Current status of insect pest resistance in Bt cotton

Introduction

For nearly three decades, transgenic crops expressing insecticidal proteins from the soil bacterium B. thuringiensis (Bt) have stood as a definitive cornerstone of global agricultural biotechnology (Gould, 1998; Tabashnik et al., 2008) [1,2] . Since its commercial introduction in 1996, Bt cotton (Gossypium hirsutum L.) has been adopted by millions of farmers worldwide (James, 2016) [3] . It has provided highly effective, target-specific control of devastating lepidopteran pests, including the pink bollworm (P. gossypiella), American bollworm (Helicoverpa armigera Hubner) and tobacco budworm (Heliothis virescens Fabricius) (Benedict et al., 1996) [4] . The socio-economic and environmental benefits of this adoption have been profound. The technology is characterized by massive reductions in broad-spectrum chemical insecticide applications (Fleming et al., 2018) [5] , enhanced populations of beneficial non-target arthropods (Lu et al., 2012) [6] and increased regional yields across both smallholder and large-scale farming systems (Cattaneo et al., 2006 [7] ; National Academies of Sciences, Engineering and Medicine [NASEM], 2016; Gassmann and Reisig, 2023) [8] . Despite this historic success, the fundamental biological vulnerability of Bt technology remains an evolutionary inevitability: field-evolved resistance (Comas et al., 2014) [9] . As the spatial and temporal scale of Bt cotton cultivation expanded globally, target insect populations were subjected to relentless, widespread selection pressure (Tabashnik et al., 2013) [10] . Today, the global landscape of Bt cotton efficacy is highly fragmented (Tabashnik et al., 2017) [11] . While some regions continue to enjoy prolonged pest suppression through advanced multi-toxin pyramiding and structural seed-blend mixtures (Quan et al., 2023) [12] , others are grappling with severe practical resistance episodes that threaten to return farmers to the chemical pesticide treadmills of the past (Kranthi et al., 2005) [13] . This rapid resistance development is driven by highly complex, multi-layered regulatory and genetic defences within target pests. These defences span target-site mutations such as disruptions in midgut cadherin receptors, ABCC2 and ABCA2 transporters, along with altered midgut protease profiles and accelerated epithelium regeneration driven by internal immune signalling pathways (Fabrick et al., 2023) [14] . Beyond the pink bollworm, an increasingly urgent parallel case is emerging in the polyphagous corn earworm/bollworm, Helicoverpa zea, in which practical resistance to Cry toxins and the erosion of susceptibility to Vip3A a now constitute one of the most closely watched resistance fronts in U.S. cotton and maize agroecosystems (Yang et al., 2021; Kerns et al., 2023; Yang et al., 2024) [15,16,17] . To counter these adaptive mechanisms, current structural shifts in Insect Resistance Management (IRM) emphasize deploying next-generation pyramided multi-toxin cultivars (such as those introducing Vip3A along side Cry1A c and Cry2A b ) and exploring innovative seed-mixture strategies (or "Refuge-in-a-Bag") to ensure field-level compliance without requiring active farm layout modifications (Ives et al., 2011; Quan and Wu, 2023) [18,12] . In parallel, plant-mediated RNA interference is advancing from proof-of-concept toward a genuinely complementary mode of action that can be pyramided with Bt toxins to counter resistance that has already fixed in some field populations (Ni et al., 2017; Qi et al., 2024) [19,20] .

Global spectrum of resistance: regional case studies

The evolutionary trajectory of insect resistance to Bt cotton varies drastically by geography (Bourguet et al., 2005) [21] . These differences are largely unlinked to the biology of the insects themselves; rather, they are dictated by regional agricultural infrastructures, farm sizes and varying levels of compliance with insect resistance management (IRM) frameworks (Onstad, 2014; Sisterson et al., 2005) [22,23] .

