Identification of amine receptors from a swallowtail butterfly, Papilio xuthus L.: cloning and mRNA localization in foreleg chemosensory organ for recognition of host plants

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. Some biogenic amine analogs have been characterized as oviposition stimulants. We have cloned three amine receptors, serotonin, tyramine, and dopamine, from cDNA derived from foreleg tarsus of P. xuthus, and determined structures of both cDNA and genomic genes. The phenylethylamine (tyramine and dopamine) receptors were expressed preferentially in brain and chemosensory organs. Moreover, we observed the localized expression of dopamine receptors at the base of tarsal chemosensilla by in situ hybridization. These results suggest that amine receptors in tarsal chemosensilla have a functional role in chemoreception for host plant recognition.     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

Purification and Characterization of Biliverdin-binding Protein from Larval Hemolymph of the Swallowtail Butterfly,Papilio xuthus L.

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The N-amino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.     

Multiple matings increase the fecundity of the yellow swallowtail butterfly,Papilio xuthus L., in summer generations

Females of the yellow swallowtail butterfly, Papilio xuthus,were reared in the laboratory. They were divided into four groups held under different mating conditions: nonmating (virgin) and mated once, twice, and three times. The number of eggs in the ovaries was counted by dissection. Virgin females produced increasing numbers of mature eggs, up to about 30, in the week following emergence. When the female had mated once, the number of mature eggs was significantly higher than that of virgin females by the second day after emergence. However, the double- and triplemated females did not increase the number of eggs in each state further than the singlemated females. The double-mated females deposited significantly more eggs than the singlemated females in the laboratory. The triplemated females also deposited more eggs on the day after the third mating than the doublemated females. Thus, multiple matings increased the number of eggs deposited. The change in the hatchability and the morphology of the spermatophore in the bursa copulatrix suggested that the sperm from the last mating had precedence.     

Purification and characterization of biliverdin-binding protein from larval hemolymph of the swallowtail butterfly, Papilio xuthus L

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The Namino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.     

柑橘凤蝶细胞克隆株RIRI-PX1-C24的生物学及重组蛋白表达特性

【目的】研究柑橘凤蝶Papilio xuthus细胞系RIRI-PX1的单细胞克隆株RIRI-PX1-C24的生物学和重组蛋白表达特性。【方法】用野生型苜蓿银纹夜蛾核型多角体病毒(wild-type Autographa californica multiple nucleopolyhedrosis virus, wt-AcMNPV)侵染RIRI-PX1-C24与RIRI-PX1,检测细胞对野生型病毒的敏感性;使用重组绿色荧光蛋白杆状病毒(AcMNPV-GFP)、重组β-半乳糖苷酶杆状病毒(AcMNPV-Gal)以及重组分泌型碱性磷酸酶杆状病毒(AcMNPV-SEAP)分别侵染细胞系RIRI-PX1-C24和RIRI-PX1,在之后的24, 48, 72, 96, 120, 144和168 h检测3种重组蛋白的表达量;通过简单重复序列区间(inter simple sequence repeat, ISSR)标记比较RIRI-PX1-C24与RIRI PX1的遗传相似性。【结果】亲本细胞系 RIRI-PX1和克隆株RIRI-PX1-C24均能被wt-AcMNPV侵染,其中RIRI-PX-C24对wt-AcMNPV的敏感性较RIRI-PX1有显著提高。3种重组蛋白均能在2个细胞系中表达,其中重组绿色荧光蛋白在RIRI-PX1-C24中的表达水平远高于在亲本细胞系RIRI-PX1中的表达水平,但重组β-半乳糖苷酶蛋白和重组分泌型碱性磷酸酶蛋白在RIRI-PX1-C24中的表达水平较在RIRI-PX1中的无显著提高。用10条ISSR引物进行的RIRI-PX1-C24和RIRI-PX1 2个细胞系的指纹图谱分析中,4条引物扩增条带在这2个细胞系间存在差异;2个细胞系的遗传相似性范围在0~83.33%之间,说明克隆株及其亲本细胞系在基因型上存在差异。【结论】通过对柑橘凤蝶细胞系RIRI-PX1进行单细胞克隆,确实获得了使重组绿色荧光蛋白表达水平有显著提高的克隆株RIRI-PX1-C24,其利用价值仍需进一步的研究。     

Structure and function of papiliocin with antimicrobial and anti-inflammatory activities isolated from the swallowtail butterfly, Papilio xuthus

