Arboreal control of Wasmannia auropunctata Introduction Wasmannia auropunctata or the Little Fire Ant (LFA) is an invasive ant species native to South America. Over the last century it has spread occupy a pan-tropical distribution and is recorded in mainland USA (Smith 1929), Africa (Mikheyev et al. 2009; Wetterer et al. 1999), Australia and the Pacific region (Wetterer and Porter 2003). In the last two decades, Little Fire Ants appear to be spreading more rapidly than before. Since the 1990s, new infestations have been discovered in Vanuatu (Vanderwoude 2007), Cairns, Tuvalu, Hawai i (Conant and Hirayama 2000; Krushelnycky et al. 2005) and more recently, Papua New Guinea (Vanderwoude 2008). First reported in 1999 on the east side of Hawaii and Kauai (Conant and Hirayama 2000), it is regarded as one the worlds 100 worst invasives (Lowe et al. 2000). Little Fire Ants over-run urban (Fernald 1947) and forest ecosystems developing three dimensional super-colonies that occupy both the ground and arboreal strata (Le Breton et al. 2004). They impact wildlife populations (Jourdan 1997; Le Breton et al. 2003; Walker 2006), domestic animals (Theron 2005), and public health. There is considerable evidence that Little Fire Ants often sting domestic and wild animals on the eyes, causing keratopathy (Theron 2005). They are a serious urban (Fernald 1947) and agricultural pest, enhancing homoptera populations, stinging agricultural workers (Spencer 1941) and interfering with beneficial insects introduced for biological control (Fabres and Brown 1978). As LFA spread, all agricultural industries in Hawai i will be affected to varying degrees, especially perennial tree cropping industries such as coffee, macadamia, citrus, banana and tropical fruits. Additionally, LFA has the potential to impact on the tourism industry through decreased enjoyment of outdoor recreation opportunities for visitors and residents alike. Although eradication efforts have been underway for the small population of LFA on Kauai, LFA has spread rapidly on east Hawaii (Hawaii Ant Group 2007). Control of nests on the ground has been achieved through the use of granular baits developed for another invasive species, Solenopsis invicta (Red Imported Fire Ant). While there is scope for improvement in ground-level control, there is currently no effective method to control LFA in arboreal situations. Developing a practical method for control of Little Fire Ants in arboreal situations is therefore essential before a response to this invasive species can be considered. The use of baits to control ants is widely regarded as best practice (Williams 1983), because it minimizes the use of pesticides and provides effective colony-level control. A bait is comprised of an attractant, a carrier and an active ingredient. Insecticides suitable for use in baits should exhibit delayed toxicity, good efficacy when diluted by trophylaxis, non-repellency and ease of formulation with carriers and attractants. (Stringer et al. 1964). In the 50 years or so of ant bait development, only a handful of actives have proved useful. However, in recent years, a number of new actives have entered the market including fipronil and indoxacarb (Stanley 2004). Today, there are a number of effective bait products available giving the practitioner a wider range of choices. With some exceptions, they are formulated in a very similar way: using pre- 1
gel defatted corn grit as the carrier and once refined soya oil as the attractant. The actives available fall into two main groups: toxins and juvenoid analogues. The toxins work by killing or interfering with neural pathways or metabolism while the juvenoids (Insect Growth Regulators) interfere with reproduction and chitin formation (Miyamoto et al. 1993). Baits with a combination of both actives have also been developed (Drees and Barr 1997). Previous efforts to control W. auropunctata in Hawaii in various situations clearly demonstrate this species is difficult to control with conventional bait products. Three factors contribute to this. First, the Hilo area experiences an average annual rainfall of 300cm, making treatment with dry granules problematic (Souza et al. 2008). Second, W. auropunctata nest in both the arboreal and ground strata and ground-applied baits do not appear to be effective for the arboreal component (Souza et al. 2008). Finally, even after repeated weekly or two-weekly treatment with granular baits, ant activity quickly returns