This work was supported by a grant from the Monsanto Corn Rootworm Knowledge Research Program, grant number AG/1005 (to MDL and YC) and start-up funds to MDL from NCSU. FC was supported by grants from Monsanto&#39;s Corn Rootworm Knowledge Research Program (AG/1005) and the National Science Foundation, grant number MCB-1244772 (to MDL). The authors declare no competing interests. FC and MDL conceived and designed the experiments; FC, PW and SP performed the experiments; FC and MDL analyzed the results; and FC, NG and MDL wrote the manuscript. We thank Teresa O&#39;Leary, William Klobasa, and Stephanie Gorski for their expert assistance in screening WCR. We also thank Dr. Wade French (USDA-ARS, North Central Agricultural Research Laboratory, Brookings, SD) for shipments of eggs to establish lab colonies and providing rearing protocols.

1. Colony-level Rearing of WCR Adults
<ol>
	<li>Obtain a sufficient quantity of WCR adults (500 - 1,000) from a reliable company or research laboratory (see Table of Materials for an example) and place in a 30 cm3 cage. 
	NOTE: Use of a non-diapausing strain is highly recommended.</li>
	<li>Prepare WCR artificial diet following manufacturer&#39;s protocol and pour a 1 cm-thick layer into a 38 oz container (see Table of Materials). After mixing, store unused diet at 4 &#...

<ol>
	<li>Gray, M. E., Sappington, T. W., Miller, N. J., Moeser, J., Bohn, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+and+Invasiveness+of+Western+Corn+Rootworm:+Intensifying+Research+on+a+Worsening+Pest.">Adaptation and Invasiveness of Western Corn Rootworm: Intensifying Research on a Worsening Pest.</a> Annual Review of Entomology. 54, 303-321 (2009).</li><li>Gassmann, A. J., Petzold-Maxwell, J. L., Keweshan, R. S., Dunbar, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-evolved+resistance+to+Bt+maize+by+western+corn+rootworm.">Field-evolved resistance to Bt maize by western corn rootworm.</a> PLoS One. 6 (7), e22629 (2011).</li><li>Gray, M. 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The authors have nothing to disclose.

Although microinjection of WCR with dsRNAs for the purpose of RNAi has been reported9, this is the first protocol to establish best practices for microinjecting precellular WCR embryos, a critical process for conducting germline transformation and/or CRISPR/Cas9 genome editing in this species. Successful microinjection of WCR embryos is dependent on many factors, as is transformation efficiency. Discussed below are some of the major issues impacting the outcome of using this protocol for germline ...

Western corn rootworm (WCR), Diabrotica virgifera virgifera, is an important pest of corn1. Interestingly, WCR appears to overcome control measures more rapidly than most agricultural pests since they not only adapt physiologically but also behaviorally2,3,4,5,6. Over the past decade, RNA interference (RNAi), a powerful functional genomic tool, has been investigated as a potential control method for WCR7,8, and has also been used as a means to study gene function9. However, while RNAi is frequently performed by microinjection of double-stranded RNA (dsRNA) in other species, injection-based RNAi is rare in WCR. In fact, there are only a few reports of RNAi via microinjection of dsRNA into WCR9. The reason is that WCR can attain high-levels of gene knockdown via ingestion of dsRNA7,10, even permitting the study of embryonic effects by feeding dsRNA to the mother11. While this method makes WCR an excellent subject for functional genomic analysis via RNAi, it has slowed progress on the development of methodologies for embryonic microinjection in this species.
Despite the power of RNAi, there are a few drawbacks. For example, not all genes respond equally to RNAi. This variability can make interpreting the results of a functional genomics assay more difficult. Also, RNAi is transient and does not generate heritable mutations. On the other hand, germline transformation, another functional genomic tool, can generate heritable mutations via insertion of a marked transposable element into the genome12,13. This makes germline transformation an excellent tool for creating mutant strains for use in long-term genetic studies. Transformation can also be used to rescue a mutation by delivering a functional copy of the gene to a mutant genome14. Moreover, germline transformation is the cornerstone of a wide variety of molecular genetic techniques. In addition to knocking out and/or rescuing gene function, germline transformation can be used for enhancer trapping15, gene trapping16, Gal4-based ectopic expression17, and genome-wide mutagenesis18.
More recently, tools that enable sophisticated genome editing&#160;have been developed. These tools include Transcription Activator-Like Effector Nucleases (TALEN) and the CRISPR/Cas9-nuclease system19,20. The advantage of these new tools is that they offer researchers the ability to induce a double-stranded DNA break at almost any location within almost any genome. Once induced, these breaks can be repaired through either non-homologous end joining, which can introduce deletions or insertions in the targeted gene, or through homology-directed repair, which in the presence of an engineered construct, can catalyze the replacement of entire genes or genetic regions21,22. However, these methods require microinjection of DNA, RNA and/or protein into very young embryos.
Importantly, unlike the dsRNAs used for RNAi, the nucleic acids and proteins used for germline transformation and genome editing cannot easily cross cell membranes. Therefore, microinjection of plasmid DNAs, mRNAs, and/or proteins must take place during the syncytial blastoderm stage (i.e. before the insect embryo cellularizes). This makes the timing of injections a critical factor. For example, in Drosophila melanogaster, embryos need to be injected within two hours after egg lay12. Therefore, development of a successful embryonic microinjection protocol for WCR must take into account the best conditions for female egg laying, as well as the best method for collecting sufficient quantities of precellular embryos.
An effort to bring a wide range of molecular genetic tools to bear on WCR biology requires developing methods for collecting and microinjecting precellular WCR embryos. Here we provide detailed instructions, along with tips and tricks, to help others use transformation-based techniques in WCR research. In addition to extending transgenic technologies to WCR for use in functional genomic studies, these techniques also enable powerful new pest control strategies such as gene drive23,24 to be tested in this economically important pest.

