Since the role of botanicals (including those based on PEOs) as biopesticides has been well-established in the literature, the commencement of a new era involves newer technologies being explored, researched, and implemented. As of late, significant progress has been made in the development of novel formulations for plant-based biopesticides. With the disappearance of conventional formulations, which included liquid formulations (e.g., emulsions and suspensions) or dry formulations (e.g., dust, powders, granules, wettable powders, and water-dispersible granules), this increases the attractiveness of substituting newer technologies with biopesticides ^[1][2][3][143,144,145]. Newer biopesticides with improved stability, shelf life, and consumer safety are highly beneficial due to their competitive advantage over conventional counterparts ^[3][145]. Beyond the above sections, which extensively describe the bioactivities of PEOs, their industrial usage is limited due to constraints such as low solubility, low bioavailability, and high volatility. Therefore, to improve PEO bioactivity and stability, many researchers have suggested the encapsulation of PEOs in chemical matrices that act as carriers and facilitate the controlled release of oils. For the development of stable emulsions, nano- and microformulations based on the encapsulation of a plant’s active components onto a matrix appear to be highly promising ^[4][5][52,146].

Nanosized emulsions, being kinetically stable, are superior to microsized ones due to their low volatility, long-lasting stability, and requirement of a lower surfactant concentration, unlike microemulsions. Moreover, they have negligible flocculation and allow for the controlled release of active compounds that possess bioherbicidal, biofungicidal, and bioinsecticidal properties ^[6][7][147,148] (Table 1). Since PEOs are insoluble in water, they require large quantities of toxic solvents for their dissolution. To overcome this problem, studies advocating the use of solvent-free methods for the nanoencapsulation of PEOs are proving to be significant milestones in research ^[8][149]. Thus far, a wide range of polymeric carriers have been developed for the encapsulation of PEOs, which offer multiple benefits, most importantly the controlled release of PEOs during storage or application ^[9][150]. Encapsulated particles or composites can be prepared through emulsification, coacervation (with gelatin or gum arabic), spray drying (with maltodextrin), complexation (with cyclodextrin), ionic gelation (with chitosan), nanoprecipitation (with poly DL-lactide-co-glycolide, i.e., PLGA), and film hydration ^[10][151]. Lopes et al. ^[11][152] reported protected release and better cytotoxicity in plant extracts encapsulated with chitosan or lipid-based carriers against Sf9 insect cell lines in comparison to commercially available pesticides. Likewise, the antifungal activity of PEO from “Palmarosa” encapsulated in nanostructured lipid carriers has been reported against Aspergillus nomius Kurtzman, B.W. Horn, and Hesselt in both in vitro (mycelia growth inhibition) and in situ (against precontaminated Brazil nuts) set-ups ^[12][153]. Nanoemulsions of S. hortensis PEO were herbicidal on the selected weeds and caused membrane disruption and physiological malfunction in these plants ^[13][154]. The size and stability of nanoemulsions are largely determined by the source of the plant. For instance, nanoemulsions of PEOs from Artemisia sp. remained stable even after 28 weeks of storage when a 3:1 concentration of oil and surfactant was used ^[14][155].

Table 1 provides useful insights into the research relating to the encapsulation of PEOs in different carriers along with their mechanism of action on target cells. Regarding PEO-based biopesticides, novel formulations formed by mixing the active principles of oil with other compounds are being popularized. For instance, pure “azadirachtin” alone was less effective than the one formed in a neem oil medium ^[15][156]. Additionally, the biopesticidal properties of nanoformulations made from both pure PEOs and their constituents are quite impressive ^[8][149].

Studies on the importance of using PEOs as biopesticides are now moving at a rapid pace thanks to advances in bioinformatics. Bioinformatics tools can map the interactions of PEOs or their active constituents with their biological targets and treat multifactorial diseases, indicating the wide applications of this field in pharmacology ^[28][169]. Lately, by using different computational tools or in silico methodologies, many researchers have made significant contributions to this field. For example, Loza-Mejía et al. ^[29][170] conducted an in silico study involving docking-based virtual screening to determine the insecticidal potential of active compounds from the genus Calceolaria against insect target proteins. Likewise, the interaction of a principal compound from the wood extracts of Tabebuia heptaphylla with the target, i.e., ILE125 amino acid from A. aegypti odorant binding protein (AaegOBP1 receptor), confirmed the repellent activity of the extracts against A. aegypti ^[30][171]. The ability of active compounds from “Negramina” PEO to kill aphids was validated through their interaction with the insect transient receptor potential (TRP) channels ^[31][172], whereas the therapeutic potential of peppermint (M. piperita) against grey mold (B. cinerea) was confirmed by docking studies ^[32][173]. In another docking study, the role of insect tyramine receptors in mediating the responses of PEOs against insects was described ^[33][174]. Contrary to these studies, Sierra et al. ^[34][175] reported the detoxification response of mosquitoes against PEO constituents by molecular docking. The detoxification of a toxic xenobiotic component, p-cymene, by chemosensory proteins (CSP) present in A. aegypti larvae confirmed the role of CSPs in hampering the natural larvicidal activity of E. camaldulensis PEO ^[35][176]. Thus, the emergence of in silico studies constitutes perhaps the most significant research on biopesticides because they require fewer resources to hypothesize future physiological research and give researchers the option of conducting multiple trials to determine the appropriate interactions.

Insect transcriptome profiling has become one of the most utilized approaches in recent years for examining the molecular basis of the insecticidal activity of numerous PEOs ^[35][36][176,177]. Several molecular targets of interest for plant pathologies have been discovered through expression studies using RNA sequencing. This novel technology has completely revolutionized transcriptome analysis, allowing the quantification of gene expression and the identification of new genes at an unprecedented pace.