India: the epicentre of practical resistance

India represents the most critical warning sign for transgenic crop durability (Gutierrez et al., 2015) [24] . The country rapidly adopted first-generation single-toxin cotton (Bollgard I, expressing Cry1A c ) in 2002, followed by second-generation dual-toxin cotton (Bollgard II, expressing Cry1A c + Cry2A b ) in 2006 (Kranthi, 2014; Fabrick et al., 2023) [25,14] . Within a few years of intensive cultivation, populations of the pink bollworm (P. gossypiella) developed widespread, high-level practical resistance to Cry1A c , followed closely by cross-resistance and complete field breakdown of Cry2A b in major cotton growing states such as Gujarat, Maharashtra and Andhra Pradesh (Dhurua et al., 2011; Naik et al., 2018) [26,27] . Multi-location monitoring across the north, central and south cotton zones between 2007 and 2023 has confirmed that P. gossypiella has firmly established itself as a devastating midand late-season pest, with green-boll damage on Bt genotypes rising sharply after 2014 in central and southern India and after 2018 in the north (Kumar et al., 2025a, 2025b; Fabrick et al., 2023) [28,29] . This was the first documented field resistance of pink bollworm to Cry1A c in Bollgard cotton reported anywhere globally, established through F2 screening and diagnostic-dose bioassays across Gujarat during 2009-2010 (Mohan et al., 2016) [30] , and subsequent national surveillance confirmed its spread and rising economic impact across all three Indian cotton zones (Nagrare et al., 2023) [31] . The primary driver behind this rapid breakdown was a systemic failure of the highdose/refuge strategy (Fabrick et al., 2023) [14] . India's agricultural landscape is dominated by millions of smallholder farmers operating on fragmented plots of land (Choudhary et al., 2015) [32] . Implementing and monitoring structured, non-Bt cotton refuges proved socio-economically and logistically unfeasible (Mohan et al., 2019; Fabrick et al., 2023) [33,14] . Furthermore, the unauthorized sale of unapproved, unmonitored F2-generation Bt seeds and the prolonged survival of cotton stalks in fields past the normal season created a continuous, sub-lethal selection pressure that accelerated the fixation of resistance alleles (Kranthi, 2014; Venugopalan et al., 2009) [25,34] . As a consequence of total Bt failure against pink bollworm, growers have increasingly had to revert to heavy chemical interventions such as modern synthetic insecticides, including chlorantraniliprole, spinosad and indoxacarb, to salvage lint yields (Rakhesh et al., 2024) [35] . Meanwhile, the American bollworm (H. armigera), though still relatively suppressed compared to the pre-Bt era, has shown steady decreases in baseline susceptibility, demanding constant vigilance (Kranthi et al., 2005) [13] .

China: natural refuges and controlled susceptibility

In stark contrast to India, China has successfully maintained the field susceptibility of P. gossypiella to Cry1A c cotton for nearly three decades (Wu et al., 2008) [36] . This success stems from a unique "natural refuge" system (Wu et al., 2002; Fabrick et al., 2023) [14,37] .

Unlike the vast monocultures seen in western countries, China's small-scale agricultural system features high crop diversity (Cui et al., 2019) [38] . Cotton fields are intrinsically interplanted alongside non-Bt host crops of P. gossypiella and H. armigera, such as corn, peanuts, soybeans and vegetables (Lu et al., 2010; Wan et al., 2012) [39, 40] . These diverse, non-Bt crops generate an abundance of homozygous susceptible (S S

