Niemann-Pick Type C2 Proteins Research Articles

Niemann-Pick Type C2 Proteins in Aedes aegypti: Molecular Modelling and Prediction of Their Structure-Function Relationships.

Aedes aegypti is a major vector that transmits arboviruses through the saliva injected into the host. Salivary proteins help in uninterrupted blood intake and enhance the transmission of pathogens. We studied Niemann-Pick Type C2 (NPC2) proteins, a superfamily of saliva proteins that play an important role in arbovirus infections. In vertebrates, a single conserved gene encodes for the NPC2 protein that functions in cholesterol trafficking. Arthropods, in contrast, have several genes that encode divergent NPC2 proteins. We compared the sequences of 20 A. aegypti NPC2 proteins to the cholesterol-binding residues of human and bovine, and fatty-acid-binding residues of ant NPC2 protein. We identified four mosquito NPC2 proteins as potential sterol-binding proteins. Two of these proteins (AAEL006854 and/or AAEL020314) may play a key role in ecdysteroid biosynthesis and moulting. We also identified one mosquito NPC2 protein as a potential fatty-acid-binding protein. Through molecular modelling, we predicted the structures of the potential sterol- and fatty-acid-binding proteins and compared them to the reference proteins.

Molecular identification and functional analysis of Niemann-Pick type C2 proteins，carriers for semiochemicals and other hydrophobic compounds in the brown dog tick, Rhipicephalus linnaei

Ticks are important vectors of many pathogens with tremendous impact on human and animal health. Studies of semiochemical interactions and mechanisms underlying chemoreception can provide important tools in tick management. Niemann-Pick type C2 (NPC2) proteins have been proposed as one type of chemoreceptor in arthropods. Here, we cloned two NPC2 genes in the brown dog tick, Rhipicephalus linnaei, the tropical lineage previously named R. sanguineus sensu lato and characterized them functionally.R.linNPC2a and R.linNPC2b genes were found to be expressed at each developmental stage with the highest level in adult males. By using quantitative real-time PCR we revealed expression in multiple tissues, including midgut, ovary, salivary glands and legs. Ligand binding analysis revealed that R.linNPC2b bound a wide spectrum of compounds, with β-ionone, α-amylcinnamaldehyde, 2-nitrophenol and benzaldehyde displaying the strongest binding affinity (Ki < 10 μM), whereas R.linNPC2a showed a more narrow ligand binding range, with intermediate binding affinity to α-amylcinnamaldehyde and 2-nitrophenol (Ki < 20 μM). Molecular docking indicated that the amino acid residue Phe89, Leu77 and Val131 of R.linNPC2a and Phe70, Leu132 and Phe73 of R.linNPC2b could bind multiple ligands. These residues might thus play a key role in the identification of the volatiles. Our results contribute to the understanding of olfactory mechanisms of R. linnaei and can offer new pathways towards new management strategies.

Expression and functional analysis of Niemann-Pick C2 gene in Phytoseiulus persimilis.

As a new protein class of semiochemical binding and transporting, Niemann-Pick proteins type C2 (NPC2) in arthropods have received more attentions in recent decade. However, the gene function has not been studied in phytoseiid mites with biocontrol potential. In the current study, we cloned a NPC2 gene PpNPC2a from the transcriptome of Phytoseiulus persimilis Athias-Henriot. By encoding 181 amino acids with a conserved ML domain, PpNPC2a was found a homolog of NPC2-1 in Galendromus occidentalis Nesbitt. We then measured the spatio-temporal expression of PpNPC2a in P. persimilis, and found the highest expression in female adults compared to other stages. Due to the tiny body size of predatory mites, we only examined tissue expressions in two sections: the anterior part (gnathosoma and the first pair of legs) and the posterior part (idiosoma without the first pair of legs). Higher transcription of PpNPC2a was found in the posterior part. To investigate the potential function of PpNPC2a in P. persimilis, we interfered gene expression in female adults by feeding dsRNA, which resulted in a decrease of relative expression by 59.1 and 78.2% after 24 and 72h, respectively. Compared with the control, dsNPC2a-treated P. persimilis were insensitive to the scent of leaves or plants infested by spider mites, suggesting a role of PpNPC2a in response to plant volatiles. However, the dsNPC2a-interfered mites could still respond to four representative compounds of herbivore-induced plant volatiles, including 4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT), methyl salicylate (MeSA), β-caryophyllene and linalool. In short, our results indicated PpNPC2a may be involved in the chemosensory process of P. persimilis in response to whole-plant volatiles.