Papiliocin is a novel 37-residue cecropin-like peptide isolated recently from the swallowtail butterfly, Papilio xuthus. With the aim of identifying a potent antimicrobial peptide, we tested papiliocin in a variety of biological and biophysical assays, demonstrating that the peptide possesses very low cytotoxicity against mammalian cells and high bacterial cell selectivity, particularly against Gram-negative bacteria as well as high anti-inflammatory activity. Using LPS-stimulated macrophage RAW264.7 cells, we found that papiliocin exerted its anti-inflammatory activities by inhibiting nitric oxide (NO) production and secretion of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2, producing effects comparable with those of the antimicrobial peptide LL-37. We also showed that the innate defense response mechanisms engaged by papiliocin involve Toll-like receptor pathways that culminate in the nuclear translocation of NF-κB. Fluorescent dye leakage experiments showed that papiliocin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of papiliocin in 300 mm dodecylphosphocholine micelles by NMR spectroscopy, showing that papiliocin has an α-helical structure from Lys(3) to Lys(21) and from Ala(25) to Val(36), linked by a hinge region. Interactions between the papiliocin and LPS studied using tryptophan blue-shift data, and saturation transfer difference-NMR experiments revealed that Trp(2) and Phe(5) at the N-terminal helix play an important role in attracting papiliocin to the cell membrane of Gram-negative bacteria. In conclusion, we have demonstrated that papiliocin is a potent peptide antibiotic with both anti-inflammatory and antibacterial activities, and we have laid the groundwork for future studies of its mechanism of action.     

Regulatory mechanisms in phenotypic plasticity of diapause and nondiapause pupal colouration of the swallowtail butterfly Papilio machaon

Nondiapause pupae of Papilio machaon L. exhibit pupal colour diphenism comprising green–yellow and brown–white types. To understand the regulatory mechanism underlying the control of pupal colouration in P. machaon, the effect of environmental cues on diapause and nondiapause pupal colouration is investigated. When larvae reared under short‐day and long‐day conditions are allowed to pupate in sites with a smooth surface and a yellow background colour, all diapause pupae exhibit a brown–white type and 89.5% of nondiapause pupae exhibit a green–yellow type, respectively. With rough‐surface pupation sites, all diapause pupae exhibit brown–white and intermediate types, whereas a large proportion of nondiapause pupae exhibit brown–white and intermediate types, although some exhibit a green–yellow type. When extracts prepared from the head‐thoracic and thoracic‐abdominal regions of larval central nervous systems are injected into the ligated abdomens of P. machaon short‐day pharate pupae, all recipients exhibit a brown–white colouration. Furthermore, when each extract is injected into the ligated abdomen of Papilio xuthus L. short‐day pharate pupae with orange‐pupa‐inducing factor activity, recipients injected with the head‐thoracic extract exhibit the brown type, whereas those injected with the thoracic‐abdominal extract exhibit an orange colour. The results indicate that the response to the environmental cues of pupation site in P. machaon changes according to the photoperiodic conditions experienced during larval stages, and that at least two hormonal factors producing brown–white pupae are located in the larval central nervous system, with the secretion of these factors being regulated by the recognition of environmental cues in long‐day larvae.     

Identification of cytochrome P450 and glutathione-S-transferase genes preferentially expressed in chemosensory organs of the swallowtail butterfly, Papilio xuthus L

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. We undertook an expressed sequence tag (EST) analysis to identify the chemosensory-related genes that are expressed in chemosensilla on the tarsus of P. xuthus. Several genes that showed similarity with biotransformation enzymes were identified from the ESTs. Among them, a cytochrome P450 and a glutathione-S-transferase (GST) were preferentially expressed in the chemosensory organs. We have determined the structure of both cDNA and genomic sequences encoding these enzymes and designated the P450 as CYP341A2, a novel member of CYP341A subfamily, and the GST as GST-pxcs1, respectively. We observed a localized expression of CYP341A2 at the base of tarsal chemosensilla by in situ hybridization. These results suggest that these degrading enzymes play a role in the chemosensory reception for host plant recognition.     