) individuals that emerge naturally and mate with the rare homozygous resistant (R R ) individuals emerging from Bt fields (Wang et al., 2018) [41] . Long-term multi-decadal panel evaluations confirm that this natural refuge mechanism, paired with dual-strategy resistance management, has prevented field control failures of target lepidopteran pests across key production zones (Quan et al., 2023) [12] . While localized resistance alleles have occasionally risen in areas such as the Yangtze River Valley due to shifting cropping patterns, aggressive management transitions towards multi-gene traits such as stacking GFM Cry1A b /c with CpTI (cowpea trypsin inhibitor) genes successfully suppressed target populations while significantly slashing historical insecticide footprints (Jin et [42,43,14] . Screening of field-collected pink bollworm from the Yangtze River Valley confirms that, where resistance alleles do arise, recessive cadherin mutations remain the dominant mechanism and their frequency is kept low by the natural refuge, reinforcing the durability of this regionally distinct management model. It is important to note that this durability is not uniform across pest species within China: long-term bioassay monitoring of Helicoverpa armigera field populations from 2005 to 2017 detected a significant, sustained increase in Cry1A c resistance frequency in northern China, in contrast to the sustained susceptibility documented for pink bollworm over the same period (Zhang et al., 2019) [44] . This divergence has prompted the development of dedicated resistance-monitoring protocols for pyramided (Cry1A c + Cry2A b , or Cry1A c + Vip3A) Bt cotton specifically targeting H. armigera, reflecting a recognition that China's natural-refuge success with pink bollworm cannot be assumed to extend automatically to every lepidopteran pest sharing the same cotton landscape (Ruan et al., 2024) [45] .

The United States and Australia: eradication and advanced pyramiding

The United States and Australia represent highly regulated, industrialized agricultural systems that have successfully delayed resistance through strict legal mandates and rapid technological transitions (Tabashnik et al., 2010) [46] . In the southwestern United States and northern Mexico, a historic milestone was achieved with the complete eradication of the pink bollworm from all cotton-producing regions (Carriere et al., 2018; Tabashnik et al., 2023) [47,48] . This was accomplished through a tightly coordinated, multi-tactic area-wide IPM program that blended near-100 per cent Bt cotton adoption with the sterile insect technique (SIT), intensive pheromone mating disruption and strict structured refuge enforcement (Fabrick et al., 2023) [14] . Currently, the primary target pests in the US are H. zea Boddie (bollworm/corn earworm) and H. virescens (tobacco budworm) (Welch et al., 2021) [49] . Because H. zea has inherently lower baseline susceptibility to Cry proteins and rapidly developed field resistance to dual-toxin cottons (Bollgard II and WideStrike), the US and Australia have aggressively transitioned to third-generation multitoxin pyramids (Fleming et al., 2018) [5] . Technologies such as Bollgard 3, WideStrike 3 and TwinLink Plus layer a third, structurally distinct vegetative insecticidal protein (Vip3A) alongside standard Cry proteins to eliminate individuals resistant to older Cry toxins (Santiago et al., 2023) [50] . However, resistance monitoring continues to be critical. Multi-year diet-bioassay surveillance across Arkansas, Louisiana, Mississippi, Tennessee and Texas documented a significant decline in H. zea susceptibility to Vip3A a between 2016 and 2020, including field-derived strains with up to 13-fold resistance relative to susceptible laboratory reference strains an early warning that field-evolved resistance to this last-effec tive Cry-independent toxin is already underway (Yang et al., 2021) [17] . Extended sentinel monitoring using Bt sweet corn across twentyfive US states and five Canadian provinces between 2020 and 2022 confirmed widespread resistance to Cry1A b , Cry2Ab 2 and Cry1A.105, together with measurable shifts in Vip3A a allele frequencies, underscoring that resistance evolution in H. zea is a moving, multi-toxin problem rather than a single-gene event (Dively et al., 2023) [51] . In addition, localized populations of secondary or sporadic pests such as the beet armyworm (Spodoptera exigua Hubner) have demonstrated the capacity to evolve highly potent, autosomal recessive resistance to Vip3A a proteins under intense selection pressures (Reisig et al., 2026) [52] , reinforcing that even the newest single mode of action is not immune to the same evolutionary dynamics that eroded Cry1A

Molecular and genetic mechanisms of resistance

The biochemical arms race between target lepidopterans and Bt proteins occurs primarily at the midgut membrane interface (Jurat-Fuentes et al., 2021; Fabrick et al., 2023) [14,53] . For a Bt toxin to kill a larva, it must undergo proteolytic activation in the midgut, bind to highly specific epithelial receptors and oligomerize to form lethal, membrane-permeable pores (Heckel et al., 2007; Soberón et al., 2007; Fabrick et al., 2023) [54,55,14] .

