Sublethal Effects of Chlorantraniliprole on the Mobility Patterns of Sitophilus spp.: Implications for Pest Management

Simple Summary This study investigates the sublethal effects of chlorantraniliprole, an insecticide known for its low toxicity to mammals and selectivity towards non-target organisms, on two significant stored-product pests, Sitophilus oryzae (L.) and Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). Through contact toxicity assays, differences in susceptibility between the two species are observed, with S. zeamais showing higher sensitivity. Subsequent analysis reveals altered mobility behavior in chlorantraniliprole-exposed groups compared with controls, particularly in S. oryzae, which displays reduced number of food approaches and altered locomotion patterns. In contrast, S. zeamais exhibits increased walking time and decreased immobility periods under sublethal concentrations. These findings underscore the importance of considering sublethal effects in understanding the overall impact of chlorantraniliprole on stored-product pest populations. Further exploration into the long-term consequences of sublethal exposure is recommended to enhance pest management strategies. Abstract Chlorantraniliprole, an anthranilic diamide insecticide, has emerged as a promising solution for controlling agricultural pests because of its low mammalian toxicity and selectivity towards non-target organisms. This study investigated the sublethal effects of chlorantraniliprole on the mobility behavior of two significant stored-product pests, Sitophilus oryzae (L.) and Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae). Contact toxicity assays revealed varying susceptibility levels between the two species, with S. zeamais showing higher sensitivity. Subsequent analysis of mobility behavior, both in the presence and absence of food, indicated significant differences between chlorantraniliprole-exposed and control groups. While S. oryzae exhibited altered locomotion patterns and a decreased number of food approaches at sublethal concentrations, S. zeamais displayed increased walking time and reduced immobility periods. These findings highlight the importance of considering sublethal effects in understanding the overall impact of chlorantraniliprole on stored-product pests. Further research into the long-term consequences of sublethal exposure is warranted to inform more effective pest management strategies in storage.


Introduction
Sitophilus oryzae (L.) and Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), commonly known as the rice weevil and the maize weevil, respectively, are two major primary pests in the global agriculture industry [1,2].These pests typically thrive in tropical, subtropical, and temperate climate zones.They have been documented to infest various commodities, including grains, pulses, processed cereal products, nuts, pasta, and fruits [3,4].Their similar morphologies and life cycles are often associated with the marked preferred commodity they develop in, with S. oryzae primarily infesting wheat and S. zeamais predominantly affecting maize [4,5].Both insects infiltrate grains to deposit their eggs, resulting in a decrease in the quality and weight of the commodities.This process is often accompanied by a foul odor, rendering the product unsuitable for commercial use, while it frequently attracts and contributes to the proliferation of secondary pests [6,7].
For decades, numerous synthetic insecticides have been employed in the control of stored-product pests and the deterioration they induce [8].Organophosphorus insecticides have been widely used across the globe to manage agricultural pests.For example, chlorpyrifos stands as one of the frequently utilized organophosphorus compounds, earning classification as a moderately hazardous substance concerning human and animal health and, consequently, the environment [9,10].In addition to organophosphates, several pyrethroid compounds have been employed in grain storage facilities to safeguard against pests in various countries [11].Pyrethroids are extensively employed in agricultural and public health because of their relatively low mammalian toxicity, great insecticidal efficacy at low concentrations, and quick knockdown effects [12][13][14].The impacts of several pyrethroids, notably, deltamethrin and cypermethrin, have been investigated concerning stored-product pests, primarily through grain treatment [11,[15][16][17].
Besides inducing mortality in storage pests, synthetic pesticides also induce sublethal effects that can influence the biology and behavioral characteristics of insects [18,19].Some of the sublethal effects include aggressive behavior and a negative impact on courtship, lifespan, fecundity, fertility, locomotion, and morphology [20][21][22].Insects have developed a range of behavioral reactions to insecticidal substances, leading to their reduced efficacy [23].For instance, exposure to dry insecticide residues impacted the movement ability of Halyomorpha halys Stål (Hemiptera: Pentatomidae), a major agricultural pest in the United States [24].Specifically, it was suggested that organophosphate insecticides had a neutral effect on the horizontal mobility of H. halys, whereas pyrethroids drastically inhibited the horizontal mobility of adult insects.Furthermore, Morrison et al. [25] demonstrated that treating commodities such as wheat, rice, and corn with label doses of synthetic compounds reduced movement by 50-88% in adults exposed to each insecticide formulation compared with untreated controls.
Chlorantraniliprole (3-bromo-N-[4-chloro-2-methyl-6-[(methylamino) carbonyl] phenyl]-1-(3-chl-oro-2-pyridinyl)-1H-pyrazole-5-carboxamide) is an anthranilic diamide insecticide, exerting its effects through the modulation of insect ryanodine receptors.This modulation results in the depletion of intracellular calcium stores, leading to disrupted muscle regulation, paralysis, and, ultimately, the demise of the insect [26].Chlorantraniliprole exhibits low mammalian toxicity and selectivity towards non-target organisms (i.e., pollinators, parasitoids, and predators) [27].To date, chlorantraniliprole has been assessed and proven effective in inducing mortality against a broad spectrum of insect pests of agricultural significance across various orders, including beetles, moths or butterflies, true bugs, flies, termites, and thrips [28][29][30][31][32].The insecticidal effectiveness and suppression of progeny by chlorantraniliprole have been previously evidenced for S. oryzae, exhibiting effectiveness in immediate and delayed mortality [33,34], along with its efficacy and persistence against S. zeamais when combined with lambda-cyhalothrin [35].The sublethal effects of this promising compound have been studied for several coleopteran pests [36][37][38][39][40][41][42].Nevertheless, the sublethal effects of chlorantraniliprole are yet to be determined for Sitophilus spp., among other significant stored-product pests.Hence, the current study aims to ascertain, for the first time, the impact of sublethal concentrations of chlorantraniliprole on the mobility behavior of two important stored-product pests, S. oryzae and S. zeamais, to comprehensively explore all facets of the efficacy of this compound in combatting significant stored-product pests.

