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Article

Formicidal Potential of Thymol Derivatives: Adverse Effects on the Survival and Behavior of Acromyrmex balzani

by
Jaciele O. Dantas
1,
Sócrates C. H. Cavalcanti
2,
Ana Paula A. Araújo
3,
Jefferson E. Silva
1,
Thaysnara B. Brito
2,
Valfran S. Andrade
1,
Heloisa S. S. Pinheiro
1,
Swamy R. S. A. Tavares
1,
Arie F. Blank
4 and
Leandro Bacci
4,*
1
Postgraduate Program in Agriculture and Biodiversity, Universidade Federal de Sergipe, São Cristóvão 49100-000, Sergipe, Brazil
2
Department of Pharmacy, Universidade Federal de Sergipe, São Cristóvão 49100-000, Sergipe, Brazil
3
Department of Ecology, Universidade Federal de Sergipe, São Cristóvão 49100-000, Sergipe, Brazil
4
Department of Agronomic Engineering, Universidade Federal de Sergipe, São Cristóvão 49100-000, Sergipe, Brazil
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(7), 1410; https://doi.org/10.3390/agriculture13071410
Submission received: 8 May 2023 / Revised: 27 June 2023 / Accepted: 12 July 2023 / Published: 16 July 2023
(This article belongs to the Special Issue Sustainable Crop Production and Pest Control)
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Abstract

:
Leaf-cutting ants are important pests of agricultural and forest crops. Currently, few insecticides are registered for the control of these insects. Natural bioactive molecules can serve as models for the synthesis of new insecticidal compounds. Such ant killer products must be sustainable and efficient, considering not only lethal effects, but also sublethal effects, which can interfere with behavior and communication between colony members. In this study, we analyzed the toxicity of the monoterpene thymol and its derivatives, as well as the sublethal effects of these compounds on the behavior of the leaf-cutting ant Acromyrmex balzani. These effects were compared with the conventional synthetic insecticide deltamethrin. Although deltamethrin showed higher toxicity (LD50 = 0.87 × 10−5 µg/mg), all other tested compounds increased ant mortality, with thymyl chloroacetate being the most toxic derivative (LD50 = 1.41 µg/mg), followed by thymol (LD50 = 2.23 µg/mg). These three most toxic compounds interfered differentially in the behavior of ants. Thymyl chloroacetate caused increased self-cleaning and reduced allogrooming, which may be related to an attempt to avoid contamination between nestmates. In general, thymol caused greater avoidance among ants, reduced walking speed and caused disorientation in workers. On the other hand, thymyl chloroacetate (LD30) considerably increased the walking speed of the group, without changing the orientation of the individuals. Such changes may interfere with basic activities such as foraging and altering colony cohesion via different mechanisms. Thus, despite the desirable effects of deltamethrin on ant control, this insecticide is highly toxic and should be discontinued soon. Our results show that thymol and its structural modification in thymyl chloroacetate may represent potential ant killers to be used in the management of A. balzani.

1. Introduction

One of the great challenges of modern agriculture is to select new pesticides that are efficient against pests and, at the same time, minimize environmental risks to ecosystems [1]. Pest control can occur via different modes of action, either by causing primary physiological changes that result in mortality or by promoting secondary effects that compromise the performance of insects over time. Such sublethal effects may result from alterations in physiological and behavioral characteristics and in intra and interspecific communication [2]. Sublethal effects may be particularly important for eusocial insects (e.g., ants, bees, wasps, and termites) for which colony death depends on information exchange and the spread of toxic substances among nestmates. One of the difficulties in controlling leaf-cutting ants, for example, is the lack of knowledge about their complex social structure, in addition to their collective defenses (e.g., prophylactic behaviors) that can reduce control efficiency [3,4]. Behaviors carried out among ants such as cleaning, trophallaxis and other types of physical contact, such as antennation [5,6], can contribute to a faster distribution of insecticides within the colony [7,8].
Leaf-cutting ants of the genus Acromyrmex are distributed in the Neotropics, being abundant mainly in habitats altered by Eucalyptus and sugarcane plantations. In these areas, ants cause considerable damage [3,9,10] culminating in the intensive use of chemical control [11]. The damage caused by these ants is due to the cutting of plant material [12] that is transported to the interior of the nest for the cultivation of the symbiotic fungus, which is the food source of the colony [3,4]. Since ants are selective in relation to the plants they collect [13,14], products from the secondary metabolism of plants or molecules derived from these compounds may be promising for the management of these insects.
Botanical insecticides have demonstrated biological activity and, in some cases, are considered environmentally sustainable for insect control compared to conventional synthetic insecticides [15]. Thus, natural products can have a direct effect on pest control or even serve as a model for the synthesis of new bioactive compounds. In fact, many pesticides widely used in the market were synthesized from natural products, such as pyrethroids and neonicotinoids [16].
In addition to its direct bioactivity, thymol is also considered a good precursor molecule for the synthesis of new derivatives, since its chemical structure can be modified into others with greater structural stability [17], mainly through substitution reactions. As an aromatic compound, thymol does not easily undergo addition reactions, since the ring unsaturations are in resonance, which makes the compound less reactive and, consequently, more stable [18,19,20]. The replacement of a radical in the chain of a compound can improve its biological activity, resulting in new synthetic derivatives [21]. Similar compounds that present punctual alterations in a molecule or structure may differ in their physical–chemical properties and consequently in their biological activities [22] and may represent alternatives to conventional insecticides [23].
Thus, in view of the difficulty of controlling leaf-cutting ants and the potential of thymol for the synthesis of new stable molecules; in the present study, we synthesized seven ester and ether of thymol derivatives and evaluated the bioactivity of these compounds on A. balzani workers. Bearing in mind that contact and communication between colony members and foraging patterns can interfere with colony cohesion; we analyzed, in addition to survival, the sublethal effects that can alter the individual and collective behaviors of ants.

