Omics approaches to unravel insecticide resistance mechanism in Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae)

Introduction

Bemisia tabaci (Gennadius) (BtWf) or silverleaf whitefly is globally known as one of the most devastating pests due to their invasive nature. Initially, BtWf was identified to originate from the subtropical regions and tend to inhabit a temperate environment (Perring et al., 2018). Subsequently, this species has been distributed worldwide because of its polyphagous diet and rapid breeding (Abubakar et al., 2022; Ghahari et al., 2009). Among 40 morphologically indistinguishable cryptic species of BtWf, Middle East-Asia Minor one species (Biotype B or MEAM1), and Mediterranean species (Biotype Q or MED) are the most invasive and prominent genetic groups due to their global invasion (Abubakar et al., 2022; Horowitz et al., 2020; Li et al., 2021; Wang et al., 2011; Watanabe et al., 2019). Whiteflies use their unique modified mouthparts to pierce (Fig. 1) and suck the sap from plant foliage (Onstad & Knolhoff, 2022). Such feeding behavior leads to honeydew accumulation on the foliage surface, inviting mold growth and thereby affecting the plant’s photosynthetic efficiency (Farina et al., 2022; Perring et al., 2018). This damage gradually weakens infected plants, reducing their yield and eventually killing the plants (Farina et al., 2022; Perring et al., 2018). Furthermore, according to Sani et al. (2020), BtWf can vector and transmit more than 457 viruses, earning it the title of “super-vector” (Ghosh, 2021; Gilbertson et al., 2015). Begomovirus has been the most actively transmitted by BtWf, causing 20% to 100% crop yield losses amounting to billions of USD (Gangwar & Gangwar, 2018; Sani et al., 2020). Consequently, the BtWf invasion could threaten global farming and agricultural industries, particularly for Solanaceae species such as chilies and tomatoes (Farina et al., 2022; McKenzie et al., 2014).

Even though several alternatives have been introduced in pest management, such as biological control measures, the use of chemical insecticides remains the most efficient and cost-effective method (Guo et al., 2017; Siddiqui et al., 2023). However, BtWf has evolved resistance to a wide spectrum of insecticides because of overused and prolonged insecticide exposure (Horowitz et al., 2020; Siddiqui et al., 2023). During the late 1970s and early 1980s, insecticides such as pyrethroids have been increasingly used to replace organophosphates and organochlorines due to the BtWf resistance (Head & Savinelli, 2008). In response, neonicotinoids such as thiamethoxam and imidacloprid, a new class of insecticide that targets the insect nervous system (Naveen et al., 2017) was introduced in the late 1990s to further curb the pest infestation (Fig. 2).

Unfortunately, the Bemisia family has been reported to be resistant in approximately 650 cases which involve over 60 different active components, including thiamethoxam and imidacloprid (Horowitz et al., 2020; Naveen et al., 2017), and this progressive insecticide resistance in BtWf has raised concern worldwide. According to Khan et al. (2020), insects such as BtWf can resist at the metabolic level (metabolic resistance) by expressing enzymes to break down and metabolize insecticides before reaching or binding to the target molecular site. These enzymes are usually involved in the detoxification of xenobiotics, which are comprised of cytochrome P450s (CYP), carboxylesterases (COE), glutathione S-transferases (GST), and ABC transporters (ABC) (Chen et al., 2016; Wang et al., 2023; Yang et al., 2013a). Another known resistance mechanism is called target-site resistance caused by modified (mutated) insecticide binding sites in the host organism (Horowitz et al., 2020). These two resistance mechanisms suggest that regulation at the molecular level is key to unraveling the underlying BtWf resistance to develop new effective eradication strategies in the future.

High-throughput systems biology approaches such as genomics, transcriptomics, proteomics, and metabolomics offer systems-level, wholistic molecular insights into any biological entity including BtWf and its response to insecticides (Friboulet & Thomas, 2005; Ullah et al., 2022). However, there are yet no recent reviews on this topic and therefore we have gathered studies reported in the last 12 years (up to 2023) to shed some light on the applications of omics in understanding BtWf resistance to insecticide (Table 1). Therefore, this review aims to elucidate the mechanisms behind BtWf insecticide resistance, focusing on their detoxification system as revealed by recent multi-omics analyses. Furthermore, we detail the stages of the insect resistance mechanisms and describe current trend strategies such as RNAi-and CRISPR-based gene editing to combat BtWf.

