Mortality and Foraging Rates of Argentine Ant (Hymenoptera: Formicidae) Colonies Exposed to Potted Plants Treated with Fipronil 1

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1 Mortality and Foraging Rates of Argentine Ant (Hymenoptera: Formicidae) Colonies Exposed to Potted Plants Treated with Fipronil 1 Heather S. Costa and Michael K. Rust Department of Entomology University of California Riverside, California USA J. Agric. Urban Entomol. 16(1): (January 1999) ABSTRACT Individual colonies of Argentine ants, Linepithema humile (Mayr), were collected and maintained in plastic containers and randomly selected to receive a potted oleander (Nerium oleander L.) plant with one of eight different soil treatments of fipronil, diazinon, or an untreated control. Foraging and mortality rates were observed in each colony. Reductions in the rates of ants foraging on plants were similar with all treatments; however, the mortality rates of the colonies varied among treatments. For example, 1 wk after exposure to treatments, the foraging rates on plants in all treatments except the control had dropped to zero. Soil-mix treatments of fipronil had killed >90% of worker ants at 1 wk, whereas broadcast treatments of fipronil and diazinon killed <50% of workers. The soil-mix treatments of fipronil killed all queens in 4 wk, whereas broadcast treatments of fipronil took 8 wk to kill all queens. The diazinon broadcast treatment did not kill any queens. Only fipronil soil-mix treatments prevented ants from establishing colonies in the pot. These results suggest that when studying pesticide effectiveness against Agrentine ant in the field, equivalent short-term reductions in foraging rates should not be assumed to indicate equivalent mortality of ant colonies. KEY WORDS bioassay Argentine ant, Hymenoptera, Formicidae, fipronil, nursery, Ant infestations are a widespread problem for the nursery industry. Infestations of container plants with any ant species can result in delay or rejection of inter-regional shipments due to quarantines presently in place for fire ants (Solenopsis spp.) and other pests. In addition, some ant species, including Argentine ants, Linepithema humile (Mayr) ( Iridomyrmex humilis), tend colonies of homopteran pests such as aphids, scales, and whiteflies. The ants collect their honeydew as a food source and actively interfere with natural enemy activity against homopteran pests, resulting in larger populations of pests when ants are present (DeBach 1958, Addicott 1979, Bristow 1984). Preventing ants from establishing colonies in nursery stock is an important means for deterring the movement of exotic pest ant species. Lewis et al. (1992) 1 Accepted for publication 11 September

2 38 J. Agric. Urban Entomol. Vol. 16, No. 1 (1999) reported that nursery stock accounted for 5% of the interceptions of imported fire ant, Solenopsis invicta Buren, at border stations in California. These may be significant because they may often represent intact colonies. In fact, the one eradication effort of S. invicta in California was at a nursery in Santa Barbara in Commercial shipments of plants contributed to the widespread distribution of L. humile in the United States (Newell & Barber 1913, Smith 1965). Linepithema humile was introduced into France in orchids and ferns from South America (Passera 1994). The major ant pest reported in nursery crops in southern California is the Argentine ant. Argentine ants are difficult to control for several reasons. Little is known about the foraging habits of the species. They are polydomous, meaning that individual colonies of ants over a large area are actually interrelated, and individual ants can easily move from one nest site to another if a colony is disturbed (Markin 1968). This makes it particularly difficult to evaluate the effectiveness of materials used to control Argentine ants. Observations of reduced foraging rates of ants in treated sites may not give a true indication that ants have been controlled. Ant populations may move from the treated area and shift their foraging range into new or other existing nest sites. The distinction of repellency effects from mortality effects can be particularly important in nursery situations where ants may move to other locations within the same nursery and continue to be a pest problem after treatment. Thus, to evaluate the efficacy of pesticides for Argentine ant control, it will be necessary to distinguish the relative contributions of repellency and mortality to reductions in foraging rates in treated areas. In our studies, individual ant colonies were isolated to observe colony mortality and behavior in response to exposure to insecticides. Using these methods, we determined if reduced foraging rates resulted from avoidance of treated areas or from mortality of colonies due to treatments. Materials and Methods Potted oleander plants (Nerium oleander L.) were infested with black scale, Saissetia oleae (Olivier), to produce a source of honeydew that would encourage ants to forage across treated soil. Two-week-old rooted oleander cuttings were infested with black scale by introducing scale crawlers onto seedlings. One month later, after scale populations had established, the infested oleander seedlings were transplanted into 15-cm-diameter pots with treated or untreated soil. Eight treatments were used in this study: an untreated control; diazinon (5G) broadcast on the soil surface at the labeled rate of 0.49 g (AI)/m 2 ; three rates of soil mixes of fipronil 61508A (0.05 G) (Chipco, Rhone-Poulenc, Research Triangle Park, North Carolina) at 5, 10, and 25 ppm (AI); a soil mix of fipronil 60818A (0.1G) (Rhone-Poulenc, North Carolina) at 10 ppm (AI); and two rates of fipronil 60818A broadcast on soil surface at rates of 14 and 28 g (AI)/ha. Soil treatments were applied by mixing granular fipronil formulations with moist soil on a weight to volume ratio (g [AI]/cm 3 soil). The formulations for broadcast treatments were mixed with 30 cm 3 of air-dried soil to allow even sprinkling on the soil surface. The soil used was sterile University of California Type III soil mix with a dry weight density of 0.77 g/cm 3 of soil (1330 cm 3 of soil per pot).

