Final Report for the Hawaii Invasive Species Council FY2021
Project Title
Biopesticides delivered with water-storing hydrogels for control of invasive yellow crazy ants
Principal Investigator
Jia-Wei Tay, College of Tropical Agriculture and Human Resources, University of Hawaii at
Manoa
Funding Amount$31,548
Project Period
1/1/2021 to 4/30/2022

Introduction
The yellow crazy ants (Anoplolepis gracilipes) cause severe impacts in natural and some
agricultural systems. They also displace native insects, causing damage to biodiversity andecosystem sustainability in natural settings (Hoffmann and Saul 2010; Plentovich et al. 2018). In
addition to causing severe impacts on biological diversity, yellow crazy ants are considered
agricultural pests in Hawaiʻi that damage crops (e.g., bananas) with their formic acid secretions(Nelson and Taniguchi 2012).
Broad spectrum insecticide sprays present a hazard to humans and environmental health. Liquid
sucrose bait, infused with a low concentration of insecticide, is a highly effective alternative
treatment option for invasive ants. However, current approaches require substantial economic
investment in plastic bait dispensers and continual maintenance. Water-storing hydrogels provide
the sucrose solution ants prefer in a no-spills, easy-to-deploy format, circumventing the need forbait dispensers. The deployable hydrogels individually act as micro-sized controlled-release bait
dispensers, allowing sugar-feeding ants to feed directly on them (Tay et al. 2017, 2020). Recent
experimental studies reported that hydrogel baits incorporated with small amounts ofneonicotinoids successfully managed Argentine ant and yellow crazy ant populations in
Hawai‘i’s natural areas (Krushelnycky 2019), as well as Argentine ant populations in California
citrus (McCalla et al. 2020). However, neonicotinoids are under increasing scrutiny, because
they can present a threat to pollinators. There is a critical need for effective and safe insecticidal
compounds in ant baiting systems.
We initially proposed to use spinosad as an active ingredient in hydrogel baits. Althoughspinosad is a natural substance made by a soil bacterium and is classified as a reduced-risk
pesticide by the US Environmental Protection Agency (EPA), we did not use spinosad in thisstudy due to its toxicity to bees and other pollinators. Hence, we switched to using boric acid in
the hydrogel baits. We chose boric acid as it is practically nontoxic to birds, fish, and aquatic
invertebrates and relatively nontoxic to beneficial insects (US EPA 1993). According to the EPA
toxicity rating standard (acute oral toxicity), boric acid is rated in the “toxicity category III,”indicating it is only slightly toxic for vertebrates (US EPA 2006). With its sufficient solubility inwater to be prepared in 10-25 percent sucrose solution, boric acid has shown to be a highlyeffective toxicant when it is incorporated in a liquid bait (Choe et al. 2021).
The specific objectives in this study are: (1) to determine the attractiveness of improved alginatehydrogel beads containing humectant towards yellow crazy ants; and (2) to determine the fieldefficacy of boric acid delivered with improved alginate hydrogels baits against yellow crazy ants.
Although we switched to a different insecticide and incorporated a field study, we did not needto make significant changes in the budget.
Technical Approach
Objective 1: The attractiveness of fresh and aged alginate hydrogels formulated with 25%
sucrose and 0, 10, 20, or 30% humectant (we did not disclose the name of the humectant
compound in this report as we are in the process of publishing this study) was tested withlaboratory colonies of yellow crazy ants. In each trial, 150 worker ants were introduced into an
experimental arena, which consisted of a polyethylene container (20 cm x 15 cm) whose innersides were coated with a film of insect barrier to prevent escape. To prepare the aged hydrogels,
fresh hydrogels were left exposed at ambient room conditions for 3, 7, 14 and 28 days. In eachexperiment, four individual beads of the same age formulated with 0, 10, 20 and 30% humectant
were simultaneously placed 2 cm apart in a square configuration on a petri dish cover at thecenter of the experimental arena. Digital pictures of the arena were taken at 5, 10, 20 and 30
minutes after beads were introduced, and numbers of ants feeding on individual beads at eachtime interval were subsequently counted. The experiments were replicated four times using four
different ant colonies. One-way ANOVA and Tukey’s HSD test at the 0.05 level of significancewere used to compare the ant counts among the different concentrations of humectant at eachtime point.
Objective 2: Each experimental site was treated once with ~500 g of improved or standardhydrogel baits at an application rate of 10 g/m2. The hydrogel baits were hand-distributed in ~10
piles of ~50 g each, separated from each other by at least several meters and placed along
multiple active ant trails on the soil. Field sites were monitored on day 0 (pre-treatment), andweeks 1, 2, 3, 4, 6 and 8 post-treatment using monitoring traps, which consisted of 50-ml
centrifuge tubes containing a cotton ball soaked with 25% sucrose solution. At each site, 10monitoring traps were placed along the foraging trails. After 30 minutes, the traps werecollected, capped, and the number of ants in each trap was recorded. Numbers of ants weresummed across the 10 monitoring traps at each site on each monitoring date. The difference intotal ant counts among monitoring time points within the standard and improved bait treatments
were assessed by Friedman test.
Results and Deliverables
Results from Objective 1: For fresh hydrogel beads, no significant differences (P > 0.05) were
found between numbers of yellow crazy ants attracted to standard hydrogel beads and any of theformulations containing humectant at 5 and 10 minutes post-introduction. At 20 minutes post-introduction, significantly fewer ants were attracted to beads containing 30% humectant
compared to all other formulations. At 30 minutes post-introduction, however, numbers of ants
attracted to 30% humectant were only significantly lower than numbers attracted to standard
beads with no humectant (Figure 1A).
For hydrogels aged for three days, there were no significant differences in mean numbers ofyellow crazy ants attracted to any of the formulations at any point in time (Figure 1B).
For hydrogels aged for seven days, significantly more yellow crazy ants were attracted to thehydrogel beads containing 10, 20, and 30% humectant compared to the standard hydrogel beads
at 30 min post-introduction (Figure 1C). The standard beads aged for seven days appeared to behardened and contain minimal moisture in contrast to the improved beads which were flexible,
suggesting more water retention.
No yellow crazy ants were attracted to the standard hydrogel beads aged for 14 and 28 days
whereas ant counts were significantly greater (P < 0.05) on all hydrogel beads
containing 10, 20, and 30% humectant at all time intervals (Figures 1D & E). This further
supports the inference that 10-20% humectant composition does not influence bait attractiveness.
Improved hydrogel beads that were aged remained flexible and transparent in appearance, unlike
the standard hydrogel beads which hardened and became opaque as they aged (Figure 2).
Results from Objective 2: After a single field application using the standard hydrogel baits (nohumectant), yellow crazy ant numbers in the monitoring traps were significantly reduced
compared to the pre-treatment data by two weeks post-treatment. However, ant numbers
gradually rebounded and by four weeks post-treatment were not significantly different from pretreatment levels (Figure 3A). In contrast, for a single treatment using the improved hydrogel
baits (containing 20% humectant), ant numbers in the monitoring traps remained low and weresignificantly different from pre-treatment levels from week two through week eight posttreatment (Figure 3B), indicating considerable residual activity of the improved hydrogel bait. At
three weeks post-treatment, ant numbers in the monitoring traps were reduced by 66%, thehighest percent reduction observed during the study period (Figure 3B). Concurrently, numbersof ants in monitoring traps at the two untreated control sites did not show any decline during thestudy period, and instead exhibited a 3.2-22.8% increase during weeks 1-8 relative to pretreatment levels.
From the results, we conclude that the improved boric acid alginate hydrogel baits (with ahumectant) are more effective in yellow crazy ant management, as compared to boric acidhydrogel baits without a humectant. In the future, a variety of other sugar-loving ant species,
including incipient invaders, can be similarly targeted with this approach.
Other deliverables from this project include oral presentations at the 2022 National Conferenceof Urban Entomology and 2022 Hawaii Pest Control Association annual conference. The
relevant results and outcomes will also be shared and distributed through the Principal
Investigator’s website (manoa.hawaii.edu/ctahr/urbanlab). Part of the data and results will be
used for a peer-reviewed journal publication.

