AbstractEfforts to prevent losses and counterattack insect dammaging in crops by means of chemical control often contribute to the selection of resistant populations. Resistance to agricultural insecticides have been reported since the early 20th century. In the last decade, some products have been developed with the aim of mitigating the harmful effects of field management on producers and the environment. However, insecticides with different modes of action (MoA) have been reported as prone to generating field resistance and, within a few years, losing their effectiveness. The difficulties in the control management are aggravated due to the occurrence of insect’s resistance to various insecticides. Certain active substances can induce molecular modifications, including point mutations, in the genome of pest insects, are associated to resistance in field populations. The resistance mechanisms categorized by genetic mutations are particularly useful as they enable the analysis of the evolution and spread of the corresponding resistance. Additionally, it serves as a basis for the development of molecular markers that can be used for early detection of the resistance in early infestation phases. Accordingly, it allows diagnosis for preemptive decisions to alternate between products with different MoA and their application methods, contributing to practices that enhance sustainable production management initiatives. Along with the need to reduce pesticides, as ruled by international conventions, diagnostic methods for detecting insecticide resistance are increasingly in demand, especially because of the decreasing effectiveness of insecticides. Some diagnostic methods for this purpose are under development and a few already being offered as services to worldwide cultivated crops. In this review, we will focus on the main diagnostic methodologies using molecular markers, their advancements, and the outlook for the development of resistance diagnostic tools. Furthermore, we discuss the impact of insecticide resistance on coffee, a global market agricultural commodity, towards implementing effective management strategies to maintain the efficacy of chemical products and promote sustainable agricultural and pest control practices.The global view of inseticides in agricultureChemicals were historically the primary method of pest control in global agriculture and remain nowadays at this same status. The use of pesticides allows for less damage of cultivated species and, consequently, increased production. Nonetheless, the use of large quantities of these molecules, estimated at approximately 2 million tons annually [1], implies in economical drawbacks. According to data from the Food and Agriculture Organization of the United Nations (FAO) for 2021-2022, Brazil is the leading country in general pesticide use for agriculture, with 719.51 thousand tons, followed by the United States with 417.39 thousand tons and Indonesia with 283.30 thousand tons [2].Included in this account, insecticides represent a heavy burden to producers, besides having direct consequences for health and the environment. The impacts of insecticides on human health are more severe when workers are directly exposed to the products, considering that the knowledge of health risks is still insufficient for workers to change their handling practices [3]. It is proven that pesticide poisoning can cause effects on the human body such as diarrhea, headaches, dizziness, and in more severe cases, it can contribute to the development of neurological problems and carcinogenic diseases [4], [5]. Beyond human health, the environmental impacts of insecticide application are also severe, as the lack of specificity affects non-target insects that play important roles in ecosystems, such as pollinators [4] and other animals like birds and fish [6], [7]. Additionally, insecticides contaminate water [8], soil [9] and food [10], [11], causing damage to the entire trophic chain. Finally, the excessive use of insecticides can lead to the development of resistant target insect populations.The insecticide resistance problemThe first reports of insecticide resistance appeared around 1914, where resistance to sulfur was observed in scale insects. [12]. Resistance to insecticides remained at low frequency until the introduction and expansion of synthetic organic insecticides such as DDT, cyclodienes and organophosphates, between the 40s and 50s [13]. From the 60s onwards, insecticide resistance began to be considered an impact fator of the use and effectiveness of a wide range of chemical compounds. New chemicals were introduced onto the market in attempt to circumvent the situation, although, in many cases they had the same chemical class as those already existing [14]. In this way, in 1984 the Insecticide Resistance Action Committee (IRAC) was formed together with other organizations in attempt to establish adequate management of resistance. In 1998, IRAC began to develop a classification scheme for agricultural chemicals based on the so-called Modes of Action (MoA), a tool that initially classified acaricides, that is currently extended to cover biologicals and insecticides as the main basis for managing resistance [13].The most recent reports on the global use of insecticides