Insecticidal Formulations
The formulation of chlorantraniliprole, Altacor WG (wettable granules), with 350 g/kg active ingredient (a.i.) was used for the experiments.The formulation was purchased from Greece by Dupont (Athens, Greece).

Sitophilus oryzae and Sitophilus zeamais Rearing
The insect species used in the experiments originated from cultures maintained at the Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, Greece, since 2003, for approximately 240 generations.The colonies were kept in total darkness at 30 • C and 65% relative humidity (RH) [43,44].The rearing medium comprised uncontaminated hard wheat kernels [45] and maize kernels [46] devoid of infestation or impurities and had not undergone any prior treatment for S. oryzae and S. zeamais, respectively.

Bioassays on Contact Toxicity
To evaluate the contact toxicity of chlorantraniliprole against the adults of S. oryzae and S. zeamais, six series of chlorantraniliprole dilutions (i.e., 0.01, 0.025, 0.05, 0.1, 0.25, and 0.5 a.i./cm 2 ) were prepared in distilled water to assess the LC 10 , LC 30, and LC 50 concentrations.Filter papers (Whatman No. 1, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) were placed separately on the bottom of Petri dishes, which measured 8 cm in diameter and 1.5 cm in height.Using a micropipette, each filter paper was impregnated with 1 mL of each chlorantraniliprole solution corresponding to the above dilutions and left to dry for 120 min at 30 • C. A total of 5 replications (5 replications × 6 concentrations = 30 dishes per species) were performed for each concentration.Consequently, 20 adults of S. zeamais or S. oryzae, less than two weeks old, were placed in each dish.Following that, the dishes were placed in incubators set at 30 • C and 65% RH and continuous darkness.The count of deceased adults of S. zeamais and S. oryzae in the dishes was conducted 72 and 96 h post-exposure, respectively.Control dishes were also prepared by impregnating filter papers with distilled water only, as described above (5 replications, 5 dishes per species).

Sublethal Effects on Mobility
The adults of S. oryzae and S. zeamais used in the following experiments were exposed to LC 10 and LC 30 of chlorantraniliprole for 24 h, following the procedure described in Section 2.3.

Mobility in the Absence of Food
A plastic, 80 mm in diameter Petri dish (50.27 cm 2 in surface) was used for the experiment.Filter paper (Whatman No. 1) was placed on the bottom of each dish.Following this, a single adult of either S. oryzae or S. zeamais from the control group, LC 10 -exposed group, or LC 30 -exposed group was introduced into the center of the dish (arena).Each adult was allowed a three-minute acclimatization period within the arena.Subsequently, the following traits were visually recorded: (1) "walking (s)"-the duration of locomotion, (2) "stops (s)"-the duration of immobility, (3) "climb up (s)"-the duration of attempts to ascend the arena walls, (4) "upturned (s)"-the duration of time spent on the insect's back, (5) "short stops (n)"-the number of interruptions in movement followed by resumption, (6) "climb up (n)"-the number of attempts to ascend the arena walls, (7) "upturned (n)"the number of instances the beetle was found on its back, (8) "flying attempts (n)"-the number of flight attempts into the arena, and ( 9) "flying"-the duration of flight within the arena [47].These observations were conducted for 15 min.Each adult exposed to chlorantraniliprole LC 10 , LC 30 , or the control underwent thirty replicates.Separate arenas were utilized for each replication, species, and exposure.The experiments were conducted between 8 a.m. and 8 p.m. at a temperature of 30 • C [47].