Figure 1: Number (mean ± SEM) of yellow crazy ant feeding on hydrogels conditioned indifferent concentration of humectant over time tested with (A) fresh hydrogel, (B) 3 days aging
hydrogel, (C) 7 days aging hydrogel, (D) 14 days aging hydrogel, (E) 28 days aging hydrogel.

Number of ants

Figure 2: Attractiveness study with hydrogel beads aged for 28 days that contained sucrose
solution and 0%, 10%, 20%, or 30% humectant at 5-minute post introduction into test arena.

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Figure 3: Pre- and post-treatment of yellow crazy ant visits (mean ± SEM) at three sites treatedwith (A) hydrogel baits containing 2% boric acid, (B) hydrogel baits containing 20% humectant
and 2% boric acid.

References
Choe, D. H.*, Tay, J. W. *, Campbell, K., Park, H., Greenberg, L., and Rust, M. K. 2021.
Development and demonstration of low-impact IPM strategy to control Argentine ants in urban
residential settings. Journal of Economic Entomology 114: 1752-1757 * Equal contribution.
Hoffmann, B. D., and Saul, W. C. 2010. Yellow crazy ant (Anoplolepis gracilipes) invasions
within undisturbed mainland Australian habitats: no support for biotic resistance hypothesis. Biol
Invasions 12: 3093-3108.
Krushelnycky, P. 2019. Evaluation of water-storing granules as a promising new baiting tool forthe control of invasive ants in Hawaii. Report of Year 1Activities to the Hawaii Invasive SpeciesCouncil.
McCalla, K., Tay, J. W., Mulchandani, A., Choe, D. H., and Hoddle, M. 2020. Biodegradable
alginate hydrogel bait delivery system effectively controls high-density populations of Argentine
ant in commercial citrus. Journal of Pest Science 93: 1031-1042.
Nelson S., and Taniguchi G. 2012. Ant damage to banana fruits by abdominal secretions.
Honolulu (HI): University of Hawaii. 5 p. (Insect Pests; IP-29).
Plentovich, S., Russell, T., and Fejeran, C. C. 2018. Yellow crazy ants (Anoplolepis gracilipes)
reduce numbers and impede development of a burrow-nesting seabird. Biol Invasions 20: 77-86.
Tay, J. W., Hoddle, M., Mulchandani, A., and Choe, D. H. 2017. Development of an alginatehydrogel to deliver aqueous bait for pest ant management. Pest Management Science 73: 20282038.
Tay, J. W., Choe, D. H., Mulchandani, A., and Rust, M. K. 2020. Hydrogels: from controlled
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US EPA (Environmental Protection Agency). 1993. “Boric acid.” R.E.D. Facts. Office ofPesticide Programs, Washington, DC.
https://archive.epa.gov/pesticides/reregistration/web/pdf/0024fact.pdf
US EPA (Environmental Protection Agency). 2006. Report of the food quality protection act(FQPA) tolerance reassessment eligibility decision (TRED) for boric acid/sodium borate salts.
Environmental Protection Agency, Prevention, Pesticides and Toxic Substance, United States.
https://archive.epa.gov/pesticides/reregistration/web/pdf/boric_acid_tred.pdf