2. Materials and Methods

2.1. Synthesis of Synthetic Derivatives

The synthesis of thymol derivatives was carried out in the Pharmaceutical Chemistry Laboratory of the Universidade Federal de Sergipe (UFS), São Cristóvão-SE (10°54′ S; 37°04′ W, 7 m altitude), Brazil. Derivatives were synthesized by nucleophilic nuclear substitution and esterification reactions. Thymol ester derivatives followed the following methodologies: (i) thymyl acetate [24], (ii) thymyl chloroacetate, thymyl trichloroacetate, and thymyl propionate [25], and (iii) thymyl benzoate (Schotten-Baumann reaction) described in [26] thymol ether derivatives (thymyl methyl and thymyl ethyl) were synthesized by the method of Coolen et al. [27] (Figure 1). The esters were synthesized in anhydrous medium or sodium chloride, using THF (tetrahydrofuran) as solvent [21]. The reactions were monitored by thin layer chromatography (TLC), checked under 256 ηm ultraviolet light and compared with the starting material (thymol). The obtained compounds were purified using sílica gel 60 in the stationary phase and pure hexane in the mobile phase. Melting points were determined on a Logen Scientific melting point apparatus without correction. The chemicals used in the reactions and the thymol monoterpene were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

2.2. Insects

Leaf-cutter ant workers A. balzani were collected in 20 nests located on the UFS Campus. Approximately 500 insects were collected from each nest. The collected ants were standardized by size (average length 6 mm), placed in plastic containers (50 cm × 20 cm) and kept under controlled conditions (25 ± 2 °C; 70 ± 5% RH and 12 h photoperiod) for acclimatization for 24 h until the bioassays. During this period, only distilled water was provided to the workers.

2.3. Bioassays

All bioassays were carried out in climate-controlled rooms with controlled conditions (25 ± 2 °C; 70 ± 5% RH and 12 h photoperiod) at the Integrated Pest Management Laboratory at UFS. The compounds used were diluted in acetone solvent (Panreac, UV-IR-HPLC-GPC PAI-ACS, 99.9% purify). Preliminary tests showed that this solvent does not interfere with the survival and behavior of ants.
In the acute toxicity bioassays, the treatments used were the base molecule thymol and its synthetic derivatives (thymyl acetate, thymyl chloroacetate, thymyl trichloroacetate, thymyl propionate, thymyl benzoate, thymyl methyl, and thymyl ethyl). In the behavioral bioassays, thymol and its most toxic synthetic derivative (thymyl chloroaceate) were used. In all bioassays, the insecticide deltamethrin (Decis® 25 EC, 25 g i.a./L, emulsifiable concentrate, Bayer SA®, Gujarat, India) was used as positive control and acetone as a negative control. Deltamethrin was chosen because it is an active ingredient used to control ants of the genus Acromyrmex.
To determine the doses (µg of compound/mg of ant) to be applied, fifty workers of A. balzani were placed individually on a precision analytical balance (Shimadzu, Kyoto, Japan, AUW220D) to obtain the average mass of the insects. The compounds were applied topically (1 µL) in the prothoracic region of A. balzani workers with the aid of a Hamilton microsyringe with a capacity of 10 µL (Hamilton®, Reno, NV, USA). To facilitate the application, the insects were previously immobilized for 90 s in a freezer at −19 °C (Eletrolux®, Curitiba, PR, Brazil) in order to reduce their activities. Preliminary tests showed that this methodology does not affect the survival and behavior of ants.

