Expression of odorant-binding proteins and chemosensory proteins in some Hymenoptera

The expression of chemosensory proteins (CSPs) and odorant-binding proteins (OBPs) in individuals of different castes and ages have been monitored in three species of social hymenopterans, Polistes dominulus (Hymenoptera, Vespidae), Vespa crabro (Hymenoptera, Vespidae) and Apis mellifera (Hymenoptera, Apidae), using PCR with specific primers and polyclonal antibodies. In the paper wasp P. dominulus, OBP is equally expressed in antennae, wings and legs of all castes and ages, while CSP is often specifically present in antennae and in some cases also in legs. In the vespine species V. crabro CSP is antennal specific, while OBP is also expressed in legs and wings. The three CSPs and the five OBPs of A. mellifera show a complex pattern of expression, where both classes of proteins include members specifically expressed in antennae and others present in other parts of the body. These data indicate that at least in some hymenopteran species CSPs are specifically expressed in antennae and could perform roles in chemosensory perception so far assigned only to OBPs.     

Odorant-binding proteins and chemosensory proteins in pheromone detection and release in the silkmoth Bombyx mori

The genome of the silkmoth Bombyx mori contains 44 genes encoding odorant-binding proteins (OBPs) and 20 encoding chemosensory proteins (CSPs). In this work, we used a proteomic approach to investigate the expression of proteins of both classes in the antennae of adults and in the female pheromone glands. The most abundant proteins found in the antennae were the 4 OBPs (PBP, GOBP1, GOBP2, and ABP) and the 2 CSPs (CSP1 and CSP2) previously identified and characterized. In addition, we could detect only 3 additional OBPs and 2 CSPs, with clearly different patterns of expression between the sexes. Particularly interesting, on the other hand, is the relatively large number of binding proteins (1 OBP and 7 CSPs) expressed in the female pheromone glands, some of them not present in the antennae. In the glands, these proteins could be likely involved in the solubilization of pheromonal components and their delivery in the environment.     

花椒窄吉丁触角转录组及嗅觉相关基因分析

【目的】建立花椒窄吉丁Agrilus zanthoxylumi成虫触角转录组数据库,挖掘嗅觉相关基因,为今后研究其触角的化学感受机制及生物防控提供理论支撑。【方法】采用高通量测序平台IlluminaNovaSeq 6000对花椒窄吉丁雌雄成虫触角进行转录组测序,用Trinity软件对获得的高质量reads进行序列拼接与组装;使用BLAST软件将触角转录组数据比对NR, NT, Swiss-Prot, GO, KEGG, BLASTX,eggNOG, Pfam, TmHMM, SignalP, KO, Map, BLASTP和RNAMMER公共数据库;基于初步筛选到的花椒窄吉丁候选气味结合蛋白(odorant binding protein, OBP)和化学感受蛋白(chemosensory protein, CSP)以及其他鞘翅目昆虫的同源蛋白的核苷酸序列,利用MEGA软件进行系统进化分析。运用RPKM (reads perkilobase per million mapped reads)值对嗅觉相关基因表达量进行分析。【结果】花椒窄吉丁雌雄成虫触角转录组测序共获得36 209条基因和90 982条转录本,其N₅₀分别为2 103和2 523 bp,组装完整性较高。注释到NR数据库的基因最多(41.62%),其中与赤拟谷盗Tribolium castaneum相似基因所占比例最高(19%)。在GO数据库中比对到11 614个基因,按功能分为细胞组分、分子功能与生物学进程三大类57个分支,其中分子功能大类中的结合(70.57%)与催化活性(45.51%)相关基因占比最多;KEGG代谢途径分析表明,7 427条基因参与了5类代谢通路,其中涉及信号转导的基因最多,为815条;筛选到7个候选OBP基因和5个具有全长开放阅读框的CSP基因,其编码蛋白均具有化学感受蛋白家族的典型特征。系统进化分析表明,花椒窄吉丁OBPs和CSPs分别与苹果小吉丁A. mali的OBPs和CSPs氨基酸序列一致性最高。RPKM值表明,嗅觉相关基因AzanOBP1和AzanOBP2在雌成虫触角中不表达,在雄成虫触角中微量表达;AzanOBP3在雄成虫触角中高丰度表达。【结论】首次获得了花椒窄吉丁成虫触角转录组数据,筛选到了花椒窄吉丁OBP, CSP, Or, IR和SNMP等的嗅觉相关基因。推测触角中高丰度表达的OBPs对雄成虫识别同类异性释放的信息素或寄主植物释放的挥发物起关键作用。研究结果可为花椒窄吉丁化学感受基因功能分析及嗅觉感受机制研究奠定分子基础。     

