In January 2024, after an intensive four-year review, the US Environmental Protection Agency (EPA) granted registration to the novel bio-insecticide Calantha [
1,
2]. Developed by Medford, MA, USA-based GreenLight Biosciences, Calantha is the first RNA interference (RNAi)-based pesticide to reach the market.
‘‘We are very excited about this development because it finally opens the door for RNAi pesticides,” said Juan Luis Jurat-Fuentes, professor of entomology and plant pathology at the University of Tennessee in Knoxville, TN, USA. Uniquely for an insecticide, which are generally toxic to a broad variety of insect species and other organisms, Calantha specifically targets the Colorado potato beetle (
Leptinotarsa decemlineata), a highly destructive pest that can reduce crop yields by up to 80% and cause annual losses estimated to exceed 500 million USD in value [
3,
4].
The fundamental concept of RNAi as a mechanism regulating gene activity was first described in a February 1998 paper pub-lished in
Nature [
5]. The research was recognized as a ground-breaking discovery with the awarding of the 2006 Nobel Prize in Physiology or Medicine to the paper’s first and senior authors, Andrew Fire and Craig Mello [
6], now professors at Stanford University (Palo Alto, CA, USA) and the University of Massachusetts Chan Medical School (Worcester, MA, USA), respectively. Their work showed that gene expression can be silenced through the cel-lular uptake of double-stranded RNA (dsRNA).
Here is how it works: At its simplest level, genes encoded in DNA are transcribed by cellular machinery into messenger RNA (mRNA), which attaches to a ribosome to be translated into the proteins that are the building blocks of cells and their functioning. When dsRNA is taken up by a cell, however, it induces a process that degrades intracellular mRNA. The dsRNA is first recognized by the ribonuclease enzyme Dicer, which cleaves it into small pieces of RNA. These RNA pieces are then bound by the RNA-induced silencing complex (RISC), which searches the cell for matching mRNA sequences and destroys them, effectively inhibiting the synthesis of the protein coded by that mRNA and potentially disrupting essential cellular processes.
Researchers hypothesize that the mechanism originally evolved as a defense system against dsRNA viruses [
7]. In any case, one of the ideas the research suggested was that the right dsRNA, if taken up by an insect’s cells, could kill agricultural pests [
8]. By introducing dsRNA complementary to mRNA that codes for a protein critical for survival, the insect’s cells will find and destroy that mRNA. “The idea is that the cell thinks that this is a virus and tries to degrade everything that looks like it,” Jurat-Fuentes said. “It essentially induces the cells to commit suicide.”
The Colorado potato beetle (
Fig. 1), notorious as a pest resistant to most existing insecticides, made a logical target for developing an RNAi-based insecticide for the commercial market. “This beetle has managed to develop resistance to almost every insecticide introduced to manage them—after a few years of use, they just no longer work,” said Subba Reddy Palli, professor of entomology at the University of Kentucky, Lexington, KY, USA. “The latest insecticides these beetles developed resistance against are neonicotinoids. It is a good time to introduce a new product that works through a different target site and mode of action.”
The beetle inflicts extensive damage by defoliating nightshade crops such as potatoes, tomatoes, eggplants, and bell peppers. With a broad geographical distribution across the Northern Hemisphere, the pest is resistant to more than 50 chemical insecticides, representing all major types [
4], [
9]. To control it, farmers need to apply insecticides multiple times each season, trying to minimize the damage by alternating among pest control options with different modes of action. “Farmers have desperately needed something that works in a different way,” Jurat-Fuentes said. “RNAi is a completely new mode of action. It is not easy to find biological pesticides completely different from everything we have already developed.”
Calantha’s dsRNA ingredient, called Ledprona, targets an essential protein called the proteasome subunit beta type-5, coded by the
PSMB5 gene [
10]. This protein is an essential component of the cell’s waste-removal system, removing unneeded and toxic proteins that the cell tags for destruction. Without proteasome subunit beta type-5, the waste removal mechanism no longer functions, and the toxins collect and kill the cells—and insects—within two to three days [
11].
Importantly, the dsRNA sequence of Ledprona is complementary to the proteasome subunit beta type-5 in the Colorado potato beetle, meaning it has few off-target effects in other insect species. “With that specificity, you can kill the species you want to kill, but not closely related or non-target species,” Jurat-Fuentes said. “It is amazing that we can design that level of specificity. There is no other pesticide that does that. That gives it a very nice safety profile.” GreenLight Biosciences’ studies have shown that Calantha affected only two other beetles, also agricultural pests. Earthworms and pollinators like bees and butterflies and other beneficial insects like green lacewings and ladybugs were unaffected [
1], [
2].
