Progress 01/01/22 to 01/12/23
Outputs
Target Audience:The target audience for the project are scientists inacademic, government, and private sector laboratories exploring the development of new insecticidal products and technologies. The project results will help inform theirR&D efforts andprovide the broader scientific community new insights on mechanisms of signaling pathways for ecdysis andadvance adverse outcome pathway(s) for a key developmental pathway in Insecta. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The PD has trained a full-time research technician on the project. Additionally, five undergraduate students gained research experience by working on parts of the project. Several of the species reared for the project were utilized in a STEM Education Field Trip, where K-12 students had the opportunity to learn and observe the different insect orders. At Iowa State University, an undergraduate student in Biology and a graduate student in toxicology were trained on the project.The project also provided the PD the opportunity to present at three conferences and a departmental seminar: Krishnan N, Gorman CL, Bradbury SP, Jurenka RA. 2022. Elucidating the mechanism through which low-dose neonicotinoid and pyrethroid insecticides disrupt insect ecdysis. Society of Environmental Toxicology and Chemistry, Pittsburgh, PA. Krishnan N, Bradbury SP. 2022. Assessing risk of insecticide exposures to Lepidoptera: molecular to population-level effects. American Chemical Society, Chicago, IL. Krishnan N, Jurenka RA, Bradbury SP. 2022. Characterizing a novel adverse outcome pathway for neonicotinoid insecticides. North Central Branch meeting of Entomological Society of America, Minneapolis, MN. Krishnan N, Bradbury SP, Jurenka RA. 2022. Characterizing a novel adverse outcome pathway for neonicotinoid insecticides in Lepidoptera and Coleoptera. Entomology seminar series, University of Minnesota, Virtual. How have the results been disseminated to communities of interest?The presentations made by the PD at symposiums in national conferences were attended by people working in state and federal agencies, pesticide manufacturing companies, and academia: Krishnan N, Gorman CL, Bradbury SP, Jurenka RA. 2022. Elucidating the mechanism through which low-dose neonicotinoid and pyrethroid insecticides disrupt insect ecdysis. Society of Environmental Toxicology and Chemistry, Pittsburgh, PA. Krishnan N, Bradbury SP. 2022. Assessing risk of insecticide exposures to Lepidoptera: molecular to population-level effects. American Chemical Society, Chicago, IL. Krishnan N, Jurenka RA, Bradbury SP. 2022. Characterizing a novel adverse outcome pathway for neonicotinoid insecticides. North Central Branch meeting of Entomological Society of America, Minneapolis, MN. What do you plan to do during the next reporting period to accomplish the goals?We are currently treating final instar Colorado potato beetle larvae with dietary imidacloprid to determine if the larval to pupal transition stage produces AE. Following this, we will undertake topical and dietary studies withDrosophilalarvae and topical studies withDrosophilapupae. Treated diets collected from these studies and from corn earworm dietary bioassays will be analyzed to verify the nominal concentrations. Under Objective c, we will finish developing methods to successfully undertake GABA antibody staining in Lepidoptera/Coleoptera.We will then employ these antibodies to follow the release of GABA molecules at different time points before, during, and after ecdysis in control and neonicotinoid-treated individuals. We will continue to utilize the commercial CCAP antibody with different protocols, including removing the blood brain barrier sheath that surrounds the central nervous system. This might help the CCAP antibody penetrate the nervous system and bind to CCAP.Positive results will test our hypothesis of CCAP involvement in AE and elucidate the mechanisms through which CCAP is regulated in Lepidoptera/Coleoptera. Outside of these experiments, we will initiate the writing of two papers for publication before the next reporting period. The papers would report findings from our lepidopteran and coleopteran studies.
Impacts
What was accomplished under these goals?