Identification and genomic structure of chemosensory proteins (CSP) and odorant binding proteins (OBP) genes expressed in foreleg tarsi of the swallowtail butterfly Papilio xuthus

Chemoreception is a key feature for selection of host plants by phytophagous insects. Female swallowtail butterflies recognize their host plants using chemosensilla present on foreleg tarsi. We constructed a cDNA library of female tarsi and a genome library of Papilio xuthus. We identified 11 chemosensory protein (CSP) genes and three odorant binding proteins (OBP) genes from the cDNA library and eight additional CSP genes from the genome library using the ESTs as probes. A sequence similarity tree of insect CSPs showed that lepidopteran CSPs constructed big branches of the order. Small numbers of CSPs have been identified from the whole genomes of several insect orders which belong to branches separated from those of Lepidoptera. The CSP gene family of Lepidoptera may have diverged in at least two steps, the first on a small scale and the second on a large scale before and after the diversification of insect orders, respectively. Seventeen of 19 CSP genes of P. xuthus clustered in a specific region of the genome, suggesting that they were diversified by gene duplication from a common ancestral gene.     

Tarsal contact chemoreceptors of the black swallowtail butterfly Papilio polyxenes: responses to phytochemicals from host- and non-host plants

Abstract Tarsal contact chemoreceptors of the black swallowtail butterfly Papilio polyxenes (Lepidoptera: Papilionidae) were stimulated with leaf-surface extracts and ethanolic extracts of whole leaves of a host-plant (Daucus carota) and a non-host (Brassica oleracea). Both leaf extracts evoked large numbers of spikes but stimulated different receptor neurones. It is concluded that a large difference exists between the sensory responses to host and non-host extracts. Two chemicals, luteolin 7-0-(6"-0-malonyl)-β-D-glucopyranoside and trans- chlorogenic acid, known to be present in the host and known oviposi-tion stimulants for P.polyxenes , were also tested and shown to be active. No responses were found to luteolin 7-O-β-D-glucoside or to luteolin 7-O-β-D-glucuronide. These flavonoids occur in D.carota foliage, but do not stimulate oviposition.     

Molecular and functional characterization of three odorant‐binding proteins from the wheat blossom midge,Sitodiplosis mosellana

Sitodiplosis mosellana, a periodic but devastating wheat pest, relies on wheat spike volatiles as a cue in selecting hosts for oviposition. Insect odorant‐binding proteins (OBPs) are thought to play essential roles in filtering, binding and transporting hydrophobic odorant molecules to specific receptors. To date, the molecular mechanisms underlying S. mosellana olfaction are poorly understood. Here, three S. mosellana antenna‐specific OBP genes, SmosOBP11, 16 and 21, were cloned and bacterially expressed. Binding properties of the recombinant proteins to 28 volatiles emitted from wheat spikes were investigated using fluorescence competitive binding assays. Sequence analysis suggested that these SmosOBPs belong to the Classic OBP subfamily. Ligand‐binding analysis showed that all three SmosOBPs preferentially bound alcohol, ester and ketone compounds, and SmosOBP11 and 16 also selectively bound terpenoid compounds. In particular, the three SmosOBPs had high binding affinities (K_i < 20 μmol/L) to 3‐hexanol and cis‐3‐hexenylacetate that elicited strong electroantennogram (EAG) response from female antennae. In addition, SmosOBP11 displayed significantly higher binding (K_i < 8 μmol/L) than SmosOBP16 and 21 to 1‐octen‐3‐ol, D‐panthenol, α‐pinene and heptyl acetate which elicited significant EAG response, suggesting that SmosOBP11 plays a major role in recognition and transportation of these volatiles. These findings have provided important insight into the molecular mechanism by which S. mosellana specifically recognizes plant volatiles for host selection, and have facilitated identification of effective volatile attractants that are potentially useful for pest monitoring and trapping.     

Molecular characterization of iron binding proteins, transferrin and ferritin heavy chain subunit, from the bumblebee Bombus ignitus

Transferrin and ferritin are iron-binding proteins involved in transport and storage of iron as part of iron metabolism. Here, we describe the cDNA cloning and characterization of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH), from the bumblebee Bombus ignitus. Bi-Tf cDNA spans 2340 bp and encodes a protein of 706 amino acids and Bi-FerHCH cDNA spans 1393 bp and encodes a protein of 217 amino acids. Comparative analysis revealed that Bi-Tf appears to have residues comprising iron-binding sites in the N-terminal lobe, and Bi-FerHCH contains a 5'UTR iron-responsive element and seven conserved amino acid residues associated with a ferroxidase center. The Bi-Tf and Bi-FerHCH cDNAs were expressed as 79 kDa and 27 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that Bi-Tf exhibits fat body-specific expression and Bi-FerHCH shows ubiquitous expression. The expression profiles of the Bi-Tf and Bi-FerHCH in the fat body of B. ignitus worker bees revealed that Bi-Tf and Bi-FerHCH are differentially induced in a time-dependent manner in a single insect by wounding, bacterial challenge, and iron overload.     