Applied at a concentration of about 10 g per hectare, only 5 cm
3 of Calantha is required to manage an area equivalent in size to an American football field (about 90 m × 50 m) [
3]. Extensive trials using standard application equipment have confirmed the product’s efficacy and ease of use [
4], showing it to be just as effective as existing chemical insecticides against the Colorado potato beetle [
1]. Regarding price, a GreenLight spokesperson said that Calantha is “priced competitively to other premium Colorado potato beetle control products.”
GreenLight claims its novel, cell-free RNA production platform enables it to produce the dsRNA in Calantha at a projected cost of less than 1 USD per gram [
12]. Unlike conventional RNA synthesis that involves
in vitro transcription and fermentation, the company’s platform is non-transgenic—it does not involve inserting a gene into a bacteria or yeast or other organism to produce the RNA. This means its RNA products avoid the regulatory issues that apply to genetically modified organisms. It also means that production takes hours instead of weeks and is readily scalable, with the company’s 1600 m
2 facility in Rochester, NY, USA, currently capable of producing 500 kg of RNA annually [
12]. Each specific RNA product needs only a new DNA template. The process breaks down RNA from a polymeric RNA substrate such as yeast into nucleoside building blocks that are then rebuilt in the specific RNA product of interest with the DNA template and proprietary enzymes. “There were a lot of concerns that this technology would never become cost competitive with chemical pesticides or other biological pesticides,” Jurat-Fuentes said. “There have been a lot of advancements in the production of double-stranded RNA, so much so that it has gotten to a point that now it is cost-effective.”
There are, however, some downsides. The timing of application with Calantha is critical. Farmers must spray it over their crops when the Colorado potato beetle eggs are about halfway through their hatching phase [
4]. Calantha does not damage the beetle’s eggs or pupal stage. For best results, the beetles must ingest the dsRNA while they are still immature, at the first or second instar larval stages (
Fig. 2). Larvae that ingest it darken, stop eating and developing, and eventually shrivel up and die [
1]. Unlike chemical pesticides, which kill insects quickly, Calantha takes longer to kill the beetles. “Chemical pesticides act fast, almost immediately. You get death in hours,” said Jurat-Fuentes. “RNAi takes days to kill the insect.” While Calantha may also kill more mature larvae and adult beetles, they may still be capable of severely defoliating crops before they die [
10]. The product also degrades quickly after its spray-on application to the crop’s foliage, typically within three days. “If I spray today and it rains, the insecticide may wash away and not work very well,” Palli said. “If it is very sunny, ultraviolet (UV) light may degrade it.”
But perhaps the biggest potential downside of Calantha is the same one that plagues other pesticides—resistance. In a study published in 2021, Jurat-Fuentes and his team exposed successive generations of Colorado potato beetles to high levels of dsRNA in the lab [
13]. The insects developed resistance within ten generations, surprisingly by developing a mechanism whereby their cells no longer took up dsRNA. Another group of researchers reported similar findings in 2018 in a study of resistance to dsRNA in western corn rootworms [
14]. These results imply that once such resistance develops, the insects will also be resistant to any other RNAi-based insecticides. “No matter what gene you target with the dsRNA, the insect will be resistant to it,” Jurat-Fuentes said. “With RNAi, it looks like once the insect becomes resistant, that is it. You can no longer use that technology until you overcome the reduced uptake.”
Fortunately, further work published in 2023 by Jurat-Fuentes and colleagues showed that beetles resistant to RNAi remain susceptible to other chemical and biological pesticides [
15]. Farmers already know that susceptibility varies from product to product—to combat resistance, they use pesticide rotation as an agricultural best practice, a rotation to which Calantha is a welcome addition in the case of the Colorado potato beetle. “If you hit them with pesticides that work different ways, the insect has to generally evolve at least two different mutations to become resistant to both,” Jurat-Fuentes said. “If you rotate pesticides, you can extend their useful life.”
Despite the questions that remain about resistance, other RNAi-based pesticides and other agricultural products are in the works. GreenLight Biosciences is currently developing pesticides that target fall armyworms and other lepidopteran (moths and butterflies) pests,
Varroa mites (which plague honeybees) [
3], and thrips [
16]. Academic researchers have tested the technology as a tool to control Asian citrus psyllids, bark beetles, mosquitoes, and various other insect pests [
9]. Additional companies actively pursuing RNAi technology for crop protection applications include BASF (Ludwigshafen, Germany), Bayer AG (Leverkusen, Germany), Corteva (Indianapolis, IN, USA), and Syngenta (Basel, Switzerland) [
17]. “GreenLight Biosciences and others are already thinking about using RNAi to control weeds, plant diseases, and many other pests,” Jurat-Fuentes said. “RNAi products have a lot of potential—we are just at the very beginning of it.”
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