The genome of the rice planthopper egg parasitoid wasps Anagrus nilaparvatae casts light on the chemo- and mechanosensation in parasitism

BackgroundMymaridae is an ancient insect group and is a basal lineage of the superfamily Chalcidoidea. Species of Mymaridae have great potential for biological control. Anagrus nilaparvatae, a representative species of Mymaridae, is ideal for controlling rice planthopper due to its high rate of parasitism and ability to find hosts efficiently in paddy ridges and fields.ResultsUsing both PacBio single-molecule real-time and Illumina sequencing, we sequenced and assembled the whole genome of A. nilaparvatae, a first for the family Mymaridae. The assembly consists of 394 scaffolds, totaling 488.8 Mb. The assembly is of high continuity and completeness, indicated by the N50 value of 25.4 Mb and 98.2% mapping rate of Benchmarking Universal Single-Copy Orthologs. In total, 16,894 protein-coding genes in the genome were annotated. A phylogenomic tree constructed for A. nilaparvatae and other 12 species of Hymenoptera confirmed that the family Mymaridae is sister to all remaining chalcidoids. The divergence time between A. nilaparvatae and the other seven Chalcidoidea species was dated at ~ 126.9 Mya. Chemoreceptor and mechanoreceptor genes are important in explaining parasitic behavior. We identified 17 odorant binding proteins, 11 chemosensory proteins, four Niemann-Pick type C2 proteins, 88 olfactory receptors, 12 gustatory receptors, 22 ionotropic receptors and 13 sensory neuron membrane proteins in the genome of A. nilaparvatae, which are associated with the chemosensory functions. Strikingly, there is only one pickpocket receptors and nine transient receptor potential genes in the genome that have a mechanosensory function.ConclusionsWe obtained a high-quality genome assembly for A. nilaparvatae using PacBio single-molecule real-time sequencing, which provides phylogenomic insights for its evolutionary history. The small numbers of chemo- and mechanosensory genes in A. nilaparvatae indicate the species-specific host detection and oviposition behavior of A. nilaparvatae might be regulated by relatively simple molecular pathways.

Molecular identification and functional analysis of Niemann-Pick type C2 protein in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

• Niemann-Pick type C2 gene (McinNPC2) was cloned from Macrocentrus cingulum . • McinNPC2 is likely to be OBPs and the nearest evolutionary relation genes as OBPs were clustered. • McinNPC2 may be related protein as a new carrier of aroma compounds. • McinNPC2 may play roles in perception of plant volatiles. Niemann-Pick type C2 (NPC2) proteins in arthropods have been extensively differentiated and possibly duplicated according to environmental conditions and are probable to have different functions. The participation of NPC2 proteins in chemical communication in arthropods brings new objectives in environmental-friendly strategies for pest population control. In this study, NPC2 gene in Macrocentrus cingulum (McinNPC2) was newly identified by rapid amplification cDNA ends (RACE) technology. McinNPC2 amino acid sequence alignment with other representative NPC2 annotates to evaluate the highly conserved consensus amino acids, but with odorant binding proteins in M. cingulum show that only one consensus amino acid. Primary six-cysteine structures that are same to odorant binding proteins in M. cingulum were observed in McinNPC2. Phylogenetic analysis of McinNPC2 indicated that the nearest monophyletic group forming one clade with high posterior probability values clusters as Cyphomyrmex costatus (CcosNPC2) whereas the nearest evolutionary relation group as some odorant binding proteins. Moreover, quantitative real-time PCR (qPCR) measurements show that the McinNPC2 gene expression level in various tissues of the female is significantly and ubiquitously higher than in male, whereas the highest expression level in female antennae. We further explore the binding characterization of recombinant McinNPC2 to candidate odor molecules and did the modeling and docking simulations. The results showed ligands binding specificity and docking tests results indicate that β-ionone, an aroma compound commonly found in essential oils, can strongly bind with McinNPC2 . In conclusion, we proposed that McinNPC2 may be involved in chemical communication and play roles in perception of plant volatiles.

