for the regulatory agencies to consider. To refine our current ERA framework, we need to gain a better understanding of 1) the molecular mechanism of RNAi (mode of action) in transgenic crops, especially the processing of insect-active dsRNAs in plants; 2) the differential responses toward dsRNA-mediated RNAi among insects; and 3) the genomic information of both target and non-target organisms. Peer-reviewed publications 11 published (2 review and 9 research manuscripts), 4 submitted, and 1 in preparation.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Rodrigues, TB, C Khajuria, HC Wang, N Matz, DC Cardoso, FH Valicente, X Zhou, B Siegfried. 2014. Validation of reference housekeeping genes for gene expression studies in Western corn rootworm (Diabrotica virgifera virgifera). PLoS One. 9(10): e109825.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Yang, CX, HP Pan, Y Liu, X. Zhou. 2014. Selection of reference genes for expression analysis using quantitative real-time PCR in pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae). PLoS One. 9(11): e110454.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Yang CX, Pan HP, Liu Y, X. Zhou. 2015. Stably expressed housekeeping genes across developmental stages in the two-spotted spider mite, Tetranychus urticae. PLoS One. 10(3): e0120833.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Yang CX, Pan HP, Liu Y, X. Zhou. 2015. Temperature and development impacts on housekeeping gene expression in the cowpea aphid, Aphis craccivora, Koch (Hemiptera, Aphidiae). PLoS One. 10(6): e0130593.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Velez A M., J Jurzenski, N Matz, X Zhou, HC Wang, M Ellis, BD Siegfried. 2015. Developing an in vivo toxicity assay for honey bees, Apis mellifera L., to dietary RNAi. Chemosphere. 144:1083-90.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Xu, L. H., Zeng, B. S., Norland, J. E., Huang, Y. P. and X. Zhou. 2015. The coming of RNA-based pest controls. Journal of Plant Protection. 42, 673-690.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Pan, H. P., X. W. Yang, B. D. Siegfried and X. Zhou. 2015. A comprehensive selection of reference genes for RT-qPCR analysis in a predatory lady beetle, Hippodamia convergens (Coleoptera: Coccinellidae). PLoS One. 10: e0125868.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Pan, H. P., X. W. Yang, K. Bidne, R. L. Hellmich, B. D. Siegfried and X. G. Zhou. 2015. Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator." PLoS One. 10: e0129482.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Roberts, A. F., Y. Devos, G. N. Y. Lemgo and X. Zhou. 2015. Biosafety research for non-target organism risk assessment of RNAi-based GE plants. Front Plant Sci. 6:958.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Yang CX, Pan HP, Zhang DY, Zhang ZH, Siegfried BD, Liu Y, X. Zhou. 2015. Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae). Scientific Reports. 5: 18201.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Huipeng Pan, Linghua Xu, Jeffrey Edward Noland, Hu Li, Blair D. Siegfried, Xuguo Zhou. 2016. Assessment of potential risks of dietary RNAi to a soil micro-arthropod, Sinella curviseta Brook (Collembola: Entomobryidae). Frontiers Plant Science. 7: 1028.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Pan HP, Yang XW, Bidne K, Hellmich RL, Siegfried BD, X. Zhou. In vivo RNAi toxicity assay suggests negligible impacts of ingested dsRNAs on monarch butterfly, Danaus plexippus (L.).
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Chunxiao Yang, Huipeng Pan, Kunzheng Deng, Hongjun Zhang, Deyong Zhang, Yong Liu, Liangying Dai, Xuguo Zhou. 2016. Selection and evaluation of reference genes for RT-qPCR analysis in the seven-spotted ladybeetle, Coccinella septempunctata.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Xiaowei Yang, Huipeng Pan, Xuguo Zhou. Identification of reliable and condition specific reference genes for RT-qPCR in the invasive ladybeetle Harmonia axyridis.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Jeffrey E. Noland, Huipeng Pan, Blair D. Siegfried, Xuguo Zhou. In vivo toxicity of dsRNA demonstrates negligible impact on Folsomia candida Willem (Collembola: Isotomidae) gene expression and life history traits.
• Type: Journal Articles Status: Other Year Published: 2016 Citation: Huipeng Pan, Xiaowei Yang, Blair D. Siegfried, Xuguo Zhou. Impact of in vivo RNAi toxicity assay on ladybeetles suggests significantly impacts of ingested dsRNAs on ladybeetles.

