Resistance monitoring of Leucinodes orbonalis Guenée populations to commonly used insecticides on Brinjal (Solanum melongena L.)

Introduction

A dramatic example of how insecticide resistance can undermine pest management programs was observed in a major brinjal-producing area where cypermethrin, once highly effective against Leucinodes orbonalis Guenée, progressively lost its field efficacy over a five-year period of continuous use, ultimately necessitating a complete revision of the chemical management strategy [1,2] . Such instances of resistance-mediated management failure highlight the critical importance of proactive resistance monitoring as an integral component of sustainable pest management programs [3] . Insecticide resistance in arthropod pest populations develops through the selection of genetically based mechanisms that reduce the susceptibility of individuals to toxic concentrations of insecticidal compounds [4] . The primary mechanisms of resistance include enhanced metabolic detoxification through elevated activities of cytochrome P450 monooxygenases, esterases, and glutathione S-transferases; target site modifications that reduce the binding affinity of insecticide molecules; reduced cuticular penetration; and behavioral avoidance of treated surfaces [5,6] . The brinjal shoot and fruit borer L. orbonalis is subjected to particularly intensive insecticide selection pressure due to the high frequency of applications (often 15 to 30 sprays per cropping season in intensively managed systems) and the heavy reliance on a limited number of insecticide classes for its management [7] . These conditions create an environment highly Conducive to resistance development, yet systematic monitoring of resistance levels in field populations of L. orbonalis has received comparatively limited scientific attention [8,9] . The availability of standardized bioassay protocols and biochemical enzyme activity assays provides robust tools for detecting and quantifying resistance development before it reaches levels that cause visible management failures in the field [10] . Early detection of resistance through proactive monitoring enables the implementation of preventive resistance management measures, including insecticide rotation, mixtures, and integration with non-chemical control tactics [11] . The present research was designed to assess the resistance status of multiple L.

Methods

Dose-response bioassays were conducted using the leaf-dip method following the protocol established by the Insecticide Resistance Action Committee (IRAC). For each insecticide, seven logarithmically spaced concentrations plus a water control were tested against third-instar larvae from each population. Brinjal leaf discs (5 cm diameter) were dipped in insecticide solutions for 30 seconds, air-dried for 60 minutes, and placed individually in ventilated plastic containers. Ten third-instar larvae were placed on each treated leaf disc, with four replications per concentration per population. Mortality was assessed at 48 hours, and larvae unable to exhibit coordinated movement when prodded were considered dead. Biochemical enzyme activity assays were performed on surviving adults from each population using standardized spectrophotometric methods. General esterase activity was measured using alpha-naphthyl acetate as substrate, mixed-function oxidase (MFO) activity was estimated using 7-ethoxycoumarin O-deethylase (ECOD) assay, and glutathione S-transferase (GST) activity was determined using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Protein content was measured by the Bradford method and enzyme activities were expressed as specific activities per milligram of protein. Selection experiments were conducted by exposing each population to the LC30 concentration of cypermethrin or emamectin benzoate for five consecutive generations (P through F5). Survivors were reared to the next generation, and bioassays were performed at each generation to track changes in susceptibility over time. Data from dose-response bioassays were analyzed using probit regression to estimate LC50 values and their 95 percent fiducial limits. Resistance ratios were calculated as LC50 of field population divided by LC50 of the susceptible reference colony. Enzyme activity data were analyzed by one-way ANOVA and correlated with resistance ratios using Pearson's correlation coefficient.

Results

The dose-response bioassays revealed significant variation in insecticide susceptibility among the six L. orbonalis field populations for all three insecticides tested.

