Papilio Xuthus Research Articles

GDnet-IP: Grouped Dropout-Based Convolutional Neural Network for Insect Pest Recognition

Lightweight convolutional neural network (CNN) models have proven effective in recognizing common pest species, yet challenges remain in enhancing their nonlinear learning capacity and reducing overfitting. This study introduces a grouped dropout strategy and modifies the CNN architecture to improve the accuracy of multi-class insect recognition. Specifically, we optimized the base model by selecting appropriate optimizers, fine-tuning the dropout probability, and adjusting the learning rate decay strategy. Additionally, we replaced ReLU with PReLU and added BatchNorm layers after each Inception layer, enhancing the model’s nonlinear expression and training stability. Leveraging the Inception module’s branching structure and the adaptive grouping properties of the WeDIV clustering algorithm, we developed two grouped dropout models, the iGDnet-IP and GDnet-IP. Experimental results on a dataset containing 20 insect species (15 pests and five beneficial insects) demonstrated an increase in cross-validation accuracy from 84.68% to 92.12%, with notable improvements in the recognition rates for difficult-to-classify species, such as Parnara guttatus Bremer and Grey (PGBG) and Papilio xuthus Linnaeus (PXLL), increasing from 38% and 47% to 62% and 93%, respectively. Furthermore, these models showed significant accuracy advantages over standard dropout methods on test sets, with faster training times compared to four conventional CNN models, highlighting their suitability for mobile applications. Theoretical analyses of model gradients and Fisher information provide further insight into the grouped dropout strategy’s role in improving CNN interpretability for insect recognition tasks.

A Female-Biased Chemosensory Protein PxutCSP19 in the Antennae of Papilio xuthus Tuned to Host Volatiles and Insecticides.

Chemosensory protein (CSP) genes significantly enriched in the female antennae are potential molecular candidates for mediating female oviposition behaviors. In this study, we presented the interaction mechanisms of a female-antenna-biased PxutCSP19 in Papilio xuthus to 47 host volatiles, four biopesticides and 24 synthetic insecticides. Using a bioinformatics-based homology search, 22 genes orthologous to PxutCSP19 were identified from 22 other Papilio butterflies with high sequence identities to each other (73.20~98.72%). Multiple alignment analyses revealed a particularly extended N-terminus of Papilio CSP19s (an average of 154 residues) compared to insects' typical CSPs (approximately 120 residues). The expression profiles indicated that PxutCSP19 was significantly enriched in the female antennae, with a 31.81-fold difference relative to the male antennae. In ligand-binding assays, PxutCSP19 could strongly bind six host odorants with high affinities, ranging from dissociation constant (Ki) values of 20.44 ± 0.64 μM to 22.71 ± 0.73 μM. Notably, this protein was tuned to a monoterpenoid alcohol, linalool, which generally existed in the Rutaceae plants and elicited electrophysiological and behavioral activities of the swallowtail butterfly. On the other hand, PxutCSP19 was also capable of binding eight insecticides with stronger binding abilities (Ki < 12 μM) compared to host odorants. When an extended N-terminal region of PxutCSP19 was truncated into two different proteins, they did not significantly affect the binding of PxutCSP19 to ligands with high affinities, suggesting that this extended N-terminal sequences were not involved in the specificity of ligand recognition. Altogether, our study sheds light on the putative roles of PxutCSP19 enriched in the female antennae of P. xuthus in the perception of host volatiles and the sequestering of insecticides, and it complements the knowledge of butterfly CSPs in olfaction and insecticide resistance.

RNAi efficiency is enhanced through knockdown of double-stranded RNA-degrading enzymes in butterfly Papilio xuthus.