Progress 09/01/14 to 08/31/15

Outputs
Target Audience:Regulatory agencies and government Scientists from both academia and industry Stakeholders including farmers, farm communities, and private sectors General public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?PARTICIPANTS: This proposal represents a collaborative effort among scientists from the University of Kentucky (PI Zhou) and the University of Nebraska (Co-PI Siegfried). Richard Hellmich (USDA-ARS) has been providing monarch butterfly eggs for the development of RNAi toxicity assay. Funding from this proposal currently supports two post-doctoral associates at the University of Kentucky and at the University of Nebraska, respectively, and a temporary laboratory technician ( an exchange graduate student from the China Agricultural University) at the University of Kentucky. How have the results been disseminated to communities of interest?1. Dr. Andrew Robert, the director of the Center for Environmental Risk Assessment, International Life Sciences Institute Research Foundation, Dr. Ray Layton, a Research Fellow for Environmental Safety at the DuPont Pioneer, and PD Zhou secured a conference grant entitled "Susceptibility of non-target organisms to gene silencing or suppression triggered by environmental exposure to a pesticidal RNA moiety" from the Biotechnology Risk Assessment Grant (BRAG), NIFA-USDA. Funding was used to support the 13th International Symposium on the Biosafety of Genetically Modified Organisms, which was held at Cape Town, South Africa between November 9 and 13, 2014. 2. This year, we have published seven peer-reviewed journal articles on the selection of appropriate reference genes for Quantitative real-time PCR (qRT-PCR) analysis for both target and non-target arthropods. Also, results from the early-tier risk assessment of honeybee, Apis mellifera, have been accepted by journal of Chemosphere. The ecological risk assessment of dietary RNAi on monarch butterfly, Danaus plexippus, has been submitted. A review paper entitled "Biosafety research for non-target organism risk assessment of RNAi-based GE plants" resulted from the international symposium at South Africa has been submitted to Frontiers in Plant Science. 3. Results from the proposed research have been presented at the national and international conferences and research institutions. What do you plan to do during the next reporting period to accomplish the goals?We have completed the early-tier risk assessment of GE RNAi maize on one species of soil decomposers, Sinella curviseta, and we are currently preparing the manuscript for the subsequent submission in 2015. To complete our evaluation of the biological impacts of dsRNAs on soil decomposers, we have now focused on the risk assessment of dietary RNAi on the other springtail species, Folsomia candida. The entire project should be concluded by 2016.

Impacts
What was accomplished under these goals? PROGRESS: 2014/09 TO 2015/08 OUTPUTS: This year we have accomplished the majority of our goals in the proposed research. We now have established standardized protocols for the dietary RNAi toxicity assays for non-target arthropods representing a diverse ecological functions, including incidental pollen feeders, honeybee, Apis mellifera, and monarch butterfly, Danaus plexippus, bio-control agents, convergent lady beetle, Hippodamia convergens, multicolored Asian lady beetle, Harmonia axyridis, and pink spotted lady beetle, Coleomegilla maculata, and soil decomposers Sinella curviseta, and Folsomia candida (Objective 2). We have continued our efforts on the development of MIQE (Minimum Information for publication of Quantitative real-time PCR Experiments) guidelines for the non-target arthropods to validate the results from our dietary RNAi toxicity assays at the transcription level. Finally, we have completed the early-tier risk assessment of GE RNAi maize on two species of incidental pollen feeders (honeybee, Apis mellifera, and monarch butterfly, Danaus plexippus), three species of bio-control agents (convergent lady beetle, Hippodamia convergens, multicolored Asian lady beetle, Harmonia axyridis, and pink spotted lady beetle, Coleomegilla maculate), and one species of soil decomposers (Sinella curviseta) (Objective 3). .