Mode of action of Bt Cry toxins in insects

1) Ingestion and Activation: Insect larvae ingest Cry protoxins, which are processed by midgut proteases into activated toxins (Fabrick et al., 2023) [14] . 2) Receptor Binding: The toxins bind to midgut receptor proteins such as cadherins and ABC transporters; reduced binding is a major mechanism of resistance (Fabrick et al., 2023) [14] . 3) Pore Formation: In the leading pore formation model, binding causes toxins to aggregate into oligomers that insert into cell membranes to form pores (Fabrick et al., 2023) [14] . 4) Lysis and Death: Uncontrolled influx of cations and water causes midgut cell swelling and lysis, killing the larva via acute tissue damage, starvation or septicemia (Fabrick et al., 2023) [14] .

Receptor alteration in Cry1A c resistance

The most common and intensely studied mechanism of resistance to Cry1A c involves mutations that disrupt target-site receptor binding (Zhao et al., 2003; Fabrick et al., 2023) [56,14] . Cadherin (Cad): Deletions or point mutations in genes encoding midgut cadherin proteins prevent Cry1A c from binding, halting the toxin activation process (Gahan et al., 2001; Fabrick et al., 2023) [57,14] . Whole-genome and transcript-level work on field-derived pink bollworm continues to refine this picture: an in-frame 234-bp inser-tion in exon 12 of the cadherin gene PgCad 1 (the r 14 allele) was shown to delete 36 amino acids from cadherin repeat 5 without altering overall transcript abundance, conferring 237-fold resistance to Cry1A c and modest cross-resistance to Cry2A b in a recessively inherited pattern direct molecular confirmation that structural loss of a single extracellular repeat, rather than loss of the gene itself, is sufficient to defeat toxin binding (Wang et al., 2020) [58] . ABCC2 Transporters: Functional genomic studies have proven that mutations in the ATP-binding cassette transporter family C2 (ABCC2) are primary drivers of field-evolved Cry1A c resistance across multiple genera, including Pectinophora, Helicoverpa and Heliothis (Wang et al., 2020) [58] . These mutations typically introduce premature stop codons or structural misfoldings that eliminate the extracellular loops where the toxin anchors (Fabrick et al., 2023) [14] .

Distinct pathways in Cry2A b and Vip3A resistance

Crucially, Cry2 and Vip3 proteins do not share the same midgut binding sites as Cry1 proteins, which forms the logical foundation of gene pyramiding (Chakroun et al., 2016) [59] . ABCA2 Transporters: Resistance to Cry2A b is strongly linked to mutations in the ABCA2 transporter rather than ABCC2 (Fabrick et al., 2015) [60] . Field-selected strains of Cry2A b -resistant pink bollworm in India share nearly identical molecular defects in the ABCA2 gene as laboratory-selected strains in the US, highlighting a globally conserved evolutionary path to resistance (Fabrick et al., 2023) [14] . Vip3A Binding: While widespread practical resistance to Vip3A in the field remains comparatively low (Welch et al., 2021; Fabrick et al., 2023) [49,14] , the genetic and biochemical basis of emerging Vip3A a resistance is now being resolved in unusually fine detail in H. zea, the pest for which this trait carries the greatest present-day stakes. Reduced binding of Vip3A a to midgut receptors, together with altered proteolytic processing of the toxin, was identified as a resistance mechanism distinct from the cadherin-and ABC-transportermediated pathways that dominate Cry resistance, confirming that Vip3A truly acts through an independent route at the receptor level (Kerns et al., 2023) [15] . Screening of five independent field-derived H. zea strains further showed that Vip3A a resistance is controlled by a single, recessive, autosomal gene in each strain, but that at least three distinct genetic loci can underlie this phenotype across populations evidence that resistance is arising through parallel, independent mutational routes rather than spreading from one source, which complicates diagnostic monitoring and management (Yang et al., 2024) [16] . Consistent with this, a polygenic-resolution analysis of wild H. zea populations found signatures of ongoing selection at multiple genomic regions associated with Cry resistance, suggesting that resistance evolution in this pest is more genetically complex than the sin-gle-locus recessive model that historically described pink bollworm (Taylor et al., 2024) [61] . A complementary genetic mapping study likewise identified a resistance locus for Cry1A c in H. zea that does not correspond to the classical cadherin or ABC-transporter genes implicated in other lepidopterans, reinforcing that this species may be assembling resistance through a broader and less predictable genetic toolkit (Benowitz et al., 2022) [62] . Together, these findings indicate that early-warning resistance already documented in H. zea (Yang et al., 2021) [17] is mechanistically heterogeneous, and that pyramids relying on Vip3A as a fail-safe against Cry-resistant genotypes should not be assumed to retain a single, stable point of vulnerability.