Comparative morphological and transcriptomic analyses reveal chemosensory genes in the poultry red mite, Dermanyssus gallinae

Detection of chemical cues via chemosensory receptor proteins are essential for most animals, and underlies critical behaviors, including location and discrimination of food resources, identification of sexual partners and avoidance of predators. The current knowledge of how chemical cues are detected is based primarily on data acquired from studies on insects, while our understanding of the molecular basis for chemoreception in acari, mites in particular, remains limited. The poultry red mite (PRM), Dermanyssus gallinae, is one of the most important blood-feeding ectoparasites of poultry. PRM are active at night which suck the birds' blood during periods of darkness and hide themselves in all kinds of gaps and cracks during the daytime. The diversity in habitat usage, as well as the demonstrated host finding and avoidance behaviors suggest that PRM relies on their sense of smell to orchestrate complex behavioral decisions. Comparative transcriptome analyses revealed the presence of candidate variant ionotropic receptors, odorant binding proteins, niemann-pick proteins type C2 and sensory neuron membrane proteins. Some of these proteins were highly and differentially expressed in the forelegs of PRM. Rhodopsin-like G protein-coupled receptors were also identified, while insect-specific odorant receptors and odorant co-receptors were not detected. Furthermore, using scanning electron microscopy, the tarsomeres of all leg pairs were shown to be equipped with sensilla chaetica with or without tip pores, while wall-pored olfactory sensilla chaetica were restricted to the distal-most tarsomeres of the forelegs. This study is the first to describe the presence of chemosensory genes in any Dermanyssidae family. Our findings make a significant step forward in understanding the chemosensory abilities of D. gallinae.

&lt;p class="Body"&gt;&lt;strong&gt;Molecular characterization of three Niemann–Pick type C2 proteins in the predatory mite &lt;em&gt;Neoseiulus barkeri &lt;/em&gt;Hughes (Acari: Phytoseiidae)&lt;/strong&gt;&lt;/p&gt;

In arthropods, a host of studies focused on insect olfactory systems, where most functional proteins including odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) have been identified. However, in other arthropods such as ticks and mites, genes encoding for OBPs and CSPs are absent. The Niemann-Pick type C2 (NPC2), as a third class of binding proteins, was indicated as potential carriers for semiochemicals and participants in chemical communication. Here, we cloned three full-length NPC2 genes (NbNPC2-1, NbNPC2-2 and NbNPC2-3) from a predatory mite Neoseiulus barkeri Hughes (Acari: Phytoseiidae) and investigated the expression levels of these genes by quantitative real-time PCR. These NPC2 proteins possessed a conserved motif of six cysteines paired in three disulfide bridges with a coincidence to insect OBPs. The 3D molecular modeling of NbNPC2 showed ligand-binding pocket of NbNPC2-1 was composed of a flexible β-structure that contributed to binding to a wide range of potential semiochemicals. Three NPC2 genes all possessed one signal peptide revealed their specific character as secretory proteins. Three NPC2 genes in N. barkeri displayed the highest expression levels in adult males, suggesting a putative role in detecting of the female sex pheromones and highly specific binding to female sex pheromone. The expression levels were lowest in the stage of eggs and increased with developmental stage growing, indicating that NPC2 genes were continuously expressed in post-embryonic stages. NbNPC2-1 in the stage of adult females was expressed 6 and 4 times higher than that of larva and nymph, suggesting NbNPC2-1 might contribute an important role in odor recognition of adult females to hunt for suitable areas for the oviposition and predation. These results showed that NPC2 genes might be involved in chemical communications of adult males, and that NbNPC2-1 preformed specified olfactory functions in adult females. This study provided a foundation for research to determine the roles of NPC2 proteins acting as odorant carrier and involved in chemical communication in phytoseiid mites.