to pre-treatment levels after treatments cease (Souza et al. 2008; Taniguchi 2008). A paste bait, able to be applied to the trunks of trees as well as the ground has been suggested as a possible solution to these problems (Souza et al. 2008). Methods and materials Site The experiment was located in a field of cultivated banana at the University of Hawaii College for Tropical Agriculture and Horticultural Research station in Hilo, Hawaii. Banana clumps had been planted on a 7 metre grid, and each clump consisted of 6-10 stems at varying stages of development (Figure 1). An abundance of dead leaves and other detritus had accumulated at the base of each clump and all clumps were heavily infested with Little Fire Ants (Figure 2), thus presenting a worst-case scenario for infestation. A 30cm swath between clumps was treated with Talstar (bifenthrin 100g/litre as a suspension concentrate) mixed 10 ml product per litre water, in order to prevent ants from neighboring clumps recruiting to lures and confounding results. Figure 1. simplified diagram of banana plantation at UH Farm 2
Figure 2. image of typical banana clump Treatments A total of seven treatments were applied: a control (nil), three paste baits made with a 7:9 peanut butter and water mix to which an emulsifier and various active ingredients were added, and three commercially available granular formulations. Three treatments were made from the same bait matrix [PB]: a 7:9 mix of smooth peanut butter and water with an emulsifier to stabilize the mixture. To this mixture were added the active ingredients as follows: 1. Boric acid to produce a bait containing boric acid 1% a.i. by weight 2. Avaunt 6g/kg to produce a bait containing indoxacarb 0.18% a.i. 3. Termidor 0.5g/kg to produce a bait containing fipronil 0.005% a.i. Three conventional bait treatments were included as follows: 4. Advance carpenter ant bait (abermectin 0.11%) 5. Amdro fire ant bait (hydramethylnon 0.739%) 3
6. Advion fire ant bait (indoxacarb 0.045%) 7. control (nil treatment) Design The experiment was designed as a complete-blocks design with four replicates. Treatments were randomly allocated to plots within blocks. Plot-treatment allocations are appended. Measurements Measurement of ant activity were made as follows. A chopstick smeared with a thin layer of peanut butter was placed at four locations in each plot: two on the ground at the base of the banana stems and two on banana stems at the base of a selected leaf. Chopsticks were exposed for 60 minutes before ants recruiting to each chopstick were counted. A digital photograph was taken of each exposed chopstick and the images used later to count ants that had recruited to the lure. Full recruitment was considered to occur when more than 50 ants were present at a lure and these were recorded as 50. The counts on the two ground chopsticks and the two tree chopsticks were combined, providing a maximum count of 100 for both ground and tree recruitment. Thus, ant activity was defined as the total number of ants counted at two chopstick lures and may be a value between 0 and 100. Chopsticks were always placed at the same location in subsequent measures. Analysis Recruitment at ground level, tree level, and tree+ground combined were tested for differences from the control by analysis of variance. Measurements were square-root (n+1) transformed to normalize the data (Southwood 1978). Duncan s multiple means test was used to determine differences between treatments. The computer program ASSISTAT (Silva and Azevedo 2006) was used for ANOVA and Duncan s calculations. Results Ant activity was measured on 16 June 2009 prior to treatments being applied. An analysis of variance and Duncan s multiple means test demonstrated no pre-treatment differences between blocks (DF=3,16 F=1.3482 (ground), F=0. 6477 (tree)) or between treatments (DF=6,16 F=0.6757 (ground), F=0. 4015 (tree)). On 30 June, treatments were applied to each plot. For commercial baits (treatments 4-6) 20 grams of bait granules were spread evenly over the ground and litter within each plot. The paste baits were applied using a pneumatic texture gun (Vanderwoude and Nadeau 2009) to the crown and trunk of each banana plant. A quantity of paste bait was also applied to the ground and litter within each plot. Approximately 120 g PB mixture was applied to each plot. 4