Environmental and agronomic drivers of resistance

While insect genetics dictate how resistance occurs, environmental and plant-physiochemical factors dictate how fast it spreads (Tabashnik and Carriere, 2020). A core assumption of the highdose/refuge strategy is that the transgenic plant must express a consistently high concentration of the toxin throughout its lifecycle to guarantee the death of heterozygous (R S

) individuals (Onstad, 2014) [22] . Field observations demonstrate that this assumption is frequently violated by abiotic stress (Razzaq et al., 2023) [63] .

Temporal fluctuations and phenological declines

Bt toxin expression is highly dynamic and correlates strongly with the physiological age of the cotton plant (Kranthi et al., 2005) [13] . Toxin concentrations are highest during the early vegetative stages but experience a profound, natural decline during late-stage flowering and boll development (Holman et al., 2026) [64] . This drop in expression creates a dangerous "sub-lethal dose window" late in the season. During this phase, the toxin concentration falls to a level where it fails to kill heterozygous (R S

) larvae, allowing individuals with single copies of a resistance allele to survive, reproduce and rapidly increase the frequency of resistance alleles within the local gene pool (Tabashnik and Carriere, 2020).

Abiotic stress-induced gene silencing

Extreme climatic events severely compromise the insecticidal efficacy of Bt cotton (Razzaq et al., 2023) [63] . Under prolonged environmental stressors such as high-temperature spikes (greater than 40 °C), severe drought or waterlogging, the transgenic plant's metabolic activity shifts toward survival, frequently leading to a down-regulation of the CaMV-35S promoter driving the Bt gene (Razzaq et al., 2023) [63] . This reduction in protein synthesis results in sharp drops in Cry1A c and Cry2A b concentrations within key reproductive structures such as squares and bolls, leaving them vulnerable to late-season pest complexes.

Tissue-specific heterogeneity

Bt proteins are not distributed evenly throughout the plant (Holman et al., 2026) [64] . While young terminal leaves express robust levels of toxins, reproductive tissues particularly the boll endocarp, petals and square bracts often express substantially lower baseline concentrations (Holman et al., 2026) [64] . This spatial heterogeneity allows opportunistic pests such as S. litura or H. armigera to feed on microhabitats within a single Bt plant where the toxin dose is sub-lethal, effectively providing them with a "built-in" spatial refuge that inadvertently screens for tolerant phenotypes (Kranthi et al., 2005) [13] .

Next-generation insect resistance management (IRM)

To safeguard the future of transgenic cotton and prevent a widespread resurgence of synthetic chemical pesticide use, IRM frameworks must evolve beyond simple, single-toxin paradigms (Tabashnik et al., 2013) [10] .

Evolution of the refuge strategy: from structured to seedblends

The traditional structured refuge model, which requires farmers to plant a clean, distinct block of non-Bt cotton (usually 5% to 20% of the total area), has failed in smallholder-dominated regions due to socio-economic constraints and low compliance (Sisterson et al., 2005) [