Antennal transcriptome analysis and expression profiles of putative chemosensory soluble proteins in Histia rhodope Cramer (Lepidoptera: Zygaenidae)

Histia rhodope Cramer (Lepidoptera: Zygaenidae) is one of the most destructive defoliators of landscape tree Bischofia polycarpa (Levl.) Airy Shaw in China stretching to other Southeast Asia regions. Olfactory genes, encoding proteins such as odorant carrier proteins believed to initiate olfactory signal transduction in insects, have been acknowledged to be novel targets for pest control. In this study, we established antennal transcriptome of H. rhodope and ultimately identified 19 odorant binding proteins (OBPs), 23 chemosensory proteins (CSPs) and 4 Niemann-Pick type C2 proteins (NPC2s). The 19 OBPs, 6 CSPs and 4 NPC2s were assessed to validate the differential expressions between sexes, and between olfactory and non-olfactory tissues. 8 OBPs and 2 CSPs exhibited male-biased antennae expression, while 6 OBPs, 2 CSPs and HrhoNPC2a exhibited female-biased antennae expression. Moreover, 17 OBPs, 4 CSPs and 2 NPC2s were predominantly expressed in the antennae compared with non-olfactory tissues. HrhoOBP1 and HrhoOBP8 were predominantly expressed in the antennae and heads, HrhoCSP8 and HrhoCSP14 were highly expressed in abdomens and legs, HrhoNPC2c was highly expressed in abdomens, while HrhoNPC2d was expressed in all tissues. Phylogenetic analysis revealed that most H. rhodope proteins were closely related to proteins from other moths. Moreover, compared with other nocturnal moths, acting as a diurnal moth, we found that H. rhodope may have lost a PBP gene. Our results provide important molecular information for further studies on olfactory mechanisms of H. rhodope.

Sterol transfer by atypical cholesterol-binding NPC2 proteins in coral-algal symbiosis.

Reef-building corals depend on intracellular dinoflagellate symbionts that provide nutrients. Besides sugars, the transfer of sterols is essential for corals and other sterol-auxotrophic cnidarians. Sterols are important cell components, and variants of the conserved Niemann-Pick Type C2 (NPC2) sterol transporter are vastly up-regulated in symbiotic cnidarians. Types and proportions of transferred sterols and the mechanism of their transfer, however, remain unknown. Using different pairings of symbiont strains with lines of Aiptasia anemones or Acropora corals, we observe both symbiont- and host-driven patterns of sterol transfer, revealing plasticity of sterol use and functional substitution. We propose that sterol transfer is mediated by the symbiosis-specific, non-canonical NPC2 proteins, which gradually accumulate in the symbiosome. Our data suggest that non-canonical NPCs are adapted to the symbiosome environment, including low pH, and play an important role in allowing corals to dominate nutrient-poor shallow tropical seas worldwide.