<table border="0" cellpadding="0" cellspacing="0" width="1155">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Qualitative Filter Paper (Black)</td>
			<td style="width:189px;">Ahlstrom</td>
			<td style="width:287px;">8613-0900</td>
			<td style="width:339px;">For egg microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 oz Containers&#160;</td>
			<td style="width:189px;">Anny&#39;s Plastic Tableware</td>
			<td>ASET101</td>
			<td>Egg container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila Agar Type II</td>
			<td style="width:189px;">Apex</td>
			<td style="width:287px;">66-103</td>
			<td>Substrate for WCR egg-laying dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Featherweight Forceps&#160;</td>
			<td>Bioquip</td>
			<td style="width:287px;">4748</td>
			<td>Handling larvae and pupae&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clorox Regular-Bleach1</td>
			<td style="width:189px;">Clorox</td>
			<td style="width:287px;">44600-30770</td>
			<td>Wash corn and dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trucker&#39;s Favorite Yellow</td>
			<td>Coor Farm Supply</td>
			<td>502</td>
			<td>Corn for feeding WCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microinjection System (Homemade)</td>
			<td style="width:189px;">NA</td>
			<td style="width:287px;">Parts &#38; instructions available upon request</td>
			<td>Controls injection pressure (0-20 psi)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Elmer&#39;s Non-toxic Glue</td>
			<td>Elmer&#39;s Products, Inc.</td>
			<td style="width:287px;">E304</td>
			<td>Glue eggs on filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">epTIPS Microloader Tips</td>
			<td style="width:189px;">Eppendorf</td>
			<td style="width:287px;">C2554691</td>
			<td>Backfilling needle loading tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Tissue Culture Dishes</td>
			<td style="width:189px;">Falcon</td>
			<td>25383-103</td>
			<td>Sprouting corn /150 x 25 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand&#160;Quantitative-Grade Filter Paper Circles</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td>S47576C</td>
			<td>Making agar dish for egg-lay/9cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sparkleen</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">04-320-4</td>
			<td>Wash dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plain Microscope Slides</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">12-549-3</td>
			<td>Holding filter paper and eggs for microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Western Corn Rootworm w/o Pollen Substitute</td>
			<td style="width:189px;">Frontier Agricultural Sciences&#160;</td>
			<td>F9766B</td>
			<td>WCR adult artificial diet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton Balls, Large</td>
			<td>Genesee Scientific</td>
			<td>51-101</td>
			<td>Close the flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Globe Scientific 3.0 mL Small Bulb Transfer Pipettes</td>
			<td>Globe Scientific</td>
			<td>137035</td>
			<td>Collect and transfer eggs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica M165 FC Fluorescence Stereomicroscope&#160;</td>
			<td style="width:189px;">Leica</td>
			<td>M165 FC&#160;</td>
			<td>WCR screening</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DsRed Filter Set for Fluorescence Stereomicroscope</td>
			<td style="width:189px;">Leica</td>
			<td>DSR</td>
			<td>Excitation filter: 510-560 nm, emission filter: 590-650 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGFP filter</td>
			<td style="width:189px;">Leica</td>
			<td>GFP2</td>
			<td>Excitation filter: 440&#8211;520 nm, emission filter: 510 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BugDorm</td>
			<td>MegaView Science</td>
			<td>DP1000_5P</td>
			<td>Cage for adult colony</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Miniature Paint Brush</td>
			<td style="width:189px;">MyArtscape</td>
