RNA Interference for Insect Pest Management

Ana Maria Velez, Asst. Professor; Entomology Department UNL Author

11/06/2023 Added

73 Plays

Description

RNA interference (RNAi) is a gene-silencing mechanism conserved across eukaryotic organisms. RNAi has been widely used as a research tool to study gene function. Over the past decade, RNAi has been investigated for agricultural applications, including managing plant insect pests. During this presentation, I will provide an overview of the current state of using RNAi for insect pest management.

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[00:00:00.750]The following presentation
[00:00:02.220]is part of the Agronomy and Horticulture Seminar series
[00:00:05.790]at the University of Nebraska-Lincoln.
[00:00:08.490]Good afternoon and welcome to the Department of Agronomy
[00:00:11.040]and Horticulture Seminar series.
[00:00:13.320]It is my pleasure to introduce Dr. Ana Maria Velez Arango.
[00:00:17.520]Dr. Velez Arango received her Bachelor's of Science degree
[00:00:20.460]from Pontificia Universidad Javeriana
[00:00:23.790]and her MS in Entomology
[00:00:25.260]from the National University of Columbia.
[00:00:28.140]She received her PhD here
[00:00:29.490]at the University of Nebraska-Lincoln in 2013
[00:00:32.250]working on resistance to Cry1F in fall armyworms.
[00:00:36.510]Dr. Velez Arango joined the Department of Entomology in 2013
[00:00:40.230]as a postdoctoral researcher
[00:00:42.210]and as a research assistant professor in 2016.
[00:00:45.720]As of 2023,
[00:00:47.310]Dr. Velez Arango serves as an associate professor
[00:00:49.800]in the Department of Entomology
[00:00:51.450]with research and teaching responsibilities.
[00:00:54.210]Her work focuses on understanding how insects respond
[00:00:56.940]and adapt to chemical stressors
[00:00:58.560]and specifically on current and emerging technology
[00:01:00.960]used for insect pest management
[00:01:03.060]such as transgenic plants that express toxins
[00:01:05.520]from bacillus thuringiensis, BT and RNA interference.
[00:01:09.900]Her labs work consider these methods at the molecular,
[00:01:12.750]organismal and population level.
[00:01:15.570]Dr. Velez Arango has produced 29 peer reviewed articles
[00:01:18.450]along with 10 invited reviews and book chapters.
[00:01:21.090]She also owns six patents related to her work in RNAi.
[00:01:25.170]Dr. Velez Arango received the DuPont Young Professor Award
[00:01:28.260]in 2016
[00:01:29.820]and was recognized
[00:01:30.900]as distinguished Multicultural CASNR alumni in 2019.
[00:01:35.010]Dr. Velez Arango will be presenting
[00:01:36.480]on RNA Interference for Insect Pest Management.
[00:01:39.720]Thank you Christian for the introduction.
[00:01:41.790]Absolutely my pleasure.
[00:01:43.277]I don't think, I think I got it here.
[00:01:46.040]Okay before I start, I wanna thank Dr. Ashley Stengel
[00:01:50.400]for inviting me to give this presentation.
[00:01:53.430]And what I wanted to do with this presentation
[00:01:55.710]is to make it basic enough
[00:01:57.840]for those of you who don't have a molecular background
[00:02:00.540]but give enough detail for those of you
[00:02:02.310]that have a molecular background.
[00:02:05.010]And also not a lot of time to present everything
[00:02:08.340]that I have done with RNAi
[00:02:10.140]so I'm just kind of showing a little bit
[00:02:12.510]and I want to give you some history on this technology.
[00:02:16.590]Before I start and I forget,
[00:02:18.660]I wanna acknowledge the main people that has helped me
[00:02:21.990]with this research.
[00:02:23.250]Dr. Blair Siegfried who was the one
[00:02:25.560]first that introduced me to this work.
[00:02:28.230]Dr. Ken Narva that used to work in Dow AgroSciences,
[00:02:31.560]now he works at Greenlight Biosciences
[00:02:33.960]and he's still one of my biggest mentors
[00:02:36.990]and Dr. Elaine Fishilevich,
[00:02:38.580]who also was a big mentor on RNAi for me.
[00:02:41.310]Now she works with the human component of
[00:02:43.137]for RNAi therapeutics.
[00:02:45.480]And then the work that I will present today
[00:02:47.670]are from my students and postdocs.
[00:02:49.590]So Mariana Sanchez, Matthew Welter, Dr. Molly Darlington
[00:02:54.090]and Dr. Suresh Varsani
[00:02:55.707]and then my collaborators from Bayer CropScience,
[00:02:58.560]Dr. Will Moar and Chitvan Khajuria
[00:03:01.260]and Kristopher Silver from Kansas State University.
[00:03:07.457]A brief outline of what I will talk today.
[00:03:09.240]First, I want talk to you about how RNAi was discovered,
[00:03:13.350]some of the general applications
[00:03:15.000]because it's not only used for pest management.
[00:03:17.730]A RNAi for Insect Pest Management, so how it works,
[00:03:21.210]some of the delivery strategies
[00:03:22.860]and then just narrow down and focus on my work
[00:03:25.470]on Western Corn Rootworm Management,
[00:03:27.390]some history, trait identification, mode of action
[00:03:30.660]and then field population susceptibility.
[00:03:34.380]So in terms of the discovery,
[00:03:36.060]it was discovered
[00:03:38.370]like a lot of things in science by mistake.
[00:03:41.820]So there was researchers in California
[00:03:43.740]trying to produce petunia that were more purple
[00:03:47.010]and what they find is that they were actually producing
[00:03:50.760]flowers that were white
[00:03:52.020]or like with these patterns with white.
[00:03:55.560]They didn't know what was happening.
[00:03:59.070]And then eventually, they start finding this in fungi
[00:04:02.610]and then C. Elegans
[00:04:04.500]and it was Andrew Fire and Craig Mello
[00:04:07.200]that finally describe it in C. elegans
[00:04:10.260]as a post-transcriptional gene silencing.
[00:04:12.780]So instead of putting more of that messenger RNA
[00:04:16.170]that will produce the purple,
[00:04:17.910]so the messenger RNA like kind of like the COVID protein,
[00:04:20.520]what they were actually getting is this
[00:04:23.040]and I'll talk more a little bit of how that work
[00:04:24.783]or a double stranded RNA.
[00:04:26.880]And all of these work on the description of RNA interference
[00:04:30.060]gave them the Nobel Prize in Physiology and Medicine
[00:04:32.670]in 2006.
[00:04:36.060]So when we talk about RNA, we need to go back
[00:04:39.120]to the Central Dogma of Biology
[00:04:41.370]and I think maybe the laser here will work.
[00:04:44.370]Yeah, so we need to go back to the Central Dogma of Biology
[00:04:48.480]where you have your DNA,
[00:04:49.560]then the nucleus is transcribing to messenger RNA.
[00:04:53.160]Then this messenger RNA is translated into a protein.
[00:04:56.160]So basically what we do with RNA interference
[00:04:58.560]is we degrade this messenger RNA
[00:05:00.510]and there is no production of protein.
[00:05:02.520]If you remember with the COVID vaccines,
[00:05:05.280]we were introducing messenger RNA
[00:05:07.500]to produce the spike proteins
[00:05:09.210]to then generate the immunity.
[00:05:11.460]In this case, we're actually eliminating
[00:05:14.220]innate messenger RNAs.
[00:05:20.220]So it is a post-transcriptional gene silencing mechanism
[00:05:24.060]that is conserved across all eukaryotic organisms.
[00:05:27.480]And then the mRNA degradation prevents
[00:05:29.820]the formation of that protein
[00:05:31.320]and we describe this as gene silencing.
[00:05:35.400]So there is three different pathways of gene silencing
[00:05:39.150]that we have studied:
[00:05:40.410]the siRNA pathway, miRNA pathway and piRNA pathway.
[00:05:45.300]All of these pathways are important
[00:05:47.010]for gene regulation in our bodies.