Niemann-Pick proteins type C2 are identified as olfactory related genes of Pardosa pseudoannulata by transcriptome and expression profile analysis

In arthropods, the large majority of studies on olfaction have been mainly focused on insects, whereas little on Arachnida, even though olfaction is very important in arachnid behavior. Pardosa pseudoannulata is one of the most common wandering spiders in rice fields, as the important natural enemy against a range of pests. However, little is known about the potential chemosensory proteins involved in olfactory behavior of these spiders. Niemann-Pick proteins type C2 (NPC2) as a new class of binding and transport proteins for semiochemicals in arthropods especially ticks and mites has received more attention in recent years. In this study, six NPC2s namely PpseNPC1-6 were newly identified in the appendages of P. pseudoannulata based on transcriptome data. A phylogenetic analysis indicated that all of P. pseudoannulata NPC2s were clustered together forming one clade with high posterior probability values. In addition, the sequences shared the same subclade with the NPC2 sequences of ticks and scorpion. The motif-patterns indicated that PpseNPC2-5 had the common pattern with the two-spotted spider mite Tetranychus urticae and the ant Trachymyrmex cornetzi. Furthermore, quantitative real-time PCR (qPCR) measurements were conducted to evaluate the expression profile of these genes in various tissues of P. pseudoannulata. It was found that most NPC2s (PpseNPC2-1, PpseNPC2-2, PpseNPC2-5 and PpseNPC2-6) were highly expressed in adult pedipalps and chelicerae. Owing to the functional olfactory organs in Chelicerata of pedipalps, our results supported a putative role of NPC2s as new odorant carriers in P. pseudoannulata.

Odorant-Binding Proteins as Sensing Elements for Odour Monitoring.

Odour perception has been the object of fast growing research interest in the last three decades. Parallel to the study of the corresponding biological systems, attempts are being made to model the olfactory system with electronic devices. Such projects range from the fabrication of individual sensors, tuned to specific chemicals of interest, to the design of multipurpose smell detectors using arrays of sensors assembled in a sort of artificial nose. Recently, proteins have attracted increasing interest as sensing elements. In particular, soluble olfaction proteins, including odorant-binding proteins (OBPs) of vertebrates and insects, chemosensory proteins (CSPs) and Niemann-Pick type C2 (NPC2) proteins possess interesting characteristics for their use in sensing devices for odours. In fact, thanks to their compact structure, their soluble nature and small size, they are extremely stable to high temperature, refractory to proteolysis and resistant to organic solvents. Moreover, thanks to the availability of many structures solved both as apo-proteins and in complexes with some ligands, it is feasible to design mutants by replacing residues in the binding sites with the aim of synthesising proteins with better selectivity and improved physical properties, as demonstrated in a number of cases.

Molecular characterization and expression analysis of putative odorant carrier proteins in Adelphocoris lineolatus

Abstract The alfalfa plant bug Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae), a serious pest of several agricultural crops, is extremely attracted by flowering plants to perform season host switching. Generally, chemoreception of A. lineolatus plays a crucial role in detecting food sources, locating mates and oviposition sites. In addition, odorant carrier proteins are believed to participate in the initial chemical communication and perception in insects. In this work, we identified 31 putative odorant carrier protein genes from the antennal transcriptomes of A. lineolatus, including 17 new odorant binding proteins (OBPs), 10 new chemosensory proteins (CSPs) and four Niemann-Pick type C2 proteins (NPC2s). Phylogenetic analysis demonstrated that the OBP and CSP genes from four mirid bug species (A. lineolatus, Adelphocoris suturalis, Apolygus lucorum and Lygus lineolaris) had a relatively close evolutionary relationship. Regular patterns and key conserved motifs of OBPs and CSPs in Hemiptera insects are also identified by using Multiple EM for Motif Elicitation (MEME) tool. Tissue expression profiles analysis showed that nine of the 17 OBPs, 10 CSPs and two of the four NPC2s were primarily or uniquely expressed in the adult antennae suggesting their putative roles in chemoreception. Our study provides the first insight into the roles of odorant carrier proteins in chemoreception of A. lineolatus, which will help to facilitate biological functions of odorant carrier proteins and develop novel insect behavioral regulation strategy in future.

Antennal Protein Profile in Honeybees: Caste and Task Matter More Than Age.

