Location: Biological Control of Pests Research
2016 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
By analyzing the discharge collected from the stinger apparatus (milking) and the contents of poison and Dufour’s glands of little black ants, seven new compounds were found, including amines, alkaloids and terpenes. Two terpenes, ß-springene and neocembrene, were found only in the defensive secretion of queens. All amines and alkaloids were from the poison gland and terpenes from the Dufour’s gland. This research provides the first evidence for the natural occurrence of three amines.
In addition to the little black ants, the chemical profiles of defensive secretions of 3 other ant species were determined. Sixteen natural products were obtained from collaborators, including 9 botanical extracts, 6 ant extracts and one synthetic naturally occurring compound. Toxicity bioassay is ongoing for these materials and compounds.
An innovative method for analyzing trace volatile compounds in insects was developed. Insects rely heavily on chemical signals for their communication, particularly the social insects, such as ants and termites. These signals serve as a "language" to mediate interactions among themselves and with other organisms. These compounds are very useful in developing environmentally-friendly methods in pest management. They can be used to monitor insect populations, to mass trap insects, and to disrupt insect mating behavior. Unfortunately, chemical analysis of these signal compounds have often been proven to be a great challenge, partially because these chemicals exist at very low concentration in insects. A sample preparation method was developed that has significantly improved the efficiency of extraction of insect volatiles using solid-phase microextraction (SPME) techniques. This method can be used in analysis of signal compounds not only in ants, but also in many other pest insects and will greatly facilitate the identification of volatile compounds that mediate insect behaviors. Based on this new method, a series of volatile compounds were identified from the red and black imported fire ants and many have never been identified previously. The identification of these volatiles is an important first step in determining their functional role in ant society. This method has also been used to determine volatiles from other ants, aiming to find toxins and repellants for invasive ants.
The red imported fire ant and the black imported fire ant were two important invasive ants. Although the black imported fire ant was introduced and established in the United States more than one decade earlier than the red imported fire ant, the latter has gradually displaced the black imported fire ant throughout most of its distribution. However, the black imported fire ant still persist in areas along the northern boundary of the distribution range of the red imported fire ants, including northern Alabama, north Mississippi, and southern Tennessee. In 1958, the USDA Animal and Plant Health Inspection Service (APHIS) enacted a Federal Imported Fire Ant Quarantine (7CFR301) for both species and their hybrid. Tyramides were recently reported to exist in males of many ant species in the subfamily Myrmicinae, including the red imported fire ants. Transfer of tyramides from male to newly mated queen was demonstrated in the red imported fire ants. However, the males of the black imported fire ants have not been examined. Five tyramides in males of the black imported fire ants were identified and quantified. These tyramides may have diverse physiological and behavioral functions in an ant society and they may have a potential to be used in fire ant management.
Studies were conducted to examine the effectiveness of three different fungi on fire ants. High concentrations of each fungal spore were suspended and sprayed onto filter paper and placed into cages containing red imported fire ant workers (Solenopsis invicta). The cages were monitored to determine mortality rate in the presence of any post mortem sporulation. Based on the results from this experiment a strain of Beauvaria bassiana was chosen as the best candidate for further studies. Spores of this fungus were stored in liquid or sprayed onto a substrate using a fluid bed dryer to determine spore survival which was favored at cool temperatures for a period of 2 months.
Targets for RNA must be carefully chosen, because there is currently no evidence that RNAi can be used to effectively silence focal genes in the targeted species, RIFA. A proof of concept pilot experiment is needed showing that dsRNA treatment reduces the level of gene expression (relative to appropriate controls) in the target species. Even better would be some evidence that this altered gene expression also produces a phenotypic effect. The development of an RNAi experiment is highly complex. Care must be taken in both the design of dsRNA reagents and the analysis of potential off-target effects. A subset of insect genes from the model insect, Drosophila melanogaster, identified as critical to eukaryotic life, was compared with the RIFA genome. The list included 458 genes. Of these, 28 were absent from RIFA. 85 were nearly identical at the amino acid level (sufficiently identical that BLAST comparison result indicated a score of 0, indicating homology). Additionally, 104 genes were similar with a score of lower than 1.00E-100, indicating a high likelihood of homology. Targets will be chosen from RIFA genes that are expected to be lethal and are not so highly conserved with other organisms that non-target effects are expected. The list can be sorted and expanded to include preliminary assessments of additional comparisons with RNAi results from other insects such as the model insect Tribolium castaneum, and with other genomes such as Apis mellifera.
RIFA colonies were established from locally identified specimens collected during a flood. These colonies were housed in containment and with protocols designed for long-term colony maintenance. Colonies were collected in March and four healthy cultures have been maintained to date. Female alates have been isolated and those that have lost wings will be maintained as potential colony foundresses. Larvae, pupae, and workers from each colony were isolated and specimens of total RNA and genomic DNA were prepared from each, and are stored for transcriptome sequencing.
Many insecticides used in current ant bait products are not water soluble. In order to incorporate a water insoluble active ingredient, surfactants are usually needed. Unfortunately, surfactants very often deter ant feeding. It was found that certain organic solvents, under certain concentrations, did not deter ant feeding. Such solvents can be used in sugar water to increase the solubility of the active ingredient. This provides a great tool to optimize the ant bait matrixes.
For evaluation of dsRNA preparations, a water soluble carrier is needed to ensure delivery to laboratory specimens in bioassays of RNAi efficacy. Four different sugars at multiple concentrations have been tested in unreplicated pilot tests to identify candidate solutions and delivery methods. Selecting appropriate colony subcultures and presentation of materials will be critical. Further testing of attraction and consumption will be performed to ensure ingestion of dsRNA preparations.
Accomplishments
None.
Review Publications
Wang, L., Elliott, R.B., Jin, X., Zeng, L., Chen, J. 2015. Antimicrobial properties of nest volatiles in red imported fire ants, Solenopsis invicta (Hymenoptera: Formicidae). Naturwissenschaften. 102:66.
Wang, L., Chen, J. 2015. Fatty amines from little black ants, Monomorium minimum, and their biological activities against red imported fire ants, Solenopsis invicta. Journal of Chemical Ecology. 41:708-715.
Wang, L., Zeng, L., Chen, J. 2015. Sublethal effect of imidacloprid on Solenopsis invicta (Hymenoptera: Formicidae) feeding, digging, and foraging behavior. Environmental Entomology. 44:1544-1552.
Wang, L., Zeng, L., Chen, J. 2015. Impact of imidacloprid on new queens of imported fire ants, Solenopsis invicta (Hymenoptera: Formicidae). Scientific Reports. 5:Aricle number:17938 doi:10.1038/srep17938.
Allen, M.L. 2016. Characteristics of a laboratory strain of Coleomegilla maculata with a novel heritable wing spot pattern trait. Advances in Entomology. 4:47-60.
Allen, M.L. 2016. Gold: a unique pigmentation defective laboratory strain of the lady beetle. Advances in Entomology. 4:11-18.
Chen, J. 2016. Toxicity and efficacy of two emulsifiable concentrates of 2-tridecanone against red imported fire ants. Advances in Entomology. 4(1):37-46.