The efficiency of RNA interference (RNAi) has always limited the research on the phenotype innovation of Lepidoptera insects. Previous studies have found that double-stranded RNA-degrading enzyme (dsRNase) is an important factor in RNAi efficiency, but there have been no relevant reports in butterflies (Papilionoidea). Papilio xuthus is one of the important models in butterflies with an extensive experimental application value. To explore the effect of dsRNase in the RNAi efficiency on butterflies, six dsRNase genes (PxdsRNase 1-6) were identified in P. xuthus genome, and their dsRNA-degrading activities were subsequently detected by ex vivo assays. The result shows that the dsRNA-degrading ability of gut content (<1 h) was higher than hemolymph content (>12 h). We then investigated the expression patterns of these PxdsRNase genes during different tissues and developmental stages, and related RNAi experiments were carried out. Our results show that different PxdsRNase genes had different expression levels at different developmental stages and tissues. The expression of PxdsRNase2, PxdsRNase3, and PxdsRNase6 were upregulated significantly through dsGFP injection, and PxdsRNase genes can be silenced effectively by injecting their corresponding dsRNA. RNAi-of-RNAi studies with PxEbony, which acts as a reporter gene, observed that silencing PxdsRNase genes can increase RNAi efficiency significantly. These results confirm that silencing dsRNase genes can improve RNAi efficiency in P. xuthus significantly, providing a reference for the functional study of insects such as butterflies with low RNAi efficiency.

Morphology and spectral sensitivity of long visual fibers and lamina monopolar cells in the butterfly Papilio xuthus.

Extensive analysis of the flower-visiting behavior of a butterfly,Papilio xuthus, has indicated complex interaction between chromatic, achromatic, and motion cues. Their eyes are spectrally rich with six classes of photoreceptors, respectively sensitive in the ultraviolet, violet, blue, green, red, and broad-band wavelength regions. Here, we studied the anatomy and physiology of photoreceptors and second-order neurons ofP. xuthus, focusing on their spectral sensitivities and projection terminals to address where the early visual integration takes place. We thus found the ultraviolet, violet, and blue photoreceptors and all second-order neurons terminate in the distal region of the second optic ganglion, the medulla. We identified five types of second-order neurons based on the arborization in the first optic ganglion, the lamina, and the shape of the medulla terminals. Their spectral sensitivity is independent of the morphological types but reflects the combination of pre-synaptic photoreceptors. The results indicate that the distal medulla is the most plausible region for early visual integration.

‘Color’ processing in the butterfly visual system

The swallowtail butterfly, Papilio xuthus, has excellent color discrimination abilities, and its visible light spectrum is notably wide. We discuss the neural basis of color vision in P. xuthus, highlighting some of the evolutionary adaptations in this species in relation to other insects. These adaptations include inter-photoreceptor (PR) interactions that produce spectral-opponent PRs, and complex higher order color-coding neurons.

Butterfly Wing Translucence Enables Enhanced Visual Signaling.

The light reflected by the dorsal side of butterfly wings often functions as a signal for, e.g., mate choice, thermoregulation, and/or predator deterrence, while the ventral wing reflections are generally used for crypsis and camouflage. Here, we propose that transmitted light can also have an important role in visual signaling because, in many butterfly species, the dorsal and ventral wing sides are similarly patterned and locally more or less translucent. Extreme examples are the Japanese yellow swallowtail (Papilio xuthus Linnaeus, 1758) and the Yellow glassy tiger (Parantica aspasia Fabricius, 1787). Their wings exhibit a similar color pattern in reflected and transmitted light, which allows enhanced visual signaling, especially in flight. Contrasting cases in which the coloration and patterning of dorsal and ventral wings strongly differ are the papilionid Papilio nireus Linnaeus, 1758, and the pierid Delias nigrina Fabricius, 1775. The wings observed in reflected or transmitted light then show very different color patterns. Wing translucence thus will strongly affect a butterfly's visual signal.

Spider mites avoid caterpillar traces to prevent intraguild predation

The phytophagous spider mites Tetranychus kanzawai and Tetranychus urticae can be as small as < 0.5 mm; thus, they are often incidentally consumed along with food plant leaves by voracious lepidopteran larvae (hereafter, ‘caterpillars’). Therefore, the ability to avoid such intraguild predation should confer a selective advantage to mites. We experimentally demonstrated that adult females of both mite species avoided settling on food plant leaves with traces of all tested caterpillar species (Bombyx mori, Papilio xuthus, Spodoptera litura and Theretra oldenlandiae). We examined additional interactions using B. mori and T. kanzawai and found that B. mori trace avoidance by T. kanzawai lasted for more than 48 h. Tetranychus kanzawai also avoided B. mori traces on plant stems, along which mites access leaves. Moreover, T. kanzawai avoided acetone extracts of B. mori traces applied to filter paper, indicating that chemical substances of caterpillar traces are responsible for the avoidance. This study is the first demonstration of a repellent effect of herbivore trace chemicals on heterospecific herbivores. Although spider mites have developed resistance against many synthetic pesticides, these results predict that natural compounds simulating caterpillar traces may repel spider mites from agricultural crops.