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: 1. Rodrigues, TB, C Khajuria, HC Wang, N Matz, DC Cardoso, FH Valicente, X Zhou, B Siegfried. 2014. Validation of reference housekeeping genes for gene expression studies in Western corn rootworm (Diabrotica virgifera virgifera). PLoS One. 9(10): e109825.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: 2. Yang, CX, HP Pan, Y Liu, X. Zhou. 2014. Selection of reference genes for expression analysis using quantitative real-time PCR in pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae). PLoS One. 9(11): e110454.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: 3. Pan HP, Yang XW, Siegfried BD, X. Zhou. 2015. A comprehensive selection of reference genes for qPCR analysis in a predatory lady beetle, Hippodamia convergens (Coleoptera: Coccinellidae). PLoS One. 10(4): e0125868.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: 4. Pan HP, Yang XW, Bidne K, Hellmich RL, Siegfried BD, X. Zhou. 2015. Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator. PLoS One. 10(6): e0129482.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: 5. Yang CX, Pan HP, Liu Y, X. Zhou. 2015. Stably expressed housekeeping genes across developmental stages in the two-spotted spider mite, Tetranychus urticae. PLoS One. 10(3): e0120833.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: 6. Yang CX, Pan HP, Liu Y, X. Zhou. 2015. Temperature and development impacts on housekeeping gene expression in the cowpea aphid, Aphis craccivora, Koch (Hemiptera, Aphidiae). PLoS One. 10(6): e0130593.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: 7. Developing an in vivo toxicity assay for honey bees, Apis mellifera L., to dietary RNAi. 2015. Velez A M., J Jurzenski, N Matz, X Zhou, HC Wang, M Ellis, BD Siegfried. Chemosphere.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: 8. Yang CX, Pan HP, Zhang DY, Zhang ZH, Siegfried BD, Liu Y, X. Zhou. 2015. Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae). Scientific Reports.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: 9. Pan HP, Yang XW, Bidne K, Hellmich RL, Siegfried BD, X. Zhou. 2015. In vivo RNAi toxicity assay suggests negligible impacts of ingested dsRNAs on monarch butterfly, Danaus plexippus (L.).
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: 10. Roberts, A., Y Devos, G Lemgo, X Zhou. 2015. Biosafety research for non-target organism risk assessment of RNAi-based GE plants. Frontiers in Plant Science.

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: Regulatory agencies and government Scientists from both academia and industry Stakeholders including farmers, farm communities, and private sectors General publics Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This proposal represents a collaborative effort among scientists from the University of Kentucky (PI Zhou) and the University of Nebraska (Co-PI Siegfried). Richard Hellmich (USDA-ARS) has been providing monarch butterfly eggs for the development of RNAi toxicity assay. Funding from this proposal currently supports two post-doctoral associatesat the University of Kentucky and at the University of Nebraska, respectively, and a temporary laboratory technician at the University of Kentucky. How have the results been disseminated to communities of interest? Regulatory agencies and government Scientists from both academia and industry Stakeholders including farmers, farm communities, and private sectors General publics What do you plan to do during the next reporting period to accomplish the goals? We are wrapping up the project by finishing up the dietary RNAi toxicity assays on the remaining incidental pollen feeder, monarch butterfly D. plexippus, and soil decomposers, springtails Folsomia candida and Sinellacurviseta.