Developmental expression of odorant‐binding proteins and chemosensory proteins in the embryos of Locusta migratoria

We have investigated the development of chemosensilla and the secretion of odorant‐binding proteins (OBPs) and chemosensory proteins (CSPs) in the embryo of Locusta migratoria manilensis. We first report the changes of each sensillum in embryo just preceding hatch in detail and show that different sensilla have different developmental processes. Trichogen cells are first involved in forming the structure of pegs, and then, after retraction, they start secreting OBPs and CSPs in the sensillar lymph. The synthesis of LmigOBP1 starts during the embryogenesis about 0.5 h preceding hatching, specifically in sensilla trichodea and basiconica of the antenna. LmigOBP2, instead, was only found in the outer sensillum lymph (oSl) of sensilla chaetica of the antenna, while we could not detect LmigOBP3 in any type of sensilla of the antenna. The ontogenesis of CSPs in the embryos is similar to that of OBPs. Expression of CSPI homolog in Locusta migratoria is detected using the antiserum raised against SgreCSPI. CSPI is specifically expressed in the outer sensillum lymph of sensilla chaetica of the antenna, and anti‐LmigCSPII dose not label any sensilla of the embryos. These data indicate that in locusts, OBPs and CSPs follow different temporal expression patterns, and also that OBPs are expressed in different types of sensilla. © 2009 Wiley Periodicals, Inc.     

Expression pattern analysis of odorant‐binding proteins in the pea aphid Acyrthosiphon pisum

Odorant‐binding proteins (OBPs) are soluble proteins mediating chemoreception in insects. In previous research, we investigated the molecular mechanisms adopted by aphids to detect the alarm pheromone (E)‐β‐farnesene and we found that the recognition of this and structurally related molecules is mediated by OBP3 and OBP7. Here, we show the differential expression patterns of 5 selected OBPs (OBP1, OBP3, OBP6, OBP7, OBP8) obtained performing quantitative RT‐PCR and immunolocalization experiments in different body parts of adults and in the 5 developmental instars, including winged and unwinged morphs, of the pea aphid Acyrthosiphon pisum. The results provide an overall picture that allows us to speculate on the relationship between the differential expression of OBPs and their putative function. The expression of OBP3, OBP6, and OBP7 in the antennal sensilla suggests a chemosensory function for these proteins, whereas the constant expression level of OBP8 in all instars could suggest a conserved role. Moreover, OBP1 and OBP3 are also expressed in nonsensory organs. A light and scanning electron microscopy study of sensilla on different body parts of aphid, in particular antennae, legs, mouthparts, and cornicles‐cauda, completes this research providing a guide to facilitate the mapping of OBP expression profiles.     

Intriguing similarities between two novel odorant-binding proteins of locusts

Two novel odorant-binding proteins (OBPs) of locust, LmigOBP2 and LmigOBP3 are very different from each other and from the previously reported LmigOBP1 in their amino acid sequences. Moreover, OBP3 contains three additional cysteines, a fact not previously recorded in standard length OBPs. However, these two proteins exhibit remarkably similar binding affinities to a set of organic compounds. Such behaviour is supported by three-dimensional models, showing very similar folding for LmigOBP2 and LmigOBP3, but clearly different for LmigOBP1. Also several amino acid residues lining the binding pockets of the three proteins appear conserved in LmigOBP2 and LmigOBP3, but not in LmigOBP1. Western blot experiments revealed the presence of LmigOBP2 in antennae, mouth parts and cerci, but could not detected LmigOBP3 in any of these tissues. In immunocytochemistry, antibodies against LmigOBP2 strongly stained the outer lymph of sensilla chaetica of the antennae, in contrast with LmigOBP1, previously reported in sensilla basiconica.     

A Proteomic Investigation of Soluble Olfactory Proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.     