show this ranking is led by Indonesia, the United States, China and Brazil, with 116,405 thousand tons, 72,985 thousand tons, 70,126 thousand tons and 59,587 thousand tons, respectively [2]. Consequently, known resistance in a wide variety of pest insects are closely related to the most cultivated crops in these countries. In Indonesia, the main reports of resistance are in Spodoptera frugiperda , with resistance to insecticides in the chemical groups of organophosphates, avermectins, pyrethroids, spinosyns, and diamides, and in Plutella xylostella , with resistance detected to organophosphate and pyrethroid insecticides [15]. In Brazil, there are insecticide resistance reports for many of the insects figuring on the top of the agricultural pests and diseases government list presenting high phytosanitary risk to production [16]. Among these insects, Bemisia tabaci is noted with reports of resistance to tetranortriterpenoids, diamides, carbamates, phenylthiourea, neonicotinoids, and ketoenol [17];Helicoverpa armigera and Spodoptera frugiperda , with reports of resistance to organophosphates, carbamates, pyrethroids, oxadiazines [18], [19], [20]; Tuta absoluta [21], [22], [23] and Plutella xylostella [24], [25], [26], [27] to organophosphates.The United States government also has a ranking of the pests and diseases that pose a significant threat to the country’s agriculture [28]. Among the listed insects, there are reports of insecticide resistance in Spodoptera litura , to organophosphates, carbamates, pyrethroids, and oxadiazine [29], Thaumatotibia leucotreta, resistant to benzoylureas [30], Helicoverpa armigera , resistant to pyrethroids, cyclodienes, and organophosphates [20],Tecia solanivora, resistant to carbamates and organophosphates [31] and Tuta absoluta, resistant to organophosphates, carbamates, pyrethroids, avermectins, oxadiazine, benzoylureas, and spinosyns [21].To aggravate the situation, it has been shown that insecticide resistance mechanisms are affected by climate change [32] and that these changes are related to the increased risk of pest invasion areas worldwide [33], [34], [35], [36].Therefore, the challenge of monitoring resistance is greater than just accounting reports of already installed resistance, which would allow a better control of its development and dissemination. Thus, management techniques and technologies that prioritize environmental concerns are essential for ensuring a more sustainable future, and diagnosing resistance can be a crucial action [37], [38], [39], [40].Besides the agricultural problems posed to crop pests, the consequences of the development of insecticide-resistant insect populations are also closely related to human health. Just as it is an environmental concern, resistance in non-target insect populations is also a critical issue for human health, as there are cases of resistance in insect vectors of important human diseases, such as mosquitoes that transmit dengue, zika, chikungunya, yellow fever, and malaria [41], [42], [43]. Finally, other consequences of resistance development are still uncertain, as in non-target insects, such as bees, ants, and termites [44].The main forms of insecticide resistanceIn order to perform diagnosis, the primordial question is to determine the most probable mechanism conferring the resistance to a given insect challenged with a specific insecticide. Resistance development is an evolutionary phenomenon arising from a set of dynamic biochemical actions and reactions that are triggered, for example, by the continuous use of chemical products that result in significant metabolic and genetic changes to organism populations over time as a response to selective pressures. As new MoAs and its associated technologies emerge, the path to discovery is deepened to understand the genetic changes responsible for resistance [47]. At the present, insect resistance mechanisms are categorized into different groups: cuticular resistance, behavioral, metabolic, sequestration and “target-site” mutations.Cuticular resistance occurs due to modifications in the insect’s cuticle that can prevent insecticide penetration. This mechanism involves thickening of the cuticle, which acts as a physical barrier, and changes in cuticle composition, such as increased waxes, which can reduce insecticide absorption [47], [48], [49], [50], [51]. Although this mechanism protects the insect from a wide range of insecticides, it generally confers low levels of resistance when considered in isolation [52].Behavioral resistance, often underestimated due to uncertainty about whether it should be considered a true form of resistance, is defined by the insects’ ability to avoid contact with the toxin [53], [54]. Changes in behavior, such as avoiding treated areas or altering feeding patterns, can reduce exposure to the insecticide. This mechanism of resistance has been reported for a wide range of chemical classes, including organochlorines, organophosphates, carbamates, and pyrethroids [55].Metabolic resistance can be characterized as a detoxification process, meaning the ability to metabolize and/or neutralize chemicals as a defense mechanism, present in insects and plants, through enzymes that degrade or modify toxic compounds, rendering them ineffective [56]. This process involves three main classes of enzymes: 1) Cytochrome P450 Monooxygenases (P450s), which oxidize organic compounds, making them more water-soluble and more easily excreted. 