Invasive insects cost the global economy around USD 70 billion per year. Moreover, increasing agricultural insect pests raise concerns about global food security constraining and infestation rising after climate changes. Current agricultural pest management largely relies on plant breeding—with or without transgenes—and chemical pesticides. Both approaches face serious technological obsolescence in the field due to plant resistance breakdown or development of insecticide resistance. The need for new modes of action (MoA) for managing crop health is growing each year, driven by market demands to reduce economic losses and by consumer demand for phytosanitary measures. The disabling of pest genes through sequence-specific expression silencing is a promising tool in the development of environmentally-friendly and safe biopesticides. The specificity conferred by long dsRNA-base solutions helps minimize effects on off-target genes in the insect pest genome and the target gene in non-target organisms (NTOs). In this review, we summarize the status of gene silencing by RNA interference (RNAi) for agricultural control. More specifically, we focus on the engineering, development and application of gene silencing to control Lepidoptera through non-transforming dsRNA technologies. Despite some delivery and stability drawbacks of topical applications, we reviewed works showing convincing proof-of-concept results that point to innovative solutions. Considerations about the regulation of the ongoing research on dsRNA-based pesticides to produce commercialized products for exogenous application are discussed. Academic and industry initiatives have revealed a worthy effort to control Lepidoptera pests with this new mode of action, which provides more sustainable and reliable technologies for field management. New data on the genomics of this taxon may contribute to a future customized target gene portfolio. As a case study, we illustrate how dsRNA and associated methodologies could be applied to control an important lepidopteran coffee pest.

Chemical control methods to prevent crop damage have long been directly implicated in the selection of lepidoptera insect populations resistant to insecticides. More recently, new products featuring different modes of action (MoA), developed to mitigate the negative effects of control management on both producers and the environment, are rapidly losing efficacy due to the emergence of resistant insects. Among these, certain resistances are associated with molecular changes in the genomes of pest insects that are valuable for developing molecular markers for diagnostic tools, particularly the point mutations. Molecular diagnosis represents an innovative solution for insecticide resistance management (IRM) practices, allowing for the effective monitoring of insecticide resistance. This approach facilitates decision making by enabling the timely alternation between different modes of action (MoAs). In this context, this review focuses on the major lepidopteran pests that affect globally significant crops, discussing the impacts of insecticide resistance. It gathers literature on diagnostic methods; provides a comparative overview of the advantages of different techniques in terms of efficiency, cost, precision, sensitivity, and applicability; and highlights several novel diagnostic tools. Additionally, this review explores the coffee leaf miner, Leucoptera coffeella, as an applied model to illustrate potential approaches for more effective and sustainable control strategies.

Global crop yields are estimated to be reduced by 30–40% per year on account of plant pests and pathogens. Agricultural insect pests raise concerns about constraining global food security and climate changes contributing to the rise of infestation. The current management relies on plant breeding, associated or not with transgenes and chemical pesticides. Both approaches face serious technology obsolescence on the field due to resistance breakdown or development of insecticide resistance. The need for new Modes of Action (MoA) approaches in managing crop health grows each year, driven by market demands to reduce economic losses and phytosanitary requirements to meet the consumer perception. Disabling pest genes by sequence-specific expression silencing is considered a promising tool in the development of environment and health respectful biopesticides. The specificity conferred by long dsRNA-base solutions give support to minimizing effects on off-targeted genes in the insect pest genome and the target gene in non-target organisms (NTOs). In this review, we summarize the current status of gene silencing by RNA interference (RNAi) for agricultural control. More specifically, we focus on the engineering, development and application of gene silencing to control Lepidoptera by the employment of non-transforming dsRNA technologies. Despite some delivery and stability drawbacks of topical applications, we reviewed works showing convincing proof-of-concept results that point to imminent innovative solutions. Considerations about the regulamentation of the ongoing research on dsRNA-based pesticides to produce commercialized products for exogenous application are discussed. Academic and industry initiatives reveal a worthy effort to accomplish controlling Lepidoptera pests with this new mode of action to provide more sustainable and reliable technologies to field management. New data on genomics of this taxon encourage the increment of a customized target genes portfolio. As a case of study, we illustrate how dsRNA and associated methodologies could be applied to control an important Lepidopteran coffee pest.

Background, Leucoptera coffeella (Guerin-Meneville, 1842) is a moth species (Lyonetiidae, Lepidoptera) pest that causes severe losses to coffee crops. Further information about its genomic data is required to allow molecular strategies for the development of sustainable pesticides and to gain in-depth knowledge on phylogenetics. However, the closest complete genome available is within the superfamily level (Yponomeutoidea). Here we report the generation of the first long-read genome, transcriptome and proteome results of L. coffeella and the in silico analysis performed in these molecular levels to investigate genes involved in the siRNA processing. Results, PACBio and paired-end Illumina combined DNA sequencing from pupae samples resulted in more than 436 Gb subreads and 31Mb reads with N50 read length of 15,512 nt, mean read length 13.8 Kb and max read length 420.7 Kb. Additionally, 20Gb data of short DNA sequencing was combined to produce 1,984 contigs comprising 397 Mb in total. The longest and shortest scaffold sizes are 10,809,567 nt and 15,247 nt, respectively (mean size 200,178 nt). The N50 scaffold was 275,598 nt and the GC content was 36.10%. Predicted coding DNA sequences counted 39.930 gene models. Searching of 5286 BUSCO groups revealed 91.7 percent of completeness (single and duplicated genes combined) compared to lepidoptera genomes (lepidoptera_odb10). Flow cytometry showed the 1C DNA content is approximately 295 Mb. RNA-Seq from seven development stages resulted in 28294 identified transcripts. Additionally, proteomics from immature stages resulted in 2045 proteins matching the gene models. Conclusions, This first nuclear genome of the Lyonetiidae family brings valuable molecular resources to study Lepidoptera genomes. Genome, transcriptome and proteome sequencing to raise genome annotation precision may resolve uncovered taxonomic issues. In addition, these combined approaches provide insights into plant-insect interaction players, as horizontally transferred genes (HGT) and endosymbionts. Put together, the generated data enables the development of molecular tools towards sustainable biotechnology solutions for lepidopteran pest control.

There is a strong demand for sustainable, durable, safe, and specific solutions against agricultural pests due to breeding limitations and chemical control constraints. Biotechnological approaches to insect pests require phylogenetic information at the molecular level for RNAi application and other tailored strategies, such as insecticide resistance molecular markers. Sequencing technologies have advanced in resolution, speed, and cost-effectiveness to offer striking impact on genomics and transcriptomics. To provide deep and reliable data to high-throughput studies, state-of-the art technologies require high purification and integrity of the submitted samples. However, the limited number of existing protocols to obtain high mass nucleic acids from insects render difficult to obtain elevated purity and integrity standards. Species characteristics, as scales covered bodies of Lepidoptera and mining eating habits, hinder the quality of nucleic acid samples. Additionally, small size individuals are limited to yield sufficient amounts to robust base sequence coverage. The coffee leaf miner (CLM) Leucoptera coffeella is microlepdoptera insect responsible for severe leaf damage and huge yield losses to the coffee crop. Here we present simple and reproducible procedures we used to successfully obtain the whole-genome long-read PACBIO and Illumina (DNA) and transcriptome (RNA) of L. coffeella for de novo assembly. Therefore, we developed customized protocols applicable to insects to the extraction of genomic High Molecular Weight DNA and total RNA suitable for downstream high standard sequencing applications and other sensitive molecular analyses.

Efforts 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...