Impacts
What was accomplished under these goals? This year we have been focusing on 1) establishing standardized validation protocols for the dietary RNAi toxicity assays developed in last year(Objective 2); and 2) carrying out the early-tier risk assessment of arthropod-resistant transgenic RNAi maize on selected non-target arthropods (Objective 3). To validate newly developed dietary RNAi toxicity assays, we have established standardized Quantitative real-time PCR (qRT-PCR) analysis protocols for both target and non-target insect species following the MIQE(Minimum Information for publication of Quantitative real-time PCR Experiments) guideline. qRT-PCR is a rapid and reliable method for the detection and quantification of gene expression during different biological processes. To ensure the accuracy, a critical component in qRT-PCR analysis is to normalize data by measuring in parallel the expression of a reference gene from the same samples. Housekeeping genes, involved in basic cellular functions, are typically maintaining stable and constitutive expression in all cells and regardless of physiological conditions. We currently have concluded reference gene selection studies on the incidental pollen feeders, honeybee, Apis mellifera, and monarch butterfly, Danaus plexippus, bio-control agents, ladybugs, Harmonia axyridis and Hippodamia convergens, and several species of sap-sucking insects. The inclusion of aphids and spider mites, the prey of bio-control agents and the vehicle of insect-active dsRNAs, is to lay a foundation for the future dietary RNAi toxicity assays with live preys instead of the synthesized dsRNAs. The combined results from our dietary RNAi toxicity assays suggested that 1) no significant biological effects of either honeybee or Western corn rootworm, Diabrotica virgifera virgifera (Dvv), vATPase dsRNAs on honeybees, although transcriptional impacts do exist; 2) dsRNA is stable under the conditions of exposure in both larval and adult bee RNAi toxicity assays. In addition, in the presence of the honeybees, the dsRNA is stable; 3) Dvv vATPase dsRNA was cross-active at the transcriptional level with vATPases from both ladybeetles; and 4) biological impacts,however, were only observed in H. convergens, indicating a differential response to the ingested dsRNA in ladybeetles from the same family.Different non-target species, even within the same taxonomic group, respond to dsRNA-mediated RNAi treatment differently. For example, among the two ladybug species, H. convergens is significantly more responsive toward dsRNA-mediated RNAi treatments than H. axyridis, and displayed strong cross-reaction with the Western corn rootworm dsRNAs. Surprisingly, in comparison to H. convergens, H. axyridis vATPase has more 21mer matches with Western corn rootworm vATPase. This result is not consistent with the notion that the 21mer matches (the number of the exact same 21 nucleotide sequence matches between the target and non-target species) dictates the sensitivity to dsRNA-mediated RNAi treatments. Results from bees and ladybugs suggest that RNAi crops, under the worst case scenario, can potentially impact non-target organisms at the transcription level. However, additional assessments are warranted to investigate the impacts at the organismal level, including both mortality and life history traits.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Rodrigues, TB, C Khajuria, HC Wang, N Matz, DC Cardoso, FH Valicente, X Zhou, B Siegfried. 2014. Validation of reference housekeeping genes for gene expression studies in Western corn rootworm (Diabrotica virgifera virgifera). PloS One. 9(10):e109825.
• Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Yang, CX, HP Pan, Y Liu, X. Zhou. 2014. Selection of reference genes for expression analysis using quantitative real-time PCR in pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae). PLoS One. 9(11):e110454.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Pan,HP, XW Yang, K Bidne, R Hellmich, B Siegfried, X Zhou. Selection of reference genes for qRT-PCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator. Gene.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Pan, HP, XW Yang, K Bidne, R Hellmich, B Siegfried, X Zhou. A comprehensive selection of reference genes for quantitative real-time PCR analysis in a predatory natural enemy, Hippodamia convergens (Coleoptera: Coccinellidae). Journal of Pest Science.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Yang, CX, HP Pan, Y Liu, X. Zhou. Stably expressed housekeeping genes across developmental stages in the two-spotted spider mite, Tetranychus urticae. PLoS One.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Yang CX, HP Pan, Y Liu, X. Zhou. 2014. Temperature and development impacts on housekeeping gene expression in the cowpea aphid, Aphis craccivora Koch (Hemiptera, Aphidiae) PLoS One.

Progress 09/01/12 to 08/31/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This proposal represents a collaborative effort among scientists from the University of Kentucky (PI Zhou) and the University of Nebraska (Co-PI Siegfried). Richard Hellmich (USDA-ARS) has been providing monarch butterfly eggs for the development of RNAi toxicity assay. Funding from this proposal currently supports two post-doctoral associates at the University of Kentucky and at the University of Nebraska, respectively, and a temporary laboratory technician at the University of Kentucky. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We are on track to complete all the stated objectives which will provide answers to questions directly pertaining to the development of a risk assessment framework for the RNAi crops. The newly established in vivo RNAi toxicity assay for each surrogate species will allow us to define the test conditions (worst case scenario), and determine the appropriate endpoint measurements for the risk assessment of RNAi crops. The research will directly assist in the formulation of ecological risk assessment framework for RNAi transgenic crops by establishing in vivo RNAi toxicity assays for relevant surrogate species representing a diverse community of non-target arthropods within the RNAi maize agroecosystem.

