INTRODUCTION

Production of cereals plays a crucial role in the global food supply, representing between 15% and 20% of protein intake in either humans or animals (^{Safdar et al., 2023}). In Colombia, more than 2 million tons were produced in 2021; however, 9 million tons were imported, according to the National Federation of Cereal, Legume, and Soybean Growers (^{Fenalce, 2023}). Due to their seasonal nature and permanent consumption, storage is a requirement (^{Richards et al., 2002}).

In this context, the lesser grain borer, Rhyzopertha dominica (F.), stands out as a primary pest (^{García-Lara et al., 2007}). This insect, belonging to the Bostrichidae family, is 2 to 3 mm long and 0.8 to 1 mm wide, with a color ranging from reddish brown to dark brown (^{Fisher, 2013}). Its thorax shows a crenulated dome-shaped anterior margin, and the thorax and elytra have distinctive dimples. The antenna is short, with ten segments and a loose, three-jointed terminal club (^{Fisher, 2013}; ^{Majeed et al., 2015}).

It is a polyphagous and cosmopolitan insect (^{Edde, 2012}), noted for its robust mandibles in both larvae and adults, which are adapted to feed on the germ and endosperm of grains (^{Fisher, 2013}), exhibiting a high fecundity rate and a wide range of temperature and humidity (^{Chanbang et al., 2008}). It produces adverse changes in the composition of grains, such as loss of weight, germination capacity, nutritional value, and malodor, resulting in significant damage to cereals such as wheat, corn, and rice (^{Chanbang et al., 2008}; ^{Perišić et al., 2021}), with losses ranging from 10% to 23% of the total cereal production stored in Colombia (^{Malagon & Trochez, 1985}).

There are different control measures based on Integrated Pest Management (IPM) (^{Abrol, 2013}), such as cultural control, physical control, biological control, and chemical control based on the application of pesticides; however, the latter is usually the most effective method and has specific measures for its use (^{Carvalho, 2017}). In Colombia, chemical control is the main method for controlling R. dominica, and only two classes of molecules are authorized: organophosphates (pirimiphos-methyl) and pyrethroids (deltamethrin and bifenthrin), and also the fumigant phosphine, according to the Colombian Agricultural Institute (^{ICA, 2022}).

Resistance of R. dominica to phosphine has been reported (^{Collins et al., 2002}; ^{Edde, 2012}; ^{Yang et al., 2018}). Active ingredients, like pyrethroids used in stored grain pest control, have exhibited control failures and resistance in evaluations conducted in eastern Australia and two populations in Colombia (^{Daglish & Nayak, 2018}; ^{Salcedo-Ortega et al., 2021}). In addition, there is evidence of genetic resistance to pyrethroids in other stored grain pests, such as Sitophilus spp. ^{Haddi et al. (2018)} found mutations in the site of action of pyrethroids (knockdown resistance [kdr]) in Brazilian populations. The objective of the present research was to evaluate the efficacy of two insecticides, deltamethrin and deltamethrin, combined with piperonyl butoxide (Delmetrin and K-Obiol) in the control of R. dominica in six populations from Colombia. The purpose is to establish scientific bases to guide the management and prevention of R. dominica resistance to chemical insecticides and contribute to the rational and efficient use of pesticides in the storage of cereals in Colombia.

MATERIAL AND METHODS

Rearing of Rhyzopertha dominica. Populations of R. dominica were collected from mills in: Puerto Lopez (4°5'56.8'' N 72°57.388'' W) and Granada (3°32'46.5'' N 73°42.412'' W) from the department of Meta, El Espinal (4°08'55'' N 75°52'55'' W), Neiva (2°55'38.3'' N 75°16.913' W), Pore (5°43'40.5'' N 71°59.56' W), and Córdoba (8°22′00″N 75°42′00″W) from the departments of Tolima, Huila, Casanare, and Córdoba, respectively. Insects were reared and tested under laboratory conditions in the Entomology Laboratory of the University of Tolima in the department of Tolima, Colombia. The rearing process, conducted under controlled laboratory conditions (temperature: 27 ± 2°C, relative humidity: 64.8 ± 5%, photoperiod: 12:12), adhered to specific parameters.

Insects were reared on broken rice in 4-liter plastic containers covered with pieces of organza veil secured with an elastic band, which was changed every two months to avoid contamination by fungi or other organisms. To prevent any previous insect infestation, the rice grains used for rearing and experiments were initially classified by removing damaged grains and sterilized by incubation for 72h at a temperature of -18°C. Two days before use, we brought the rice grains to laboratory temperature (^{Salcedo-Ortega et al., 2021}). The moisture content of the grains was approximately 11.5%.