A comparative transcriptome analysis of Sitophilus zeamais (Mochul’skii) in response to the PEO of Melaleuca linariifolia var. alternifolia Maiden and Betche revealed that 3562 differentially expressed genes (DEGs) were found to be involved in insecticide detoxification and mitochondrial function. Of these DEGs, 2836 genes were up-regulated, whereas 726 genes were down-regulated ^[35][176]. The mapping of DEGs identified an increase in the expression of several genes, including cytochrome P450s (CYP450), glutathione S-transferases (GSTs), carboxylesterases, ATP-binding cassette transporters (ABC transporters), and those associated with respiration and metabolism of xenobiotics ^[35][176]. However, PEO treatment reduced the expression of genes encoding enzymes involved in respiration, electron flow, and energy synthesis in the mitochondria. Another study by Liao et al. ^[37][178] reported that fumigation with the PEO of M. alternifolia induced the expression of 2208 DEGs, and the NAD⁺/NADH (nicotinamide dehydrogenase) enzyme was identified as the prime target of action for oil in insects. The oil resulted in abnormal structural changes and severe damage to the insect mitochondria and directly blocked electron transport through the mitochondrial respiratory pathway, ultimately leading to insect death. Similarly, the effect of M. alternifolia PEO on the morphology and ultrastructure of mitochondria in B. cinerea has been reported to cause mitochondrial dysfunction by reducing the activities of important mitochondrial enzymes and those involved in the tricarboxylic acid (TCA) cycle ^[38][179]. The fumigation of terpinen-4-ol (a component of M. alternifolia PEO) in S. zeamais induced the up- or down-regulation of roughly 592 DEGs in insect RNA ^[39][180]. Here, several DEGs encoding detoxification enzymes were identified, including 16 CYP450s, 14 esterases (ESTs), 10 UDP-glucuronosyltransferases (UGTs), 8 GSTs, and 2 ABC transporter genes. Of particular interest was the consistent overexpression of the genes encoding P450s, GSTs, and ESTs after terpinen-4-ol exposure, which sheds light on the crucial role of these genes in the systemic metabolic responses of insects ^[39][180].

Muturi et al. ^[40][181] assessed the larvicidal activity of PEO from Commiphora erythraea (Ehrenb.) Engl. and its fractions against three mosquito species, namely, Culex restuans Theobald, C. pipiens L., and A. aegypti. Real-time PCR (polymerase chain reaction) analysis revealed that the expression of CYP450s (CYP6M11 and CYP6N12) and a GST gene (GST-2) involved in xenobiotic detoxification by mosquito larvae was significantly up-regulated by PEO treatment. The GST enzyme is also a potential molecular target of PEO from Cymbopogon citratus (DC.) Stapf in Asian long-horned ticks, Haemaphysalis longicornis Neumann, which induces the enzyme at a significantly higher rate following exposure to sublethal concentrations of the oil ^[41][182]. However, there is a paucity of information on the molecular mechanism underlying GST enzyme action, which, if available, may open new avenues for the development of effective pest management techniques for ticks and tick-borne diseases ^[41][182]. More recently, with the advent of RNA-Seq as the technology of choice for gene expression analysis, a set of genes associated with adrenergic signaling/Ca²⁺ channels, apoptosis, focal adhesion, cGMP-PKG (cyclic guanosine monophosphate-dependent protein kinase G) signaling, ECM (extracellular matrix)–receptor interaction, ubiquitin-mediated proteolysis, the mTOR (mammalian target of rapamycin) signaling pathway, and the longevity regulating pathway were identified in ticks exposed to C. citratus PEO and its constituent, citronellal ^[42][183]. Up-regulation of most of the genes involved in Ca²⁺ signaling (CACNAID, ADCY9, TPM1, and MYH6) and apoptosis (CYC, DRONC, CASP7, CASP9, BCL2L1, and BCL-xL) by PEO treatment was found to induce neurotoxicity and cytotoxicity in insects. Further, the toxicity of oil on insects was expected to be a product of complex factors, including oxidative stress due to increased ROS accumulation, reduced ATP levels, mitochondrial depolarization, increased intramitochondrial free Ca²⁺, and either necrotic or apoptotic death induction in ticks ^[42][183]. The activation of CYP450 genes by several terpenoids found in PEOs has also been predicted by RNA-Seq ^[35][43][176,184].

Two potentially relevant genes are cathepsin and lipase, both of which play significant roles in larval development and reproduction. The importance of these genes is evident in a study by Hegedus et al. ^[44][185], which reported that the down-regulation of CatB/CatB-like and CatL/CatL-precursor in crucifer root maggot (Delia radicum L.) and RNAi-mediated silencing of CatL-precursor in red flour beetle (Tribolium castaneum Herbst) negatively affected mid-gut metamorphosis, tissue remolding, and larval fat body decomposition in the former, while causing 100% mortality in the latter. Further, through a complete transcriptome analysis of T. castaneum, Gao et al. ^[36][177] reported that the PEO of Artemisia vulgaris L. exerted its insecticidal activity by increasing the expression of genes encoding antioxidant enzymes, copper-zinc-superoxide dismutases, heme-peroxidases, and various transcription factors in beetles. From these studies, many gene-specific responses pertaining to PEO treatment have been highlighted in insect species. But, to discover novel, efficient, and environmentally friendly alternatives for controlling insect pests, researchers in the future will have to focus on more transcriptomic studies to investigate the molecular targets of various PEOs in insects.