Survey methodology

We compiled scholarly articles from a diverse array of sources, encompassing esteemed databases such as Web of Science, Scopus, and PubMed. This process involved executing meticulous searches employing specific keywords and combinations, comprised of (“Bemisia tabaci” OR “B. tabaci”) AND (insecticide OR pesticide OR resistance) AND (omic* OR CRISPR OR RNAi) within the search field. Furthermore, our selection criteria were stringent and focused on recent publications spanning the period from 2011 to 2023. Figure 3 displayed a PRISMA model outlining the criteria for the inclusion and exclusion of research articles in the review focused on exploring the applications of multi-omics platforms in comprehending BtWf insecticide resistance. These chosen articles were aimed to furnish current emerging research trends in the application of multi-omics technology to unveil insecticide resistance in BtWf and the contemporary strategies deployed to manage their infestations. Therefore, this comprehensive review could cater the individuals who are interested in agriculture, pest management strategies, sustainable crop protection, and actively engaged in insecticide resistance mechanism research, particularly in BtWf.

Mechanism of insecticide resistance in BtWf

The detoxification systems are important aspects of the metabolic resistance mechanism in insects during invasion (Khan et al., 2020). According to Gao et al. (2022) and Yang et al. (2013b), detoxification systems in insects can be classified into three phases and each of these phases consists of a superfamily of enzymes/proteins (Fig. 4).

Phase I involves detoxification enzymes, including cytochrome P450 monooxygenase (CYP) and carboxylesterases (COE) (Lin, Yang & Yao, 2022). These enzymes work to mitigate the toxicity of a wide range of xenobiotics by reducing the biological activity of various endogenous and exogenous toxic substances through processes like oxidation, hydrolysis, and reduction (Abubakar et al., 2022; Eakteiman et al., 2018; Lin, Yang & Yao, 2022; Yang et al., 2013b) (Table 1). CYP is a heme-containing monooxygenases superfamily known for their versatility and capability to perform mixed-function oxidation, enabling the enzyme to insert an oxygen atom into the unreactive C–H bonds of organic molecules, while reducing another oxygen atom to water (Chiliza, Martínez-Oyanedel & Syed, 2020; Lu, Song & Zeng, 2021; Podgorski et al., 2022; Yadav et al., 2023). This capability allows CYPs to play a critical role in breaking down endogenous and exogenous compounds, including defense against synthetic insecticides, due to their genetic diversity and broad substrate specificity (Feyereisen, 2006; Lu, Song & Zeng, 2021; Nauen et al., 2022; Yang et al., 2013a). Meanwhile, COE has the advantage to hydrolyze the ester bond, that leads to the disruption of a significant bond that preserve and maintain the structure of certain insecticides, such as pyrethroids, organophosphates, carbamates, and even neonicotinoids (Khan et al., 2020). Therefore, both of the enzyme families were frequently targeted and constitutively overexpressed to justify their contribution to detoxifying xenobiotics and conferring insecticide resistance (Lu, Song & Zeng, 2021; Xia et al., 2019).

Phase II reactions involve the process of conjugation of xenobiotics and Phase I products into hydrophilic compounds, which are more water-soluble substances such as sugars, sulphates, phosphates, amino acids, or glutathione (Koirala, Moural & Zhu, 2022; Yu, 2008). This conjugated product exhibits high polarity compared to its precursor compound, which makes it less toxic and easily excreted from insects (Lin, Yang & Yao, 2022; Yang et al., 2013b; Yu, 2008). UDP-glucuronyl transferases (UGTs) and glutathione transferases (GSTs) are the enzyme superfamilies involved in these reactions (Koirala, Moural & Zhu, 2022; Yang et al., 2013b). For instance, UGTs enhance the water solubility of xenobiotics and insecticides by catalysing the attachment of glycosyl groups from activated sugar donors to a broad range of hydrophobic toxic substances (Li et al., 2018). Whilst GSTs catalyse the bonding of reduced glutathione (GSH) to electrophilic toxic substances produced during Phase I reactions (Meng et al., 2022). Furthermore, the GSTs are not only able to metabolize toxic substances but also help to eliminate oxidative free radicals from insecticide exposure (He et al., 2018; Meng et al., 2022; Wang et al., 2022). Evidently, several studies have shown that Phase II enzymes can conjugate major neonicotinoid insecticides such as thiamethoxam and imidacloprid in BtWf (Wang et al., 2021; Yang et al., 2013b) (Table 1).