2.3.1. Acute Toxicity

The bioassays established to determine the dose–response curves and estimate the lethal doses necessary to kill 30, 50, and 90% of the A. balzani populations were carried out in a completely randomized experimental design, with six repetitions per treatment. Each experimental unit consisted of a group of five to six workers of the same size, totaling 2313 individuals. The ants were placed in a Petri dish (Global Trade Technology, Monte Alto, SP, Brazil) (9 × 1.5 cm) lined with filter paper (Unifil) moistened with 0.6 mL of distilled water and covered with a transparent pvc film (G-util Guarufilme, Guarulhos, SP, Brazil).
Initially, three doses of compounds (1; 2.5 and 5 µg of compound/mg of insect) were tested to discover the ascending phase of the curve. Subsequently, another four to seven doses were used to adjust the data to the Probit model (probability of mortality as a function of the dose of the compounds). Mortality assessments were performed 72 h after setting up the bioassays. Individuals were considered dead when they remained motionless when touched by a bristle brush.
To determine the survival curves and estimate the lethal times necessary to kill 50% (LT50) of the A. balzani populations, the experimental procedures were the same as in the previous bioassays, with the exception of the dose used, number of repetitions, and evaluation of time. The LD90s of each treatment were used in a completely randomized design, with 15 repetitions per treatment, totaling 1019 individuals. The lethal doses necessary to kill 90% of the populations, obtained in the previous bioassays, were chosen because they are a standard of pest control efficiency in Brazil [28,29]. Initially, mortality assessments were performed every 10 min for half an hour, then every 30 min for 2 h, followed by 4 h assessments during the first 24 h. Subsequently, evaluations were performed at intervals of 6 h in the following 24 h and every 12 h until completing 100 h.

2.3.2. Individual and Collective Behavior

The bioassays were carried out to evaluate the effects of the compounds on the individual behavior of the treated A. balzani workers and the behavioral responses of a group of non-treated ants in relation to a treated one. The experimental designs were completely randomized, with 30 repetitions per treatment. Acromyrmex balzani workers were exposed to acetone (negative control) and to the compounds deltamethrin, thymol, and thymyl chloroacetate at the LDs30 and LDs50 determined in the acute toxicity bioassays. Before being exposed to the treatments, the ants were placed in a Petri dish (9 × 1.5 cm) lined with filter paper moistened with distilled water for 5 min for acclimatization.
For the individual bioassay, each experimental unit was composed of a worker, totaling 210 individuals. After acclimatization, the ants were immobilized and treated with the compounds. One minute after the treatments were applied, the self-cleaning behavior was evaluated. For the collective bioassay, each experimental unit was composed of a group of seven workers, totaling 1470 individuals. After acclimatization, one ant from each group was randomly removed, immobilized, and marked with non-toxic yellow paint (Acrilex Tintas Especiais S.A., São Bernardo do Campo, São Paulo, Brazil) in the mesothorax region, topically treated with the compounds and after 3 min of the application it was relocated in the Petri dish with the other non-treated ants. One minute after the relocation, the evaluation of cleaning, antennation, avoidance, and aggression behaviors began. Preliminary tests indicated that the paint did not affect the ant’s behavior.
In both bioassays, observations were performed for one continuous minute, with a one-minute interval between them, during a period of 10 min, totaling 5 min of observation for each insect. Overall, behaviors were recorded for 2100 min [2 bioassays × 7 treatment and dose interactions (one control and two doses of three treatments) × 30 replicates × 5 min of observation].

2.3.3. Walking Behavior

To evaluate the effects of the compounds on the walking behavior of A. balzani workers, individual, and collective walking bioassays were performed. The experimental designs were completely randomized, with 60 repetitions per treatment. Acromyrmex balzani workers were exposed to acetone (negative control) and to the compounds deltamethrin, thymol, and thymyl chloroacetate at the LDs30 and LDs50 determined in the acute toxicity bioassays. For the individual bioassay, each experimental unit was composed of a worker, totaling 420 individuals. The ants were previously immobilized, treated with the compounds, and transferred to Petri dish arenas (9 × 1.5 cm) lined with filter paper moistened with distilled water. For the collective bioassay, each experimental unit was composed of a group of four workers, totaling 1680 individuals. The four ants were marked with non-toxic paint in the colors blue, lilac, green, and yellow (Acrilex Tintas Especiais S.A., São Bernardo do Campo, São Paulo, Brazil) in the posterior and medial region of the thorax, treated after 3 min of paint application and placed in the arena. Preliminary tests indicated that the paint did not affect the ant’s behavior.
One minute after placing the ants in the arena, recording began for a period of 10 min using a video camera (Panasonic SD5 Superdynamic—model WV-CP504), equipped with a Spacecom lens (1/3″ 3–8 mm) attached to a computer. The distance covered (cm), velocity (cm/s) and meander (°/cm) were captured in the Ethovision XT software (version 8.5; Noldus Integration System, Sterling, VA, USA), and the data were analyzed using the Studio 9 Program (Pinnacle Systems, Mountain View, CA, USA).

