The ability to perceive and differentiate various chemical stimuli present in a set environment is paramount to an organism’s success (1–4). Insects, the most successful group of animals on Earth, have developed a sophisticated olfactory system that has widely contributed to this success. Insect olfactory systems are known for their remarkable sensitivity and the ability to integrate odorant blends through distributed specificity of receptor tuning profiles (5–7). The classification and integration of these profiles in different portions of “odor space” rely on structures like the mushroom body and lateral horn of the protocerebrum, enabling precise discrimination of pheromone blends or subtle differences in plant odor blends (5, 8). Insect olfaction is composed of several transmembrane receptors and soluble and insoluble proteins, which collaborate harmoniously to receive, process, interpret, and ultimately react to external stimuli (3). The key olfactory proteins involved in this process include odorant binding proteins (OBPs), odorant receptors (ORs), ionotropic receptors (IRs), odorant degrading enzymes (ODEs). and sensory neuron membrane proteins (SNMPs) (3). ORs form a heteromeric complex with a ubiquitous coreceptor coined odorant receptor co-receptor (Orco) that is omni-present in every functional OR complex and is highly conserved among all insects (3). In general, exogenous odorants or volatiles enter the sensillum lymph through cuticular pores and are subsequently bound and solubilized by OBPs, wherein this OBP-odorant complex is transported across the sensillum to a candidate OR for transduction (3, 9) ( Figure 1 ). Once the OBP-odorant complex (or the odorant alone) is bound to a receptive OR, a transduction cascade is triggered, which leads to action potentials transmitting from olfactory receptor neurons to the higher integration centers within the protocerebrum. Odorants must be deactivated rapidly by ODEs or scavengers once this occurs, otherwise efficiency of olfactory processes will be impaired via prolonged exposure of the respective odorant inducing overstimulation. Numerous lines of evidence suggest that many ODEs such as cytochrome P450s, glutathione S-transferases (GSTs), carboxyl/cholinesterases (CCEs) are involved in degrading volatile molecules during the deactivation process (3, 10–13). Some studies indicate that prior to degradation by ODEs, pheromones undergo deactivation through their binding to OBPs (e.g., pheromone binding proteins, PBPs). Additionally, these OBPs serve as scavengers, contributing to the decline of the receptor potential after stimulus offset. This implies the existence of a broader molecular mechanism beyond enzymatic degradation (3, 14–17).

Within the realm of olfaction processing, OBPs play a vital role as the primary mediators connecting the external environment with ORs (7, 9). OBPs are frequently necessary for safeguarding exogenous hydrophobic volatiles against degradation prior to their interaction with the corresponding ORs. This protection occurs following the initial uptake, binding, and transportation of these volatiles within the aqueous sensillum lymph. The delivery of the exogenous volatiles to the OR triggers an elicited response, allowing for the recognition of volatiles from hosts or natural enemies and identification of pheromones of potential mates. Following the stimulation of ORs by exogenous molecules, OBPs may also participate as molecular traps, preventing neuron oversaturation (1–3, 17–20). In addition, evidence shows that OBPs may play essential roles in cleaning the perireceptor space from undesirable xenobiotics, including plant volatiles and pesticides. This function potentially contributes to host plant adaptation and pesticide resistance (20–27). Despite their primary role as olfactory proteins, recent research has identified OBPs to be involved in a variety of physiological roles in insects outside of olfactory tissues, owing in part to their structural variability and efficacy in the transporting, sequestering, and concealing of various hydrophobic molecules (2, 3, 9, 28–30).

Roughly half of insect species are phytophagous, forming a close relationship with the host plants they feed and interact with (31). During the coevolution of insects and plants over hundreds of millions of years, insects have evolved diverse mechanisms to adapt to numerous xenobiotics (12, 13, 32–34). Olfaction in insects may serve as an “Achilles heel” - a target for plant defense because of its remarkable sensitivity, critical importance, and vulnerability (22). OBPs serve as the primary point of contact for the insect olfactory system with xenobiotics, playing a principal role in modulating chemical communication and defense. Here, we initially summarize the classification and structural properties of OBPs in various insect orders. Then we focus on the variety of functional roles of OBPs in insect communication and adaptation to xenobiotics. Our review concludes with prospective thoughts on future studies that could expand our knowledge of OBPs and their diverse functions in chemical ecology and xenobiotic adaptation.