Recruitment of ants to peanut butter lures virtually ceased for all treatments within two days. Ant activity in the control plots remained steady exceeding the 50 ants per chopstick count for almost all measures. By 11 August, six weeks post treatment, distinct differences between treatments were apparent (DF=6,18 F=3.9741 (ground), F=5.3279 (tree) F=4.7922 (combined)), (Figure 3, 4). For measures of ant activity on the ground, in trees or a combination of both, ant activity in the indoxacarb and fipronil paste bait plots were significantly less than the control and the Amdro treated plots were midway between these and the control (Table 1). Ant activity in the boric acid paste, Advion and Advance bait plots were not different from the control six weeks after treatment. Treatments were re-applied on 12 August, 1 September, 6 November and 9 December as weather conditions allowed. Despite application of a chemical barrier between plots on 23 June, 31 August, 2 November and 7 December, it appeared that ants repeatedly breached this barrier throughout the course of this experiment. This became most apparent 4-6 weeks following each application. Results for subsequent re-treatment of plots were therefore compromised and further statistical analysis was not attempted. All ant activity data up to 28 days following each experimental treatment were combined and these are presented in an x-y scatterplot (days since treatment vs ant activity) with polynomial lines of best fit included (Figure 6 (tree) and Figure 7 (ground)). 100 90 80 number of ants 70 60 50 40 30 ground Boric Acid Indoxacarb Fipronil Advance Amdro Advion Control 20 10 0 26-Jun 1-Jul 6-Jul 11-Jul 16-Jul 21-Jul 26-Jul 31-Jul 5-Aug 10-Aug 15-Aug date Figure 3. Ant recruitment to peanut butter lures placed on the ground. Treatments applied 30 June. 5
100 90 80 70 tree number of ants 60 50 40 30 20 Boric Acid Indoxacarb Fipronil Advance Amdro Advion Control 10 0 26-Jun 1-Jul 6-Jul 11-Jul 16-Jul 21-Jul 26-Jul 31-Jul 5-Aug 10-Aug 15-Aug date Figure 4. Ant recruitment to peanut butter lures placed in trees. Treatments applied 30 June. 100 90 80 number of ants 70 60 50 40 30 20 combined Boric Acid Indoxacarb Fipronil Advance Amdro Advion Control 10 0 26-Jun 1-Jul 6-Jul 11-Jul 16-Jul 21-Jul 26-Jul 31-Jul 5-Aug 10-Aug 15-Aug date Figure 5. Ant recruitment to peanut butter lures placed in trees and on the ground. Treatments applied 30 June. 6
100 80 number of ants 60 40 boric acid indoxacarb fipronil Advance Amdro Advion control 20 0 0 5 10 15 20 25 days after treatment Figure 6. Mean number of ants counted in trees as a function of time since treatment for all treatment applications combined (polynomial line of best fit) 100 80 number of ants 60 40 boric acid indoxacarb fipronil Advance Amdro Advion control 20 0 0 5 10 15 20 25 days after treatment Figure 7. Mean number of ants counted on the ground as a function of time since treatment for all treatment applications combined (polynomial line of best fit) 7
Table 1. Differences between treatments (Duncan s multiple means test, P=0.05) on measures of ant activity six weeks after initial treatment (like suffixes indicate no significant differences between treatments) Treatment Mean ant activity 6 weeks post treatment on ground Mean ant activity 6 weeks post treatment in trees Boric acid + PB 89 a 91 a 90 a Indoxacarb + PB 47 b 7 b 27 b Fipronil + PB 39 b 25 b 32 b Advance 100 a 68 a 84 a Amdro 56 ab 46 ab 51 ab Advion 100 a 83 a 92 a Control 100 a 92 a 96 a Mean ant activity 6 weeks post treatment (combined) Discussion For commercially available baits and for the boric acid+pb bait, ant activity in plots was not different from the control six weeks after the application of treatments. These results are consistent with those reported by Souza et. al. (2008) and Taniguchi (2008) for the granular baits, and demonstrate the difficulty in controlling this species. Of the commercial baits, Amdro was the most effective treatment. Application of paste baits mixed with indoxacarb or with fipronil resulted in significantly reduced ant activity up to six weeks post-treatment, both in trees and at ground level. Indoxacarb and Fipronil are both effective bait toxicants, and although there was no equivalent commercial bait containing fipronil tested in this experiment, indoxacarb is also the active ingredient in Advion fire ant bait. The indoxacarb+pb bait performed significantly better than Advion in this trial. This suggests that the PB bait matrix, the ability to apply PB bait to vegetation, or both factors, improved efficacy. The commercially available