3 COSTA & RUST: Mortality and Foraging Rates of Argentine Ants 39 Twenty-four individual colonies of Argentine ants were assembled and maintained in plastic containers (25 by 30 by 10 cm) in a walk-in rearing room (30 ± 2 C and a photoperiod of 16:8 [L:D] h). Each colony was provided with an artificial nest constructed from a plaster-filled petri dish (10 cm diameter) formed with an 4.5 cm diameter by 0.5 cm deep circular area in the center of the dish to serve as an artificial nest (unpublished data). Dishes were covered with a black cardboard disk that could be removed to observe the number of ants and queens in a nest. Moisture was applied to nests through wicks embedded in the plaster. In addition to an artificial nest, each colony container also was provided with one water supply wick, a small dish of 5% sucrose solution over cotton, and one dish of fly pupae. About worker ants and several queens (4.6 ± 0.4, mean ± SE) were introduced into each container. After ants had acclimated for 1 mo, one infested oleander plant was introduced into each ant colony. Treatments were arranged in a completely randomized design; ant colonies were randomly selected to receive oleander plants in pots treated with one of eight different treatments. Three replicates of each treatment were used. Foraging. To estimate the foraging rates of ants in each colony after treatment, the number of ants foraging up the stem of the oleander plants in 1 min was monitored and compared among treatments. Stems were observed for at least three, 1-min counts per plant at 1, 2, 3, 4, 5, 7, 9, 14, 21, 28, 35, and 42 d after treatment. Mortality. To estimate mortality rates of worker and queen ants, dead ants were removed from colonies 1, 2, 3, 4, 7, 9, 14, 21, 28, 35, 42, 49, and 52 d after treatment by using an aspirator. Collected samples were examined under a stereo microscope to identify body fragments. Only fragments that included an ant head capsule were counted. Previous studies determined that head capsules of ants remain even if the bodies are consumed by other ants (unpublished data). Queens were differentiated from workers based on head capsule size. After 52 d, all ants were removed from nests, plants, and soil to determine the total number of ants remaining in each colony. The cumulative percentage of ant mortality for each date was calculated. Mortality counts of worker and queen ants were analyzed separately. Soil exposure bioassay. At the end of the mortality trial, soil samples were collected from the top, middle and bottom edges of pots and stored in petri dishes to test the residual activity of soil treatments. Sixteen weeks after treatment, ants were exposed for 24 h to each soil sample. Ninety-two milliliter vials were coated with Teflon and a 2-cm-diameter section of moist filter paper was placed on the bottom of each vial. Soil (1.2 cm 3 ) was added to cover the filter paper, and 10 ants were introduced into each vial. Vials were covered with parafilm and held at ambient temperature for 24 h. Contents were emptied into cool water to count the number of live ants. The mean number of ants surviving after 24 h was compared among treatments. All data were analyzed using one-way analysis of variance (ANOVA) (Minitab Statistical Software) followed by a Fisher protected least significant difference (LSD) test for comparison of means. Percentages were arcsine transformed before analysis.