[00:05:49.890]But when we talk about the siRNA pathway,
[00:05:52.830]that is the one that we use or we explore
[00:05:54.870]for pest management
[00:05:57.390]is also used or is triggered by exogenous
[00:06:01.440]double stranded RNA or dsRNA.
[00:06:03.420]And I will be referring to dsRNA a lot in this presentation
[00:06:06.930]and this evolve as a defense mechanism
[00:06:09.150]against nucleic acid
[00:06:11.490]so like a transposable elements and viruses.
[00:06:17.580]And as we get at the end of the presentation,
[00:06:19.500]we'll get back to this
[00:06:20.490]because it gets them more complicated than we thought.
[00:06:23.160]So in terms of applications in general for research,
[00:06:26.220]it has been used for a long time for functional genomics.
[00:06:29.430]So just to do that genotype to phenotypes
[00:06:32.640]to understand what is the roles of those genes.
[00:06:35.400]For gene therapy is silencing of viruses
[00:06:38.310]or genes associated with diseases.
[00:06:40.830]So I mentioned Elaine Fishelevich.
[00:06:42.870]She now works in a company that they are developing
[00:06:46.170]new technology or a new therapy.
[00:06:48.030]I think it's now in the market for rare genetic diseases.
[00:06:52.860]And then in agriculture for control of pest and pathogens,
[00:06:55.800]so insects, nematodes, fungi and viruses
[00:06:58.860]for to improve plant nutrition
[00:07:00.840]and therapeutic for bees for viruses and Varroa mites.
[00:07:04.830]But for this talk, I'll mainly talk about insects.
[00:07:10.620]So how it works for insects.
[00:07:12.930]What we do is we knock down or silence those two terms
[00:07:16.470]are used interchangeably.
[00:07:18.960]Genes with double stranded RNA
[00:07:21.300]and then this generates a lethal or a sublethal effect
[00:07:24.180]that will be useful to control those insects best.
[00:07:27.720]So what are the advantages of this new technology?
[00:07:30.750]First is really sequences specific.
[00:07:32.760]So when we're talking about managing insect pests,
[00:07:35.820]it depends on the sequence.
[00:07:37.590]So it will target a really specific gene species.
[00:07:40.150]So it has high selectivity
[00:07:42.240]and has low environmental persistence.
[00:07:44.700]Those are two things that we are always looking for
[00:07:47.820]when we're thinking in terms of insect pest management.
[00:07:50.910]Disadvantages is that as we started exploring
[00:07:54.240]more of this technology,
[00:07:55.410]we find that there is variability
[00:07:57.180]between different insects
[00:07:58.530]so it doesn't work well in our insect species.
[00:08:02.190]It's lower mortality.
[00:08:03.780]So if you think of synthetic insecticides,
[00:08:05.610]you can find mortality 24 to 48 hours.
[00:08:08.310]Then we would move to BTs.
[00:08:10.470]Mortality happens in around seven days.
[00:08:13.050]With RNAi, it happens in 10 days.
[00:08:15.030]They start feeling sick earlier
[00:08:16.590]and slow the rate of consumption
[00:08:19.200]but it takes about 10 days for them to die.
[00:08:23.100]And then this is kind of like a...
[00:08:27.570]we have higher selectivity
[00:08:29.100]but we can target them less pest
[00:08:31.020]with this specific technology.
[00:08:35.280]Some of the delivery strategies that are being explored
[00:08:39.120]will depend on the insect
[00:08:40.770]and the efficiency for that specific insect.
[00:08:43.590]I cannot go into a lot of detail.
[00:08:45.150]This can be a whole new presentation.
[00:08:47.910]But we define it in with these terms
[00:08:50.250]so host-induced gene silencing or HIGS,
[00:08:53.160]that will be in a transgenic plant
[00:08:55.260]or it's also being explored in a genetically modified yeast
[00:08:59.730]for mosquito control.
[00:09:00.870]So they just put pellets in water
[00:09:03.090]for controlling mosquito larva,
[00:09:05.190]spray-induced gene silencing or SIGS,
[00:09:07.950]virus-induced gene silencing or VIGS
[00:09:10.800]and then trunk injections and root absorption.
[00:09:13.920]And then the trunk injections and root absorptions,
[00:09:17.460]those are more for insects that feed on sap like aphids.
[00:09:22.050]But for this talk, I mainly talk about the transgenics
[00:09:25.830]and the SIGS or spray-induced gene silencing.
[00:09:33.030]So in terms of hosting those gene silencing,
[00:09:35.370]the first report that this could be done was in 2007
[00:09:39.630]and was in this paper in "Nature"
[00:09:41.280]a biotechnology that is really famous
[00:09:43.710]and they show that with Western corn rootworm,
[00:09:45.780]they could express in corn this gene for the V-ATPase.
[00:09:50.400]And then what happens that is more important
[00:09:52.260]when we're thinking in terms of pest management
[00:09:54.360]is that it reduced the damage of the insect.
[00:09:56.850]And I'll go into a lot of more detail on
[00:09:59.700]Western corn rootworm biology.
[00:10:01.770]So that was the first demonstration of HIGS in 2007.
[00:10:06.270]And then there is another product
[00:10:09.600]and let's just go back there for a second.
[00:10:12.420]And these two products that I'm going to talk
[00:10:14.310]are the ones, those that are like either
[00:10:16.980]recently in the market or will be soon in the market.
[00:10:20.310]And I'll talk more about the HIGS
[00:10:22.590]later in the presentation.
[00:10:24.150]The other one is the spray-induced gene silencing.
[00:10:27.480]This is being developed by GreenLight Biosciences
[00:10:31.140]for Colorado potato beetle.
[00:10:33.210]And this product really relevant to the presentation today
[00:10:37.350]is open for EPA comments,
[00:10:39.840]so from the public for regulatory approval.
[00:10:42.780]So it will likely be in the market hopefully next year.
[00:10:49.500]So something to think about in term of this
[00:10:51.630]deliver strategies that originally when we was discovered,
[00:10:55.440]everybody thought this is amazing.
[00:10:57.240]We're going to solve all the pest management issues.
[00:11:00.780]This looks awesome.
[00:11:02.400]Then what we started to find is that it's,
[00:11:04.250]it doesn't look as good.
[00:11:06.090]We find that for Coleoptera or for beetles,
[00:11:08.310]they are really, really susceptible.
[00:11:10.050]We are still trying to understand
[00:11:11.430]why they are so susceptible to it,
[00:11:14.040]but then it's just really variable in other organisms.
[00:11:18.030]Lepidoptera doesn't work at all in some species
[00:11:23.490]and I can go into a whole presentation talking about that.
[00:11:26.243]Hemiptera, Orthoptera, Diptera and Hymenoptera
[00:11:30.570]is give and take, is really variable.
[00:11:33.660]The other factors that could affect it is a species.
[00:11:37.710]So even between different species, it might change.
[00:11:40.590]Life stages.
[00:11:42.450]They also vary in terms of susceptibility.
[00:11:45.480]Tissues and even genes can have different effectiveness
[00:11:49.560]between the species.
[00:11:52.470]And when we think into SIGS
[00:11:54.780]or the strain-induced gene silencing,
[00:11:57.780]we need to take in the formulation and stability
[00:12:01.980]in the environment
[00:12:02.820]because we will have UV light, rain and microorganisms.
[00:12:06.570]So for that, they are using a lot of different things
[00:12:09.090]to stabilize those products
[00:12:10.650]like bacterials, virus or yeast,
[00:12:12.510]liposomes, peptides and polymers and nanoparticles.
[00:12:16.140]This is an area of research that is growing
[00:12:19.050]but also for HIGS or host-induced gene silencing,
[00:12:23.370]we also need to think in terms of the internal
[00:12:27.570]physiology of the insect
[00:12:28.920]that might affect the effectiveness
[00:12:30.570]of that double strand RNA.
[00:12:31.980]So for example, in Hemiptera insects
[00:12:34.080]that I mentioned earlier,
[00:12:35.520]they have of RNAs or enzymes that can degrade
[00:12:38.670]these double strand RNA
[00:12:40.110]in their enzymes in the saliva.
[00:12:41.880]So that's one of the reasons why it doesn't work that well.
