Location: Biological Control of Pests Research
2017 Annual Report
Objectives
Objective 1: Discover new efficacious natural products, including fire ant-specific pathogens, for Integrated Pest Management (IPM) of invasive ant species.
Sub-objective 1A: Evaluate naturally occurring toxins for invasive ant control.
Sub-objective 1B: Evaluate entomopathogenic agents for invasive ant control.
Objective 2: Determine gene function and utilize existing genomic resources to develop gene disruption approaches for mitigating the impact of invasive ants.
Sub-objective 2A: Perform database comparisons to identify candidate genes for disruption.
Sub-objective 2B: Functionally characterize specific candidate genes targeted for disruption.
Objective 3: Improve existing and design new biopesticide delivery systems as part of Integrated Pest Management (IPM) programs for invasive ant species.
Sub-objective 3A: Enhance the efficacy of biopesticides by improving adjuvants and synergists.
Approach
Current practice for controlling invasive pest ants depends heavily on synthetic insecticides, which have hazardous impacts beyond their intended uses. New safer and more sustainable technologies are needed to improve pest ant management. This project plan describes research to develop new products for managing invasive ants, particularly the imported fire ant, Solenopsis invicta, and the tawny crazy ant, Nylanderia fulva. This research will focus on 1) identifying new biopesticides, including naturally occurring toxins and biological control agents; 2) identifying and characterizing targets and methods for gene disruption; and 3) developing and improving delivery systems to maximize the efficacy of newly developed and existing biopesticides. This research will produce innovative products and methods for managing invasive pest ants.
Progress Report
In addition to 3 ant species, Monomorium pharaonic, Monomorium chinense, Tapinoma melanocephalum, chemical profiles of eastern subterranean termites, Reticulitermes flavipes; and spotted lady beetles, Coleomegilla (C.) maculate, the Bagrada bug, Bagrada (B.) hilaris, were determined. Numerous terpenes were determined in ants in the genus of Monomorium. Our research demonstrated that Monomorium ants may be one of the most potent terpene producing animals in nature.
In addition to precocinelline that has been long known as a defensive compound in ladybeetles, 2,4,6-trimethylpyridine was identified in C. maculate. It is the first demonstration that 2,4,6-trimethylpyridine, a common precursor of many other substituted pyridines used in commerce, occurs in nature. Toxicity bioassays showed that 2,4,6-trimethylpyridine is a potent contact and fumigation toxin to the red imported fire ants. A series of sulfur containing compounds were identified in the invasive stink bug, B. hilaris. Contact and fumigation toxicities of 3 isothiocyanates, allyl isothiocyanate (AI), 2-phenylethyl isothiocyanate (2-PI) and 3-(methylthio)propyl isothiocyanate (3-MPI) were determined for the red imported fire ants and tawny crazy ants.
Many benzoates occur in nature. Although insecticidal properties of certain benzoates have been reported for pest insects, toxicities of benzoates to the red imported fire ants and tawny crazy ants have never been determined. They have never been used in ant control. Contact and fumigation toxicity of 15 benzoates were assessed. Among all 15 tested benzoates, methyl benzoate is the most potent fumigant and benzyl benzoate and hexyl benzoates are two most potent contact toxins. The contact toxicity of benzoates was positively related to the length of alcohol chains. In contrast, the fumigation toxicity was negatively related to the length of alcohol chains. A formulation using benzyl benzoate has been developed and is being tested in the field. Many benzoates are used as flavor ingredients for human consumption, indicating their low mammalian toxicity. Our study has demonstrated the potent toxicity of these naturally occurring compounds against fire ants and the feasibility of using these compounds in fire ant management.
Electroantennogram (EAG) responses of red imported fire ants and black imported fire ants to 50 selected compounds were evaluated. Twenty five compounds elicited EAG response from workers and female alates and 15 had response from males. The behavioral effect of those positive compounds are being evaluated. Female specific compounds that elicited EAG response from males will be screened for sex pheromones. These compounds may have diverse effect on fire ants and may act as attractants or repellants. They can be potentially used in fire ant management.
Studies were conducted to determine growth inhibition of 2 different natural toxins from Camelina sativa on selected strain of Beauvaria bassiaba to be used as synergists to control the red imported fire ant (Solenopsis invicta). Beauvaria bassiana NI8 was exposed to Sinapic acid and Chlorogenic acid. Sinapic acid was mixed at a 0.22% level directly into potato dextrose agar (PDA) media (48°C) and poured into plates. Chlorogenic acid was mixed at a level of 0.07% directly into PDA media (48°C) and also poured into plates. Control plates were standard PDA. A suspension of NI8 spores was made in 0.2% Ethoxylated tridecyl alcohol (TDA) (HLB8) and then streaked across the plates, 10 repetitions of each treatment and control. Plates were allowed to incubate at 25°C for 16 hours prior to reading. Plates were viewed under an inverted microscope for germination. The mean percent spore germination in Sinapic acid-PDA plates was 68.5% and 68.6% Chlorogenic acid-PDA plates, while 70.4% in the control-PDA plates.
To identify targets that may be of primary use as invasive ant control in the field, two sets of expressed genes (Ribonucleic acid (RNA) transcripts) were sequenced. Three replicates of Red Imported Fire Ant (RIFA) worker larvae and worker pupae were compared, and genes associated with food processing by larvae and development from larva to adult were compiled into a spreadsheet indicating levels of expression and degree of variation by stage. A wide range of both characterized and uncharacterized differentially expressed genes were identified, involving many key physiological processes.
Eleven genes that were consistently expressed in the larva and pupa forms were identified as possible reference genes for double stranded ribonucleic acid(dsRNA) experiments. These will be considered along with published recommendations. RIFA colonies have been maintained since March 2016, and three of four colonies are thriving. A method was devised for delivering and measuring liquid intake. The colonies were all tested (four replicates) for sugar solution preferences using glucose, fructose, sucrose, and equimolar concentration of all three. Additionally, pilot studies designed toward delivering insect homogenate in liquid form have been completed. The first gene for which primers have been designed and ordered is one that is expected to provide a phenotypic change in the cuticle, and will be useful as a positive control. Primers from 4 exons were designed for dsRNA preparation and expression detection. Delivery by microinjection is being considered for dsRNA designed for chemical ecology studies as well as pest control. Equipment has been transferred and experimental setup is forthcoming. Based on preliminary and replicated tests of liquid feeding, dsRNA in solution may be delivered in sucrose or mixed sugar solution at a wide range of concentrations not less than 1% and not saturated. Based on preliminary tests, a dilution of insect homogenate may serve as a delivery medium for dsRNA. Based on pilot test, delivery of control dsRNA solution to subset of colony (“colonoid”), dsRNA is not, in and of itself, lethal. That being said, colonoid response included some mortality, and production of a single alate female. Based on preliminary results, dsRNA assays using colonoids will require at least 30 days to yield results.
Accomplishments
1. Benzoates for fire ant control. Mound treatment is commonly used in managing fire ants. A major concern for such treatment is the potential detrimental effect of insecticides to the environment. Environmentally friendly products are desired. Based on an extensive screen, ARS scientists at Stoneville, Mississippi, found two benzoates are potent contact toxins to fire ants. These benzoates are used as flavor ingredients for human consumption, indicating their low mammalian toxicity. These compounds occur naturally in numerous plants, indicating they may also be safe to the environment. It may be feasible to use these compounds in fire ant management to reduce the negative impact of fire ant chemical control.