2.4. Statistical Analysis

Mortality data were submitted to Probit analysis to determine the dose-mortality curves for each treatment using the PROC PROBIT procedure in SASTM (SAS Institute, 2008). From these curves, the lethal doses necessary to cause 30, 50, and 90% mortality (LD30, LD50, and LD90) and their respective confidence intervals (CI95%) at 95% probability were obtained. The LDs were compared using the criterion of non-overlapping confidence intervals with the origin of the interval.
Survival analyses were performed using Kaplan–Meier estimators using the Log-Rank test (SigmaPlot, version 14). From this analysis, the survival curves and lethal times necessary to cause 50% mortality of individuals (LT50) and their respective confidence intervals (CI95%) at 95% probability were obtained. The curves were compared using the Holm–Sidak multiple comparison method at a significance level of 0.05 and amalgamated when there was no difference (SigmaPlot, version 14). The LT50 was compared using the criterion of non-overlapping confidence intervals with the origin of the interval.
Individual and collective behavior and walking behavior data (n = 60) of the ants exposed to the compounds were initially submitted to the Shapiro–Wilk (p > 0.05) and Brown–Forsythe (p > 0.05) tests to verify the normality of the data and the homogeneity of the variance, respectively. As these variables failed the tests, the data were initially submitted to non-parametric Kruskal–Wallis analysis of variance (p < 0.05) to verify if there are differences between the medians of the doses tested. When there was no difference, the LD30 and LD50 data were amalgamated. Then, a Kruskal–Wallis test was performed, followed by the Dunn’s multiple comparison method (p < 0.05) to compare the effect of treatments in relation to the control, within each dose. And finally, the doses of the same treatment were compared using the Kruskal–Wallis test followed by Wilcoxon (p < 0.05).

3. Results

3.1. Toxicity

All synthetic derivatives were toxic to A. balzani workers by contact, with LD50 ranging from 1.41 to 5.67 µg/mg (Figure 2). However, these doses were higher than that observed for deltamethrin (LD50 = 0.87 × 10−5 µg/mg). Thymyl chloroacetate was the most toxic synthetic compound for ants. The necessary dose of this compound to kill 50% of populations was 1.41 µg/mg (CI95%: 0.95–1.81), therefore, 37% less than the dose of the base molecule thymol 2.23 µg/mg (CI95%: 1.63–2.75) (Figure 2). The highest toxicity of thymyl chloroacetate was maintained at the dose considered as standard control (LD90 = 5.83 µg/mg; CI95%: 4.42–9.30) in relation to thymol (LD90 = 9.24 µg/mg; CI95%: 6.67–17.21) and other synthetic derivatives, which showed LD90 ranging from 12.68 to 30.75 µg/mg. The other synthetic compounds showed doses (LD50 and LD90) higher than thymol (Figure 2).
The survival of A. balzani workers exposed to the LDs90 of deltamethrin, thymol, and their synthetic derivatives was significantly reduced over time (Log-rank test: χ2 = 537.2, d.f. = 5, p < 0.001) (Figure 3). There was no difference between the survival curves of thymyl propionate and thymyl ethyl (p = 0.78) and thymyl acetate and thymyl methyl (p = 0.25). Acromyrmex balzani workers showed a rapid reduction in survival when exposed to these compounds. In less than 10 h, more than 80% of individuals died (LT50 = 7.1 h, CI95%: 4.5–9.7 and LT50 = 9.5 h, CI95%: 6.4–12.6, respectively) (Figure 3). Likewise, the survival curves of A. balzani workers exposed to thymol and thymyl trichloroacetate did not differ (p = 0.36), as well as the curves of thymyl chloroacetate and thymyl benzoate (p = 0.70). The curves of these compounds showed mean lethal times of 16.8 h (CI95%: 13.2–20.4) and 34.1 h (CI95%: 30.8–37.3), respectively (Figure 3). Deltamethrin, on the other hand, caused slower mortality in A. balzani workers. It took 40.1 h (CI95%: 34.6–45.5) for this insecticide to kill 50% of the population (Figure 3).

3.2. Individual and Collective Behavior

In general, deltamethrin, thymol, and its synthetic derivative thymyl chloroacetate altered the individual and collective behaviors of A. balzani workers in relation to the control (Figure 4 and Figure 5).
Data on individual self-cleaning behavior of A. balzani workers varied between doses (H = 10.28, d.f. = 1, p = 0.001). Workers showed a greater number of self-cleaning procedures when exposed to deltamethrin, at both doses, and to the compound thymyl chloroacetate at LD50 (Figure 4). Ants treated with thymol, however, did not change this behavior in relation to the control (Figure 4).
The collective cleaning behavior of A. balzani workers did not vary between doses (H = 2.78, d.f. = 1, p = 0.095). Cleanliness of treated subjects was reduced in thymol and thymyl chloroacetate treatments. This behavior did not change when subjects were treated with deltamethrin (Figure 5A).
Collective antennation behavior varied between doses (H = 37.06, d.f. = 1, p < 0.001). The number of antennations was reduced in subjects treated with the highest dose of all compounds. LD30 did not affect this behavior (Figure 5B). The collective avoidance behavior of A. balzani workers did not vary between doses (H = 2.32, d.f. = 1, p = 0.128). Subjects treated with thymol were avoided by non-treated subjects. However, deltamethrin and thymyl chloroacetate did not affect this behavior (Figure 5C). Collective aggresive behavior did not vary between doses (H = 3.57, d.f. = 1, p = 0.051). Only individuals treated with deltamethrin were more aggressive than non-treated individuals. The aggressiveness of non-treated A. balzani workers compared to those treated with thymol and thymyl chloroacetate did not differ from the control (Figure 5D).