4 40 J. Agric. Urban Entomol. Vol. 16, No. 1 (1999) Results Foraging. When plants were initially moved into the colonies, ants in all treatments began excavating soil from pots within the first 30 min. Many began moving pupae from artificial nests into pots. By the next morning ants had modified their foraging behavior. In all soil-mix treatments of fipronil, the foraging rate of ants on plants dropped to zero within 1 d, and ants completely avoided the pots throughout the remainder of the experiment (Table 1). Diazinon broadcast treatments also were effective in significantly reducing foraging rates of ants to zero by day 1; however, foraging rates and soil colonization in diazinon-treated pots began to increase after 30 d (Table 1). Foraging on plants was significantly reduced with broadcast treatments of fipronil 2 d after exposure, and dropped to zero by day 4 (Table 1). Unlike with fipronil soil-mix treatments, however, the ants continued to excavate and inhabit the pots throughout the experiment by entering through the drain holes on the bottom of the pots. Mortality. Twenty-four hours after exposure, all soil-mix treatments of fipronil killed significantly more workers than did controls or broadcast treatments of fipronil or diazinon, effectively killing 93% of workers in about 1 wk (Table 2; Fig. 1). Broadcast applications of fipronil killed a significant number of workers 2 wk after exposure, and killed almost all workers by wk 5 (Table 2; Fig. 2). The higher concentration of broadcast fipronil took longer to kill ant colonies, suggesting it may have been more repellent to ants than the lower broadcast concentrations. There was significantly higher mortality in diazinon treatments compared with controls 2 d after exposure. About 40% of the worker ants were killed by diazinon in 1 wk; however, the percentage of mortality did not increase much more after that time. Queen mortality was significantly higher in ant colonies exposed to soil-mix treatments compared with controls, with all queens killed 4 wk after exposure (Table 3). Broadcast treatments of fipronil took longer to show effects on queens, but killed a significant number of them 5 wk after exposure compared with controls, and killed almost all queens by wk 8 (Table 3). In contrast, the diazinon broadcast treatment did not kill any queens during the study (Table 3). Phytotoxicity was observed on the three oleander plants treated with the highest level of fipronil 61508A soil mix (25 ppm). Leaves appeared dried and curved longitudinally. There was no apparent effect of any of the treatments on the black scale population. Soil exposure. Exposure of ants to soil samples from all soil-mix treatments, including samples from the top, middle, and bottom sections of the pot, resulted in significant ( 80%) mortality of ants within 24 h (Table 4). None of the soils from broadcast treatments caused significant mortality of ants within the 24-h exposure. Discussion In our laboratory studies, ant colonies were provided with everything necessary for survival and did not need to forage on plants. Ants, however, initially attempted to nest in the potting soil and forage on plants. Our foraging rates were lower than rates reported for field trials on trees when a similar method of monitoring was used (Shorey et al. 1992). This is to be expected because our laboratory

5 Table 1. Mean number of Argentine ants foraging up plants in 1 min at 1 d, 4 d, and 1, 2, 4, and 8 wk after treatment. Number of ants foraging per min (mean ± SE) b Time after initial exposure Treatment a 1d 4d 1wk 2wk 4wk 8wk Control (Untreated) 2.1 ± 0.7a 1.9 ± 0.5a 1.1 ± 0.1a 1.8 ± 0.8a 1.75 ± 0.6a 2.5 ± 1.8a BC diazinon, 4,900 g (AI)/ha 0 ± 0 0 ± 0b 0 ± 0b 0 ± 0b 0.1 ± 0.1b 1.1 ± 0.5ab SM fipronil 61508A, 5 ppm (AI) 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b SM fipronil 61508A, 10 ppm (AI) 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b SM fipronil 61508A, 25 ppm (AI) 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b SM fipronil 60818A, 10 ppm (AI) 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b BC fipronil 60818A, 14 g (AI)/ha 2.2 ± 0.5a 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b 0 ± 0b BC fipronil 60818A, 28 g (AI)/ha 3.5 ± 1.4a 0 ± 0b 0.1 ± 0.1b 0 ± 0b 0 ± 0b 0 ± 0b a BC, broadcast application; SM, soil-mix application. b Means within a column followed by the same letter are not significantly different (P < 0.05) (ANOVA, Fisher LSD). COSTA & RUST: Mortality and Foraging Rates of Argentine Ants 41

