Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
Entomology
Non Technical Summary
Hemipteran vectors are economically important plant-sucking pests within agricultural systems and their populations are expected to increase due to shifting global weather patterns that accelerate insect population growth. To bolster the toolbox for controlling hemipteran vectors, we have focused on identifying novel physiological pathways thatsimultaneously inhibit feeding, halt pathogen transmission, and induce mortality.We previously demonstrated that chemical inhibitors of inward rectifier potassium (Kir) reduce function of aphid salivary glands, eliminate phloem feeding events, and led to death through starvation.However, significant gaps in knowledge remain regarding the toxicological relevance of hemipteran Kir channels and further, the salivary gland remains an unexploited target site for hemipteran control.Thus, the goal of this proposal is to test the hypothesis that inducing salivary gland failure through Kir channel inhibition represents a viable approach to reducing hemipteran-mediated feeding damage in agricultural systems. To achieve this goal, we propose the following objectives: 1) test the efficacy of structurally diverse Kir channel inhibitors to inhibit and/or alter salivary secretions to prevent plant feeding by hemipteran vectors; 2) determine the ability of Kir channel inhibitors to prevent pathogen transmission and acquisition by hemipteran vectors; 3) enhance plant systemicity of Kir channel inhibitors through novel botanical solubilizers. Collectively, these data will further validate hemipteran Kir channels and the salivary gland as tractable targets for agrochemical development. We aim to increase the sustainability of the agricultural industry through management of hemipteran populations and thus, this project supports the priorities listed in the program code A1112.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
0%
Goals / Objectives
General Hypotheses:We hypothesize Kir channels are a tractable target site to induce salivary gland failure that will lead to feeding cessation and/or reduced virus transmission in two economically important hemipteran vectorsThere is a need for insecticides that reduce hemipteran populations while simultaneously preventing feeding and pathogen transmission.Considering this, theoverarching goalof this investigation is to test the hypothesis that modulation of hemipteran Kir channels will reduce saliva and protein secretion during feeding, which will prevent plant feeding and transmission of plant pathogens. To achieve our goal, we will:Test the hypothesis that structurally diverse Kir inhibitors reduce and/or alter salivary secretions and plant feeding of hemipteran vectors;.This objective will further validate Kir channels as a molecular target to prevent salivation and feeding in aphids and will provide the first insights into the ability to target salivary gland Kir channels to reduce damages stemming from stink bug feeding.This study will also provide novel insights into the mechanistic underpinnings driving enzyme concentrations in watery- and gel- saliva of aphid and stink bugs and the varying plant responses to the saliva.Test the hypothesis that exposure to Kir channel modulators during feeding will reduce the acquisition and transmission of insect-vectored plant pathogens;Objective 2 will systematically test the ability of Kir channel inhibitors to regulate acquisition and transmission of aphid- and stink bug- vectored plant pathogens.These data will be of significant interest to the agricultural industry because an insecticide that induces mortality and alters pathogen dynamics does not currently exist.Optimize the formulation for increased bioavailability and enhanced systemic activity of lipophilic Kir channel modulators. Objective 3 will aim to employ novel technology that enables non-water soluble compounds, such as Kir inhibitors, to be used in hydroponic systems and enable systemicity in plant tissues.Data collected in this objective will provide insights into the potential to control hemipteran pests after hydroponic treatment of host plants, which is of obvious interest to the agriculture industry.
