INTRODUCTION
Invasive ant species are commonly recognized as pests that are harmful to ecosystems, agriculture, and human activity in their introduced regions, as typified by the red imported fire ant Solenopsis invicta Buren (Jemal and Hugh-Jones 1993; Gutrich et al. 2007; Wylie et al. 2020) and Argentine ant Linepithema humile Mayr (Holway et al. 2002; Roura-Pascual et al. 2010). To control and/or eradicate invasive ant species, chemical control methods are typically applied (Inoue et al. 2015; Sakamoto et al. 2017, 2019). The toxicity of the active ingredient and/or insecticide concentration to control pests is generally evaluated based on acute toxicity (24-96 h) tests at the individual level, in which binary conditions (survival versus death) are assessed in test organisms every 24 h after toxic exposure (Hayasaka et al. 2015; Rondeau et al. 2015; Sakamoto and Goka 2021; United States Environmental Protection Agency 2021). Thus, behavioral changes in test species immediately after exposure have not been extensively reported to date (except for the knockdown in S. invicta (Li and Zeng 2013)).
Based on the knowledge that ants self-organize through interactions between nestmates (Hölldobler and Wilson 1990), it is possible that behavioral changes at the individual level may affect collective (i.e., colony-level) behavior and decisions in the way of some signal and/or means. This notion implies that individual-level behavioral changes (when exposed to the insecticide) play an important role in the interactions between individuals and subsequently lead to colony-level responses to environmental impacts. However, previous reports assessing behavioral changes in ants immediately after chemical exposure are limited despite the importance of such information.
In this study, we observed that L. humile workers displayed an unusual behavior, namely, mandible-opening behavior (Fig. 1), when exposed to the insecticide fipronil.
MATERIALS AND METHODS
In this study, we observed behavioral differences between fipronil-exposed Linepithema humile workers and nontreated (exposed to only sucrose water) workers. Linepithema humile workers were collected in Kobe City, Hyogo Prefecture, Japan (34°41′50.1′′N 135°13′51.4′′E unamura et al. 2007; Seko et al. 2021) in April 2021 and reared for one week in a thermostatic incubator (LH-308CT, Nippon Medical & Chemical Instruments Co., Ltd., Japan) at 25 °C with a 12:12 h (L:D) photoperiod. Commercial fipronil [Prince Flowable, fipronil/water and surfactant (5:95, v/v), Kumiai Chemical Industry Co., Ltd., Japan] was used in this study. On the experimental day, 10 L. humile workers were placed in a plastic petri dish (45 mm in diameter) and fed 1.5 mg of 25% sucrose water that was spiked with 1.5 mg of a given (0.005%: 50 ppm) fipronil solution per individual. The test concentration followed the recommended concentration of a commercial fipronil-containing insecticide called ‘Argentine ant Sugoto Taiji Ekizai’ (Fumakilla Ltd., Japan). Controls were prepared in the same manner but were provided only 25% sucrose water. Then, behavioral changes in fipronil-treated workers versus those fed only sucrose water were visually monitored for 2 h. To assess the effect of fipronil exposure on behavioral changes among L. humile workers, we conducted Fisher’s exact test (α < 0.05) in R ver. 3.6.1 (R Core Team, 2019).
RESULTS
In the fipronil-treated group, in addition to behavioral failure and death, mandible-opening behavior (Fig. 1) lasting from a few seconds to several tens of seconds was observed. The mandible-opening behavior was repeated after an interval of a few seconds until the L. humile workers died. Behavioral changes were significantly more frequent in the fipronil-treated group than in the control group (p = 0.0007, Fisher's exact test) (Fig. 2). Furthermore, the workers that opened their mandibles did so immediately after insecticide exposure (within 10 minutes) and continued to display the behavior until death. The other workers (2/10 fipronil-treated workers) died within 10 minutes after insecticide exposure without displaying mandible-opening behavior.
Fig. 1.
Photographs of Linepithema humile workers. (A) L. humile workers were fed 25% sucrose water (control) and (B) 0.005% (50 ppm) fipronil solution (treatment).
Fig. 2.
Comparison of the rate of mandible-opening behavior between the fipronil-treated (fed 0.005% (50 ppm) fipronil solution sucrose water; treatment) and nontreated (fed only sucrose water; control) groups of Linepithema humile workers. Asterisks indicate significant differences (Fisher's exact test; ***: p < 0.001).
DISCUSSION
We observed an unusual behavior (i.e., mandible-opening behavior) displayed by L. humile workers immediately after insecticide fipronil exposure. Although researchers have previously assessed the sublethal effects of toxic compounds at low concentrations on ant behavior and/or reproductive ability (e.g., Thiel and Köhler 2016; Pan et al. 2017), the behaviors of ants subjected to toxics at lethal concentrations have been rarely assessed in detail to date, excluding the research of Li and Zeng (2013).
It has generally been recognized in many animals, including ant species, that individuals tend to display unusual behaviors such as convulsions, agonal movements, abnormal excretion, and regurgitation when exposed to lethal doses/concentrations of toxins (i.e., toxic symptoms; Buczkowski and Schal 2001; Li and Zeng 2013; Sunamura et al. 2021). Therefore, the mandible-opening behavior observed in this study also might represent a toxic symptom. However, the relationship between toxic exposure and the mandible-opening behavior of ants has not been studied to date (implied in Li and Zeng (2013)), thus suggesting that the toxic symptom has been unrecognized and overlooked until now. In addition, we cannot deny the possibility that workers opening their mandibles to send signals to others (i.e., secretion of ants' pheromones) (Sasaki et al. 2014). Ants typically have secretory glands, which secrete pheromones, including alarm pheromones, at the base of their mandibles (Sasaki et al. 2014). Furthermore, pheromones are often secreted with mandible-opening/faring behavior (Sasaki et al. 2014). Based on the above, it is possible that the L. humile workers send signals to other nestmates via the secretion of any pheromones after fipronil exposure. Accordingly, understanding the response of ant colonies to insecticide exposure is indispensable to assess not only the susceptibility to toxic chemicals at the individual level but also behavioral changes and subsequent effects at the colony level.