			<td>MAS-102-MINI</td>
			<td>Liner 2/0&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Joystick Micromanipulator</td>
			<td style="width:189px;">Narishige</td>
			<td style="width:287px;">MN-151</td>
			<td>Micromanipulator and needle holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Microscope XY Stage</td>
			<td style="width:189px;">Olympus</td>
			<td style="width:287px;">265515</td>
			<td style="width:339px;">Microscope stage (adapted for use with stereoscope)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grade 90 Cheesecloth</td>
			<td style="width:189px;">Online Fabric Store</td>
			<td>CHEE90</td>
			<td>For egg-lay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic paraffin film&#160;</td>
			<td style="width:189px;">Pechiney Plastic Packaging</td>
			<td>PM-996</td>
			<td>Seal agar dish after microinjection/Roll size 4 in. x 125 ft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Percival Incubator</td>
			<td style="width:189px;">Percival&#160;</td>
			<td style="width:287px;">I41VLH3C8</td>
			<td>WCR growing chamber (insect rearing incubator)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narrow Mouth Erlenmeyer Flasks</td>
			<td style="width:189px;">VWR</td>
			<td>4980-300-PK</td>
			<td>Water container for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Griffin Low Form Beakers</td>
			<td style="width:189px;">VWR</td>
			<td>1000-600-PK</td>
			<td>For egg wash</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Braided Cotton Rolls</td>
			<td>Richmond Dental Cotton Co.</td>
			<td>605-3599</td>
			<td>Use in water supply for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (35x10mm)</td>
			<td>SIGMA</td>
			<td>CLS430588-500EA</td>
			<td>For diet plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol Red</td>
			<td>Sigma</td>
			<td style="width:287px;">143-78-8</td>
			<td>Microinjection buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser-based Micropipiette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-2000/G</td>
			<td>Needle puller/Heat = 335, FIL = 4, VEL = 40, DEL = 200, PUL = 100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Topsoil</td>
			<td>The Scotts Company</td>
			<td style="width:287px;">71130758</td>
			<td>Soil for cron growing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reloc Zippit 2 Mil Zipper Bags</td>
			<td>United States Plastic Corporation</td>
			<td>48342</td>
			<td>Bag agar dish after microinjection/5x7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (100x15m)</td>
			<td style="width:189px;">VWR</td>
			<td style="width:287px;">89038-968</td>
			<td>Making agar dish for egg-lay/ 100 x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128E506</td>
			<td>WCR single pair mating chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">38 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128NC888</td>
			<td>Larvae rearing box/38 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChoiceHD 16 oz. Microwavable Translucent Plastic Deli Container</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128HRD16</td>
			<td>Larvae rearing box/16 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Microinjection Scope&#160;</td>
			<td style="width:189px;">Wild Heerbrugg</td>
			<td style="width:287px;">WILD-M8</td>
			<td style="width:339px;">Microinjection scope outfited with an XY stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td>Microinjection needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Adult Western Corn Rootworms</td>
			<td style="width:189px;">North Centeral Aqricultural reasearch Laboratory</td>
			<td style="width:287px;">Non-diapase strain</td>
			<td>Request from Dr. Bryan Wade French&#39;s lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plasmid DNA Midi Kit</td>
			<td style="width:189px;">Qiagen</td>
			<td>12143</td>
			<td>Purification of injection-ready plasmid DNAs</td>
		</tr>
	</tbody>
</table>

microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

An important consideration when developing this protocol was the stage of embryonic development at the time of microinjection. Specifically, germline transformation requires microinjection of DNAs prior to embryonic cellularization. This is because DNAs cannot easily cross cell membranes. Attempts to visualize the stages of WCR embryonic development using a nuclear stain were unsuccessful because dechorionation of WCR embryos essentially dissolves all of the protective membranes responsib...

Watch this Scientific Journal Video about Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing at JoVE.com

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

The overall goal of this Microinjection protocol is to modify the Western Corn Rootworm genome. This method can help answer key questions regarding Western Corn Rootworm biology and their ability to quickly overcome pest control methods such as BT Toxin and RANI. Besides Western Corn Rootworms, this method can also be applied to similar species, such as Southern and Northern Rootworms.I first had the idea for this method when I was asked to help with injection based RANI in Western Corn Rootworm back in the 90's. Generally, individuals new to this method will struggle. Because rearing Western Corn Rootworms in the laboratory is labor intensive.And survival rates remain low using current technology. Obtain between 500 and 1, 000 non-diapausing rootworm adults from a reliable company or research laboratory. And house them at 30 cubic centimeter cages.Store the standard rootworm artificial diet in food storage containers about one centimeter deep. Unused diet can be stored at four degrees Celsius for up to two months. To feed the rootworm, load 100 centimeter Petri dishes with 10 to 15 grams of diet.Load one dish of food into each rootworm cage. And replenish it when the supply runs low or the diet dries out. Also keep a 300 milliliter water reservoir, covered by a cotton ball, in each cage to serve as a water source.Change the water when it runs low. Maintain the caged rootworm at 26 degrees Celsius with 60%humidity and a 14/10 light cycle in an insect rearing incubator. Set the lights off time later in the day such as at midnight so eggs are laid before morning work hours.First, collect the embryos. For an egg collection surface, load a 100 millimeter Petri dish with 30 milliliters of 1%agar and water. Next, place a single layer of filter paper on the agar, followed by four layers of cheese cloth.Each layer should be cut to size. Next, as late in the day as possible, place the egg collection plate into the rootworm cage and cover it with a tinfoil tent. Then on the following morning, remove the egg collection chamber.Use forceps to pick up one layer of cheese cloth at a time and place into 500 milliliter beaker loaded with water. Wash the eggs off of the cheese cloth by swirling in the water. Then, use a bulb pipet to transfer the eggs to another beaker of water as a wash step.Perform two or three washes in this manner. Then, deposit the cleaned eggs onto filter paper with as little water as possible. Next, prepare a slide by taping a piece of black filter paper flat to the slide to improve contrast.Then drizzle fine lines of non-toxic glue onto the paper that will be used to secure the eggs. Keep the glue below the girth of an egg. While the glue is wet, use a fine brush to place the eggs onto the glue line.Keep at least one egg's distance between each egg. Prepare multiple lines of eggs per slide. The eggs should now be injected within 30 minutes.Now, combine the helper plasmid, donor plasmid, and phenol red buffer. Use high quality, super coiled plasmids made with an endotoxin-free commercial kit. Fortex briefly to mix.And then spin down particulates for three minutes at maximum speed. Next, backfill pulled borosilicate glass needles with 0.5 to 1 microliters of injection solution. If any bubbles form, be sure to remove them.Then, attach the injection needle to the micromanipulator. Now, if necessary, under the microscope, gently break off the sealed needle tip using a fine pair of forceps or by gently touching the tip into a glass barrier. Try to make the smallest possible opening that releases injection solution.Before injecting the rootworm eggs, use a fine paintbrush to carefully wet the surface of one to three prepared eggs with sterile water. Then, inject the wetted embryos. Gently insert the needle into the middle of the embryo, and inject a small volume of solution.Start with using 10 to 13 psi of pressure and adjust the pressure as needed. Dry embryos has a distinct round shape, by pre-wetting each embryo with water will change their shape and soften the eggshell thus allowing the needle to easily penetrate. A successfully injected embryo will have a small amount of red color.If an egg was damaged, looks peculiar, or cannot be injected then crush it or remove it from the slide. After all eggs are injected, move the filter paper with the eggs on it to a 1%agar dish. Then, seal the dish with paraffin film and incubate it at 26 degrees Celsius until larvae hatch.12 days after injection, start checking for larvae everyday for the next two weeks. Transfer the larvae using a fine brush to a rearing box with sprouted corn and soil to rear them under standard conditions. Germline transformation of rootworm was attempted by co-injecting a transposase carrying plasmid with a transposon carrying plasmid into pre-cellular embryos.In the 26 survivors, each EFP expression was used to screen for donor element insertion. Although thoracent progeny were identified, screening rootworm embryos with the intense light source proved to be lethal. The next round of germline transformation utilized a red florescent protein gene driven by a constitutively active promoter from Drosophila melanogaster.Survival rates improved, but only one DS red positive larvae was recorded. Next, embryos were micro-injected with buffer alone or with helper and thorescently marked donor DNA plasmids. A similar ratio of embryos hatched when injected with plasmids or buffer alone.And interestingly, only about half of un-injected embryos hatched. This put the lethality of manipulating the embryos for the injection as high as performing the injection. After watching this video, you should have a good understanding of how to micro inject Western Corn Rootwall embryos.Once mastered two to three hundred embryos can be injected per hour using this technique. This method is indebted to techniques developed for germline transformation of Drosophila melanogaster which serve as templates applicable to many other species. Following this procedure, other methods like CRISPR/Cas9 based genome editing can be performed to make targeted mutations in Western Corn Rootwall's genome.In order to answer specific questions about genome function.

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7.3K vistas.  North Carolina State University. The overall goal of this Microinjection protocol is to modify the Western Corn Rootworm genome. This method can help answer key questions regarding Western Corn Rootworm biology and their ability to quickly overcome pest control methods such as BT Toxin and RANI. Besides Western Corn Rootworms, this method can also be applied to similar species, such as Southern and Northern Rootworms.I first had the idea for this method when I was asked to help with injection based RANI in Western Cor...