Molecular characterization,expression pattern and ligand‐binding properties of the pheromone‐binding protein gene from Cyrtotrachelus buqueti

Pheromone‐binding proteins (PBPs) play important roles in the information exchange between insect sexes, specifically in the process of transporting fat‐soluble odour molecules from the external environment to olfactory receptors through the olfactory sensillum lymph. The PBP functions in this process may explain the sex pheromone identification mechanism used by insects, laying a theoretical foundation for the prevention and control of pests by interfering with olfactory recognition. In the present study, a PBP gene of Cyrtotrachelus buqueti (GenBank accession number: KU845733) is cloned for prokaryotic expression. Using N‐phenyl‐1‐naphthylamine as the fluorescent probe in a competitive binding assay, the ability of CbuqPBP1 to bind 12 sex pheromone analogues and three volatiles of Neosinocalamus affinis shoots is examined. Of the 12 C. buqueti sex pheromone analogues, dibutyl phthalate gives the greatest displacement (inhibitory constant value of 11.1 μm ), whereas the other sex pheromone components show much smaller displacements. Consistent with other PBPs, the three plant volatiles (linalool, benzaldehyde and indole) show only a limited displacement of CbuqPBP1. However, the binding abilities of 1 : 1 ratios of each of the three plant volatiles with dibutyl phthalate show increases of 62.3%, 65.1% and 51.7% over the binding abilities of the three plant volatiles alone. CbuqPBP1 has dual roles in the processes of sensing sex pheromones and plant volatiles.     

Molecular modeling of the binding modes of the iron‐sulfur protein to the Jac1 co‐chaperone from Saccharomyces cerevisiae by all‐atom and coarse‐grained approaches

The iron‐sulfur protein 1 (Isu1) and the J‐type co‐chaperone Jac1 from yeast are part of a huge ATP‐dependent system, and both interact with Hsp70 chaperones. Interaction of Isu1 and Jac1 is a part of the iron‐sulfur cluster biogenesis system in mitochondria. In this study, the structure and dynamics of the yeast Isu1–Jac1 complex has been modeled. First, the complete structure of Isu1 was obtained by homology modeling using the I‐TASSER server and YASARA software and thereafter tested for stability in the all‐atom force field AMBER. Then, the known experimental structure of Jac1 was adopted to obtain initial models of the Isu1–Jac1 complex by using the ZDOCK server for global and local docking and the AutoDock software for local docking. Three most probable models were subsequently subjected to the coarse‐grained molecular dynamics simulations with the UNRES force field to obtain the final structures of the complex. In the most probable model, Isu1 binds to the left face of the Γ‐shaped Jac1 molecule by the β‐sheet section of Isu1. Residues L₁₀₅, L₁₀₉, and Y₁₆₃ of Jac1 have been assessed by mutation studies to be essential for binding (Ciesielski et al., J Mol Biol 2012; 417:1–12). These residues were also found, by UNRES/molecular dynamics simulations, to be involved in strong interactions between Isu1 and Jac1 in the complex. Moreover, N₉₅, T₉₈, P₁₀₂, H₁₁₂, V₁₅₉, L₁₆₇, and A₁₇₀ of Jac1, not yet tested experimentally, were also found to be important in binding. Proteins 2015; 83:1414–1426. © 2015 Wiley Periodicals, Inc.     

Expression pattern and ligand‐binding properties of odorant‐binding protein 13 from Monochamus alternatus hope

Odorant‐binding proteins (OBPs) are believed to play an important role in olfactory recognition. In this study, expression pattern and fluorescence binding characteristics of MaltOBP13 from the Japanese pine sawyer beetle, Monochamus alternatus Hope, were investigated via qPCR analysis of MaltOBP13 mRNA level and binding assay of MaltOBP13 and ligands. qPCR monitoring indicated MaltOBP13 mainly expressed in newly emerged males, particularly highly expressed in the last abdominal segment of males, and the expression level was significantly higher in 13‐day‐old mated adults than those of other stages. To further understand the function of the MaltOBP13 protein in odorant reception, the binding affinity of recombinant MaltOBP13 to ligands was tested by fluorescence binding assays with N‐phenyl‐1‐naphthylamine as a fluorescent probe. The results of this assay indicated that MaltOBP13 exhibited a high binding affinity for pine volatiles and binding capacity was higher in acidic conditions than in neutral environment, indicating a possible role in finding host plants.