Control failure assessment. The control failure consisted of exposing unsexed adults of R. dominica from the six populations collected to the doses indicated on the label of commercial products containing the pyrethroid deltamethrin. The commercial products used were Deltametrin® 2.50EC (C.I. Entquim Ltda, Bogotá D.C., Colombia, 60 ml/1000 kg of grain) and K-Obiol® EC25 (Bayer Crop Science, Bayer S.A., Bogotá, DC, 20 ml/1000 kg of grain). According to the Colombian Institute of Agriculture (ICA), the 80% mortality threshold was employed as the basis for control failure assessments.

We soaked 300 g of paddy rice with the indicated dose of the insecticide in 9 ml of water and 0.5% Agrotin® SL adjuvant (Bayer Crop Science, Bayer S. A., Bogotá, DC, Colombia). After shaking the rice for three minutes to ensure the impregnation of all grains, it was air-dried for one hour and then separated into groups of 30 g in 250-ml glass bottles. The control involved paddy rice impregnated with water and Agrotin 0.5%. Each treatment consisted of ten replicates. We evaluated mortality rates (considering organisms without any movement after 5 minutes of observation as dead) at 24, 48, 72, and 144 hours after exposing R. dominica to each treatment.

Residual effects and survival bioassay. In this trial, we applied the commercial doses to rice, as previously mentioned. We assessed adult survival until reaching a 90% mortality rate. We maintained consistency with the protocol detailed above for the bioassay. The control group received a treatment composed exclusively of water and 0.5% Agrotin. We recorded mortality daily for 35 days. A total of 100 adult insects were used for each combination of population and insecticide concentration.

Data Analysis. A two-way analysis of variance (ANOVA) was performed for the control failure tests, with insecticides as independent variables, followed by a Tukey mean comparison test. The results of the survival bioassays were subjected to survival analysis, which was performed using Kaplan-Meier estimators (Log-rank method). Insects that remained alive at the end of the bioassay were censored for analysis. The overall similarity between survival times and median survival (LT₅₀ values) was tested using the log-rank test χ², and pairwise comparisons between curves were performed using the Holm-Sidak test (P < 0.05).

RESULTS AND DISCUSSION

Control failure assessment. The cumulative mortalities of the two insecticides evaluated did not reach 80% during 144-h, with 48-h mortality rates ranging from 2% to 12% (Table 1).

In El Espinal, a cumulative mortality of 17% at 48-h was observed with the application of deltamethrin + piperonyl butoxide (Table 1), as had been reported previously for a population from the same place by ^{Salcedo-Ortega et al. (2021)}. Similarly, in the Neiva population, a mortality rate of 21% was recorded, indicating a behavior comparable to that observed in El Espinal. This similarity could be attributed to El Espinal and Neiva showing similar agricultural conditions and being located near comparable ecological environments.

Significant differences in insecticide effectiveness were only evident after 144-h in the populations of Cordoba, Granada, and Pore (F= 48.48, df= 2, P < 0.0001) (Table 1). In this context, the combination of deltamethrin and piperonyl butoxide showed greater efficacy, achieving mortality rates of 44%, 36%, and 48%, respectively, compared to the exclusive use of deltamethrin (Table 1). Piperonyl butoxide exhibits a synergistic effect, which acts as an inhibitor of the metabolic detoxification of insects (^{Shakya et al., 2022}). It binds non-covalently to the cytochrome P450 enzyme (^{Hodgson & Levi, 1999}), altering the function of the oxidases responsible for detoxification and affecting their ability to metabolize molecules such as pyrethroids (^{Fouad et al, 2023}). Frequently, a reduction in susceptibility to deltamethrin, attributed to the effect of piperonyl butoxide on stored grain pests, has been observed ( ^{Arthur, 1994}; ^{Arthur et al., 2020}; ^{Fouad et al., 2023}; ^{Salcedo-Ortega et al., 2021}).

Table 1 Cumulative mortality (%) of Rhyzopertha dominica, means with a common letter are not significantly different (p > 0.05). 