Discussion

The findings of this research provide the first systematic assessment of insecticide resistance status in L. orbonalis populations from diverse brinjal-producing areas in Peru, yielding important insights for resistance management planning. The overall summary of the resistance monitoring data indicates that pyrethroid resistance is developing in populations subjected to intensive selection pressure, while newer-chemistry insecticides retain high levels of efficacy across all sampled populations. The agreement between the present biochemical findings and previous reports on metabolic resistance mechanisms in other lepidopteran species supports the generality of esterase and oxidase-mediated detoxification as primary resistance pathways in this pest order. However, there is a disagreement with some earlier suggestions that L. orbonalis is inherently slow to develop resistance due to its limited host range and generation time constraints, as the selection experiments demonstrated detectable resistance development within just five generations [5,6] . The explanations for the differential resistance development rates among the three insecticides tested relate to their distinct modes of action, metabolic susceptibility to detoxification enzymes, and the duration of selection pressure in field populations. Cypermethrin, as a sodium channel modulator, is particularly susceptible to metabolic detoxification through esterase hydrolysis and oxidative metabolism, both of which were elevated in resistant populations [7,8] . In contrast, emamectin benzoate targets glutamate-gated chloride channels through a mechanism that appears less readily circumvented by the common metabolic resistance pathways, explaining its sustained effectiveness [9,10] . The broader implications of these findings for resistance management strategy are significant. The detection of moderate-level pyrethroid resistance in intensively managed populations serves as an early warning signal that current management practices in these areas are unsustainable and require modification [11,12] . The implementation of insecticide rotation programs alternating between pyrethroids, diamides, spinosyns, and avermectins based on their distinct IRAC classification groups can distribute selection pressure across multiple target sites and metabolic pathways, thereby decelerating resistance evolution [13,14] .

Resistance management recommendations

Based on the resistance monitoring results obtained in this research, a set of evidence-based resistance management recommendations is proposed for *L. orbonalis* control programs in brinjal production systems. First, pyrethroid insecticides including cypermethrin should be restricted to a maximum of two applications per cropping season in areas where resistance ratios exceed 2.0, and alternative insecticide classes should be employed for the remaining spray applications. Second, rotation programs should incorporate insecticides from at least three different IRAC mode of action groups within each cropping season, ensuring that no single target site is subjected to more than two consecutive selection events. Third, annual resistance monitoring using standardized bioassay protocols should be established for key brinjal production areas to track changes in susceptibility and enable timely adjustments to management recommendations.

Cross-resistance patterns

Preliminary assessment of cross-resistance patterns among the three insecticides tested suggests limited cross-resistance between cypermethrin and the newer-chemistry compounds (emamectin benzoate and indoxacarb), consistent with their distinct modes of action and different susceptibility to metabolic detoxification pathways. Population F, which exhibited the highest cypermethrin resistance ratio (5.0), showed only moderate resistance ratios for emamectin benzoate (2.6) and indoxacarb (2.2), indicating that metabolic mechanisms conferring pyrethroid resistance provide only partial protection against these alternative insecticide classes. This limited cross-resistance is encouraging for resistance management, as it confirms the continued viability of rotation strategies employing these three insecticide groups for managing populations with existing pyrethroid resistance.

Conclusion

The main contribution of this research lies in establishing the first baseline resistance monitoring dataset for *L. orbonalis* populations from diverse production areas in Peru, providing the foundation for evidence-based resistance management programming. The supporting evidence from bioassays, biochemical analyses, and selection experiments collectively demonstrates that pyrethroid resistance is actively developing in intensively managed *L. orbonalis* populations (resistance ratios up to 5.0), while newerchemistry insecticides (emamectin benzoate and indoxacarb) retain higher levels of susceptibility across all tested populations. The significance of these findings for the field of brinjal pest management lies in the demonstration that proactive resistance monitoring can detect resistance development at an early stage, before field control failures become apparent, enabling timely implementation of preventive management strategies. Open questions remaining from this investigation include the characterization of target site resistance mechanisms that may develop in parallel with or independently of metabolic resistance, the population genetic structure of *L. orbonalis* that influences gene flow and resistance spread between geographically separated populations, and the interaction between resistance management strategies and other pest management objectives such as environmental sustainability and food safety.

Acknowledgement

AcknowledgementsThe authors recognize the contributions of the laboratory research assistants and field collection teams who participated in population sampling, colony establishment, bioassay execution, and biochemical analyses throughout this investigation. The Lima Research Institute of Crop Sciences provided the laboratory infrastructure, insect rearing facilities, and analytical equipment essential for this research. Financial support was provided by the Peruvian National Council for Science, Technology and Technological Innovation (CONCYTEC) under grant number.

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