3.3. Walking Behavior

In general, deltamethrin, thymol, and its synthetic derivative thymyl chloroacetate, altered the speed and individual and collective distance covered by A. balzani workers in relation to the control (Figure 6 and Figure 7). The speed (z = −5.51, p < 0.001) and the distance covered (z = −6.49, p < 0.001) in the control were higher when the ants were alone (Figure 6A,B) compared to when they were in a group (Figure 7A,B).
The individual velocity of A. balzani workers varied between doses (H = 5.79, p = 0.016). Acromyrmex balzani workers treated individually with thymol decreased the speed, regardless of the applied dose (Figure 6A). Individuals treated with the LD30 and LD50 of this compound reduced the distance walked by 49 and 46% in relation to the control (607.4 cm), respectively (Figure 6B–D). The movement of workers treated with thymol was more erratic compared to the control, as can be seen from the representative displacement tracks (3D graphs) (Figure 6B–D). Without treatment (control), the individuals moved in a more circular fashion around the edge of the arena (Figure 6B). The opposite was observed for its synthetic derivative thymyl chloroacetate, where the workers increased the speed (Figure 6A) and the distance covered to 775.2 and 893.8 cm when exposed to LD30 and LD50, respectively (Figure 6B–D). In this case, the movement remained circular, similar to the control (3D graphics) (Figure 6B–D). However, there were differences in the effects caused between the doses. Deltamethrin, on the other hand, caused a reduction in these parameters only at the highest dose (Figure 6).
The mean collective velocity of A. balzani workers (four ants/arena) varied between doses (H = 6.17, p < 0.013). When A. balzani workers treated with thymol (LD50) were placed together in the arena, the speed and average distance covered did not differ from the control (Figure 7).
However, at LD30, the movement of ants treated with this compound was more erratic compared to the control, as can be seen by the representative displacement tracks (3D graphs) (Figure 7B,C). The compound thymyl chloroacetate altered the collective walking behavior of A. balzani workers. At LD30, the pattern observed in the individual was maintained, with an increase of two times in the average speed (Figure 7A) and 2.6 times in the distance covered (Figure 7C), in relation to the control (0.546 cm/s and 224.9 cm). When the ants were exposed to the dose necessary to kill 50% of the population, the pattern was reversed. Velocity and distance covered decreased to 0.296 cm/s and 143.9 cm (Figure 7A,B,D). The results obtained for the two deltamethrin doses did not cause significant differences in the behavior of the ants when compared to the control (Figure 7).

3.4. Meander Behavior

The individual meandering behavior of A. balzani varied between doses (H = 12.20, p < 0.001). Individuals treated with both doses of thymol showed more erratic/disordered movement with greater change in direction of movement compared to control (Figure 8A). Workers treated with the LD30 and LD50 of this compound increased meander by 66 and 103% in relation to the control (197 ± 30 cm), respectively (Figure 8A). Changes in the direction of movement of ants treated with deltamethrin and thymyl chloroacetate differ from the control (Figure 8A).
The average of collective meandering behavior (four ants/arena) of A. balzani varied between doses (H = 5.81; p = 0.016). Change in the direction of movement of the ants in relation to the control was observed when exposed to thymol and thymyl chloroacetate, both at the LD30; and for the LD30 and LD50 of deltamethrin. In this case, for thymol there was a 97% increase in meander compared to the control (385 ± 26/cm) (Figure 8B). For thymyl chloroacetate this behavior reduced significantly when compared to the control. For deltamethrin, the LD50 caused a significant increase in this behavior compared to LD30 (Figure 8A).

