Effects of maximum residue limit of triflumezopyrim exposure on fitness of the red imported fire ant Solenopsis invicta

Insects and insecticide

Red imported fire ants, Solenopsis invicta, were collected from an organic farm in Conghua County, Guangzhou, China and reared at the Red Imported Fire Ant Research Centre at the South China Agricultural University. S. invicta colonies were provided with a 10% w/w sugar water solution and frozen locusts Locusta migratoria. The social form of S. invicta colonies were determined using methods of Shoemaker & Ascunce (2010), and only polygyne colonies were used in this experiment. All bioassays were conducted under laboratory conditions, and all colonies were reared in the laboratory at least 1 week prior to the start of the experiment.

The commercially available insecticide, Pyraxalt™ 10% triflumezopyrim SC (DuPont™ Crop Protection, Pudong, Shanghai, China), was used in all experiments.

Effect of triflumezopyrim on S. invicta food consumption and colony growth

The Environmental Protection Agency (EPA) of the United States establishes tolerances for the residues of triflumezopyrim in rice grains and rice hulls of 0.4 ppm and 1.0 ppm, respectively (https://www.federalregister.gov/documents/2017/10/16/2017-22356/triflumezopyrim-pesticide-tolerances, accessed on 2019-6-4). Therefore, we used 0.5 and 0.2 μg triflumezopyrim/ml for the residue tolerances of triflumezopyrim based on the regulatory requirements of the EPA.

Pyraxalt™ is advertised to provide up to 25 days of consistent hopper control (http://www.dupont.com/products-and-services/crop-protection/pyraxalt-insect-control.html, access on 2019-6-2). The estimated average life span of minor, medium, and major workers of S. invicta is 45 days, 75 days and 135 days, respectively (Calabi & Porter, 1989). In light of these two combined factors the experiment was conducted over 8 weeks.

The experimental method was reflective of the work of Pan, Lu & Wang (2017) in which a colony was divided into the same weight (>20 g), and each sub-colony contained workers, brood, larva, pupa, and at least one functional queen. Each sub-colony then received either the control 10% w/w sugar water or a single concentration of triflumezopyrim (0.2 μg/ml or 0.5 μg/ml) in 10% w/w sugar water; solutions were provided in a 15 ml glass tubes with a cotton stopple. Sub-colonies received a sufficient amount of locusts. The weight of each sub-colony was taken every 7 days to evaluate the effect of triflumezopyrim on colony growth of S. invicta. The accumulated percentage weight loss of each sub-colony was calculated after 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days and 56 days of treatment (Pan, Lu & Wang, 2017). Sugar water and locust consumption (mg sugar water or locust per g ant) was also calculated for each sub-colony at 1 day, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days and 56 days. Water evaporation was also estimated. Four colonies were used in this experiment.

The effect of triflumezopyrim on active behavior of S. invicta

We evaluated the effect of triflumezopyrim on S. invicta behavior by measuring their grasping ability and walking speed after 56 days of treatment. The experiment method was reflective of the method used by Huang et al. (2016) and Wang, Zeng & Chen (2015).

To test the grasping ability of the fire ant, 10 workers varying in size from the treatment or control sub-colony were placed on an A4-sized paper. The paper was gently turned over 180 degrees after 10 s and held in place for 10 s. Workers falling from the paper was considered to have lost their grasping ability. The grasping ability was evaluated by grasping rate using the following formula: grasping rate (%) = (number of workers possessing grasping ability/total number of workers in test) × 100%. Each sub-colony from each treatment and control group was tested three times.

To test the walking speed of the fire ants, a tray with a fire ant sub-colony was connected to a forging arena (tray, L × W × H, 41.75 × 27.5 × 12 cm) by a wood bridge. The bridge consists of two 20 cm vertical segments and one 20 cm horizontal segment. The walking speed of the workers was measured by recording the time needed for an ant to walk across the 20 cm horizontal segment of the bridge. The method was described by Wang, Zeng & Chen (2015). A total of 25 workers were measured from each sub-colony.

The effect of triflumezopyrim on the aggressiveness of S. invicta

In this experiment, we assessed the effect of sublethal doses of triflumezopyrim on the aggressive behavior of S. invicta. The experimental method was reflective of the method used by Pan, Lu & Wang (2017). The aggressiveness level of S. invicta was assessed when S. invicta individuals and colonies were confronted with another ant species, Pheidole fervida, at 56 days after treatment. Four P. fervida colonies were used.

S. invicta and P. fervida colonies were paired in inter specific individual or colony interaction tests. The inter specific interaction was quantified using a behavioral assay described by Pan, Lu & Wang (2017).

In inter specific individual aggression, interactions were scored and calculated as described by Rice & Silverman (2013) and Pan, Lu & Wang (2017). Four replicate experiments were performed for three pairs of colonies with each receiving a dose of triflumezopyrim. Each replicate involved different workers.

Inter specific colony aggression was evaluated using an assay described by Pan, Lu & Wang (2017). Specially, 30 workers varying in size from pairs of treated S. invicta and untreated P. fervida colonies were randomly chosen and introduced into a petri dish. Mortality rates of S. invicta were recorded after 3 h. Three replicates were performed for three pairs of colonies receiving a dose of triflumezopyrim at the same time. Each replicate involved different workers.

Statistical analyses

All data were analyzed using Shapiro–Wilk and Levene’s tests for normal distribution and homogeneity of variances, respectively. If data were normally distributed and had similar variances, then the means of measured variables were compared by one-way analysis of variance (ANOVA). Significant ANOVA results were corrected for multiple comparisons using LSD’s method. Non-normally distributed data were analyzed using a non-parametric Kruskal–Wallis test to compare medians; differences significant at the 0.05 significance level were subjected to a Mann–Whitney test for pairwise comparisons. All statistical analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA).