Mobility in the Presence of Food
In the present experiment, the locomotion behavior of S. oryzae and S. zeamais adults exposed to chlorantraniliprole LC 10 , LC 30 , or the control was observed in the presence of food.For this purpose, 0.3 g of wheat kernels for S. oryzae and 0.3 g of maize kernels for S. zeamais were positioned at the center of the arena.Prior to the bioassays, insects were deprived of food for an entire day.Subsequently, an adult of either S. oryzae or S. zeamais, exposed to chlorantraniliprole LC 10 , LC 30 , or the control, was released near the edge of the arena (the dish's walls), and the time taken to reach the food source was recorded.Additionally, the duration spent on the food patch and the number of subsequent visits to the food were noted.Thirty replicates were conducted for each of the S. oryzae and S. zeamais LC 10 -exposed, LC 30 -exposed, or control groups, with behavioral observations conducted visually over a period of 15 min.The experiments were carried out between 8 a.m. and 8 p.m. at a temperature of 30 • C.

Statistical Analysis
Using probit analysis, sublethal concentrations corresponding to the LC 10 , LC 30, and LC 50 values of chlorantraniliprole in adults of S. oryzae and S. zeamais were determined independently with a 95% confidence interval (CI) [48,49].R statistical software (version 2.15.1) was used to determine the sublethal concentrations of chlorantraniliprole [50].Data on the impact of chlorantraniliprole on the walking behavior of S. oryzae and S. zeamais adults were transformed to log(x + 1) format to achieve standard means and normal variances [47,51].Two-way ANOVA was conducted to assess the impact of chlorantraniliprole on the mobility of both weevil species [52].The Tukey HSD test was employed to separate means at a significance level of 0.05 [53] using the statistical package JMP 16.2 [54].

Mobility in the Absence of Food
The main effect species was significant for all tested traits, while concentration was significant for upturned (n), flying (n), and flying (s).The interaction species × concentration was not significant for walking (s), stops (n), and stops (s) (Table 2).For all traits tested, no significant differences were noticed compared to controls, apart from the number of climb-ups for LC 30 -exposed adults and the duration of climbing for both the LC 10 -and LC 30 -exposed S. oryzae adults (Table 3).For the LC 30 -exposed adults, the number of times they attempted to climb the arena's walls was significantly lower compared with the adults of the control (5.4 and 10.2 for LC 30 and control, respectively).The adults treated with LC 10 and LC 30 spent significantly less time attempting to climb the walls (158.2 s and 130.2 s for LC 10 and LC 30 , respectively) than the control adults (294.1 s).Furthermore, LC 10 and LC 30 caused the exposed individuals to lie on their dorsal side for longer than the control weevils (20.0 s, 26.1 s, and 13.9 s, respectively), while flight attempts were observed only in the control group.Regarding the adults of S. zeamais exposed to LC 30 of chlorantraniliprole or control, significant differences were noticed in all the traits tested.The time the weevils spent walking was significantly increased for the LC 10 -exposed and LC 30 -exposed adults (569.5 s and 535.9 s, respectively) compared with the control adults (413.1 s).The number of times that the insects stopped walking as well as the duration the insects were motionless was significantly reduced for the LC 10 -exposed (2.2 and 81.6 s, respectively) and LC 30 -exposed adults (1.2 s and 38.8 s, respectively) compared with the control (5.3 s and 292.7 s, respectively).The duration of climbing attempts in the arena's walls showed a significant increase for the LC 30 -exposed adults (309.3 s) compared with the LC 10 -exposed (225.2 s) and control adults (202.2 s).Only the adults from the control and LC 10 groups performed flying attempts (Table 4).

Mobility in the Presence of Food
The main effect species was not significant for all three traits tested, while concentration was significant for food approach.The interaction species × concentration was significant for all three traits (Table 5).The time spent between the arena and the food source differed significantly for the LC 10 -exposed S. oryzae adults (677.2 s) and control (868.3 s), while the differences were not significant between the control and the LC 30 -exposed adults (825.7 s) (Table 6).The time that S. oryzae adults spent in food was significantly higher in LC 10 (225.2 s) than in the controls (74.6 s), whereas LC 30 (33.5 s) did not demonstrate significant differences with the control.Regarding the number of times the weevil approached the food source, a significantly lower number of visits were observed in LC 30 (0.5) compared with the control (1.5) and LC 10 (1.5).Concerning S. zeamais, no significant differences were observed among adults treated with LC 10 , LC 30 , or the control for any of the traits tested.However, the duration of the time spent between the arena and the food source was higher in LC 10 (846.1 s), followed by the control (783.9 s) and LC 30 (754.5 s), while less time was spent in the food source by the LC 10 -treated weevils (53.0 s) than the controls (102.5 s) and LC 30 -treated weevils (121.8 s) (Table 7).