Specificity of Monoterpene Interactions with Insect Octopamine and Tyramine Receptors: Insights from in Silico Sequence and Structure Comparison.

Octopamine and tyramine receptors (OARs/TARs) are interesting targets for new insecticide development due to their unique roles in insects' physiological and cellular response and their specificity to invertebrates. Monoterpene compounds that bear resemblance to the natural ligands have been shown to bind to the OARs/TARs but elicit varied responses in different insect species. Using in silico methods, we attempt to investigate the molecular basis of monoterpene interactions and their specificity in different OARs and TARs of damaging or beneficial insects. Sequence and structure comparison revealed that the OARs/TARs studied generally have more similarities in terms of structure rather than sequence identity. Together with clustering and network analyses, we also revealed that the role of IL3 might be crucial in the identification of OAR and TAR and their unique function. Among the 35 monoterpenes subjected to ensemble docking, carvacrol had the most negative average binding energies with the target insect OARs and TARs. The differences in the key interacting residues of carvacrol with insect OARs and TARs could be the origin of variation in the responses of insect species to this monoterpene. Results suggest that carvacrol may be a potential natural-product-based insecticide, targeting multiple insect pests while being nonharmful to honeybees and Asian swallowtail butterflies. This work could provide insights into the development of effective species-specific natural-product-based insecticides that are more environmentally friendly than conventional insecticides.

Compression behavior of bio-inspired lattice structures inspired by Papilio Xuthus

The main objective of this research is to study the compression behaviour of a Novel bio-inspired lattice structure from the wing structure of one of the butterfly species named Papilio Xuthus. Two major parameters are chosen from the structure to make multiple designs that may cause changes in the behaviour of the structure among all available parameters. The parametric values required for the designs were calculated with the help of Response Surface Methodology (RSM) using Minitab software. The proposed designs are modelled in Autodesk fusion 360 modelling software, and 3D printed specimens of size 40x40x40 (all in mm) are fabricated by the Vat polymerization principle based on the chosen parameters. Then, the 3D-printed specimens were tested for compression using universal testing machine equipment. The test results obtained from the testing process were evaluated, and a lattice structure with better performance was compared with the honeycomb structure.

Spider mites avoid caterpillar traces to prevent intraguild predation

The phytophagous spider mites Tetranychus kanzawai and Tetranychus urticae (Acari: Tetranychidae) can be as small as &lt; 0.5 mm; thus, they are often incidentally consumed along with food plant leaves by voracious lepidopteran larvae (hereafter, ‘caterpillars’; Shirotsuka and Yano, 2012). Therefore, the ability to avoid such intraguild predation should confer a selective advantage to mites. We experimentally demonstrated that adult females of both mite species avoided settling on food plant leaves with traces of all tested caterpillar species (Bombyx mori, Papilio xuthus, Spodoptera litura, and Theretra oldenlandiae). We examined additional interactions using B. mori and T. kanzawai and found that B. mori trace avoidance by T. kanzawai lasted for more than 48 h. Tetranychus kanzawai also avoided B. mori traces on plant stems, along which mites access leaves. Moreover, T. kanzawai avoided acetone extracts of B. mori traces applied to filter paper, indicating that chemical substances of caterpillar traces are responsible for the avoidance. This study is the first demonstration of a repellent effect of herbivore trace chemicals on heterospecific herbivores. Although spider mites have developed resistance against many synthetic pesticides (Attia et al., 2013; Van Leeuwen et al., 2010), this study showed the potential of using natural compounds simulating caterpillar traces in repelling spider mites from agricultural crops.

Motion-sensitive neurons activated by chromatic contrast in a butterfly visual system.

A pattern of two equally bright colours contains only chromatic contrast. Unlike in flies, such a pattern elicits strong optokinetic responses in the butterfly Papilio xuthus. To investigate the neural basis of chromatic motion vision, we performed single-cell electrophysiology. We found spiking neurons exhibiting direction-selective motion sensitivity in the second optic ganglion, the medulla. We analysed the response characteristics of these neurons using two-colour stripe patterns moving vertically. We systematically manipulated the intensities of the colours so that the set of presented patterns included an isoluminant condition for the butterfly. Moving patterns containing only chromatic contrast still elicited a response in the neurons. The neurons' sensitivity profile is similar to that of the behavioural responses. Post-recording dye injection revealed that the neurons have dendrites in the ventral lateral protocerebrum and axonal processes in the medulla, suggesting a feedback role. Presumably, the neurons contribute to subtracting wide-field motion to facilitate the detection of small moving targets. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Genome-Wide Analysis of Odorant-Binding Proteins in Papilio xuthus with Focus on the Perception of Two PxutGOBPs to Host Odorants and Insecticides.