[00:12:44.610]And then in terms of for example in the mid gut,
[00:12:47.040]Lepidoterans or all your caterpillars
[00:12:50.130]have a lot of RNAs in their mid guts
[00:12:52.800]that degrade that before it can be even uptaken.
[00:13:01.500]So that was just a little bit of the wrap up
[00:13:03.540]of how we're using it for insect pest management.
[00:13:09.900]Now I would like to move into talking about
[00:13:14.280]how we are using it for Western corn rootworm.
[00:13:18.720]So just a little bit of history
[00:13:20.610]for those of you that are maybe not familiar
[00:13:23.130]with the Western corn rootworm.
[00:13:25.110]The Western corn rootworm is the most important pest
[00:13:27.960]of corn in the US corn belt.
[00:13:30.510]The larvae damaged the root as you can see here.
[00:13:33.003]This is a non-infested root, an infested root.
[00:13:36.660]What happens is with high winds,
[00:13:38.700]the plants lodges and then that affects the yield of corn.
[00:13:43.379]This is now considered a $2 billion pest
[00:13:46.680]in terms of the money that need to invest in inputs
[00:13:50.910]and then yield losses.
[00:13:53.280]And to make it even worse is a highly adaptable pest.
[00:13:56.790]So these species has evolved resistance to
[00:14:00.240]all the groups of synthetic insecticides, not everywhere
[00:14:02.760]but in different places in the US.
[00:14:05.100]Cultural techniques that I think that's amazing.
[00:14:07.380]They figure out that in Illinois,
[00:14:09.510]that they can lay eggs on soybeans
[00:14:11.370]because next year is going to be corn
[00:14:14.010]and then to transgenics, so the BT protein.
[00:14:16.470]So they are just really highly adaptable.
[00:14:18.540]We still don't understand why.
[00:14:22.290]So because of that, we need new traits
[00:14:24.630]for Western corn rootworm management.
[00:14:28.410]In terms of transgenic crops for insect pest management,
[00:14:31.350]we have the first generation.
[00:14:33.120]That that first generation for rootworms came in 2003.
[00:14:37.410]That was the Bacillus thuringiensis proteins.
[00:14:41.340]The second generation really recent.
[00:14:43.650]The first product was commercialized last year in 2022.
[00:14:47.700]And then the third generations
[00:14:49.590]are proteins from other sources like other types of bacteria
[00:14:53.190]or even ferns
[00:14:54.510]that are just in the pipeline of development.
[00:15:01.770]So before I move into this,
[00:15:03.600]for those of you that are not familiar
[00:15:05.180]of how biotech products work or what is the pipeline
[00:15:08.730]or how they are developed,
[00:15:10.830]they are first discover.
[00:15:12.090]So they check a lot of different traits,
[00:15:14.130]see which ones are efficient.
[00:15:15.870]Then from those pick a couple to explore more.
[00:15:19.050]Then they do target characterization,
[00:15:21.420]more into detail on how that will work
[00:15:23.730]and maybe greenhouse experiments.
[00:15:25.440]Eventually express it in plants planted in the field
[00:15:28.950]and see how it will work in different environments.
[00:15:31.830]Another big component is the ecological risk assessment
[00:15:35.010]that has two components
[00:15:36.900]and I want you to consider that for biotech traits,
[00:15:39.630]these ecological risk assessment is way more stringent
[00:15:42.810]than any of the synthetic pesticides or even biologicals.
[00:15:47.460]And these two components are the effects
[00:15:49.290]on non-target organisms including humans resistance.
[00:15:52.440]And then eventually the product is released.
[00:15:55.110]And during this process, we also study the mode of action
[00:15:59.010]to both understand how could potentially affect
[00:16:01.740]other organisms
[00:16:03.120]and how resistance could evolve.
[00:16:06.450]So I'm going to talk a little bit about that.
[00:16:10.680]So just going back, I told you about 2007
[00:16:13.980]where the first publication came up into the market,
[00:16:17.220]into say that it will work against Western corn rootworm.
[00:16:22.410]There was different genes that they tested
[00:16:24.660]and they finally found that a really effective gene
[00:16:27.330]is this gene, the DvSnf7.
[00:16:30.870]This Snf7 gene is part of the endosomal
[00:16:33.660]sorting complex pathway
[00:16:35.940]and basically to put it in layman terms,
[00:16:38.010]it helps the cell take all the garbage out.
[00:16:41.070]So one interesting thing is that when you give these
[00:16:43.050]for example to the rootworms adults
[00:16:44.670]is that they start getting stinky as they starting to die
[00:16:47.580]because their cells cannot longer
[00:16:50.400]excrete all the things from their cells.
[00:16:53.340]So this product is by Bayer CropScience
[00:16:56.790]and is branded as a SmartStax PRO.
[00:16:59.620]It has three different modes of action
[00:17:02.280]that DvSnf7 double strand RNA
[00:17:04.680]and then two different BT proteins
[00:17:07.230]we do two different modes of actions.
[00:17:09.450]We're stacking it with three modes of action
[00:17:11.820]because as I mentioned, they're highly adaptable.
[00:17:14.070]So this is a way that we can maybe delay
[00:17:16.050]that resistance evolution.
[00:17:18.120]This product was registered by EPA in 2017
[00:17:22.200]and commercially released in 2022.
[00:17:24.957]And you might wonder why the big gap
[00:17:27.840]between that in part was COVID.
[00:17:30.120]But the other part is that when they're going to release
[00:17:32.520]any transgenic product,
[00:17:33.930]it needs to be approved in other countries for export.
[00:17:38.610]So it wasn't approved in China until 2021
[00:17:41.907]and that was one of the reasons why
[00:17:43.620]it wasn't commercial available until 2022.
[00:17:49.230]There is another product by Corteva Agriscience
[00:17:52.470]that express this gene, the SSJ1 or snakeskin.
[00:17:56.760]This gene has a different,
[00:17:58.080]completely different type of mode of action.
[00:18:00.000]Is really important for the binding between cells.
[00:18:04.230]So it just at the end you have like,
[00:18:06.660]you see that they can get separated.
[00:18:09.810]This is currently under regulatory revision.
[00:18:19.080]And then an important part to explain,
[00:18:21.330]I mentioned that ecological risk assessment.
[00:18:23.820]So for transgenics, we have a really defined framework
[00:18:28.860]for ecological risk assessment defined by the EPA.
[00:18:32.460]As I say, more stringent than for any other products
[00:18:35.190]for insect pest management
[00:18:37.050]and how this work is that
[00:18:39.300]or how it work initially for BTs is that you define
[00:18:42.180]a problem where you talk about your protection goals.
[00:18:45.870]Lower protection goals are usually maintained
[00:18:48.390]different ecological functions like pollination,
[00:18:50.790]predation and degradation of things in the soil.
[00:18:53.550]And then what are the endpoints?
[00:18:54.840]And by endpoints I mean mortality is going to affect
[00:18:57.810]fertility or other endpoints for other organisms.
[00:19:01.710]We eventually have to test those things
[00:19:04.680]and we do it in a tier approach where we do a tier one
[00:19:07.740]with a laboratory worst case scenario
[00:19:09.870]with the highest dose possible
[00:19:11.460]or like maybe 10 times higher than what you will express
[00:19:14.370]in the field.
[00:19:15.270]If you don't find effects here in the tier one,
[00:19:17.700]is deemed safe and then approved for that specific organism.
[00:19:22.650]We're going organism by organism.
[00:19:24.780]If they find there is something there,
[00:19:26.580]then they move to a tier two
[00:19:28.050]where is an extended laboratory semi-field,
[00:19:30.660]a more realistic dose to what they will encounter
[00:19:32.850]in the field.
[00:19:34.140]If they don't find anything else, they stop there.
[00:19:37.260]But if they find something else of concern,
[00:19:39.330]they move to a tier three
[00:19:40.770]that is more a field approach.
[00:19:42.210]Usually we don't get a lot into this piece.
[00:19:46.020]Then you do analysis of exposure.