Then, the detoxification process advances to Phase III, the final phase, which involves the elimination or removal of conjugated xenobiotics and metabolites from the cell (Yang et al., 2013b). While some metabolites may have been made less toxic through the reactions in Phases I or II, they can also be directly excreted without undergoing further chemical alteration (Hilliou et al., 2021). This phase predominantly relies on ATP-binding cassette (ABC) and other transmembrane transporters. ABC transporters are proteins that have the function of transporting various types of substances, such as amino acids, peptides, sugars, and hydrophobic compounds across the membrane (Campbell et al., 2023). These ABC proteins are active transporters that rely on ATP for transporting substrate across the lipid membranes (Amezian, Nauen & Van Leeuwen, 2024; Denecke et al., 2021). The superfamily of ABC transporters in insects can be categorized into eight subfamilies (ABCA to ABCH) with diverse ATP-binding sites (Merzendorfer, 2014; Wu et al., 2019). Several members of the insect ABC transporter superfamily (ABCA, ABCB, ABCC, and ABCG) significantly contribute to insecticide resistance by actively preventing the accumulation of insecticides and xenobiotics within their cells (Denecke et al., 2021; Wu et al., 2019).

Omics analyses reveal key regulatory mechanisms of BtWf resistance

Strategies in combatting BtWf

The application of insecticides does not appear to be a perfect solution for eradicating BtWf infestation as previous studies have shown its ability to resist insecticides. Auspiciously, multi-omics analysis provides a new paradigm to understand the mode of action for this resistance in the bigger picture. Henceforth, one of the proposed strategies to manage the BtWf infestation is by inhibiting their ability to detoxify and neutralize the xenobiotics. The multi-omics analyses have identified several highly expressed genes and proteins responsible for detoxification activity against various insecticides, such as abamectin, cyantraniliprole, pymetrozine, imidacloprid, chlorpyrifos, bifenthrin, and thiamethoxam (Guo et al., 2020a; Wang et al., 2020a; Yang et al., 2013c).

Initially, gene silencing systems were used as a tool to functionally determine and validate heterologous expressions and correlations of multi-omics hypothesized findings of detoxifying genes and enzymes from resistance BtWf (Homem & Davies, 2018). But as the gene silencing technologies evolved, advanced molecular editing procedures such as RNA interference-(RNAi) and CRISPR-(Clustered Regularly Interspaced Short Palindromic Repeats) based gene editing played a critical and beneficial role in knocking down the genes involved in xenobiotics detoxification and resistance to control such pests (He et al., 2019, 2018; Seman-Kamarulzaman et al., 2023; Suhag et al., 2021; Zahoor et al., 2021).

The success of the RNAi technique relies on delivering dsRNA into insects, which can be accomplished through methods such as microinjection, oral feeding, egg soaking, transfection, topical application, spraying with nanoparticles, leaf-mediated feeding, or using transgenic plants (Karthigai Devi, Banta & Jindal, 2023). For instance, Upadhyay et al. (2011) showed that RNAi is feasible to be applied in BtWf by feeding the pest with diets containing synthesized dsRNA and siRNA. As a result, they hypothesized that plant-mediated RNAi technology might be beneficial for controlling phloem-feeding pests, especially BtWf. Then, Eakteiman et al. (2018) effectively implemented plant-mediated RNAi on BtWf by downregulating the GST gene (BtGSTs5). These reports show that pathways that are heavily engaged in the breakdown of endogenous and foreign substances could be disrupted and suppressed for the BtWf control.

Nonetheless, delivering an effective lethal dose of dsRNA is a challenge for this strategy, which is due to the instability of unprotected dsRNA and the restricted dsRNA movement throughout the plants especially at the field level (Suhag et al., 2021). Thus, Jain et al. (2022) have improved the foliar spray-induced gene silencing (SIGS) approach by encapsulating dsRNA within a layered double hydroxide (LDH), known as BioClay. This innovative approach provides remarkable stability in environmental conditions and proves to be highly effective in selectively triggering RNAi to disrupt the entire life cycle of BtWf.