In this study, we annotated 49 odorant-binding proteins (OBPs) in Papilio xuthus, with four novel genes and seven improved sequences. Expression profiles identified numerous OBPs in antennae or reproductive tissues. Using two antenna-enriched general OBPs (PxutGOBP1 and PxutGOBP2) as targets, we screened three key compounds by a reverse chemical ecology strategy. Of these, an oviposition stimulant vicenin-2 could strongly interact with PxutGOBP1, representing a dissociation constant (Ki) value of 10.34 ± 0.07 μM. Molecular simulations and site-directed mutagenesis revealed the importance of His66, Thr73, and Phe118 between PxutGOBP1 and vicenin-2 interactions. Two other compounds, an ordinary floral scent β-ionone and a widely used insecticide chlorpyrifos, exhibited high affinities to PxutGOBPs (Ki < 13 μM). Furthermore, two mutations His66Ala and Thr73Ala of PxutGOBP1 significantly reduced the binding to chlorpyrifos. Our study provides insights into the putative roles of PxutGOBPs in odorant perception and identifies key binding sites of PxutGOBP1 to vicenin-2 and chlorpyrifos.

Comparative analysis on visual and olfactory signals of Papilio xuthus (Lepidoptera: Papilionidae) during foraging and courtship.

This study examined the roles of visual and olfactory responses during foraging and courtship in butterfly Papilio xuthus. P. xuthus showed obvious orientation to color in the range of 350–500 nm. Visits of P. xuthus females and males to blue, purple, and red artificial cloth flowers were ♀ 54.90% and ♂ 39.22%, ♀ 19.61% and ♂ 35.29%, and ♀ 9.80% and ♂ 19.61%, respectively. Application of 10% honey on these artificial flowers resulted in an increase of 3.41 and 3.26 fold in flower visits by the butterfly compared to controls. When 10% honey water was sprayed on flower branches without colorful flowers, branch visiting was very low, only seven times for females and two times for males, indicating that colors might be more critical than odor for foraging even though visual and olfactory perceptions both play important roles during foraging. During courtship, four types of chasing were observed in a natural population of P. Xuthus; the four types are males chasing females (49%), males chasing males (25%), females chasing males (13%), and females chasing females (10%). However, when odorless artificial models of butterflies were used, no significant differences were observed among these types of chasing, indicating that olfactory perception was crucial for the butterfly during courtship. Profiling volatile organic chemicals (VOCs) and individual bioassays revealed that VOCs contents of butterflies were not related to recognizing sex partners; by contrast, some level of α-farnesene, increased the frequency of male chasing female. This could be due to that α-farnesene is easy to be detected by butterflies because of its volatility and higher content in female.

Study of the heterologous gene expression characteristics of a new clone of a cell line derived from Papilio xuthus and its serum-free adaptation.

The baculovirus expression vector system using insect cells as a bioreactor has been used for in vitro expression of recombinant proteins and plays an important role in the fields of biology, agronomy, and medicine. Screening suitable host cell lines is an important part of the construction of insect cell baculovirus expression systems. In previous research, we used a single-cell cloning process with the Papilio xuthus cell line RIRI-PX1 and obtained the monoclonal cell line RIRI-PX1-C31. In this study, we compared the basic biological and recombinant protein expression characteristics of RIRI-PX1-C31 and its parent cell line RIRI-PX1 and found that the expression of recombinant β-galactosidase in RIRI-PX1-C31 was significantly higher than that in the parental cell line. Further serum-free adaptation studies confirmed that RIRI-PX1-C31 can adapt to the growth environment of Express Five Serum-free medium and that its expression level of recombinant β-galactosidase was significantly higher than that before adaptation.

Connectomics: From form to function in butterfly colour vision

A recent study has revealed how the connectivity of neurons in the lamina of the Asian swallowtail butterfly forms the basis of this insect's exceptional colour vision through two circuit motifs: colour opponency of photoreceptors and broadband colour integration by lamina neurons.