[00:19:48.540]Are the organisms,
[00:19:50.220]and we're talking a lot of different organisms,
[00:19:52.440]going to even be exposed to this trait,
[00:19:55.830]potential effects and then
[00:19:57.660]the EPA takes the risk characterization
[00:19:59.790]and defines if the product can be released.
[00:20:02.160]So this one was the framework that we use for BTs.
[00:20:05.010]We wanted to know if this will work for RNAi.
[00:20:08.640]We found after years
[00:20:10.050]that it will work for the same framework.
[00:20:12.540]And it seems from a workshop that I recently was in DC,
[00:20:16.140]that is going to work for the third generation
[00:20:18.180]of transgenics too.
[00:20:21.150]So just really briefly, I did some work
[00:20:23.370]during my postdoc working on honeybees for this.
[00:20:27.150]But this work here is for the DvSnf7
[00:20:30.000]from Bayer CropScience.
[00:20:32.220]Basically what they find super specific,
[00:20:34.890]not a lot of effects on other organisms,
[00:20:38.400]yes they find in Southern corn rootworms
[00:20:40.405]but they have an 98.8 sequence identity
[00:20:43.020]really closely related to species.
[00:20:45.390]When we talk about BTs, you're affecting the same orders.
[00:20:48.630]In this case, you're affecting maybe some organisms
[00:20:51.990]from the same tribe.
[00:20:53.880]So really getting to really a specific.
[00:20:58.080]I forget that this one doesn't click.
[00:21:02.760]And then for the ecological risk assessment
[00:21:05.160]for the DvSnf7 using that EPA framework
[00:21:07.980]that I explained earlier,
[00:21:09.720]they did a really extensive testing where they check
[00:21:13.320]multiple non-target organisms including predators,
[00:21:16.650]parasitoids, pollinators, soil biota,
[00:21:19.440]aquatic and terrestrial vertebrates
[00:21:21.720]and then look at different endpoints,
[00:21:23.640]so survival, growth, development, reproduction.
[00:21:26.340]And they found that basically there was no adverse effect
[00:21:29.340]at a or above the level of the
[00:21:32.100]maximal expected environmental concentration.
[00:21:35.400]Now going to, I don't have a slide on that,
[00:21:37.470]but they also find that there is no effects on mammals.
[00:21:40.020]Mammals and vertebrates in general
[00:21:42.030]were used to degrade nucleic acids.
[00:21:47.130]So now to finalize, I'll going to talk a little bit of
[00:21:52.680]what has been done by my students.
[00:21:55.680]And within this framework,
[00:21:58.410]we have worked a little bit in trait discovery
[00:22:01.290]and characterization, mode of action
[00:22:03.870]and the ecological risk assessment in terms of resistance.
[00:22:08.250]And what we're finding is we're opening like everything
[00:22:10.680]more of a can of worms in a lot of cases.
[00:22:15.000]So this one we, in terms of trait identification,
[00:22:18.210]Mariana Sanchez, a Master's student,
[00:22:20.310]we wanted to explore chemoreceptors
[00:22:23.010]as a species-specific genes.
[00:22:25.047]And in this case, it will be even more specific
[00:22:27.930]that some of the other genes that have been explored
[00:22:30.480]just because those genes are more
[00:22:32.100]conserved between different species.
[00:22:34.140]In this case, it will be genes that are really
[00:22:37.080]species-specific.
[00:22:38.580]So we wanted to explore these ones
[00:22:41.700]and we look at different ones
[00:22:43.890]but I'm only going to talk about gustatory receptors
[00:22:46.500]or GR genes.
[00:22:47.990]In rootworms, they have three different genes:
[00:22:50.010]gr1, gr2, gr3.
[00:22:51.810]And is thought that these genes are related
[00:22:54.420]with the detection of CO2 by larva
[00:22:57.330]that gets them into finding those roots in the soil
[00:23:00.600]once they emerge.
[00:23:05.100]So for this, we use a parental RNAi.
[00:23:08.970]I did some of this work during my PhD,
[00:23:11.760]is kind of different.
[00:23:14.370]So in this case, we did the adult females feet
[00:23:17.430]on the double strand RNA
[00:23:19.410]and then we looked at the phenotype in the offspring.
[00:23:22.830]Everything that I have been talking to this point,
[00:23:25.140]we feed it to the larva or the adult
[00:23:27.090]and they die or show the phenotype.
[00:23:29.220]In this case, we're passing it to the next generation.
[00:23:37.440]And by the way, this doesn't work in all organisms
[00:23:40.440]this well.
[00:23:42.990]So we explore those three different genes.
[00:23:45.930]We find that with gr2, we can knock down or silence
[00:23:50.100]the gene on the adults, on the eggs
[00:23:53.280]and even on the larva we saw some effects.
[00:23:56.580]We also from these larva tested on these behavioral chambers
[00:24:01.530]where we have CO2 and water.
[00:24:04.620]The CO2 was basically carbonated water and normal water.
[00:24:08.310]And we find that actually those larva
[00:24:10.470]were not able to find the CO2.
[00:24:13.350]So if they have the receptor,
[00:24:14.820]they are able to find the CO2 like if it was the root.
[00:24:18.060]If that receptor is knocked down or silenced,
[00:24:20.670]then you will find that they will not be able to find
[00:24:23.427]the CO2.
[00:24:25.200]Eventually, we didn't continue exploring this idea.
[00:24:28.140]Really good to understand gene function
[00:24:30.510]but it was really variable.
[00:24:32.070]It will be really hard to produce that in a plant
[00:24:34.900]or in a sprayable.
[00:24:37.350]So one of the things that happens
[00:24:38.670]when you're working in these kind of things,
[00:24:40.110]you might be in some of the work that I did
[00:24:42.240]during my postdoc.
[00:24:43.320]We even expressed things on plants
[00:24:45.690]in collaboration with the other sciences
[00:24:47.580]but it, the plant didn't express enough to
[00:24:49.710]be able to take it to market.
[00:24:53.010]Then the other piece is the mode of action,
[00:24:55.470]really complicated mode of action
[00:24:58.740]and we have been working for years trying to
[00:25:00.930]figure out how it works.
[00:25:02.310]It doesn't work the same in all organisms
[00:25:04.320]and what we have learned from C. elegans
[00:25:06.480]cannot necessarily move into insects.
[00:25:10.470]So I divide, I like, we like to divide the mode of action
[00:25:13.350]in three places.
[00:25:14.340]One, uptake.
[00:25:15.240]So how is that double strand RNA
[00:25:17.220]getting uptake in the mid gut of the insect
[00:25:19.470]because everything that they are getting is through feeding.
[00:25:22.800]The double strand RNA
[00:25:23.940]just because of the physical, chemical properties
[00:25:25.980]couldn't get through the cuticle of the insect.
[00:25:29.040]The other piece is the processing
[00:25:30.600]that eventually generates that silencing
[00:25:34.110]and then how it moves from cell to cell.
[00:25:37.200]From our experiments with parental RNAi,
[00:25:39.600]we know it's moving from the gut to the ovaries.
[00:25:42.000]We really don't understand why.
[00:25:43.740]So that's kind of like the three steps.
[00:25:45.600]Each one of those steps have multiple genes
[00:25:48.390]that are in both.
[00:25:50.790]So Molly, Dr. Molly Darlington during her PhD work
[00:25:55.350]on trying to understand some of the puzzle pieces
[00:25:59.070]for the uptake.
[00:26:00.480]During my postdoc, I found DCR2 and then,
[00:26:05.160]oh it's not here.
[00:26:06.120]DCN argano-2, those two important proteins in the processing
[00:26:10.710]that are part of that.
[00:26:12.720]So for uptake, not going to go into a lot of detail.
[00:26:17.310]There was a lot of work from Molly
[00:26:19.650]but she wanted to evaluate the different routes
[00:26:22.050]of cellular uptake using pharmacological inhibitors.
[00:26:25.826]RNAi of RNAi, that's not a typo.
[00:26:28.770]That's a technique that we use that we knock down the genes
[00:26:31.470]that we want to study
[00:26:32.430]and then check the gene involved in the RNAi.
[00:26:37.350]And if you want more details
[00:26:39.240]or understand that a little bit better,
[00:26:40.710]you can ask me later.