Discussion
Species of Sitophilus pose a major threat to the food industry worldwide because of their expansion capabilities through global commerce and the immense post-harvest crop losses they cause [55,56].A rising global problem lies in insecticide-resistant strains of Sitophilus spp., on both organophosphates and pyrethroids, in regions of Egypt, Brazil, Australia, and South Korea [8,[57][58][59].Furthermore, their life cycles as primary pests require larvae to develop inside the kernel, rendering their control efforts difficult depending on the substrate [60].Especially for S. oryzae, Boukouvala et al. [61] demonstrated that etofenprox used on several grain commodities provides different mortality rates for adults of this weevil.For example, the highest mortality was recorded on treated barley (95.0%), while for oats, maize, wheat, and whole rice, the treatment resulted in decreased mortality levels, varying from 56.7 to 80.6%, 21 days post-exposure.
Aside from toxicity-focused mortality bioassays, the sublethal effects of insecticidal formulations on behavioral traits, like mobility implications, may play an essential role in insect pest control [62].Several insecticides, like chlorantraniliprole, interfere with the locomotor capacities of insects, leading to their demise [63].According to the results of the present study regarding walking behavior, when both lethal concentrations were tested with the presence of food, the S oryzae group in LC 10 spent significantly less time between the arena and the food source, as well as on the food source only, compared with the controls.In contrast, the results for S. zeamais demonstrate that the LC 10 -exposed beetles spent more time between the arena and the food source and less time in the food source than the control group; nonetheless, the differences were not significant.This emphasizes that the exposure of the beetles to chloranthraniliprole did not influence the duration spent on the food source.However, evaluating whether this behavior originated from foraging or eating is outside the purview of this work and merits further research.In addition, varying walking values were observed, and no flying attempts were recorded for the exposed beetles, compared to the control.Specifically, LC 30 led to fewer climbing attempts and reduced climbing duration in S. oryzae compared with the controls.LC 10 and LC 30 caused S. oryzae adults to spend more time on their dorsal side.Sitophilus zeamais demonstrated increased walking duration with LC 10 and LC 30 , alongside fewer stops and longer stop periods compared with the controls.LC 30 significantly affected climbing and upturned behavior in S. zeamais.Flying behavior was significantly affected, eliminating it in both sublethal concentrations for S. zeamais individuals.It is therefore concluded that there is no specific trend in the response, as it seems that once these beetles were exposed to the active compound, they became more active in terms of walking, climbing, and decreasing stop durations.These findings suggest that these two sister species [64] react variably in contact with the same insecticide at sublethal concentrations.They also shed light on important negative effects of chlorantraniliprole, especially in lower concentrations, that can affect the motor activity and flying capacity of S. oryzae and eventually their population ecology.Reduced climbing and the lack of flying attempts can obstruct the overall colony development of S. oryzae and S. zeamais, and potentially other stored-product pests, through reduced foraging and mating, as well as prevent new infestations nearby [25].Contrarily to the control group, S. zeamais individuals exposed to this insecticide demonstrated increased mobility or variable flying activity and spent longer durations upturned, indicating an immediate irritation effect.
After a thorough literature review, it is deduced that there has been significant effort in understanding the flight/walking behavior after insecticide exposure in a plethora of insect pests, including species of the genus Sitophilus.For example, Morrison et al. [65] highlighted that the ability of H. halys adults to move horizontally, climb vertically, and fly was reduced by an average of 20-60% when exposed to insecticides compared with a control group.In particular, methomyl, thiamethoxam, and thiamethoxam + lambdacyhalothrin maintained over 65% climbing capacity, while thiamethoxam and bifenthrin preserved over 50% walking capacity.However, after exposure to insecticides, a considerable number of H. halys adults maintained notable mobility and flight capability, with flight being particularly noticeable immediately following exposure.In a recent study by Silva Barros et al. [66], male moths of Chloridea virescens (Fabricius) (Lepidoptera: Noctuidae) exposed to chlorantraniliprole demonstrated shorter flight distances in comparison with a control group.Furthermore, the flight speed of males was significantly reduced after chlorantraniliprole treatment.Another related study on the lethal and sublethal effects of chlorantraniliprole against Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae), a major pest of soybean crops, demonstrated that LD 50 proved effective in reducing the overall walking ability of the tested individuals.It notably reduced the velocity as well as the total distance covered by the larvae [67].Previously, Guedes et al. [68] conducted a study on the flight take-off and walking behavior of both resistant and susceptible strains of S. zeamais, demonstrating that behavioral responses to deltamethrin differed among strains regardless of concentration, with resistance to stimuli unrelated to physiological resistance.Males showed varying flight take-off rates, while females exhibited consistent mobility.The authors concluded that behavioral resistance did not always correlate with physiological resistance.According to de Araújo et al. [69], the number of take-offs observed in the tested groups of S. zeamais remained consistent when exposed to essential oils, similar to the control group.The authors noted varying levels of flight activity among different resistant strains when exposed to insecticides, highlighting the response variations among different strains of the same species [69].Although our study documented lower or zero flight activity of both Sitophilus species tested compared with the control, this important finding needs further investigation by testing more strains of S. oryzae and S. zeamais with the same and additional insecticides.Given that both species coexist in storage [70], the prohibition of their flight activity with the application of sublethal concentrations of a single insecticide minimizes the probability of their dispersal, especially S. zeamais since it is a much stronger flyer than S. oryzae [71].It has been reported that S. zeamais exhibits a heightened flight activity and natural dispersal ability within storage units, suggesting the potential for migration between storage areas and fields, and vice-versa [71][72][73].
Here, contact toxicity tests demonstrated that for the adults of S. zeamais, significantly lower concentrations of chlorantraniliprole were required to achieve mortality for LC 10 , LC 30 , and LC 50 .In a former study, Vásquez-Castro et al. [74] suggested that the increased tolerance to fenitrothion mixed with esfenvalerate exhibited in S oryzae comparatively to S. zeamais is a direct outcome of the behavioral differences in each species.Specifically, S. zeamais has a greater flight capability, promoting cross-infestations, which would lead to increased gene flow in certain populations, reducing tolerance levels for a specific insecticide [74].On the other hand, S. oryzae is mostly abundant in warehouses and is frequently exposed to pesticides, leading to selective pressures and favoring tolerance evolution [75].The differences in the susceptibility of S. zeamais compared with S. oryzae are in line with the above observations, nevertheless, irrespective of tolerance.