Reproductive and task partitioning in large colonies of social insects suggest that colony members belonging to different castes or performing different tasks during their life (polyethism) may produce specific semiochemicals and be differently sensitive to the variety of pheromones involved in intraspecific chemical communication. The main peripheral olfactory organs are the antennal chemosensilla, where the early olfactory processes take place. At this stage, members of two different families of soluble chemosensory proteins [odorant-binding proteins (OBPs) and chemosensory proteins (CSPs)] show a remarkable affinity for different odorants and act as carriers while a further family, the Niemann-Pick type C2 proteins (NPC2) may have a similar function, although this has not been fully demonstrated. Sensillar lymph also contains Odorant degrading enzymes (ODEs) which are involved in inactivation through degradation of the chemical signals, once the message is conveyed. Despite their importance in chemical communication, little is known about how proteins involved in peripheral olfaction and, more generally antennal proteins, differ in honeybees of different caste, task and age. Here, we investigate for the first time, using a shotgun proteomic approach, the antennal profile of honeybees of different castes (queens and workers) and workers performing different tasks (nurses, guards, and foragers) by controlling for the potential confounding effect of age. Regarding olfactory proteins, major differences were observed between queens and workers, some of which were found to be more abundant in queens (OBP3, OBP18, and NPC2-1) and others to be more abundant in workers (OBP15, OBP21, CSP1, and CSP3); while between workers performing different tasks, OBP14 was more abundant in nurses with respect to guards and foragers. Apart from proteins involved in olfaction, we have found that the antennal proteomes are mainly characterized by castes and tasks, while age has no effect on antennal protein profile. Among the main differences, the strong decrease in vitellogenins found in guards and foragers is not associated with age.

Cholesterol transfer at endosomal-organelle membrane contact sites.

Cholesterol is delivered to the limiting membrane of late endosomes by Niemann-Pick Type C1 and C2 proteins. This review summarizes recent evidence that cholesterol transfer from endosomes to the endoplasmic reticulum and other organelles is mediated by lipid-binding proteins that localize to membrane contact sites (MCS). LDL-cholesterol in the late endosomal/lysosomes is exported to the plasma membrane, where most cholesterol resides, and the endoplasmic reticulum, which harbors the regulatory complexes and enzymes that control the synthesis and esterification of cholesterol. A major advance in dissecting these cholesterol transport pathways was identification of frequent and dynamic MCS between endosomes and the endoplasmic reticulum, peroxisomes and plasma membrane. Positioned at these MCS are members of the oxysterol-binding protein (OSBP) and steroidogenic acute regulatory protein-related lipid-transfer family of lipid transfer proteins that bridge the opposing membranes and directly or indirectly mediate cholesterol transfer. OSBP-related protein 1L (ORP1L), ORP5 and ORP6 mediate cholesterol transfer to the endoplasmic reticulum that regulates cholesterol homeostasis. ORP1L and STARD3 also move cholesterol from the endoplasmic reticulum-to-late endosomal/lysosomes under low-cholesterol conditions to facilitate intraluminal vesicle formation. Cholesterol transport also occurs at MCS with peroxisomes and possibly the plasma membrane. Frequent contacts between organelles and the endo-lysosomal vesicles are sites for bidirectional transfer of cholesterol.

A foreleg transcriptome for Ixodes scapularis ticks: Candidates for chemoreceptors and binding proteins that might be expressed in the sensory Haller’s organ