[00:26:42.240]And then microscopy.
[00:26:43.350]So we have different ways that we can take cellular uptake.
[00:26:47.550]We can have phagocytosis, pinocytosis and receptor mediated.
[00:26:52.020]So she wanted to explore different genes involved
[00:26:54.300]in each one of these
[00:26:55.920]to better understand how it's getting inside.
[00:26:58.050]We already had an idea
[00:26:59.340]that if maybe clattering dependent endocytosis
[00:27:02.940]will play a role but the effect wasn't clear.
[00:27:05.370]So we had a suspicion that maybe
[00:27:07.350]other pathways could be involved.
[00:27:11.040]So just really sure because I cannot go into detail.
[00:27:14.460]We find that only evidence is still
[00:27:16.530]for the clathrin independent endocytosis
[00:27:18.683]still seems that maybe other pathways could be involved
[00:27:21.990]but we didn't have really strong evidence.
[00:27:24.780]But she did this beautiful experiment
[00:27:27.420]with confocal microscopy
[00:27:29.310]where she gave them this pharmacological inhibitor
[00:27:32.670]dextran sulfate.
[00:27:33.930]This dextran sulfate is important for scavenger receptors.
[00:27:39.030]She labeled her double strand RNA for Snf7 with Alexa Fluor
[00:27:43.740]so its that,
[00:27:46.650]that red that you see.
[00:27:48.570]So in here you can see the control, the dsRNA
[00:27:51.180]and you can see that that red is being uptaken by the cells.
[00:27:54.330]These ones are inside guts.
[00:27:56.790]And then when you do the Dextran Sulfate
[00:27:58.920]plus the double strand RNA,
[00:28:01.530]you don't see any red.
[00:28:02.850]So that show us really evidently that
[00:28:05.670]scavenger C receptors are definitely involved
[00:28:08.370]in the uptake of a double strand RNA in rootworms
[00:28:11.910]and gave us further evidence
[00:28:13.890]that clathrin-dependent endocytosis plays a role
[00:28:18.150]in Western corn rootworm uptake of double strand RNA.
[00:28:22.080]The other part that we're exploring,
[00:28:24.240]as I say I explored this part for the processing
[00:28:26.580]during my postdoc,
[00:28:28.410]we're trying to understand how it's moved from cell to cell.
[00:28:31.140]We have a hypothesis that is due by, done by exosomes.
[00:28:37.170]So Dr Suresh Varsani, my postdoc,
[00:28:39.870]is currently working on this project.
[00:28:42.330]We also want to explore if these exosomes
[00:28:44.880]could potentially be used
[00:28:46.290]or if we give the double strand RNA coated in exosomes,
[00:28:49.830]if that could be used to target a recalcitrant species.
[00:28:56.970]That said with mode of actions, that's a really dense work
[00:29:00.780]that I didn't wanna go into a lot of detail.
[00:29:03.360]Now let's talk more about the field susceptibility
[00:29:05.730]that is more applicable for like
[00:29:07.650]the application of this technology.
[00:29:10.770]When you work with plant-incorporated protectants,
[00:29:13.500]that's how we call any transgenics,
[00:29:15.810]we need to worry about resistance.
[00:29:17.580]Why, because when you think in terms
[00:29:19.350]of synthetic insecticides,
[00:29:20.700]you're only applying that insecticide
[00:29:22.320]when you see the pressure of the insect.
[00:29:24.570]In this case, that trait is expressed all the time.
[00:29:28.260]So the likelihood of the organism to adapt to resistance
[00:29:31.830]is even higher.
[00:29:33.900]The other part that is important that we have learned
[00:29:36.270]and I can talk with if any of us is interested,
[00:29:38.730]I can tell you more, is the high dose.
[00:29:41.070]We know that this trait is not expressed in a high dose.
[00:29:44.790]So the likelihood of evolving resistance is higher
[00:29:47.400]and that's why the SmartStax Pro product
[00:29:50.100]has two different proteins from BT
[00:29:52.530]with two different mode of actions
[00:29:53.880]plus the DvSnf7 double strand RNA.
[00:29:57.120]So because of this, the EPA requires
[00:29:59.880]insect resistance management or initially in the early days
[00:30:03.780]of be of plant-incorporated protectants,
[00:30:06.540]it was those only this insect resistance management.
[00:30:09.780]But later on, they also require that they need to use
[00:30:14.370]both traits.
[00:30:15.990]And I can, I could give a whole lecture on this,
[00:30:18.237]not going to go in a lot of details.
[00:30:20.490]The other thing that is different is that RNAi
[00:30:23.030]is also active in adults.
[00:30:25.170]With BTs, they only work on larva.
[00:30:27.210]The adults can need any other tissue,
[00:30:29.820]not going to have any effect.
[00:30:31.920]In the adults, in with RNAi,
[00:30:34.815]it will have an effect on the adults.
[00:30:36.510]So the likelihood of potential resistance might be higher.
[00:30:40.650]And we also wanted to explore that idea,
[00:30:43.080]well if it works in the adults,
[00:30:45.270]anybody that has worked with rootworms,
[00:30:47.250]they over winter,
[00:30:48.330]so you need to collect the adults this year.
[00:30:51.030]Let them over winter and you're not going to be able
[00:30:53.280]to test anything until next spring.
[00:30:55.590]So we wanted to test if maybe doing assays with the adults
[00:30:59.430]will be equivalent to what you find
[00:31:01.350]in terms of susceptibility with the larva.
[00:31:05.220]And that's the work that I'm going to describe in a second.
[00:31:08.040]But then also in 2018, Bayer CropScience
[00:31:11.370]showed that they could develop a resistant colony
[00:31:14.880]from insects collected in the field.
[00:31:17.640]So they did multiple generations of selection
[00:31:21.630]and they find out that they could generate a colony
[00:31:23.970]that it was higher than 130 times more resistant
[00:31:27.540]to the double strand RNA for DvSnf7.
[00:31:30.210]They started to look a little bit in the mechanism
[00:31:33.510]and like how it will work.
[00:31:35.370]As you can see here, this is DvSnf7.
[00:31:38.580]Red is the susceptible, blue is the resistant.
[00:31:43.350]It was resistant to a lot of other different
[00:31:46.350]double strand RNAs.
[00:31:48.060]So not good because if it evolves resistance to one trait,
[00:31:51.300]it will evolve resistance to all double stranded RNAs.
[00:31:54.630]So that kind of killed some of the excitement
[00:31:57.000]of people working with RNAi.
[00:31:59.430]And yeah, as you can see, similar to what Molly showed
[00:32:02.130]with the uptake with the inhibitor,
[00:32:05.490]that's what they found with the resistance,
[00:32:06.870]that there is no uptake.
[00:32:09.000]I think in here you just cannot see it
[00:32:11.520]but the control, you can see the red there.
[00:32:14.160]So anyway, the mechanism is due to uptake
[00:32:18.210]of the double strand RNA.
[00:32:19.980]Bayer CropScience, they probably already know the mechanism.
[00:32:22.830]Haven't published yet.
[00:32:23.880]We're waiting for
[00:32:24.930]for that publication to come out
[00:32:26.580]to understand more of details on that.
[00:32:29.790]So as part of what I mentioned,
[00:32:31.530]we wanted to stress if we can use the adults
[00:32:33.570]but also have a a better understanding of
[00:32:35.880]how field populations will be susceptible
[00:32:38.700]to double strand RNA.
[00:32:40.920]So we collaborated with Bayer CropScience
[00:32:44.154]and my student Matthew Welter, this work,
[00:32:47.040]we had populations from Nebraska, from Iowa,
[00:32:51.060]from South Dakota and Minnesota.
[00:32:53.160]Two different susceptible controls:
[00:32:55.260]crop characteristics that is a commensurable vendor,
[00:32:58.020]Bayer CropScience and then others.
[00:33:00.450]Bayer CropScience RNAi resistant strain
[00:33:04.080]and then a Cry3Bb1 resistant.
[00:33:06.810]Cry3Bb1 again is a BT protein.
[00:33:11.610]So it's not going to go in a lot of detail.