. To overcome this human variable, the seed-blend strategy also known as Refuge-in-a-Bag (RIB) has been widely deployed in industrialized nations and is under serious consideration for developing markets (Ives et al., 2011; Onstad, 2014) [18,22] . By mechanically mixing a designated percentage (e.g., 5% to 10%) of non-Bt cotton seed directly into the commercial bag, compliance becomes 100 per cent mandatory and automated (Ives et al., 2011) [18] . Every field automatically contains a random, scattered distribution of susceptible host plants, ensuring the survival of susceptible alleles without requiring conscious field modifications by the grower (Tabashnik et al., 2010) [46] . India mandated a full transition from structured refuges to RIB from the 2020 season, with commercial Bt seed packets required to contain 5-10% non-Bt refuge seed blended directly into each 475 g bag (Mohan, 2020) [65] . However, the durability of this fix depends entirely on seed-industry stewardship: an ICAR-Central Institute for Cotton Research assessment of commercial RIB packets found irregularities including admixture of Bt seed into the refuge fraction, poor germination of the non-Bt component, and asynchronous flowering between the refuge and the main Bt crop problems that, if uncorrected, would functionally recreate the same non-compliance that undermined the structured refuge it replaced (Mohan, 2020) [65] .

Multi-toxin pyramiding and novel proteins

The industry must continue transitioning away from dual-toxin models toward multi-toxin pyramids that integrate Vip3A alongside distinct Cry proteins (Santiago et al., 2023) [50] . Because Vip3A shares no structural homology or midgut binding sites with Cry toxins, the probability of an insect simultaneously mutating two or three independent receptor pathways is mathematically very low, provided that resistance to the companion Cry toxins is not already advanced in the target population (Chakroun et al., 2016) [59] . However, as the recent identification of multiple independent genetic routes to Vip3A a resistance in H. zea (Yang et al., 2024) [16] means pyramid durability can no longer be treated as guaranteed by biochemical independence alone; sustained baseline monitoring of Vip3A susceptibility is now essential wherever this trait is deployed.

Emergence of RNA interference (RNAi) and next-gen biotech

As resistance to standard crystalline proteins evolves, the integration of RNA interference (RNAi) traits represents the next major technological leap (Razzaq et al., 2023) [63] . Pyramiding a traditional Bt gene with a plant-mediated dsRNA construct targeting vital insect genes (such as v-ATPase or ribosomal proteins) introduces a completely unique, non-protein mode of action. Larvae feeding on these plants ingest the dsRNA, triggering gene silencing within their cells, bypassing midgut membrane receptor mutations entirely (Razzaq et al., 2023) [63] . Proof-of-concept work in cotton itself supports this approach: transgenic cotton engineered to express dsRNA against juvenile-hormonerelated genes (HaJHAMT and HaJHBP) in H. armigera was effective against Bt-resistant larvae and computer simulations parameterized on northern Chinese cropping conditions where millions of farmers grow Bt cotton alongside abundant non-transgenic host plants showed that pyramiding a Bt toxin with an independent RNAi trait substantially delayed the spread of resistance relative to Bt cotton deployed alone (Ni et al., 2017) [19] . More broadly, dsRNA-based biopesticide formulations are progressing rapidly toward field and commercial use across insect pest systems, though recent resistance-management analyses caution that dsRNA resistance can itself evolve relatively quickly under intense, sustained selection, primarily through reduced cellular uptake of the dsRNA molecule rather than target-gene mutation underscoring that RNAi should be deployed as one pillar of a pyramided strategy rather than as a stand-alone replacement for Bt (Qi et al., 2024) [20] .