4. Discussion

Studies have shown that altering a parent molecule can cause changes in the bioactivity of compounds [8,21,23]. In the present study, we synthesized new molecules derived from thymol and evaluated the bioactivity of these compounds on leaf-cutting ants. In general, our results show that thymol and its synthetic derivative thymyl chloroacetate have lethal effects, and, mainly, considerable sublethal effects on the ant A. balzani.
The monoterpenoids present in plant essential oils are among the most studied bioactive compounds [30] and have shown bioactivity against a wide variety of species [31,32,33,34,35,36,37,38]. Here we found that not only thymol but also its synthetic derivatives were active against A. balzani via contact exposure. The bioactivity that a chemical agent can exert on a specific biological organism is related to the hydrophobic, electronic and steric characteristics of its molecules [22]. Thus, the biological activity of a xenobiotic may be associated with the presence of a functional group, size, and degree of unsaturation [39]. Some research has suggested that thymol may potentiate ligand-gated chloride channels in the nervous system, thereby acting as a neurotoxic insecticide [40,41], probably acting as a positive allosteric modulator of A GABA receptors [42]. Type A GABA receptors are primary inhibitors of the central nervous system, binding to specific subunits of these channels [43]. Furthermore, octopaminergic receptors, which are unique to insects, are also considered to be important sites of action for the monoterpene thymol [44].
Among all derivatives synthesized, thymyl chloroacetate showed the highest toxicity against A. balzani workers. The greater formicide activity of thymyl chloroacetate when compared to thymol can be explained by the increased affinity of this compound with the site of action in the insect, improving this interaction with the receptors [45]. Costa et al. [8] also demonstrated that some indole derivatives [(1-(1H-indol-3-yl) pentan-1-one)] resulted in greater toxicity against the ant Atta opaciceps Borgmeier than the base molecule itself. Greater bioactivity of this derivative occurred due to the addition of a chlorinated group that may have potentiated its insecticidal effect. Chlorine is considered an electron-withdrawing group and a halogen compound [21]. However, here, we verified that the increase of chlorine molecules in the substituent group of the base molecule, with the formation of the thymyl trichloroacetate derivative, did not correspond to a proportional increase in the toxicity against A. balzani workers. Possibly, the addition of more Cl atoms may have contributed to annul the electron reactivation effect, modifying the molecule’s center of activity and making it less susceptible to molecular interactions.
Although thymol and its derivative thymyl chloroacetate showed a lethal effect on ants, the organosynthetic insecticide deltamethrin showed greater toxicity. On the other hand, thymyl chloroacetate showed an intermediate time to mortality (34.1 h) between deltamethrin (40.1 h) and the other compounds (<17 h). In this way, this derivative may allow the survival of individuals in a time interval sufficient for colonial interactions to allow the dissemination of the xenobiotic among nestmates. Furthermore, thymol and thymyl chloroacetate showed relevant sublethal effects from the point of view of leaf-cutting ants management.
All three of the most active compounds caused sublethal effects by altering ant’s behaviors differentially compared to the control. Such behavioral changes could contribute to management efficiency, but via different mechanisms.
Self-cleaning is a common hygiene activity performed by ants when they detect the presence of a xenobiotic [7]. On the other hand, allogrooming behavior, despite being common among eusocial insects [46], presents a certain duality and may contribute to both containment and dissemination of pathogens [47]. Here, we verified that ants treated with deltamethrin and LD50 of thymyl chloroacetate were able to perceive contaminants by increasing the number of self-cleaning procedures. However, collectively, thymyl chloroacetate, showed the opposite result, showing a reduction in the number of allogrooming. A similar result was found by Theis et al. [47] in a system of ants exposed to fungi, where the authors attributed this behavioral change to a possible attempt to contain the spread of the pathogen in the colony. This same argument could explain why ants treated with LD50 of thymyl chloroacetate increased walking speed when they were isolated, but reduced walking when they were in a group at LD50. Thus, the reduction in walking could indicate an attempt to reduce the probability of interindividual contact.
Studies have shown that self-cleaning or allogrooming behaviors, as well as physical contact, increase the dispersal of substances [5]—including insecticides—among members of leaf-cutter ant colonies [48]. In this case, the speed of colonial dissemination of thymyl chloroacetate, as well as of thymol, via allogrooming, would be reduced in relation to deltamethrin. On the other hand, the reduction of allogrooming could be favorable to the management, it is enough to make the colony more susceptible to the incidence of pathogens, which would reduce its survival. In this case, different control strategies (e.g., Chemical—thymyl chloroacetate—and biological) used jointly could have faster effects in the control of leaf-cutting ants.
Another form of dissemination of compounds occurs through physical contact between individuals, such as antennae. None of the compounds tested in the LD30 altered this parameter in relation to the control, which demonstrates that the interindividual communication was not altered. Antennation behavior is of paramount importance in the exchange of information between colony members since antennae have several sensilla with olfactory receptors. The perception of surface odors can be influenced by antenna morphology, the structure of neurons, odor-binding proteins [49], as well as the proportion of the chemical component [6,50,51]. The response of pheromone-sensitive olfactory receptors can also be mediated by biogenic amines such as dopamine, serotonin, tyramine, tyrosine, and octopamine. These amines may be related to the different behavior of ants since they have high metabolic diversity and act as neuromodulators and neurotransmitters [52,53,54]. Recent studies already demonstrate, for example, that thymol and its isomer carvacrol interfere with the level of dopamine and tyramine in ant brains, which is associated with their locomotion, as well as their aggressive behavior [55].
From the control point of view, the compounds could have different sublethal effects. Thymol caused greater avoidance among ants, as well as a reduction in speed and a greater occurrence of meandering (disorientation) in workers; this could compromise the colony’s ability to forage for resources, including on toxic baits. On the other hand, thymyl chloroacetate (LD30) considerably increased the walking speed of the group. It can be hypothesized that this increase in walking speed is an indication of the irritability of ants to these compounds. Increasing walking speed could not only increase foraging efficiency but also cause greater energy expenditure by the colony. Mannino et al. [55] also found an increase in the walking activity of the ants Formica cinerea, Tetramorium caespitum, and Myrmica scabrinodis, when exposed to thymol and carvacrol, as well as an increase in the levels of dopamine and tyramine in the brain of these ants, which is associated with their locomotion.
Although deltamethrin had a greater lethal effect and considerable sublethal effects, this insecticide is in the process of being derogated [56], being considered highly toxic to beneficial organisms and the environment [57]. Therefore, there will be a need, in the near future, to replace this compound with new molecules [10,56]. In fact, the toxicity of deltamethrin is so high for leaf-cutting ants, that it possibly ends up resulting in increased inter-individual aggressiveness, which could compromise cohesion and the performance of collective activities (e.g., foraging) by the colony.
In conclusion, our results show that thymol and its derivative, thymyl chloroacetate, have ant-killing potential, and present relevant sublethal effects to be used in the management of A. balzani. Currently, thymol derivatives are not yet commercially used for pest control, as their effects on these organisms are still being evaluated. However, our study demonstrates the great potential that these compounds have to be used in the near future for the formulation of new insecticides.