Our results corroborate the presence of control failure in all evaluated populations at the recommended label doses of both insecticides studied and the low level of protection of grains from R. dominica in Colombia. These results are in agreement with preliminary findings in the Tolima department in Colombia by ^{Salcedo-Ortega et al. (2021)} and in other research worldwide too (^{Daglish & Nayak, 2018}; ^{Kavallieratos et al., 2015}; ^{Lorini & Galley, 1998}; ^{Salcedo-Ortega et al., 2021}; ^{Trostanetsky et al., 2023}).

This resistance has been frequently attributed to the use of insecticides at high doses (^{Barathi et al., 2024}; ^{Helps et al., 2020}). In addition, this resistance can be caused by the limited rotation of the modes of action of the insecticides (^{Madgwick & Kanitz, 2024}; ^{Sparks et al., 2021}). This is compounded by the low level of authorized chemical molecules in Colombia. The lack of variety in treatments contributes to increased selection pressure (^{Thia et al., 2023}), resulting in high levels of pyrethroid resistance, as reported by ^{Daglish & Nayak (2018)} in eastern Australia. This phenomenon has been noted in other stored grain pests (^{Attia et al., 2020}; ^{Haddi et al., 2018}). However, the populations studied came from places with high use of insecticides in agricultural ecosystems, which may increase the pressure on the insects in the field.

In this context, evaluating new molecules as an alternative to pyrethroids and organophosphates is essential. Although the efficacy of organophosphates, specifically pirimiphos methyl (Actellic®), has been proven to control R. dominica (^{Salcedo-Ortega et al., 2021}), it is essential to explore other molecules to strengthen and diversify management strategies. The concern lies in the current existence of only one effective molecule on the market for the management of R. dominica in stored grains, which highlights the need for further studies on the efficacy of other molecules with different modes of action to be included in pest management and control plans.

Residual effects and survival bioassay. The results of the survival bioassays revealed significant differences in the mean survival time of R. dominica adults treated only with deltamethrin. The least susceptible population found was El Espinal, with an LT₅₀ of 29.620 (27.681-31.559) days, while the most susceptible populations, such as Pore and Puerto Lopez, exhibited mortality within days; others were positioned at intermediate levels (log-rank test, X² = 277.753, df = 5, P < 0.001) (Figure 1). It is important to note that the El Espinal population in our assays showed a lower LT₅₀ compared to the study by ^{Salcedo-Ortega et al. (2021)}.

Figure 1 Survival curves of six Colombian populations of Rhyzopertha dominica exposed to commercial pyrethroids: Deltamethrin. The curves labeled with the same letter are not significantly different according to a Holme-Sidak test (P > 0.05). 

Significant differences were observed in the treatment with deltamethrin + piperonyl butoxide, leading to the identification of three distinct groups (Log-rank test, X² = 133.866, df = 5, P < 0.001). The least susceptible group consisted of El Espinal and Neiva, followed by Córdoba, Granada, and Puerto López (Figure 2). Pore was identified as the most susceptible population, exhibiting an LT₅₀ of 7.060 (6,478-7,642) days.

Figure 2 Survival curves of six Colombian populations of Rhyzopertha dominica exposed to commercial pyrethroids: Deltamethrin + piperonyl butoxide. The curves labeled with the same letter are not significantly different according to a HolmeSidak test (P > 0.05). 

These results show different residual effects on populations between the two insecticides. El Espinal and Neiva stand out as the least susceptible populations to the insecticides evaluated. On the opposite side, Pore stands out as the most susceptible population with the lowest mean lethal time. El Espinal and Neiva are located in areas with a long history of intensive insecticide use in commercial agriculture, particularly in crops such as rice, corn, and cotton. In contrast, the region where Pore is situated has relatively recently adopted these agricultural practices. Our findings indicate a correlation between the duration of exposure to insecticides in agricultural settings and the observed susceptibility levels of R. dominica populations.

In addition, we observed that insect survival in all populations was significantly lower when treated with deltamethrin + piperonyl butoxide compared to the treatment with deltamethrin alone (Figure 2 vs. Figure 1). However, despite this reduction in survival, this treatment does not achieve effective control of R. dominica.

CONCLUSIONS

The possible resistance of R. dominica to insecticides is noted, highlighting the need for toxicological studies to determine its degree of resistance. In addition, it is important to research the efficacy of molecules with different modes of action to strengthen chemical management strategies. Likewise, the implementation of Integrated Pest Management (IPM) is essential, emphasizing alternative methods such as cultural, physical, and biological control. These findings highlight the urgency of addressing resistance and diversifying approaches for effective control of this pest.