Baseline susceptibility of rice leaf folder Cnaphalocrocis medinalis (Guenee) (Lepidoptera: Crambidae) to selected insecticides

Abstract

With the rising food production demands, pesticides remain essential for achieving high yields, particularly in staple crops like rice. Despite the known environmental hazards of chemical pesticides, their repeated use has become common practice, often leading to selection pressure and the development of insecticide resistance. This issue is exacerbated when insect pests like the rice leaffolder, Cnaphalocrocis medinalis, which undergoes several generations a year, are exposed to the same insecticide across consecutive generations. Recent failures in controlling rice leaffolder outbreaks in Kerala have raised concerns, though no formal studies have documented insecticide resistance in these populations. This study aimed to address this gap by assessing the susceptibility of rice leaffolder populations to selected insecticides and investigating potential resistance mechanisms. Field populations from five agroecological units (AEUs) in Kerala were assessed for insecticide resistance through laboratory bioassays, with WYD (AEU 20) as a susceptible reference. The PKD (AEU 23), KUD (AEU 4), and TCR (AEU 6) populations experienced significant selection pressure, leading to moderate to high resistance to the tested insecticides, surpassing the resistance observed in the ONT (AEU 3) population. Resistance to acephate (5.07- to 172.53-fold) was identified for the first time in India. High resistance to quinalphos (133.24- to 611.37-fold) and carbosulfan (25.40- to 347.96-fold) were also noted. The observed carbosulfan resistance, despite no prior use, likely stems from cross-resistance due to previous organophosphate exposure, as both target acetylcholinesterase. Continuous exposure intensified resistance to lambda-cyhalothrin (up to 763.66-fold) and fipronil (up to 154.83-fold). Diamide resistance was significant, with chlorantraniliprole (1089.63 fold) and flubendiamide (1572.64-fold), marking the first reported flubendiamide resistance in C. medinalis in India. Cross-resistance to cartap (14.85- to 23.90-fold) and emamectin benzoate (24.97- to 81.09-fold) suggested resistance mechanisms driven by non-specific detoxification pathways. Further, the study aimed to elucidate the biochemical mechanisms of resistance by assessing the activities of detoxification enzymes. Resistant populations exhibited significantly elevated activities of carboxylesterase (CarE) (1.1–1.6-fold), cytochrome P450-dependent monooxygenase CytP450) (1.5–2.5-fold), and glutathione S transferase (GST) (2.3–3.0-fold). These findings underscore the increased activity of detoxifying enzymes as a contributing factor to the resistance observed in C. medinalis further confirmed through synergism bioassays. Bioassays with synergists revealed diverse resistance mechanisms across populations, driven by variations in detoxification enzyme activity. Metabolic resistance to acephate, quinalphos, carbosulfan, and lambda-cyhalothrin was primarily associated with CarE, CytP450, and GST, either individually or in combination. Multiple enzyme involvement was evident in PKD, KUD, and TCR, while CytP450 had a dominant role in ONT, highlighting enzyme-specific contributions to insecticide resistance. However, metabolic detoxification was not the primary driver of chlorantraniliprole and flubendiamide resistance in most populations, suggesting the involvement of alternative mechanisms. Minor contributions from CarE and CytP450 were detected in PKD, while slight synergistic effects in KUD may be linked to endosymbiont mediated resistance. In the case of fipronil, resistance was mediated by CytP450 in PKD, KUD, and ONT, whereas non-metabolic mechanisms likely contributed to the high fipronil resistance observed in TCR. These findings underscore the complexity of resistance mechanisms and highlight the need for further investigation into alternative pathways for diamide and fipronil resistance. This study investigated target site insensitivity as a potential resistance mechanism to diamides and fipronil in C. medinalis by analyzing mutations in the ryanodine receptor (RyR) and resistance to dieldrin (Rdl) genes. Gene duplication and a novel I4712N mutation in transmembrane (TM) domain 3 of RyR were detected in ONT and KUD populations, while PKD and TCR populations had a nonsynonymous mutation (F4691L) and a nonsense mutation (Y4692*) in the TM2-TM3 linker of the RyR gene, which may impact diamide binding. Additionally, a K4885K synonymous mutation was identified in TCR in the TM4-5 linker. As these mutations are newly reported, functional validation is required to confirm their role in resistance. Molecular analysis of the Rdl gene identified an A282S mutation in TM 2 of all populations, including the susceptible WYD, suggesting a limited role in fipronil resistance. Notably, this study reports a novel V457F mutation in TM 4 of the Rdl gene in the resistant TCR population, which may have contributed to high fipronil resistance (154.83-fold) by altering GABA receptor function. The absence of target-site mutations for isoxazoline and meta-diamides suggests their continued efficacy against C. medinalis. Metagenome analysis identified Pantoea sp. and Wolbachia sp. as potential endosymbiont contributors to microbial detoxification of chlorantraniliprole and flubendiamide resistance in KUD. These findings provide new insights into resistance mechanisms and highlight the need for further functional confirmation. Rice leaffolder populations in Kerala have developed diverse resistance mechanisms in response to sustained insecticide pressure, exhibiting metabolic and target-site adaptations. This study presents the first detailed evaluation of insecticide resistance in C. medinalis from Kerala, uncovering alarming resistance levels to multiple insecticides, including newly documented cases for acephate, carbosulfan, lambda-cyhalothrin, and flubendiamide. Metabolic detoxification, primarily through CarE, CytP450, and GST, played a key role in resistance against organophosphates, carbamates, and synthetic pyrethroids, with multiple detoxification pathways raising concerns about cross-resistance. Mutations in the RyR and Rdl genes suggest target site insensitivity for diamides and fipronil (TCR), while the potential involvement of endosymbionts in microbial detoxification (KUD) adds another layer of complexity. These findings highlight the urgent need for proactive resistance management strategies, integrating insecticide rotation, biological control, and molecular monitoring to sustain effective pest management in Kerala’s rice ecosystems.