Author Contributions

Conceptualization, A.P.A.A., J.O.D. and L.B.; methodology, J.E.S. and L.B.; validation, L.B.; formal analysis, J.E.S. and L.B.; investigation, J.O.D., J.E.S., V.S.A., T.B.B., H.S.S.P. and S.R.S.A.T.; resources, S.C.H.C. and A.F.B.; data curation, J.O.D., J.E.S., V.S.A., T.B.B., H.S.S.P. and S.R.S.A.T.; writing-original draft preparation, J.O.D. and J.E.S.; writing—review and editing A.P.A.A. and L.B.; visualization, S.C.H.C., A.P.A.A. and A.F.B.; supervision, L.B.; project administration, L.B.; funding acquisition, S.C.H.C., A.F.B. and L.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico—Brasil (CNPq), the Fundação de Apoio à Pesquisa e a Inovação.Tecnológica do Estado de Sergipe (FAPITEC/SE)—Brasil, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001, and the Financiadora de Estudos e Projetos—Brasil (FINEP).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data sharing is not applicable to this article.

Acknowledgments

The authors acknowledge Universidade Federal de Sergipe and funding sources for the financial support in publishing this paper.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Synthesis of ester and ether of thymol derivatives [21].
Figure 1. Synthesis of ester and ether of thymol derivatives [21].
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Figure 2. Toxicity by topical application of the insecticide delltamethrin (red) (a), thymol (blue) (b), thymol ester derivatives (green) (c–g), and thymol ether derivatives (orange) (h,i) on workers of the leaf-cutting ant Acromyrmex balzani. Lethal doses (logarithmic scale) were estimated based on dose–response bioassays using Probit analysis. Shaded areas represent 95% confidence intervals. Circles indicate the means (±standard error) of mortalities observed in the bioassays. β = slope of the curve, n = number of insects tested, and LD50 = lethal dose needed to kill 50% of the population.
Figure 2. Toxicity by topical application of the insecticide delltamethrin (red) (a), thymol (blue) (b), thymol ester derivatives (green) (c–g), and thymol ether derivatives (orange) (h,i) on workers of the leaf-cutting ant Acromyrmex balzani. Lethal doses (logarithmic scale) were estimated based on dose–response bioassays using Probit analysis. Shaded areas represent 95% confidence intervals. Circles indicate the means (±standard error) of mortalities observed in the bioassays. β = slope of the curve, n = number of insects tested, and LD50 = lethal dose needed to kill 50% of the population.
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Figure 3. Survival curves and lethal time (LT50) of workers of the leaf-cutting ant Acromyrmex balzani topically exposed to LDs90 (see Figure 2) of the insecticide deltamethrin, thymol, and ester and the ether of thymol derivatives. Survival curves were compared by the Holm–Sidak method and amalgamated when there was no difference at p < 0.05. Squares indicate the mean (±95% confidence interval) of the LT50 (lethal time required to kill 50% of the population).
Figure 3. Survival curves and lethal time (LT50) of workers of the leaf-cutting ant Acromyrmex balzani topically exposed to LDs90 (see Figure 2) of the insecticide deltamethrin, thymol, and ester and the ether of thymol derivatives. Survival curves were compared by the Holm–Sidak method and amalgamated when there was no difference at p < 0.05. Squares indicate the mean (±95% confidence interval) of the LT50 (lethal time required to kill 50% of the population).
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Figure 4. Analysis of the individual self-cleaning behavior of leaf-cutter ant Acromyrmex balzani workers exposed to LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue) and the more toxic thymol derivative (thymyl chloroacetate) (green). The circles represent the 5th and 95th percentiles, the error lines the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05.
Figure 4. Analysis of the individual self-cleaning behavior of leaf-cutter ant Acromyrmex balzani workers exposed to LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue) and the more toxic thymol derivative (thymyl chloroacetate) (green). The circles represent the 5th and 95th percentiles, the error lines the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05.
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Figure 5. Analysis of collective cleaning (A), antennation (B), avoidance (C), and aggression (D) behaviors of leaf-cutting and workers Acromyrmex balzani exposed to LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue), and the more toxic thymol derivative (thymyl chloroacetate) (green). The circles represent the 5th and 95th percentiles, the error lines the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Even boxplots with light and dark colors indicate that there was no difference between doses. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05.
Figure 5. Analysis of collective cleaning (A), antennation (B), avoidance (C), and aggression (D) behaviors of leaf-cutting and workers Acromyrmex balzani exposed to LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue), and the more toxic thymol derivative (thymyl chloroacetate) (green). The circles represent the 5th and 95th percentiles, the error lines the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Even boxplots with light and dark colors indicate that there was no difference between doses. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05.