Ecologically based IPM and sucking pest vacuum management

Because Bt toxins are strictly lepidopteran-specific, their deployment has inadvertently created an ecological vacuum in cotton ecosystems (Lu et al., 2012) [6] . Secondary, non-target sucking pests such as the cotton jassid (Amrasca biguttula biguttula Ishida) and whiteflies (Bemisia tabaci Gennadius) have emerged as primary threats, necessitating chemical intervention (Nyesse et al., 2026) [66] . To prevent these secondary sprays from destroying the natural biological control agents that suppress target lepidopterans, modern IPM must incorporate highly selective, narrow-spectrum insecticides (e.g., flonicamid, afidopyropen) alongside behavioural disruptions such as pheromone trapping (Nyesse et al., 2026) [66] . Maintaining robust populations of natural predators is a critical, overlooked line of defence that eliminates rare Bt-resistant larvae surviving on transgenic plants (Lu et al., 2012) [6] . The scale of this secondary-pest pressure is illustrated by whitefly dynamics in north India, where a severe Bemisia tabaci outbreak in 2015 was followed by an even more prolonged infestation in 2022 that exceeded the economic threshold level for more than ten consecutive weeks across surveyed districts of Punjab, Haryana and Rajasthan; the analysis attributed the resurgence to a combination of susceptible cotton genotypes, delayed sowing, sub-optimal insecticide use and favourable weather rather than to Bt trait failure per se, but the episode underscores that whitefly and the cotton leaf curl virus it transmits now rival lepidopteran resistance as a threat to Bt cotton profitability in this region (Kumar et al., 2024) [67] .

Gaps in current knowledge and future directions

Despite extensive research into transgenic crop durability, several critical blind spots remain that require urgent attention from academic and industrial researchers: 1. Vip3A Baseline Susceptibility Mapping and Mechanistic Diversity: With the rapid adoption of Bollgard 3 and equivalent traits, global baseline susceptibility monitoring for Vip3A remains uneven (Welch et al., 2021) [49] . Because Vip3A a resistance in Helicoverpa zea already appears to arise from at least three distinct genetic loci across independent field populations (Yang et al., 2024) [16] . 2. Climate Change Interactions: Quantitative models mapping the precise impact of rising ambient temperatures and carbon dioxide levels on the tissue-specific expression of pyramided Bt traits remain scarce (Razzaq et al., 2023) [63] . Understanding these interactions is vital for predicting future pest outbreaks (Holman et al., 2026) [64] . 3. Smallholder-Compatible RIB Models: Research must focus on the agronomic viability of seed-blend strategies in smallholder settings, particularly regarding the risk of larval movement between adjacent Bt and non-Bt plants, which can unintentionally accelerate resistance development (Ives et al., 2011) [18] . 4. RNAi Resistance Risk Assessment: As plant-mediated RNAi traits move toward commercial pyramiding with Bt in cotton, field-realistic resistance-risk data are still limited. Laboratory selection experiments in other cropping systems show that dsRNA resistance, driven mainly by reduced cellular uptake, can evolve within relatively few generations under intense selection (Qi et al., 2024) [20] ; equivalent data for lepidopteran cotton pests under realistic field exposure regimes are needed before RNAi can be relied upon as a durable independent mode of action.

Cross-Species Refuge Adequacy:

China's natural-refuge model is well validated for pink bollworm but appears insufficient on its own for Helicoverpa armigera (Zhang et al., 2019) [44] ; multi-species resistance-monitoring frameworks that do not assume uniform refuge adequacy across the pest complex are needed. 6. RIB Seed-Quality Assurance: Where refuge-in-a-bag has replaced structured refuges, resistance-management outcomes now depend on seed-industry quality control (blend accuracy, non-Bt seed germination, and flowering synchrony) as much as on the underlying genetics; routine, independent auditing of commercial RIB packets should be considered a core component of IRM rather than a one-off compliance check (Mohan, 2020) [65] .

Conclusions

The current status of insect pest resistance in Bt cotton serves as a powerful reminder of evolutionary resilience. The divergence between India's practical resistance disasters and the sustained management successes in China and the United States demonstrates that the durability of biotechnology is fundamentally dependent on human management. Single-toxin models are thoroughly obsolete and dualtoxin systems are under severe pressure. The emerging, mechanistically diverse resistance to Vip3A a now documented in H. zea shows that even the newest single-protein technologies are not exempt from this trajectory. Sustaining the immense socio-economic benefits of Bt cotton requires a rapid, global transition toward three-protein pyramids, automated refuge mechanisms such as seed-blends, and the proactive, carefully risk-assessed incorporation of alternative modes of action such as RNAi. Ultimately, biotechnology must not be viewed as a stand-alone solution, but rather as a highly dynamic, evolving component embedded within a broader, ecologically intensive Integrated Pest Management framework.

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