Impacts
What was accomplished under these goals? We have 1) developed in vivo RNAi toxicity assay for the incidental pollen feeders, honeybee, Apis mellifera, and monarch butterfly, Danaus plexippus, bio-control agents, ladybugs, Harmonia axyridis and Hippodamia convergens, and decomposers, springtails, Folsomia candida and Sinella curviseta; 2) refined the worst case scenario for the RNAi toxicity test; and 3) investigated risk assessment endpoints and test duration for respective surrogate species. Sequence similarity, dose, and exposure are the key factors that define the worst case scenario for the RNAi-toxicity test. However, during the development of in vivo RNAi toxicity assay for the surrogate species, sequence identity, especially identical sequences of at least 21 nucleotides (20mers) between target and non-target arthropods of arthropod-active genes, might dictate the specificity of transgenic RNAi maize. Interestingly, different surrogate species, even within the same taxonomic group, respond to dsRNA-mediated RNAi treatment differently. Our preliminary results showed that ladybugs are more sensitive toward dsRNA-mediated RNAi treatment than honeybees. However, among the two ladybug species, Hippodamia convergens is significantly more responsive toward dsRNAs than Harmonia axyridis, and displayed strong cross-reaction using the Western corn rootworm dsRNA. The validity of in vivo RNAi toxicity assay was examined at the transcriptional level using a readily available qRT-PCR protocol and at the translational level using a commercially available V-ATPase polyclonal antibody. In addition to mortality, we also documented other life history traits, including body weight, development time, and fecundity, as the potential endpoints for the ecological risk assessment of RNAi crops, especially under sublethal conditions of exposure.

Publications

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

Outputs
OUTPUTS: The Aims of objective-1 include 1) selection of appropriate surrogate species for non-target arthropods that represent different habitats and diverse ecological functions within the transgenic RNAi maize system, and 2) Clone and sequence V-ATPase subunit A, which is the target sequence for transgenic maize expressing dsRNA, from surrogate species selected in Aim-1. The Working Hypothesis is that the primary structure of housekeeping genes such as V-ATPase is highly conserved across diverse taxonomic groups. For the first year, we have completed both aims. Based on reviewers' suggestions, we removed European corn borer, Ostrinia nubilalis, a non-target herbivore, from the list of surrogate species. For Aim-2, we have successfully obtained the complete open reading frames (ORFs) of V-ATPase subunit-A in selected surrogate non-target arthropods (NTAs) using RACE (rapid amplification of cDNA ends)-PCR and standardized molecular cloning techniques. A 400bp region which has the highest sequence similarity among target and nontarget surrogate species has been selected as the target region for the subsequent in vivo RNAi toxicity assay. PARTICIPANTS: This proposal represents a collaborative effort among scientists from the University of Kentucky (PD Zhou) and University of Nebraska (Co-PD Siegfried). Funding from this proposal currently supports a post-doctoral research associate at the University of Nebraska and two research technicians at the University of Kentucky. TARGET AUDIENCES: Agricultural biotechnologists in academia, industry, and federal regulatory agencies. Ultimately, information acquired from this research should be disseminated to general public through risk analysis and risk communication processes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Although we are only 1 year into a three year project, we are on track to complete all the stated objectives which will provide answers to questions directly pertaining to the potential risk of RNAi crops to non-target arthropods within the transgenic RNAi maize system. The conclusion of our initial cloning efforts of the V-ATPase subunits allows us to develop the individual in vivo RNAi toxicity bioassays for selected NTAs respectively which will provide molecular evidence for the evaluation of early-tier risk hypotheses. Our preliminary findings have been reported at the XXIV International Congress of Entomology (Zhou and Siegfried, Developing a framework for assessing the risks associated with RNAi crops on non-target organisms, Invited symposium talk, Deagu, Korea, August 19 - 25, 2012).

Publications

• No publications reported this period