[00:33:14.040]You can see that there is a lot of diversity
[00:33:16.620]in the susceptibility.
[00:33:19.560]The only one that I want you to notice here, the resistant,
[00:33:22.530]you can't even plot it there
[00:33:23.700]because you can't get a little concentration.
[00:33:26.940]For this population, you can see it's not a mistake.
[00:33:29.580]The F is up here.
[00:33:31.380]Leigh, Nebraska,
[00:33:32.550]those larva relatively not that susceptible.
[00:33:36.420]Interesting there, a lot of the field testing
[00:33:38.730]was done in Leigh, Nebraska of the
[00:33:40.650]of this product.
[00:33:45.480]Then with the adult susceptibility,
[00:33:47.220]like everything in science,
[00:33:48.420]you think that everything is going to work beautiful
[00:33:51.000]but it didn't.
[00:33:52.050]We find out there is a lot of variability
[00:33:54.360]actually in the adults.
[00:33:56.190]So we had to do more assays and understand
[00:33:58.500]that for the control population,
[00:34:00.630]you can see there is a lot of variability
[00:34:02.580]between the ages of the insects in terms of susceptibility
[00:34:05.850]and then when you move into females and males,
[00:34:08.610]it's also really different.
[00:34:11.580]We still don't understand the biological mechanism
[00:34:14.190]of this happen.
[00:34:15.023]So make the testing more challenging
[00:34:17.610]and we had to H-synchronize the insects
[00:34:20.490]to be able to do the testing.
[00:34:23.040]And in terms of the adult susceptibility,
[00:34:25.200]again a lot of variability but interestingly enough,
[00:34:29.610]the susceptibility didn't correlate
[00:34:31.440]with the larval susceptibility.
[00:34:33.780]We still don't understand why.
[00:34:35.010]So the original idea that we could use adults
[00:34:38.310]to monitor for resistance will not work.
[00:34:42.330]But we understood or learned a lot about in general,
[00:34:45.660]how larva and adults are susceptible in the field.
[00:34:50.490]We wanted to further explore these.
[00:34:54.600]So Molly did another study where we wanted to correlate
[00:34:58.200]this in terms of gene function
[00:35:01.110]or the gene expression.
[00:35:03.270]So she did in 2020, collections of those adult rootworms,
[00:35:08.310]tested susceptibility in both larva and adult
[00:35:11.400]and did a gene expression analysis of some target genes.
[00:35:18.300]And what she found out is that
[00:35:22.050]in terms of not going to go in a lot of details here
[00:35:26.100]is that there is genes that overlap between larvae and adult
[00:35:31.800]and then some that are more overexpressed in adult
[00:35:35.580]or on larva that that might explain why
[00:35:38.400]is that differential gene expression between the larvae
[00:35:41.040]and the adults
[00:35:43.140]or the differential susceptibility.
[00:35:46.140]Something that was interesting
[00:35:47.700]is that we will not looking for toll receptor genes.
[00:35:50.340]Toll receptor genes are more related with immune response,
[00:35:54.120]not with any of the RNAi pathways.
[00:35:57.810]We still don't know the linkage
[00:35:59.220]and it's something that we would like to explore
[00:36:01.110]a little bit more.
[00:36:02.940]And we find this new gene hen1
[00:36:05.880]that has never been described before.
[00:36:09.210]In terms by population effect for larvae and adults,
[00:36:12.450]again a lot of different overlapping genes
[00:36:15.390]and these gene are genes that I was showing you.
[00:36:17.640]The mode of action, I show you a couple of genes.
[00:36:20.400]A lot of those genes are correlated differences
[00:36:23.790]between adult and larva.
[00:36:26.160]Again, we have for larva some of those toll receptor genes
[00:36:30.660]that are present.
[00:36:34.380]So, we originally thought that we were only working
[00:36:36.900]with the siRNA pathway
[00:36:38.910]but now we're finding that a lot of those genes
[00:36:40.980]and there is crosstalk between the pathways
[00:36:43.740]and it got more complicated than we thought.
[00:36:46.710]And you have also these toll receptor genes
[00:36:49.410]that are potentially related with immunity.
[00:36:52.710]So just something to think about for us
[00:36:56.520]in how that will affect susceptibility in the field.
[00:37:00.000]For the next step that will happen here,
[00:37:01.980]we really wanna start understanding
[00:37:04.380]how resistance will evolve in the
[00:37:06.600]in the field.
[00:37:10.080]So just some and I'll finish really early
[00:37:13.260]take home messages.
[00:37:15.150]RNAi highly specific tool for pest management.
[00:37:19.620]Had to do a really, a short overview.
[00:37:23.040]Didn't went into a lot of detail
[00:37:24.450]but I'm happy to answer any question.
[00:37:26.460]First transgenic product
[00:37:27.810]for Western corn rootworm management
[00:37:29.550]was first commercially available in 2022.
[00:37:31.980]So really recent.
[00:37:33.270]We had only been I guess two years in the market.
[00:37:37.050]The first spray for Colorado potato beetle
[00:37:39.360]is in open process for public comment by EPA.
[00:37:44.250]If anybody's interested, send me an email
[00:37:46.980]and you wanna comment, I can send you the link for that.
[00:37:50.190]In terms of ecological risk assessment,
[00:37:52.500]like any type of insecticide that we're going to apply,
[00:37:56.700]there is a risk of resistance
[00:37:58.590]and we need to concern,
[00:37:59.670]be concerned of how we are going to protect this technology.
[00:38:02.400]In terms of effects on non-target organisms,
[00:38:05.340]the effects are minimal
[00:38:06.870]and even less than when we talk about BTs for example.
[00:38:11.610]And one big challenge that we have
[00:38:13.290]is the communication with the public.
[00:38:15.600]I remember a couple of years ago
[00:38:17.130]talking with Vice-Chancellor Mike Bain,
[00:38:19.530]that he mentioned that they did a survey in Nebraska
[00:38:23.310]and they mentioned that 60% of the population
[00:38:25.830]think that you shouldn't need DNA.
[00:38:27.840]So if we are starting from that background
[00:38:29.520]even in agricultural state
[00:38:32.190]that we have even a high use of transgenics,
[00:38:35.490]that will be a
[00:38:37.033]a really big challenge when you have a double strand RNA
[00:38:40.035]or RNA in the world.
[00:38:43.621]And with that, I will be happy to answer any questions.
[00:38:49.221]My understanding is RNAi was or
[00:38:51.300]or that process double strand RNA was how viruses
[00:38:54.630]have in plants and potentially causing effect
[00:38:57.300]of shutting down.
[00:38:58.830]That was a way of the plant protecting itself
[00:39:00.750]or whatever organism
[00:39:02.790]that was trying to defend itself.
[00:39:03.900]So if you have resistance to RNAi,
[00:39:06.000]it seems broad across several genes,
[00:39:08.970]are those resistant populations
[00:39:11.100]potentially even more susceptible to viruses?
[00:39:14.190]Has any work been done on it?
[00:39:15.750]That's a great question that we would like to answer
[00:39:18.090]and it's also that I would like
[00:39:20.610]that I curious.
[00:39:22.320]So okay, two things.
[00:39:23.460]First, viruses are not great at controlling rootworms.
[00:39:28.650]So there is not a lot of viruses that kill rootworms.
[00:39:32.820]For what reason I don't know if in the soil is harder
[00:39:35.520]and a plant pathologist can correct me if that's the case.
[00:39:39.420]Two, I have the same question
[00:39:41.220]but also how potentially the
[00:39:44.730]like maybe populations that have different exposure
[00:39:47.130]to different viruses
[00:39:48.390]or higher loads of viruses
[00:39:50.220]might make them even develop that resistance faster or not
[00:39:53.280]or more susceptible.
[00:39:54.113]We really don't know the correlation.
[00:39:56.010]We know there was a study on honeybees
[00:39:57.870]but what they did is that they injected double strand RNA
[00:40:01.230]to the bees
[00:40:02.460]and then gave them a virus.
[00:40:03.960]And actually what they that
[00:40:05.340]that they did was that make the honeybees more resistant
[00:40:09.360]to the virus.