Little is known about the molecular basis for the olfactory capabilities of the sensory Haller’s organ on the forelegs of ticks. We first expanded the known repertoire of Ionotropic Receptors (IRs), a variant lineage of the ionotropic glutamate receptors, encoded by the black-legged Ixodes scapularis genome from 15 to 125. We then undertook a transcriptome study of fore- and hind-legs of this tick in an effort to identify candidate chemoreceptors differentially expressed in forelegs as likely to be involved in Haller’s organ functions. We primarily identified members of the IR family, specifically Ir25a and Ir93a, as highly and differentially expressed in forelegs. Several other IRs, as well as a few members of the gustatory receptor family, were expressed at low levels in forelegs and might contribute to the sensory function of Haller’s organ. In addition, we identified eight small families of secreted proteins, with sets of conserved cysteines, which might function as binding proteins. The genes encoding these Microplusin-Like proteins and two previously described Odorant Binding Protein-Like proteins share a common exon-intron structure, suggesting that they all evolved from a common ancestor and represent an independent origin of binding proteins with potential roles comparable to the ChemoSensory Proteins and Odorant Binding Proteins of insects. We also found two Niemann-Pick Type C2 proteins with foreleg-biased expression, however we were unable to detect foreleg-biased expression of a G-Protein-Coupled pathway previously proposed to mediate olfaction in the tick Haller’s organ.

Niemann-Pick C2 Proteins: A New Function for an Old Family.

Niemann-Pick proteins type C2 (NPC2) are carriers of cholesterol in vertebrates, with a single member in each species. The high sequence conservation between mammals and across vertebrates is related to their common function. In contrast, NPC2 proteins in arthropods have undergone extensive duplication and differentiation, probably under environmental pressure, and are likely to have different functions. Recent studies have suggested that in arthropods these proteins might act as carriers for semiochemicals and other hydrophobic compounds. In this study we focused on the function of a specific NPC2 gene in the moth Helicoverpa armigera (HarmNPC2-1). This protein binds several flavonoids with micromolar dissociation constants. The best ligand was gossypol, present in cotton, one of the main host plants for H. armigera. Western blot revealed the presence of HarmNPC2-1 in different parts of the body, including the antennae, proboscis, and abdomen. In the antennae, in situ hybridization experiments produced strong staining in auxiliary cells at the base of sensilla trichodea, basiconica, coeloconica, and chaetica. Immunocytochemistry confirmed the expression of the protein in sensilla chaetica. Our results support a role of semiochemical carriers for NPC2 proteins in insects and indicate such proteins as new targets for insecticide-free pest population control.

Functional characterization of a Niemann-Pick type C2 protein in the parasitoid wasp Microplitis mediator.

Niemann-Pick type C2 (NPC2) is a type of small soluble protein involved in lipid metabolism and triglyceride accumulation in vertebrates and arthropods. Recent studies have determined that NPC2 also participates in chemical communication of arthropods. In this work, two novel NPC2 proteins (MmedNPC2a and MmedNPC2b) in Microplitis mediator were identified. Real-time quantitative PCR (qPCR) analysis revealed that MmedNPC2a was expressed higher in the antennae than in other tissues of adult wasps compared with MmedNPC2b. Subsequent immunolocalization results demonstrated that NPC2a was located in the lymph cavities of sensilla placodea. To further explore the binding characterization of recombinant MmedNPC2a to 54 candidate odor molecules, a fluorescence binding assay was performed. It was found MmedNPC2a could not bind with selected fatty acids, such as linoleic acid, palmitic acid, stearic acid and octadecenoic acid. However, seven cotton volatiles, 4-ethylbenzaldehyde, 3,4-dimethylbenzaldehyde, β-ionone, linalool, m-xylene, benzaldehyde and trans-2-hexen-1-al showed certain binding abilities with MmedNPC2a. Moreover, the predicted 3D model of MmedNPC2a was composed of seven β-sheets and three pairs of disulfide bridges. In this model, the key binding residues for oleic acid in CjapNPC2 of Camponotus japonicus, Lue68, Lys69, Lys70, Phe97, Thr103 and Phe127, are replaced with Phe85, Ser86, His87, Leu113, Tyr119 and Ile143 in MmedNPC2a, respectively. We proposed that MmedNPC2a in M. mediator may play roles in perception of plant volatiles.