Impact: There is a need for new insecticidal modes of action that target susceptible windows of development in insect pests. Discovery of new modes of action would help support IPM programs, delay development of insecticide resistance, and reduce non-target effects. We previously discovered that an insecticide (imidacloprid) that mimics acetylcholine caused the failure of pest moths and non-target butterfly larvae to complete pupation. We termed this response as arrested ecdysis (AE). In the current project, we are investigating the extent to which AE can be elicited in additional pest species, including beetles (red flour beetle, mealworm beetle, and Colorado potato beetle) and flies (housefly and fruit fly). Additionally, we are assessing the extent to which additional types of acetylcholine mimics cause AE. If AE is observed at doses lower than those that cause outright mortality, further elucidation of the AE mechanism can result in the discovery of new insecticides that lower environmental loading while targeting three major groups of insects that impact agricultural and livestock production. Under Objective a, we topically tested the following nAChR agonist compounds on corn earworms and fall armyworms: nicotine, nitenpyram, sulfoxaflor, acetamiprid, and thiacloprid. Additionally, we tested pilocarpine (a muscarinic acetylcholine receptor agonist) and mecamylamine (a nAChR antagonist). Similar to responses seen with imidacloprid, relatively low doses (1 to 20 µg/larva) of nitenpyram, acetamiprid, and thiacloprid caused high rates of AE in corn earworms. Fall armyworms were recalcitrant to AE at these doses, however, at 10- to 100-fold greater doses (200 µg/larva), these compounds caused low rates of AE. With sulfoxaflor and nicotine, both corn earworms and fall armyworms were largely recalcitrant to AE, even at 200 µg/larva. Since different nAChR agonists bind to different combinations of nAChR subtypes, these results suggest that AE is strongly elicited by only certain receptor subtypes. Additionally, the highest soluble concentrations of pilocarpine and mecamylamine in acetone did not cause AE in either species, suggesting that AE is likely caused by the activation of nAChRs. Under Objective b, we topically dosed both corn earworms and fall armyworms with imidacloprid (20 µg/larva), nitenpyram (20 µg/larva), and sulfoxaflor (100 µg/larva). Five larval samples for each insecticide were collected at the following time points after treatment: 0h, 6h, 24h, and after pupation or AE. Internal concentrations of the samples were determined via LC-MS. Both corn earworms and fall armyworms significantly metabolized all three compounds; on average, the 6h samples had 33-72% lower concentrations and the 24h and pupa/AE samples had 79-99% lower concentrations than the 0h samples. Additionally, we did dietary studies with the three compounds; 500 ng/g of imidacloprid and nitenpyram caused high rates of AE in corn earworms while 1000 ng/g sulfoxaflor (corresponding to the highest solvent concentration that did not cause mortality in controls) had no effect. In fall armyworms, 1000 ng/g imidacloprid, nitenpyram, and sulfoxaflor caused little to no AE. The results from this objective indicate that the differences in insecticide susceptibility between the two species is likely due to toxicodynamic factors. Under Objective c, we have not made significant progress due to the difficulty in finding a CCAP antibody that will help identify the neurons that are involved in ecdysis.A commercial antibody against CCAP failed to identify CCAP producing neurons in Lepidoptera larvae and in Coleoptera pupae. Additional inquiries to researchers previously using CCAP antibodies have not been fruitful.Alternative strategies employed include injecting CCAP into Lepidoptera larvae that had previously been treated with imidacloprid.These rescue experiments did not work, probably due to the failure of CCAP in circulation to cross the blood brain barrier.Identification of the GABA producing neurons will be undertaken in the last year of funding. Under Objective d, we topically treated the pupae of the following beetle species with imidacloprid and acetamiprid: red flour beetle, mealworm beetle, and Colorado potato beetle. All three species showed arrested adult ecdysis, with incompletely shed pupal cuticles and unexpanded adult wings. We also topically treated housefly larvae and pupae with these compounds; the larvae produced elongated pupae and the adults from the treated pupae exhibited low rates of AE. Results from this objective show that neonicotinoid-mediated AE at the adult stage can be seen in both Coleoptera (highly susceptible) and Diptera (lowly susceptible). Additionally, in housefly larvae, neonicotinoids produce an unspecified effect on pupal shape.
Publications
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