Effect of triflumezopyrim on S. invicta colony growth

There is no significant difference in the survival of S. invicta colonies among 0.5 μg/ml triflumezopyrim, 0.2 μg/ml triflumezopyrim, and the control (Fig. 1; day 7, LSD test, F_{2, 9} = 0.404, P = 0.679; day 14, LSD test, F_{2, 9} = 0.009, P = 0.991; day 21, LSD test, F_{2, 9} = 0.116, P = 0.892; day 28, LSD test, F_{2, 9} = 0.089, P = 0.916; day 35, LSD test, F_{2, 9} = 0.298, P = 0.749; day 42, LSD test, F_{2, 9} = 0.746, P = 0.501; day 49, LSD test, F_{2, 9} = 0.488, P = 0.629; day 56, LSD test, F_{2, 9} = 0.527, P = 0.608). After 56 days of treatment, the accumulated percentage weight losses were 51.40%, 50.65% and 58.46% in colonies treated with 0.5 μg/ml triflumezopyrim, 0.2 μg/ml triflumezopyrim, and controls, respectively.

Effect of triflumezopyrim on S. invicta food consumption

There is no significant difference in sugar consumption between ants treated with 0.5 μg/ml triflumezopyrim, 0.2 μg/ml triflumezopyrim, and the control (Table 1; day 1, LSD test, F_{2, 9} = 0.946, P = 0.418; day 7, LSD test, F_{2, 9} = 0.673, P = 0.534; day 14, LSD test, F_{2, 9} = 0.322, P = 0.733; day 21, LSD test, F_{2, 9} = 1.396, P = 0.296; day 28, LSD test, F_{2, 9} = 2.129, P = 0.175; day 35, LSD test, F_{2, 9} = 0.283, P = 0.760; day 42, LSD test, F_{2, 9} = 1.472, P = 0.280; day 49, LSD test, F_{2, 9} = 1.768, P = 0.225; day 56, LSD test, F_{2, 9} = 0.782, P = 0.486).

Triflumezopyrim treatments do not have an impact on locust consumption (Table 2; day 1, LSD test, F_{2, 9} = 0.004, P = 0.996; day 7, LSD test, F_{2, 9} = 0.172, P = 0.845; day 14, LSD test, F_{2, 9} = 0.539, P = 0.601; day 21, LSD test, F_{2, 9} = 1.726, P = 0.232; day 28, LSD test, F_{2, 9} = 1.443, P = 0.286; day 35, LSD test, F_2,9 = 3.691, P = 0.068; day 42, LSD test, F_{2, 9} = 0.728, P = 0.509; day 49, LSD test, F_{2, 9} = 1.940, P = 0.199; day 56, LSD test, F_{2, 9} = 0.089, P = 0.915).

Effect of triflumezopyrim on S. invicta activity behavior

After 56 days of treatment the grasping rate of workers was 88.67 ± 2.14%, 90.33 ± 1.67% and 71.67 ± 5.13% in 0.5 μg/ml triflumezopyrim, 0.2 μg/ml triflumezopyrim, and the control, respectively. The grasping rate of workers in the triflumezopyrim treatment is higher than that of the control (0.5 μg/ml triflumezopyrim and control, Mann–Whitney test, U = 27, P = 0.009; 0.2 μg/ml triflumezopyrim and control, Mann–Whitney test, U = 18, P = 0.002).

After 56 days of treatment the walking speed of workers was 9.50 ± 0.22 mm/s, 9.39 ± 0.23 mm/s and 9.41 ± 0.38 mm/s in 0.5 μg/ml triflumezopyrim, 0.2 μg/ml triflumezopyrim, and control, respectively. The walking speed of workers in 0.5 μg/ml triflumezopyrim treatment was higher than that of the control (0.5 μg/ml triflumezopyrim and control, Mann–Whitney test, U = 4044.5, P = 0.02), and the walking speed of workers in the 0.2 μg/ml triflumezopyrim treatment is not significantly different from either that in the 0.5 μg/ml triflumezopyrim or the control (0.2 μg/ml triflumezopyrim and control, Mann–Whitney test, U = 4199.5, P = 0.050; 0.2 μg/ml triflumezopyrim and 0.5 μg/ml triflumezopyrim, Mann–Whitney test, U = 4733.0, P = 0.514).

Effect of triflumezopyrim on S. invicta aggressiveness

After 56 days of treatment the aggressiveness indices of S. invicta workers was 1.58 ± 0.28, 1.31 ± 0.29 and 0.47 ± 0.20 in 0.5 μg/ml triflumezopyrim, 0.2 μg/ml triflumezopyrim, and the control, respectively. The aggressiveness index of S. invicta was significantly increased by triflumezopyrim treatment (0.5 μg/ml triflumezopyrim and control, Mann–Whitney test, U = 57.5, P = 0.004; 0.2 μg/ml triflumezopyrim and control, Mann–Whitney test, U = 75.5, P = 0.030).

In the group aggression experiment, the mortality of S. invicta workers was 6.33 ± 1.51%, 6.67 ± 1.80%, 7.33 ± 2.14% in 0.5 μg/ml triflumezopyrim, 0.2 μg/ml triflumezopyrim, and control after 56 days of treatment, respectively; there is no significant difference among triflumezopyrim treatments and the control (LSD test, F_{2, 33} = 0.77, P = 0.926).