2) Esterases, which hydrolyze esters, often deactivating chemical compounds, and 3) Glutathione S-transferases (GSTs), which conjugate chemical compounds with glutathione, facilitating their excretion [57]. The genes that confer metabolic resistance are generally different between insect species, and the production of detoxification enzymes is usually caused by the amplification of these genes [47]. Because this mechanism involves a succession of reactions, metabolic resistance is influenced by various external factors, such as the type and frequency of chemical use, climatic conditions, biological factors, agricultural and management practices, and environmental residues and contamination, which can accelerate, decelerate, or modify how it develops in populations of organisms, similar to cuticular and sequestration resistance processes.Sequestration resistance is a particular case of metabolic resistance in which insects have the ability to isolate and store insecticide molecules in specific compartments within their bodies without causing harm. This mechanism involves sequestration in vacuoles or organelles and binding to transport or sequestration proteins, which bind and inactivate the insecticides, preventing them from reaching their targets [47], [50].Target-site resistance is relevant for accurately understanding the spread of resistance genes. This mechanism involves changes in the enzymes, receptors, or structural proteins of insects, where chemicals exert their toxic action, preventing the chemical from binding effectively to its target, thereby reducing or eliminating its toxicity [50]. Insect vectors of human diseases and has frequently been studied to understand resistance in agricultural pest insects [18], [57], [58], [59].The main considered mechanisms are: 1) Genetic Mutation, which occurs from mutations that cause changes in the amino acid sequence, potentially altering the structure of the protein involved in binding with insecticide molecules. These can be single nucleotide polymorphisms (SNPs), insertions, or deletions; 2) Alteration in gene expression, either by overexpressing the amount of the target protein, diluting the chemical effect, or by underexpressing the amount of the target protein, minimizing the chemical’s action sites; 3) post-transcriptional regulation of genes involved in resistance processes and its impact on these processes, with research into non-coding RNAs being the main area of development; and 4) Post-Translational Modification, through chemical modifications to the target protein (phosphorylation, methylation, glycosylation) after its synthesis, which can alter its conformation and affinity for the chemical.Genetic mutations of the kdr target-site (knockdown resistance) in the voltage-gated sodium channel (vgsc) are extensively studied and result in resistance to pyrethroids and DDT [60], [61]. Other commonly described mutations occur in the acetylcholinesterase-1 (ace-1) gene, which confer resistance to organophosphates and carbamates [62], [63], [64] and mutations in the GABA receptor, commonly known as rdl mutation (resistance to dieldrin), which are associated with resistance to several insecticide groups [64], [65], [66]. For relatively new insecticide groups, such as diamides, the mutation occurs in ryanodine receptors (RyR) [58], [67], [68] and is frequently described in lepidopterans [18], [67], [69], [70]. A list compiled in 2020 by IRAC presents the relationship between the MoA of each insecticide and the associated target-site resistance, facilitating access to references [71].Post-Translational Modification, gene expression and post-transcriptional regulation are quite puzzling to understanding in the context of the resistance mechanisms [47], [50]. Therefore, a way to shorten this path and allow a greater understanding of the evolution and dissemination of resistance is to seek more direct processes that can be detected, diagnosed, and consequently studied with greater agility.High sensitivity and viability make the target-site resistance driven by genetic mutation of special interest in developing molecular markers [72], as it is primarily characterized by mutations in the coding region of the proteins targeted by an insecticide and, therefore, results in high levels of resistance [73]. Moreover, in many cases, these mutations in the same target gene are responsible for resistance to the same insecticide in different insect species, making molecular markers an excellent monitoring tool [74]. Thus, the identification and localization of genes associated with insecticide resistance are essential for understanding resistance mechanisms and can be effectively achieved with the help of molecular markers, which adds value to resistance management strategies [75]. Therefore, we can conclude that target-site molecular markers are the most suitable for developing products for molecular diagnostics with great market potential, as they can be used to overcome the need for time-consuming bioassays and assist in decision-making in the field, although this second step remains a challenge [72].