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Figure 6. Individual displacement behavior of leaf-cutting ant workers Acromyrmex balzani exposed for 600 s to LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue), and the most toxic thymol derivative (thymyl chloroacetate) (green) in 9 × 9 cm arenas (A). The circles in the graph represent the 5th and 95th percentiles, the error lines represent the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05. (B). Representative trails (3D graph) of the walking and displacement (cm) of workers in the control and in the LD30 (C) and LD50 (D) of the treatments. The circles in the graphs (B–D) represent the mean distances traveled by the insects and the bars the standard errors.
Figure 6. Individual displacement behavior of leaf-cutting ant workers Acromyrmex balzani exposed for 600 s to LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue), and the most toxic thymol derivative (thymyl chloroacetate) (green) in 9 × 9 cm arenas (A). The circles in the graph represent the 5th and 95th percentiles, the error lines represent the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05. (B). Representative trails (3D graph) of the walking and displacement (cm) of workers in the control and in the LD30 (C) and LD50 (D) of the treatments. The circles in the graphs (B–D) represent the mean distances traveled by the insects and the bars the standard errors.
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Figure 7. Collective displacement behavior (four ants/arena) of workers of the leaf-cutting ant Acromyrmex balzani exposed for 600 s to LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue) and the most toxic thymol derivative (thymyl chloroacetate) (green) in 9 × 9 cm arenas (A). The circles in the graph represent the 5th and 95th percentiles. The error lines the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05. (B). Representative trails (3D graph) of the walking and displacement (cm) of workers in the control and in the LD30 (C) and LD50 (D) of the treatments. The circles in the figures represent the mean distances traveled by the insects and the bars the standard errors.
Figure 7. Collective displacement behavior (four ants/arena) of workers of the leaf-cutting ant Acromyrmex balzani exposed for 600 s to LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue) and the most toxic thymol derivative (thymyl chloroacetate) (green) in 9 × 9 cm arenas (A). The circles in the graph represent the 5th and 95th percentiles. The error lines the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05. (B). Representative trails (3D graph) of the walking and displacement (cm) of workers in the control and in the LD30 (C) and LD50 (D) of the treatments. The circles in the figures represent the mean distances traveled by the insects and the bars the standard errors.
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Figure 8. Meander behavior (°/cm) individual (A) and collective (B) of workers (average of four ants/arena) of the leaf-cutting ant Acromyrmex balzani exposed for 10 min at LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue) and the more toxic thymol derivative (thymyl chloroacetate) (green) in 9 × 9 cm arenas. The circles and shaded area represent the data points and distribution. The error lines represent the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05.
Figure 8. Meander behavior (°/cm) individual (A) and collective (B) of workers (average of four ants/arena) of the leaf-cutting ant Acromyrmex balzani exposed for 10 min at LD30 (lighter colors) and LD50 (darker colors) of the insecticide deltamethrin (red), thymol (blue) and the more toxic thymol derivative (thymyl chloroacetate) (green) in 9 × 9 cm arenas. The circles and shaded area represent the data points and distribution. The error lines represent the 10th and 90th percentiles, and the edges of the box the 25th and 75th percentiles. The medians (black lines inside the boxplots) followed by an asterisk differ from the control by Dunn’s test at p < 0.05. Boxplots with different letters indicate that the mean lethal doses (LD30 and LD50) differ using the Wilcoxon test at p < 0.05.
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MDPI and ACS Style

Dantas, J.O.; Cavalcanti, S.C.H.; Araújo, A.P.A.; Silva, J.E.; Brito, T.B.; Andrade, V.S.; Pinheiro, H.S.S.; Tavares, S.R.S.A.; Blank, A.F.; Bacci, L. Formicidal Potential of Thymol Derivatives: Adverse Effects on the Survival and Behavior of Acromyrmex balzani. Agriculture 2023, 13, 1410. https://doi.org/10.3390/agriculture13071410

AMA Style

Dantas JO, Cavalcanti SCH, Araújo APA, Silva JE, Brito TB, Andrade VS, Pinheiro HSS, Tavares SRSA, Blank AF, Bacci L. Formicidal Potential of Thymol Derivatives: Adverse Effects on the Survival and Behavior of Acromyrmex balzani. Agriculture. 2023; 13(7):1410. https://doi.org/10.3390/agriculture13071410

Chicago/Turabian Style

Dantas, Jaciele O., Sócrates C. H. Cavalcanti, Ana Paula A. Araújo, Jefferson E. Silva, Thaysnara B. Brito, Valfran S. Andrade, Heloisa S. S. Pinheiro, Swamy R. S. A. Tavares, Arie F. Blank, and Leandro Bacci. 2023. "Formicidal Potential of Thymol Derivatives: Adverse Effects on the Survival and Behavior of Acromyrmex balzani" Agriculture 13, no. 7: 1410. https://doi.org/10.3390/agriculture13071410

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