Project Methods
Objective 1:?Collection of water- and gel-saliva:Aphid 'watery' saliva will be collected by allowing aphids to feed on the artificial membrane system as described in Obj. 1.2.As per Chaudhary et al.57, 100 aphids per membrane for a minimum of 800 aphids will be assayed and samples will be pooled and stored at -80°C.For gel saliva, salivary sheaths will be removed from membranes and stored at -80°C.Stink bug watery saliva will be collected by allowing stink bugs to feed on the artificial membranes for 24 h, removed from the membrane, and then chilled for 5 min on ice and placed ventral side up under a dissecting scope.Watery saliva, secreted from the tip of the proboscis as the insects return to room temperature, will be collected with a microinjector (Nanoliter 2010, WPI).Watery saliva will be stored at -80°C until needed.For gel saliva, salivary sheaths will be removed from membranes and stored at -80°C.In addition, we will increase applicability of this methods by allowing stink bugs to feed on cherry tomatoes and stylet sheaths will be removed 48 hours post feeding and stored at -80°C30.Enzyme activity assays:Watery and gel saliva collected from aphids and stink bugs exposed to Kir inhibitors will be assayed for amylase, peroxidase, polyphenol oxidase, and glucose oxidase activities as per Peiffer and Felton (2014)30.We currently measure amylase and peroxidase levels in mosquito saliva and oxidase activities in honey bee hemolymph, thus we have the necessary equipment and expertise to complete this portion of the study.Impact of water- and gel-saliva on plant defensive genes:Seeds of a commercial soybean variety will be will be planted in a greenhouse and raised following established procedures in the Davis Lab. Three weeks after emergence, test plants will be used for experiments.Concentrated water or gel saliva will be suspended in phosphate buffered saline and applied to the upper most fully expanded trifoliate test plants.All treatments will be arranged in a randomized complete block design (RCBD) with four replications.Two days after treatment, leaf samples will be randomly selected and brought back to the laboratory for testing.Extraction of total RNA and cDNA synthesis will occur as per Miranda et al. (2013)58.For reference genes, primersGmCYP2andGmELF1Afrom Miranda et al. (2013) will be used.For defense-related gene expression, primersPR1,PR3, andPR10from Upchurch and Ramirez (2011)59will be used. The qRT-PCR will follow Miranda et al. (2013)58.Electrical penetration graph (EPG) recordings:To compare vector feeding behavior on plants, EPG experiments will be conducted withA. gossypiiandP. guildiniias described in our previous publications14,60.Changes to feeding behavior after exposure to 5-10 structurally diverse Kir inhibitors (Table 1 and other unpublished scaffolds) will be compared to each compounds corresponding inactive analog and a vehicle control.Feeding waveforms identifying specific activities will be distinguished using characteristics listed in our previous publications14,60.Three test plants (1 virus-free control, 1 virus-free + Kir inhibitor, 1 virus free + inactive analog) will be placed randomly within the Faraday cage.Next, one insect per test plant will be placed on the abaxial side of a leaf and feeding behavior will be recorded for 4 h.This will be repeated 40 times, per aphid per host/Kir inhibitor/inactive analog combination.Pre-probe, xylem phase (G), E1 (sieve element salivation), and E2 (phloem sap ingestion) durations will be recorded per 4 h feeding bout.Data will be tested for normality by the Kolmogorov-Smirnov test and tested for homogeneity of variance by the Levene Test in PROC GLM using SAS software.Differences in probing behaviors will be tested by one-way ANOVA.For stink bugs, procedures will follow those described in Lucini et al.56.Objective 2:Virus source:The PLRV isolate (PLRV-MNR06) andpotato virus Y (PVY) inoculumwill be used in transmission experiments and are currently maintained by successivetransmissions toRusset Burbankin the Davis Lab at LSU.Test plants:Soybean (AG46X6) and potato (certified cv Russet Burbank seed tubers) will be planted and maintained following standard procedures.Prior to use in transmission experiments, test plants will be visually inspected and serologically tested by DAS-ELISA to confirm that they are virus-free or virus-infected.When soybean reaches V4 and potatoes are at six weeks after emergence, plants will be entered into experiments.Virus detection:To identify PLRV infection in potato test plants, leaflets will be picked and promptly rolled to extract leaf sap, which will be stored at -20°C until needed.Potato leafroll virusandPotato virus Ydetection will be performed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using polyclonal kits according to manufacturer's protocols.Absorbance will be measured at 405 nm with a spectrophotometer currently available in the Swale and Davis Labs.Plants will be considered positive if the optical density readings is greater than the mean of five negative controls (non-infected plants) plus three times their standard deviation, which is standard procedures.Reverse transcriptase-polymerase chain reaction (RT-PCR) will be conducted on all plants and aphids.Viral RNA will be extracted and RT-PCR will both be used using commercial kits following manufacturer's specifications.Primers for virus detection will be used as described by Singh et al. (1995)65.All RT-PCR products will be visualized on agarose gels stained with ethidium bromide and then photographed.Infection rates will be calculated as number of infected test plants divided by total number of test plants, expressed as a percentage.Phaseolibacter flectensbacteria:Phaseolibacter flectenswas isolated from a field collected redbanded stink bug allowed to feed on sterile soybean infusion agar as described by our previous publication55.Single colonies were streaked on nutritionally rich agar plates and incubated for 1-3 d at room temperature.A single colony from each plate was grown in LB broth overnight culture at room temperature.An aliquot of each purified strain of 800 ml with 200 ml glycerol was stored at -80°C.?Objective 3:We will treat the soil of the plant with the newly formulated KM-WSC and will assess the systemicity of the compound by taking leaf punctures of the plant and quantifying the chemical concentration at various leaf locations through high-performance liquid chromatography (HPLC) that is coupled to a mass electrospray ionization (ESI) mass spectrometry. In addition to quantification of chemical throughout the leaf tissues, we will perform EPG recordings as described in Objective 1 to measure the influence of the formulated chemical to ensure the activity between contact exposure and ingestion is not different.