Connectome of the lamina reveals the circuit for early color processing in the visual pathway of a butterfly

Connectomics has become a standard neuroscience methodology in a few model animals,1 with the visual system being a popular target of study.2-5 Combining connectomics with circuit and behavioral physiology, recent studies on the color vision of the fruit fly Drosophila melanogaster have focused on the mechanisms underlying early wavelength processing in the optic ganglia.6-8 However, the color vision capabilities of D.melanogaster are limited,9 compared with many flower-visiting insects.10,11 For example, a butterfly Papilio xuthus has six spectral classes of photoreceptors. Each ommatidium contains nine photoreceptors in one of three fixed combinations, making the eye an array of three spectrally distinct ommatidia types.12 Behaviorally, P.xuthus can detect 1nm differences in light wavelength across the spectrum from ultraviolet to red, outperforming humans.13 What is the neuronal basis of such precise color vision? How does such a system evolve? Addressing these questions requires comparative studies at the circuit level. Here, we performed a connectome analysis in the first optic ganglion, the lamina, of P.xuthus. The lamina comprises cartridges, each typically containing nine photoreceptor axons from a single ommatidium and four second-order neurons. We found abundant inter-photoreceptor connections, which are absent in the lamina of D.melanogaster. We also identified connections between neighboring cartridges, particularly those receiving inputs from spectrally distinct ommatidia. The linear summation of synaptic connections well explains the spectral sensitivity of photoreceptors and second-order neurons in the lamina.

Integrated Analysis of Transcriptome and Proteome to Reveal Pupal Color Switch in Papilio xuthus Butterflies.

Pupal color polyphenism in Papilio butterflies, including green, intermediate, or brown, is an excellent study system for understanding phenotypic plasticity. Previous studies suggested that development of brown pupae may be controlled by a hormone called pupal-cuticle-melanizing-hormone (PCMH) which is synthesized and secreted from brain-suboesophageal ganglion and prothoracic ganglion complexes (Br-SG-TG1) during the pre-pupa stage. However, detailed molecular mechanisms of neuroendocrine regulation in pupal color development remain unknown. In this study, we integrated the expression profiles of transcriptome and proteome at pre-pupa stages [2 h after gut purge (T1) and 3 h after forming the garter around the body (T2)] and pigmentation stages [10 h after ecdysis (T3) and 24 h after ecdysis (T4)] to identify important genes and pathways underlying the development of green and brown pupa in the swallowtail butterfly Papilio xuthus. Combined comparisons of each developmental stage and each tissue under green and brown conditions, a total of 1042 differentially expressed genes (DEGs) and 430 different abundance proteins (DAPs) were identified. Weighted gene co-expression network analysis (WGCNA) and enrichment analysis indicate that these DEGs were mainly related to oxidation-reduction, structural constituent of cuticle, and pigment binding. Soft clustering by Mfuzz and enrichment analysis indicate that these DAPs are mainly involved in tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. By homologous alignment, we further identified those genes encoding neuropeptides (51), GPCRs (116), G-proteins (8), cuticular proteins (226), chitinases (16), and chitin deacetylases (8) in the whole genome of P. xuthus and analyzed their expression profiles. Although we identified no gene satisfying with hypothesized expression profile of PCMH, we found some genes in the neuropeptide cascade showed differentially expressed under two pupal color conditions. We also found that Toll signaling pathway genes, juvenile hormone (JH) related genes, and multiple cuticular proteins play important roles in the formation of selective pupal colors during the prepupal-pupal transition. Our data also suggest that both green and brown pupa include complex pigment system that is regulated by genes involved in black, blue, and yellow pigments. Our results provide important insights into the evolution of pupal protective colors among swallowtail butterflies.

Swallowtail Butterflies Use Multiple Visual Cues to Select Oviposition Sites.