Insect OBPs are small water-soluble extracellular proteins, ranging from between roughly 100 to ~200 amino acid residues, with very little sequence similarity within OBPs of the same species (1). Initially described in Lepidoptera (16), these proteins were categorized into three separate subfamilies based on the amino acid sequences and differential expression patterns: pheromone binding proteins (PBPs), general odorant binding proteins (GOBPs), and antennal binding proteins (ABPs) (1, 16). However, a primary challenge with this classification methods arises from the significant variation observed in the amino acid sequences, ligand binding affinity, differential expression, and functional roles beyond Lepidoptera, extending even to functions beyond chemosensation (35, 36). Therefore, there was a pressing need for a more comprehensive and flexible classification method to accurately characterize their diverse functional roles and implications. Currently, insect OBPs are generally divided into three primary groups based on the number of conserved cysteine residues and interlocked disulfide bridges: 1) Classic OBPs (e.g. Chrysopa pallens CpalOBP4, PDB ID:6JPM), which have six conserved cysteine residues that participate in three disulfide bridges; 2) Minus-C OBPs (e.g. Apis mellifera AmelOBP14, PDB ID:3S0A), featuring four or five conserved cysteine residues and two disulfide bridges; 3) Plus-C OBPs (e.g. Anopheles gambiae AgamOBP7, PDB ID:3R1P), which possess eight or more conserved cysteine residues, four or more disulfide bridges, and a conserved proline residue ( Figure 2 ) (36). Among these groups, Classic OBPs are the most frequently identified type of OBPs in every insect genome ( Table 1 ; Figures 2 , 3 ). Phylogenic analysis of insect OBPs have shown that Classic OBPs seem to be the basal group, and other Minus-C and Plus-C groups of OBPs are subgroups of the Classic OBPs (39). This may suggest that Minus-C and Plus-C OBPs likely diverged from the Classic OBPs (39–41) ( Figure 2 ). However, the relative composition of OBPs in an insect genome can vary greatly, as some OBP groups may feature a larger expansion in one group of insects as compared to others, as has been observed in certain beetle species (35, 42–47) ( Figure 3A ; Table 1 ). There is a group of OBPs that has been termed “atypical OBPs” characterized by 10 or more conserved cysteines, a long C-terminus, a conserved proline residue, and four or more disulfide bridges, which is recorded in several mosquito and locust species, suggesting this group of genes may be recently evolved in these species (36, 48–50). Additionally, groups of insect OBPs that exist outside of the three primary structural groups can be found in certain insects, such as double domain OBPs that are found exclusively in certain wasp species (51) and Dimer OBPs that are found in some species of dipterans and lepidopterans ( Figures 3A–C ; Table 1 ) (39, 51). In certain insect groups, there is a complete absence of an entire primary group of OBPs; for instance, honey bees lack of plus-C OBPs all together ( Figure 3B ; Table 1 ) (40). The amount of OBP genes in an insect genome can vary greatly among species, ranging from as low as 7 in Ceratosolen solmsi to as high as 111 in Aedes aegypti ( Table 1 ). The reason why certain insect species possess a higher number of OBPs while others have relatively few remains unclear. However, this disparity can likely be attributed to the insects’ unique lifestyles, evolutionary processes, and wide variety of environments (39).