6 Table 2. Percentage of mortality of L. humile workers in colonies exposed to pesticide treated pots. Percentage worker mortality (mean ± SE) b Time after initial exposure Treatment a 1d 1wk 2wk 5wk Control (Untreated) 3 ± 1a 6 ± 1a 9 ± 6a 14 ± 2a BC diazinon, 4,900 g (AI)/ha 15 ± 6a 39 ± 5bc 44 ± 4b 48 ± 4b Soil-mix fipronil 61508A, 5 ppm (AI) 59 ± 3b 99 ± 1d 100 ± 0d 100 ± 0c Soil-mix fipronil 61508A, 10 ppm (AI) 46 ± 10b 99 ± 1d 100 ± 0d 100 ± 0c Soil-mix fipronil 61508A, 25 ppm (AI) 50 ± 6b 95 ± 5d 97 ± 3d 100 ± 0c Soil-mix fipronil 60818A, 10 ppm (AI) 44 ± 7b 93 ± 5d 98 ± 1d 100 ± 0c BC fipronil 60818A, 14 g (AI)/ha 4 ± 2a 50 ± 13c 70 ± 8c 100 ± 0c BC fipronil 60818A, 28 g (AI)/ha 5 ± 1a 19 ± 4ab 32 ± 1b 96 ± 3c a BC, broadcast application. b Means within a column followed by the same letter are not significantly different (P < 0.05) (ANOVA, Fisher LSD). 42 J. Agric. Urban Entomol. Vol. 16, No. 1 (1999)

7 Fig. 1. Mean percentage mortality of workers after soil-mix applications of two formulations of fipronil (61508A 0.05 G and 60818A 0.1 G) compared with a broadcast (BC) application of diazinon (5G) and an untreated control. COSTA & RUST: Mortality and Foraging Rates of Argentine Ants 43

8 Fig. 2. Mean percentage mortality of workers after broadcast (BC) applications of fipronil (60818A 0.1 G) and diazinon (5G). 44 J. Agric. Urban Entomol. Vol. 16, No. 1 (1999)

9 Table 3. Percentage of mortality of L. humile queens in colonies exposed to pesticide-treated pots. Percentage queen mortality (mean ± SE) b Time after initial exposure Treatment a 1d 1wk 4wk 8wk Control (Untreated) 0 ± 0a 8 ± 8a 8 ± 8a 8 ± 8a Broadcast diazinon, 4900 g (AI)/ha 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a Soil-mix fipronil 61508A, 5 ppm (AI) 0.22 ± 0.22a 100 ± 0c 100 ± 0b 100 ± 0b Soil-mix fipronil 61508A, 10 ppm (AI) 0 ± 0a 89 ± 11bc 100 ± 0b 100 ± 0b Soil-mix fipronil 61508A, 25 ppm (AI) 0 ± 0a 83 ± 17bc 100 ± 0b 100 ± 0b Soil-mix fipronil 60818A, 10 ppm (AI) 0 ± 0a 51 ± 26ab 100 ± 0b 100 ± 0b BC fipronil 60818A, 14 g (AI)/ha 0 ± 0a 6 ± 6a 83 ± 17b 96 ± 4b BC fipronil 60818A, 28 g (AI)/ha 0 ± 0a 0 ± 0a 28 ± 17a 100 ± 0b a BC, broadcast application. b Means within a column followed by the same letter are not significantly different (P < 0.05) (ANOVA, Fisher LSD). COSTA & RUST: Mortality and Foraging Rates of Argentine Ants 45

10 Table 4. Percentage of mortality of L. humile workers 24 h after exposure to soil samples collected from treated pots. Percentage worker mortality after 24 h (mean ± SE) b Soil depth Treatment a Top Middle Bottom Control (Untreated) 10 ± 6b 7 ± 7b 7 ± 3b BC diazinon, 4900 g (AI)/ha 17 ± 9b 23 ± 3b 13 ± 9b Soil-mix fipronil 61508A, 5 ppm (AI) 80 ± 20a 100 ± 0a 90 ± 10a Soil-mix fipronil 61508A, 10 ppm (AI) 93 ± 7a 100 ± 0a 100 ± 0A Soil-mix fipronil 61508A, 25 ppm (AI) 97 ± 3a 100 ± 0a 100 ± 0a Soil-mix fipronil 60818A, 10 ppm (AI) 97 ± 3a 100 ± 0a 100 ± 0a BC fipronil 60818A, 14 g (AI)/ha 3 ± 0.3b 7 ± 7b 3 ± 3b BC fipronil 60818A, 28 g (AI)/ha 20 ± 1.0b 13 ± 7b 13 ± 7b a BC, broadcast application. b Means within a column followed by the same letter are not significantly different (P < 0.05) (ANOVA, Fisher LSD). 46 J. Agric. Urban Entomol. Vol. 16, No. 1 (1999)