Conclusions
To our knowledge, this is the first time that the sublethal effects of chloranthraniliprole on S. oryzae and S. zeamais have been studied.The results of this investigation provide useful data regarding the susceptibility of S. zeamais in comparison to S. oryzae to low concentrations of this compound.Our research revealed significant differences in susceptibility between these congeneric species, with S. zeamais exhibiting increased motility and altered behavioral patterns compared with S. oryzae when exposed to chlorantraniliprole.This study underscores the importance of considering sublethal effects, such as affected mobility and behavior, alongside mortality rates when assessing the efficacy of insecticides on pest control.Understanding these nuances is crucial for developing effective integrated pest management strategies that minimize economic losses and mitigate the development of insecticide resistance.Furthermore, our findings highlight the need for continued research into the sublethal effects of insecticides on stored-product insect pests, particularly in species with global economic significance.

Table 1 .
Contact toxicity of chlorantraniliprole on S. oryzae and S. zeamais adults.

Table 2 .
ANOVA parameters for main effects and associated interactions for the mobility traits of S. oryzae and S. zeamais adults exposed to LC 10 and LC 30 of chlorantraniliprole (total df = 119).

Table 3 .
Mobility traits of S. oryzae exposed to LC 10 and LC 30 of chlorantraniliprole.Values are means (±standard errors).Within each column, different letters indicate significant differences (Tukey HSD test, p < 0.05).The absence of letters indicates no significant differences among values (Total df = 2.89).

Table 5 .
ANOVA parameters for main effects and associated interactions for the walking traits of S. oryzae and S. zeamais adults exposed to LC 10 and LC 30 of chlorantraniliprole in the presence of a food source (total df = 119).

Table 6 .
Effect on the walking parameters of S. oryzae with food in the center of the arena of the exposure to LC 10 and LC 30 of chlorantraniliprole.Values are means (±standard errors).Within each column, different letters indicate significant differences (Tukey HSD test, p < 0.05) (Total df = 2.89).

Table 7 .
Effect on the walking parameters of S. zeamais with food in the center of the arena of the exposure to LC 10 and LC 30 of chlorantraniliprole.Values are means (±standard errors).Within each column, different letters indicate significant differences (Tukey HSD test, p < 0.05).The absence of letters indicates no significant differences among values (Total df = 2.89).