Apart from RNAi, CRISPR technology also has emerged to be a potent solution for overcoming the challenges posed by insecticide resistance among pests. By incorporating findings from multi-omics analysis, CRISPR has the potential to enable precise and targeted modifications of the resistance-associated genes, with the prospect of reinstating susceptibility to insecticides among pests (Zahoor et al., 2021). However, the use of this gene-editing technique is still relatively new and continues to undergo development and testing, particularly in the context of BtWf insecticide resistance analysis.

In regards to BtWf, CRISPR genome editing has proven valuable in identifying a novel mutation in the acetyl-CoA carboxylase (ACC) gene that is linked with a high level of resistance to keto-enol insecticides (Lueke et al., 2020). Ketoenols function as insect growth regulators by targeting the ACC, which is encoded by a single gene in insects, and catalyse the first crucial step in fatty acid biosynthesis (Guest, Kriek & Flemming, 2020). The mutation (A2083V) was identified by utilizing five BtWf strains with varying resistance profiles and geographical locations, which subsequently validated via CRISPR-Cas 9 genome editing in Drosophila melanogaster. The substitution of alanine with valine at position 2,083 alters the enzyme’s function, contributing to the insecticide resistance phenotype (Lueke et al., 2020). Unfortunately, the genome editing validation was not performed directly in BtWf. One of the primary challenges in implementing gene editing in BtWf lies in the small size of their embryos, which makes direct injection challenging and often results in high mortality post-injection (Heu et al., 2020). Therefore, Heu et al. (2020) addressed this challenge by developing a CRISPR-Cas9 gene editing technique known as “ReMOT Control”. This procedure involves the injection of a CRISPR-Cas9 ribonucleoprotein complex directly into the ovaries of vitellogenic adult females, rather than into embryos. This approach was proven to be efficient and results in heritable gene editing in the offspring.

Additionally, CRISPR technology has advanced significantly in the model insect Drosophila melanogaster, enabling precise genome manipulation. For instance, Fusetto et al. (2017) successfully used CRISPR to knock down CYP6G1, a key enzyme in metabolizing xenobiotics like imidacloprid. Kaduskar et al. (2022) employed CRISPR to compare insecticide susceptibility by knocking down resistant (kdr) alleles in the voltage-gated sodium channels (VGSC) in D. melanogaster. Their study found that L1014F mutants were highly resistant to permethrin but susceptible to deltamethrin, I1011M mutants had moderate resistance to DDT and permethrin, and I1011V mutants remained susceptible to low concentrations of all insecticides. Kaduskar et al. (2022) also developed an allelic-drive system that reversed insecticide resistance linked to the L1014F mutation, restoring susceptibility over generations. This highlights the potential of allelic-drive technology to combat insecticide resistance. Beyond D. melanogaster, CRISPR has also been applied to other pest species like Spodoptera, Helicoverpa, Tribolium, and Agrotis (Moon et al., 2022).

Henceforth, this breakthrough opens the door to a deeper understanding of BtWf biology and offers the potential for novel solutions to insecticide resistance mechanisms. The ability to silence and modify the genome of BtWf provides means to identify genetic targets for the development of more efficient and targeted control strategies. Ultimately, this advancement may lead to more sustainable approaches for managing BtWf populations.

Conclusion and future prospects

This review details the use of systems biology omics platforms to elucidate the insecticide resistance mechanism in BtWf. Genomics and transcriptomics have proven to be the most on-demand platforms in current research due to their comprehensive, extensive data coverage and in-depth downstream analysis capabilities, particularly in the context of insecticide resistance. Despite being underutilized, proteomics and metabolomics would be advantageous and could offer crucial insights and validations into the enzymes and metabolic pathways that were closely associated with insecticide resistance in the future. Nonetheless, these omics studies highlight the key elements (genes, proteins and metabolites) of insect detoxification system, starting with Phase I detoxification of xenobiotics involving CYPs and COEs, followed by Phase II conjugation by UGTs and GSTs, and concluding with Phase III excretion of processed xenobiotics via ABC transporters. Furthermore, this integrated approach underscores the potential to discover novel insecticide resistance mechanisms beyond the detoxification system. They can uncover previously overlooked factors contributing to insecticide resistance in BtWf, such as target site resistance (including mutated genes or proteins) and inhibitors (metabolites). Accordingly, this lays the foundation for future research to pinpoint specific key elements for targeted analyses, such as RNAi and CRISPR genome editing, aimed at silencing or disrupting detoxification genes and critical enzymes involved in resistance mechanisms. Therefore, focusing on these strategies holds promising potential for managing BtWf infestations effectively.