[00:40:10.530]It's like you prompt the RNAi machinery,
[00:40:13.710]make them more
[00:40:16.470]how do you say, more tolerant to the virus.
[00:40:18.720]So we still don't even understand what is that correlation
[00:40:21.390]with a viral exposure
[00:40:22.950]and then your exposure to double strand RNA.
[00:40:25.250]So that's a great question that we still have
[00:40:27.330]not answered in terms of resistance
[00:40:29.010]and also for non-target organisms.
[00:40:37.350]That was a very nice presentation.
[00:40:39.090]I enjoyed all your visuals.
[00:40:40.830]Oh thank you.
[00:40:42.150]I have to tell you that a lot of it went way over my head
[00:40:45.450]but nonetheless, it was very interesting.
[00:40:47.670]My question is
[00:40:49.200]in a recent article in "Nebraska Farmer" came out this week,
[00:40:53.310]talked about a Bayer product with proteins and BT and
[00:40:59.790]can you tell us more about that?
[00:41:02.100]Yep. So that one is this product actually.
[00:41:11.610]So it's this Bayer product SmartStax Pro
[00:41:14.730]that has the three modes of action.
[00:41:16.320]So it has that DvSnf7 that was strand RNA
[00:41:19.860]plus the two BT proteins cry2bt1 and Cry34/35.
[00:41:25.560]In theory, all of them or no,
[00:41:27.210]we know that all of those three,
[00:41:28.890]they have different modes of action.
[00:41:30.450]So that's what we're hoping
[00:41:32.340]that it will help delay resistance.
[00:41:35.250]One issue there is that it's already,
[00:41:37.620]we have already certain populations in Nebraska
[00:41:40.500]that already have resistance to Cry2bt1
[00:41:43.107]and some of them have even resistance to Cry2bt1
[00:41:46.174]and Cry34/35.
[00:41:47.880]But those populations are the ones that we're more
[00:41:50.820]concerned about
[00:41:51.930]that they will also evolve resistance to DvSnf7.
[00:41:56.640]And what about...
[00:41:59.340]how many you, how many hybrids are...
[00:42:01.470]That's okay.
[00:42:02.303]How many hybrids or what company's license this?
[00:42:06.353]This is just Bayer. Right now just Bayer.
[00:42:10.050]Yeah and I think that a cortellis is coming with a new trait
[00:42:13.830]but it will have the same DvSnf7.
[00:42:16.710]So it will have a genetics from Corteva but it's not,
[00:42:20.490]I don't think it is
[00:42:21.720]Bob do you know or Jeff?
[00:42:23.670]I don't think it's out yet but it will have the same traits.
[00:42:26.760]But for right now the only one that is out in the market
[00:42:29.190]and the only genetics is from Bayer.
[00:42:33.950]Okay.
[00:42:38.670]Question from the...
[00:42:40.350]from online.
[00:42:44.100]There was an early concern about the uptake of 24
[00:42:47.640]to 24 base pair RNA eye molecules by insects.
[00:42:51.540]Apparently insects can only uptake larger,
[00:42:53.580]about 70 base pair duplexes.
[00:42:55.740]Hmm.
[00:42:56.573]Early works use chloroplast expression
[00:42:58.350]to generate the larger duplexes.
[00:42:59.613]It is still a concern and that's from Mitra.
[00:43:03.960]Yep. So that's a great question, Dr. Mitra.
[00:43:08.190]Yes, we find that 2120, so let's go back to this here
[00:43:17.103]in the mode of action.
[00:43:18.270]So what happened is like that double strand RNA is long.
[00:43:21.000]So what we give the insect is 240 or 150 base pairs.
[00:43:26.460]Then it gets, it gets inside.
[00:43:27.990]It is sharp in little place in little small interference RNA
[00:43:31.680]that are 21 to 25 base pairs
[00:43:34.260]and that's actually what generates the silencing.
[00:43:37.920]So at some point, we thought okay we can give them
[00:43:39.810]a bunch of 21 base pairs.
[00:43:41.490]We find out that for rootworms specifically,
[00:43:44.340]they don't take it.
[00:43:45.173]They need to take at least 60 base pairs
[00:43:47.130]will the only thing that will be uptaken by the cell.
[00:43:49.890]If you give them a 25, 21 to 24 base pairs,
[00:43:53.490]will not be uptaken by the cells.
[00:43:54.990]So will not have an effect.
[00:43:57.000]Why we don't, we still don't know.
[00:43:59.190]It might be related to those receptors
[00:44:01.560]that they have a length limit to be uptaken.
[00:44:05.577]And just a caveat there, this is
[00:44:08.280]we have only proved that for rootworms.
[00:44:10.230]They haven't been tested this for other insects,
[00:44:14.250]not that it has been published.
[00:44:16.470]A lot of this work that is
[00:44:18.090]gets makes it challenge is that companies do a lot of work
[00:44:20.640]but sometimes they don't publish.
[00:44:22.230]So you might ask the question
[00:44:23.400]and then they say oh yeah we did that
[00:44:24.660]but we never publish it.
[00:44:26.040]So not publishable yet.
[00:44:28.860]So yeah, we need to incorporate those
[00:44:31.830]longer double strand RNAs.
[00:44:33.600]In terms of the chloroplast insertion,
[00:44:37.380]they have found the product from Corteva
[00:44:39.690]actually is not producing the chloroplast.
[00:44:41.970]So somehow, still those longer sequences
[00:44:45.630]are getting inside the insect
[00:44:48.420]but for other traits or for other organisms,
[00:44:51.120]they're trying to express more in the chloroplast
[00:44:53.910]and for those of you who don't understand,
[00:44:55.350]so the plant also has that RNAi machinery.
[00:44:58.560]So if you produce a sequence,
[00:45:00.420]then the RNAi machinery from the plant will chop it.
[00:45:03.150]So the Amanda will get into the insect
[00:45:05.010]will not be as much.
[00:45:06.720]If you express it on the chloroplast,
[00:45:08.610]you're kind of protecting it from the rest of the plant
[00:45:11.370]and you're being
[00:45:13.230]or ensuring that you're getting that longer
[00:45:15.420]sequence into the insect.
[00:45:17.460]So still, the chloroplast expression
[00:45:20.100]will make it more effective.
[00:45:29.100]Thank you for this very interesting presentation.
[00:45:31.710]I do have one practical question.
[00:45:33.480]Do you have any information on the adoption rate
[00:45:35.520]of the product that was commercialized
[00:45:38.100]and are there any new concerns on the rise
[00:45:41.430]since the product went commercial?
[00:45:44.070]Not so.
[00:45:44.903]We don't really.
[00:45:45.736]I know that in 2022, it wasn't really available
[00:45:48.990]but I don't know in terms of adoption.
[00:45:50.700]I think that will,
[00:45:51.630]we will learn more of that later on.
[00:45:54.390]Jeff? Yeah.
[00:45:56.220]I will ask maybe more to the people that work with farmers.
[00:46:01.740]In terms of concerns,
[00:46:02.940]so far we haven't heard anything.
[00:46:07.080]A lot of times what happened unfortunately with companies
[00:46:10.110]is that there is a concern and they don't talk about it.
[00:46:13.410]Maybe a couple of years later
[00:46:15.660]or maybe sometimes an extension,
[00:46:17.760]a person at the university hears about it
[00:46:19.620]and then that's when we are aware of it.
[00:46:22.110]So so far, we just have had a two seasons.
[00:46:24.840]I know that in 2022, the adoption wasn't great
[00:46:28.410]because they just take it out.
[00:46:30.300]Biggest adoption interior would be this year
[00:46:32.850]but we still don't have the numbers.
[00:46:34.830]So I'm also curious to see what's going to happen
[00:46:37.230]if we're going to have any concerns.
[00:46:40.260]Okay, thank you very much for your
[00:46:41.670]Yeah, thank you. Answer.
[00:46:48.687]Okay, any further questions?
[00:46:53.711]We'll finish or we...
[00:46:54.991]I got one.
[00:46:58.620]Thank you for your presentation.
[00:46:59.670]Maybe this is also for you or for the ones in the room
[00:47:03.420]that have more contact with farmers.