Simple SummaryButterflies must not only identify host plants on which to lay their eggs—which they achieve using chemical cues—but also select suitable leaves on that plant that will support the growth of their larval offspring. Here, we asked whether swallowtail butterflies lay eggs on particular leaves of a Citrus tree and, if so, which cues they use to select the leaves. We first observed that butterflies indeed select just a few leaves on which to lay eggs. These leaf preferences were observed across many individuals, implying that they were not idiosyncratic, and the butterflies descended directly towards the leaves from some distance, suggesting that they were using visual rather than chemical cues. We then investigated which visual cues are used by the butterflies, and found that the number of eggs laid upon a leaf was correlated with its height on the tree, flatness, green reflectance, brightness, and degree of polarization. These five features may be important both for selecting young leaves and those which are situated well for egg-laying. An outstanding question for future study is how visual and chemical cues interact in this context.Flower-foraging Japanese yellow swallowtail butterflies, Papilio xuthus, exhibit sophisticated visual abilities. When ovipositing, females presumably attempt to select suitable leaves to support the growth of their larval offspring. We first established that butterflies indeed select particular leaves on which to lay eggs; when presented with a single Citrus tree, butterflies significantly favored two out of 102 leaves for oviposition. These preferences were observed across many individuals, implying that they were not merely idiosyncratic, but rather based on properties of the leaves in question. Because the butterflies descended towards the leaves rather directly from a distance, we hypothesized that they base their selection on visual cues. We measured five morphological properties (height, orientation, flatness, roundness, and size) and four reflective features (green reflectance, brightness, and degree and angle of linear polarization). We found that the number of eggs laid upon a leaf was positively correlated with its height, flatness, green reflectance, and brightness, and negatively correlated with its degree of polarization, indicating that these features may serve as cues for leaf selection. Considering that other studies report ovipositing butterflies’ preference for green color and horizontally polarized light, butterflies likely use multiple visual features to select egg-laying sites on the host plant.

계층분석방법(AHP)과 전문가 설문조사를 활용한 농업생태계 기후변화 지표생물 30종 선정

It is more efficient to select and monitor a few climate-sensitive organisms to assess the impacts of climate change on agroecosystems. In this study, indicator species of climate change in agroecosystem in South Korea were selected through analytical hierarchy process and an expert questionnaire survey. Climate sensitivity, agroecosystem symbolism, survey easiness, identification easiness, and public interest were selected as criteria for indicator species, with weights of 0.38 (±0.15), 0.23 (±0.13), 0.18 (±0.11), 0.14 (±0.09), and 0.07 (±0.06), respectively. According to these criteria, 30 indicator species were finally selected from six taxonomic groups of plants, aquatic invertebrates, bees, terrestrial beetles, butterflies, and spiders as follows. Seven plant species were selected: Taraxacum officinale, Capsella bursa-pastoris, Veronica persica, Hypochaeris radicata, Lamium amplexicaule, Trigonotis peduncularis, and Conyza sumatrensis. Seven aquatic invertebrate species were selected: Pomacea canaliculata, Cybister chinensis, Hydrochara affinis, Sternolophus rufipes, Hydaticus grammicus, Appasus japonicus, and Rhantus suturalis. Five butterfly species were selected: Eurema mandarina, Chilo suppressalis, Pieris rapae, Papilio xuthus, and Colias erate. Three spider species were selected: Argiope bruennichi, Larinioides cornutus, and Neoscona adianta. Four bee species were selected: Vespa velutha nigrithorax, V. simillima simillima, V. mandarinia, and V. simillima xanthoptera. Four species were selected for terrestrial beetles: Pheropsophus javanus, P. jessoensis, Platydracus brevicornis, and Chlaenius micans. The selected species are expected to be used as indicators for assessing the impacts of climate change in agroecosystems in the future.

Coexpression of Three Odorant-Binding Protein Genes in the Foreleg Gustatory Sensilla of Swallowtail Butterfly Visualized by Multicolor FISH Analysis.

Lepidopteran insects are mostly monophagous or oligophagous. Female butterflies distinguish their host plants by detecting a combination of specific phytochemicals through the gustatory sensilla densely distributed on their foreleg tarsi, thereby ensuring oviposition on appropriate host plants. In this study, to gain insight into the molecular mechanism underlying host plant recognition by the gustatory sensilla, using Asian swallowtail, Papilio xuthus, we focused on a family of small soluble ligand-binding molecules, odorant-binding proteins (OBPs), and found that three OBP genes showed enriched expression in the foreleg tarsus. Multicolor fluorescence in situ hybridization analyses demonstrated the coexpression of these three OBP genes at the bases of the foreleg gustatory sensilla. Further analyses on other appendages revealed that PxutOBP3 was exclusively expressed in the tissues which could have direct contact with the leaf surface, suggesting that this OBP gene specifically plays an important role in phytochemicals perception.