11 COSTA & RUST: Mortality and Foraging Rates of Argentine Ants 47 ant colonies contained <500 workers compared with unknown and relatively unlimited numbers of ants present in the field. In addition, our populations had nearby supplies of water, sugar, and protein and had no real need to concentrate their foraging efforts on plants. Although ants showed an avoidance behavior toward treated pots within a short period of time after exposure, the fipronil soil-mix treatments were still effective in killing the entire colony of ants, including queens, in 2 3 wk. This suggests that initial foraging of workers on fipronil soil-mix treated pots resulted in adequate exposure to affect the entire colony. In addition to preventing ants from foraging on potted plants, soil-mix treatments of fipronil prevented ants from establishing colonies in the pots. The use of such soil mixes may reduce the number of ant colonies being transported in commercial plants. One benefit for the consumer is that potted plants with treated soil mix will prevent ants around structures from colonizing the pots, especially L. humile. Diazinon broadcast treatments were initially effective in repelling ants and killing 40% of the worker population. Unlike fipronil treatments, however, the overall mortality rate from diazinon treatments did not continue to increase after 2 wk, and diazinon treatments did not kill any queens. This treatment may give initial relief from ant infestations, but because only workers are killed, it may result in quick resurgence and return of ants. In other systems, barriers of diazinon granules have provided repellent barriers around structures for up to 60 d (Rust & Knight 1990) and around citrus trees for 3 mo (Moreno et al. 1987). The efficacy of fipronil soil-mix treatments was maintained for at least 4 mo after application. Exposure to soil samples from all soil-mix treatments were effective in killing workers in 24 h, whereas the broadcast treatments were not effective in killing workers, even when soil was taken from the top of the pot where insecticides were concentrated. There were similar reductions in plant foraging rates of ants observed with all treatments; however, the mortality rates of the colonies varied greatly among treatments. For example, 1 wk after exposure to treatments, the foraging rates on plants in all treatments except the control had dropped to zero, which if interpreted alone might suggest equivalent levels of control among treatments. In contrast, mortality counts indicated that at that time, the soil-mix treatments had killed >90% of worker ants, whereas broadcast treatments had killed <50% of workers. When studying pesticide effectiveness in the field, short-term reductions in foraging rates should not be assumed to indicate significant mortality of ant colonies. The majority of ants may survive exposure and simply move to another site to forage. Repellency of ants from a particular crop or structure may be an adequate method of control in some cases; however, in many crop production systems avoidance of treated areas may result in equivalent or higher infestation rates by the same ant colonies in other areas of production. Acknowledgment The authors would like to thank E. Bartko, T. Y. Vo, J. Hampton-Beesley, and L. M. Hooper for technical assistance and advice. This research was funded in part by the California Association of Nurserymen, and Rhone-Poulenc, Inc.

12 48 J. Agric. Urban Entomol. Vol. 16, No. 1 (1999) References Cited Addicott, J. F A multispecies aphid-ant association: density dependence and species-specific effects. Can. J. Zool. 57: Bristow, C. M Differential benefits from ant attendance to two species of Homoptera on New York ironweed. J. Anim. Ecol. 53: DeBach, P Application of ecological information to control of citrus pests in California. Proceedings 10th International Congress of Entomology, Montreal, 1956, 3: Lewis, V. R., L. D. Merill, T. H. Atkinson & J. S. Washbauer Imported fire ants: potential risk to California. Cal. Agric. 46: Markin, G. P Nest relationship of the Argentine ant, Iridomyrmex humilis (Hymenoptera: Formicidae). J. Kans. Entomol. Soc. 41: Minitab Minitab reference manual, release 11. Minitab, Inc., State College. Moreno, D. S., Haney, P. B. & R. F. Luck Chlorpyrifos and diazinon as barriers to Argentine ant (Hymenoptera: Formicidae) foraging on citrus trees. J. Econ. Entomol. 80: Newell, W. & T. C. Barber The Argentine ant. U.S.D.A. Bull Passera, L Characteristics of tramp species, pp In D. Williams [Ed.], Exotic ants. Westview Press, Boulder. Rust, M. K. & R. L. Knight Controlling Argentine ants in urban situations, pp In R. K. Vander Meer, K. Jaffe & A. Cedeno [Eds.], Applied myrmecology, A world perspective. Westview Press, San Francisco. Shorey, H. H., L. K. Gaston, T. G. Gerber, P. A. Phillips & D. L. Wood Disruption of foraging by Argentine ant, Iridomyrmex humilis (Mayr) (Hymenoptera: Formicidae), in citrus trees through the use of semiochemicals and related chemicals. J. Chem. Ecol. 18: Smith, M. R House-infesting ants of the eastern United States. USDA Tech. Bull