baits tested in this trial are comprised of a matrix containing defatted corn grit and soya oil to which toxins and other undisclosed ingredients have been added. This bait matrix has proven to be an effective bait for Imported Fire Ants (Solenopsis invicta) and some other pest ant species. However, it is only moderately attractive to Little Fire Ants (pers ob). In preference tests, peanut butter was more attractive than soya oil to Little Fire Ants (Williams and Whelan 1992) and this may be the reason the PB matrix with indoxacarb or Fipronil added, resulted in better control than granular baits. Granular baits such as Advion, Amdro and Advance are not rain-fast, and once wet, become unattractive to foraging ants. The largest infestations of Little Fire Ants in Hawaii are found along the east coast of the Big Island. Rainfall in this district averages 120-200 inches annually and falls year-round. Granular baits are difficult to apply in these conditions as they quickly become water-logged from the constant rain. A bait manufactured on a paste matrix is likely to 8
remain effective for longer under these conditions. Paste baits dehydrate over time making them less attractive to foraging ants. To some extent, they may be rejuvenated by rainfall. Further, Little Fire Ants nest in vegetation as well as on the ground and it is likely that foragers from at least some arboreal nests will not be present on the ground at the time of bait application and therefore will survive treatment. While this may be less important for situations where a degree of population control is desired, it is a serious shortcoming for attempts at eradicating this species. Granular baits can not be effectively applied to trees while paste baits, using the methods outlined by Vanderwoude and Nadaeu (2009) can be applied efficiently to both the ground and vegetation. Paste baits therefore represent an important tool in eradications of Little Fire Ants. References Conant P & Hirayama C (2000) Wasmannia auropunctata (Hymenoptera:Formicidae): established on the Island of Hawaii. Bishop Museum Occasional Papers 64, 21-22. Drees BM & Barr CL (1997) 'Evaluation of a new insect growth regulator Pyriproxyfen (V- 71639), and other broadcast-applied bait products and product mixtures for suppression of the red imported fire ant.' T.A.M.U. Fernald HT (1947) The Little Fire Ant as a house pest. Journal of Economic Entomology 40. Hawaii Ant Group (2007) 'A plan for the prevention of establishment of new ant species in Hawaii, with special attention to the Red Imported Fire Ant (Solenopsis invicta) and the Little Fire Ant (Wasmannia auropunctata).' Hawaii Ecosystems at Risk project. Jourdan H (1997) Threats on Pacific islands: the spread of the tramp ant Wasmannia auropunctata (Hymenoptera: Formicidae). Pacific Conservation Biology 3, 61-64. Le Breton J, Chazeau J & Jourdan H (2003) Immediate impacts of invasion by Wasmannia auropunctata (Hymenoptera: Formicidae) on native litter ant fauna in a New Caledonian rainforest. Austral Ecology 28, 204-209. Le Breton J, Delabie JCH, chazeau J & Jourdan H (2004) Experimental evidence of large-scale unicoloniality in the tramp ant Wasmannia auropunctata (Roger). journal of Insect Behavior 17, 263-271. Lowe S, Browne M, Boudjelas S & De Poorter M (2000) 100 of the World s Worst Invasive Alien Species: A Selection from the Global Invasive Species Database. In. (World Conservation Union (IUCN): Auckland, New Zealand) Mikheyev AS, Bresson S & Conant P (2009) Single-queen introductions characterize regional and local invasions by the facultatively clonal little fire ant Wasmannia auropunctata. Molecular Ecology 18, 2937-2944. 9
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Walker KL (2006) Impact of the Little Fire Ant, Wasmannia auropunctata, on native forest ants in Gabon. Biotropica 38, 666-673. Wetterer JK & Porter SD (2003) The Little Fire Ant, Wasmannia auropunctata: distribution, impact and control. Sociobiology 41, 1-41. Wetterer JK, Walsh PD & White LJT (1999) Wasmannia auropunctata (Roger) (Hymenoptera: Formicidae), a destructive tramp-ant, in wildlife refuges of Gabon. African Entomology 7, 1-3. Williams D (1983) The development of toxic baits for the control of the Imported Fire Ant. Florida Entomologist 66, 162-172. Williams DF & Whelan PM (1992) Bait attraction of the introduced ant Wasmannia auropunctata (Hymeoptera: Formicidae) in the Galapagos Islands. Journal of Entomological Science 27, 29-34. 11