Despite the high diversity and variation among insect OBPs, this group of proteins has some hallmark features. In addition to the extremely conserved cysteine residues, insect OBPs typically have two to four interconnected disulfide bridges (e.g., a pattern of C1-C3, C2-C5, and C4-C6) that play a vital role in stabilizing the protein (52–58) ( Figure 2 ). Furthermore, six α-helices, which may vary in number in certain cases, synergistically work with the interlocked disulfide bridges to further enhance the protein’s stability. Specific α-helices may be involved in forming a hydrophobic cavity crucial for ligand binding activity (52, 53, 58–60). The ligand binding specificity of insect OBPs exhibits significant variation, ranging from high specificity to remarkable broadness. This diversity is influenced by the overall size and shape of the binding pocket, as well as the specific amino acids lining it (54, 55). Previous studies have demonstrated that variability in amino acid identity and length of the C-terminal region can influence ligand binding affinity. For example, in a specific case, the rearrangement of amino acids within the C-terminus region of a Drosophila melanogaster OBP (LUSH) disrupted the formation of a salt bridge, resulting in impaired binding ability to the expected ligand 11-cis vaccenyl acetate, a conspecific male sex pheromone (56). The length variation in the C-terminal region also impacts the interaction of the C-terminus with the hydrophobic binding cavity. Insect OBPs with longer C-terminus regions possess a flap that can cover the entrance of the binding cavity, whereas those with shorter C-terminus regions leave their binding cavities exposed to bulk solvent (2, 61, 62). Additional research has demonstrated that pH-induced conformational changes can impact the ligand-binding capability of specific insect OBPs (52, 63–65). Notably, Lepidopteran OBPs AtraPBP1 from Amyelois transitella and ApolPBP from Antheraea polyphemus possess a C-terminal region that plays a crucial role in pheromone binding and release, triggered by changes in pH levels (66, 67). In AtraPBP1, the polar amino acid residues Glu132 and Glu141 create two salt bridges with protonated histidine residues His 80 and His95, respectively. These two salt bridges are induced by acidic conditions, promoting the formation of a seventh helix at the C-terminal region that can compete with the ligand and trigger its release (9, 66). In contrast to OBPs in Lepidoptera, the majority of Dipteran OBPs lack a sufficiently long C-terminal region to form an additional helix able to occupy the binding cavity (9, 52). Nevertheless, many Dipteran OBPs, such as AaegOBP1 (Ae. aegypti, PDB ID:3K1E), AgamOBP1 (An. gambiae, PDB ID:2ERB), CquiOBP1 (Culex quinquefasciatus, PDB ID:3OGN), undergo pH-depended conformation changes associated with loss of binding affinity, similar to what has been observed in Lepidopteran OBPs, indicating a distinct mechanism (9). The C-terminal region of these Dipteran insect OBP proteins instead function as a “lid” over the binding cavity, a characteristic not found in other insect groups. This lid was suggested to act as a pH-sensitive hinge, moving away from the binding cavity when pH is reduced, as the OBP-odorant complex approaches the dendritic membrane (9, 52). Moreover, the ligand binding ability of an OBP may be affected by its molecular volume. For example, in the Minus-C OBP DhelOBP21 of Dastarcus helophoroides, the ligand being either too small (<100 A³) or too large (>185 A³) can disrupt its facultative binding ability (68). Additionally, hydrophobic and hydrogen bond interactions can also influence binding efficacy of an OBP, and the absence of either can lead to substantial reductions in the binding affinity of an OBP towards a ligand (68, 69). Lastly, it is worth noting that the majority of determined crystal structures of insect OBPs reveal a tendency for dimerization upon ligand binding (59, 70–73) ( Table 2 ). Insect OBP protein structures in both ligand-free apo forms and in complex with various ligands, have been determined using protein crystallography and nuclear magnetic resonance (NMR) spectroscopy (9, 74). A list of currently published insect OBP structures at the time of this publication has been provided in Table 2 . The list includes 27 individual insect OBP structures across 17 insect species, including 10 OBP structures from species in Diptera and 7 OBP structures from species in Lepidoptera. Currently, our understanding is limited, as over half of the elucidated structures (17 out of 27) come from Dipteran and Lepidopteran insects ( Table 2 ). Further research is crucial to comprehensively understand the relationship between the varied structures and functions of numerous OBPs from a wide range of insect species.

Insects encounter a diverse array of semiochemicals and xenobiotics in their environment, necessitating adaptive responses. These chemicals range from allospecific and conspecific pheromones, plant allelochemicals, volatiles, and a multitude of anthropogenic compounds, such as pesticides (34, 75–77). On one hand, insects use these chemical cues to detect their food, mates, and other substrates critical for their survival and reproduction. On the other hand, insects must evolve adaptation strategies to cope with “delicious poisons”, which are harmful compounds disguised as attractants. These chemical cues can be exploited by host plants as a defensive measure, posing survival challenges for insects (22, 78). Recent studies have demonstrated that insect OBPs play critical roles in the uptake or release of a diverse spectrum of molecules due to their stable and compact structure, high variability in binding affinity, and efficiency transportation of hydrophobic molecules (79–81). Additionally, many proteomic and transcriptomic studies focusing solely on olfactory organs, such as antennae or maxillary palps, may not identify all OBP-encoding genes within an insect genome. This suggests that certain OBPs could be exclusively expressed in non-olfactory organs and/or appendages (2, 82–85). Recently, there are many integrative reviews of insect OBPs discussing their diverse expression and functions in chemoreception and beyond (1, 2, 9, 36, 74). Therefore, in this section, our focus will be on the roles of insect OBPs in communication, host location, and their co-opted functions in pesticide adaptation.

While our understanding of insect OBPs was initially centered on olfaction, recent research conducted over the past decade has unveiled their involvement in diverse physiological processes, including communication, host location and adaptation, pesticide resistance, and reproduction. However, our comprehension of the molecular mechanisms governing OBP functions beyond olfaction remains limited due to their substantial diversity across various taxa. Recent advances in whole genomic sequences, RNA interference, gene editing, X-ray crystallography, and fluorescent competitive ligand binding assays, promise to enhance our understanding on the roles of insect OBPs towards communication and xenobiotic adaptation. This cutting-edge research will also contribute to unraveling the intricate and multifaceted mechanisms underpinning the evolutionary relationship between insects and their environment.

JA: Methodology, Visualization, Writing – original draft, Data curation, Investigation, Software. TM: Investigation, Software, Visualization, Resources, Writing – review & editing. HW: Resources, Software, Visualization, Writing – review & editing, Data curation, Methodology. FZ: Methodology, Resources, Visualization, Writing – review & editing, Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – original draft.