[00:47:05.100]And so this last technology that they mentioned
[00:47:06.930]that was released is in terms of management from the farmers
[00:47:10.080]points of view to protect let's say the technology.
[00:47:12.870]There's some constraints.
[00:47:14.520]What is for this specific technology
[00:47:16.530]if you comment on that?
[00:47:18.060]So let's step back to BT.
[00:47:20.520]So a bit like I mentioned, the insect resistant management,
[00:47:24.540]like the main thing is that we need to have a refuge
[00:47:27.120]right there.
[00:47:28.020]So the refuge means that you need to plant plants
[00:47:30.780]that are not expressing that trait.
[00:47:33.540]That way you have susceptible insects
[00:47:35.640]that then will mate with those resistance insects
[00:47:38.730]and from a lot of assumptions that we originally had,
[00:47:41.430]then you will dilute it.
[00:47:42.990]We're finding that those assumptions don't always meet
[00:47:45.450]like everything in biology.
[00:47:47.670]So then, that's why we need to do the high dose.
[00:47:52.800]Also at some point, there was no farmer compliance
[00:47:55.650]because originally, they just get a bag
[00:47:57.330]and they need to do a strips or like put it in the borders.
[00:48:01.230]Obviously farmers don't, those will get damaged.
[00:48:03.690]They didn't employ it so they employed or apply something
[00:48:06.720]called refuge in a bag
[00:48:08.220]where they give you a bag
[00:48:09.270]where it has the refuge there.
[00:48:10.980]And the refuge for example here in the Midwest,
[00:48:13.170]they completely different from Texas for example.
[00:48:16.500]Anyway, so we use the same strategies so far.
[00:48:19.500]So they still need to apply the refuge in a bag
[00:48:22.080]and form rootworms.
[00:48:23.340]Farmers also need to ensure
[00:48:25.020]that they are using IPM strategies
[00:48:27.720]and I think that's just harder to determine
[00:48:31.080]if the farmers are using the IPM.
[00:48:32.880]That's the recommendation from EPA.
[00:48:35.520]But I don't think no one can enforce
[00:48:37.800]if they are using both the...
[00:48:39.810]if they're using IPM.
[00:48:41.130]So enforce in a way, they sell the bags
[00:48:43.410]already with the refuge in the bags,
[00:48:45.270]so the percentage of seeds
[00:48:47.070]that are not expressing the trait.
[00:48:50.550]Yeah.
[00:48:56.347]Did in the process of identifying resistance,
[00:48:59.010]did they indicate any fitness costs
[00:49:01.110]associated with the resistance to RNAi or is anybody?
[00:49:04.080]It doesn't seem to have a fitness
[00:49:05.550]like everything in rootworms.
[00:49:06.597]Was that a expectation?
[00:49:09.030]With rootworms, yes.
[00:49:10.380]Like I don't know why they're so hardy
[00:49:11.790]that a lot of the things that they end up
[00:49:14.190]revolve in resistance.
[00:49:16.440]We don't find a lot of fitness costs.
[00:49:18.330]I sometime wonder if we don't,
[00:49:20.190]we maybe are measuring some fitness that they have
[00:49:22.800]but we're not measuring them.
[00:49:24.510]But yeah, rootworms just seem to be hardy at everything.
[00:49:38.160]I don't really come from a molecular background.
[00:49:41.010]So I really have a question that might
[00:49:43.140]not be the brightest one,
[00:49:44.070]but I'm really concerned about the
[00:49:46.980]gene flow from crop to the wild relatives.
[00:49:49.980]Is that like, is that a real concern?
[00:49:52.260]Can that happen and what would be the implications of that?
[00:49:55.737]So that would be the same concern that we will have
[00:49:57.660]with the BT traits.
[00:50:00.090]And I think maybe you guys are more familiar
[00:50:02.790]or maybe Bob, you're more familiar with that.
[00:50:05.280]There was not a lot of concerns in terms of the
[00:50:08.090]of the gene flow, but I don't remember.
[00:50:10.620]So like those are studies from the '90s.
[00:50:14.040]So like if there were like in some ways,
[00:50:15.960]the EPA assumes that things that were tested in the past
[00:50:19.080]for BTs will apply for things currently.
[00:50:22.470]But there is, but that's like from all the,
[00:50:25.605]in the, in a public sense,
[00:50:26.910]that's kind of like the biggest concern
[00:50:28.740]that you will renew, reduce gene
[00:50:33.317]and not even inserting the genes
[00:50:34.680]but just reducing the gene variability in closest relatives.
[00:50:40.080]But there's no wild relatives of corn
[00:50:42.750]In Nebraska.
[00:50:43.583]So it is like it will be more of a...
[00:50:44.490]Oh yeah, in New Mexico, grass stuff like that.
[00:50:51.220]But I will say in other places, if you were,
[00:50:53.640]so this technology is more relevant
[00:50:55.200]for the US Midwest for rootworms.
[00:50:57.810]If they are going to use it in another place
[00:50:59.766]where there is a concern with gene flow,
[00:51:02.040]then the regulatory agencies of that country
[00:51:04.710]will require more testing in terms of that.
[00:51:07.560]So that's part of like what happens
[00:51:09.180]with a lot of the products that going to Asia for example,
[00:51:12.390]that they need to do.
[00:51:13.740]Like companies will need to do more testing
[00:51:15.600]to demonstrate that the gene flow or like the likelihood
[00:51:18.030]of gene flow will be low.
[00:51:20.040]So that's part of the, that ecological risk assessment.
[00:51:22.800]That will be a species of concern for example.
[00:51:26.130]Yeah.
[00:51:29.340]Sure.
[00:51:32.850]My question is very practical I guess
[00:51:35.310]in that your evaluations,
[00:51:38.190]I'll say sort of stop at the laboratory or
[00:51:41.010]or maybe some field studies,
[00:51:43.410]but when it gets to Bayer or a farmer,
[00:51:47.460]that yield monitor is their yardstick
[00:51:52.020]and so what are the chances of making a
[00:51:57.240]an overinterpretation or underestimation
[00:52:00.600]based on other factors that affect yield?
[00:52:06.358]That's How do you,
[00:52:07.191]How do you deal with that?
[00:52:08.190]That's a great question
[00:52:09.540]and that just goes then to Bob and Justin (laughs) and Jeff
[00:52:13.950]that I will be concerned but yeah,
[00:52:15.540]there is always a concern of like how that will affect.
[00:52:18.360]I know that companies, what they do a lot
[00:52:20.490]is test these products
[00:52:21.780]at different environmental conditions.
[00:52:24.420]So just to be sure that they will perform the same way
[00:52:27.990]no matter if you have drought or other things.
[00:52:30.810]There is other, there was a PhD student too, not in my lab,
[00:52:34.500]that did more testing with different farmers
[00:52:36.630]and they look at field histories
[00:52:39.600]and looking at yields too.
[00:52:41.160]I don't recall all what he necessarily found
[00:52:45.270]but those are things that actually companies
[00:52:47.700]are concerned and test too.
[00:52:50.560]I, in my case, I just work up to the greenhouse
[00:52:53.730]and maybe bring the insects here
[00:52:55.557]and then give it to other people to deal with it.
[00:53:00.390]Impact shield enough, they're probably not likely
[00:53:03.480]to continue to grow it
[00:53:04.590]unless they have the concern always the yield loss.
[00:53:11.880]But for I, what I understand in terms of yields
[00:53:13.950]is as competitive to any of their other hybrids
[00:53:16.500]that they currently have.
[00:53:21.030]In terms of the trait characterization,
[00:53:22.800]that's a big part that they need to do
[00:53:24.660]because sometimes they can insert traits
[00:53:28.320]that might decrease the yield of the product.
[00:53:41.295]Thank you.
[00:53:42.128]I'd like to thank you for a very interesting presentation.
[00:53:44.160]I very much enjoyed it
[00:53:45.240]and we appreciate you taking the time
[00:53:47.010]to come and be a part of this seminar series.
[00:53:49.350]Thank you very much. Thank you.
[00:53:50.593](audience claps)

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RNA Interference for Insect Pest Management

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