Diverse Filters to Sense: Great Variability of Antennal Morphology and Sensillar Equipment in Gall-Wasps (Hymenoptera: Cynipidae)

Comparative studies on antennal sensillar equipment in insects are largely lacking, despite their potential to provide insights into both ecological and phylogenetic relationships. Here we present the first comparative study on antennal morphology and sensillar equipment in female Cynipoidea (Hymenoptera), a large and diverse group of wasps, with special reference to the so-called gall-wasps (Cynipidae). A SEM analysis was conducted on 51 species from all extant cynipoid families and all cynipid tribes, and spanning all known life-histories in the superfamily (gall-inducers, gall-inquilines, and non-gall associated parasitoids). The generally filiform, rarely clavate, antennal flagellum of Cynipoidea harbours overall 12 types of sensilla: s. placoidea (SP), two types of s. coeloconica (SCo-A, SCo-B), s. campaniformia (SCa), s. basiconica (SB), five types of s. trichoidea (ST-A, B, C, D, E), large disc sensilla (LDS) and large volcano sensilla (LVS). We found a great variability in sensillar equipment both among and within lineages. However, few traits seem to be unique to specific cynipid tribes. Paraulacini are, for example, distinctive in having apical LVS; Pediaspidini are unique in having ≥3 rows of SP, each including 6–8 sensilla per flagellomere, and up to 7 SCo-A in a single flagellomere; Eschatocerini have by far the largest SCo-A. Overall, our data preliminarily suggest a tendency to decreased numbers of SP rows per flagellomere and increased relative size of SCo-A during cynipoid evolution. Furthermore, SCo-A size seems to be higher in species inducing galls in trees than in those inducing galls in herbs. On the other hand, ST seem to be more abundant on the antennae of herb-gallers than wood-gallers. The antennal morphology and sensillar equipment in Cynipoidea are the complex results of different interacting pressures that need further investigations to be clarified.     

Three odorant-binding proteins are co-expressed in sensilla trichodea of Drosophila melanogaster

Odorant-binding proteins (OBPs) are small soluble proteins present in the aqueous medium surrounding olfactory receptor neurones. In this study we examine the expression patterns of three Drosophila OBPs (LUSH=OBP76a, OS-E=OBP83b and OS-F=OBP83a), using post-embedding immunocytochemistry. All three OBPs are co-expressed in sensilla trichodea whereas sensilla intermedia show co-expression of OS-E and OS-F only, but not of LUSH. Thus, it is confirmed that an individual sensillum can contain more than one OBP, even if it comprises only a single receptor neurone, such as the subtype T-1. In s. trichodea of lush⁻ mutants, expression of OS-E and OS-F is not impaired. No other sensillum type on antenna or maxillary palp (e.g. sensilla basiconica, sensilla coeloconica) expresses LUSH, OS-E or OS-F. Within the s. trichodea the three OBPs show the same labelling pattern: the extracellular sensillum lymph in the hair lumen and the sensillum-lymph cavities are heavily labelled. Intracellularly, the three OBPs are co-localised in a variety of dense granules in all auxiliary cells, and also in the receptor neurones. Immunocytochemical data from antennal sections of flies where lush gene expression has been tagged with the reporter gene lacZ suggest that LUSH is synthesised only in the trichogen and the thecogen cells. Thus, LUSH OBP is produced and secreted by two auxiliary cells, whereas its turnover and decomposition does not appear to be restricted to these auxiliary cells but may also occur in the tormogen and receptor cells. The immunocytochemical results are discussed with respect to current concepts of the function of odorant-binding proteins.     

木瓜榕传粉榕小蜂雌蜂触角感器的分布和超微形态

为探索木瓜榕传粉榕小蜂Ceratosolen emarginatus寄主定位机制, 应用扫描电镜和透射电镜观察了其雌蜂触角感器的类型、 分布和超微形态。结果显示： 木瓜榕传粉榕小蜂雌蜂触角呈膝状, 由柄节、 梗节和11个鞭小节组成的鞭节组成, 第3鞭小节着生一坚固的脊骨突。触角上共发现7类11种感器, 分别为毛形感器、 刺形感器、 锥形感器（包括单孔形和多孔形）、 多孔板形感器（包括长形和圆形）、 腔锥形感器（分为3种类型）、 栓锥形乳突状感器、 角锥形感器。结合表面特征和内部结构, 锥形感器、 多孔板形感器、 栓锥形乳突状感器和腔锥形感器类型1为有孔型, 为化学感器; 无孔型的毛形感器和刺形感器是机械感器, 但腔锥形感器类型2和3为本体感器或湿热压力感器; 最为特异的为角锥形感器, 其厚壁无孔, 逆向触角主轴, 为该科昆虫所特有, 推测可防止传粉榕小蜂进入榕果时滑脱。这些结果将有助于理解木瓜榕传粉榕小蜂特异性行为, 并为下一步开展电生理研究, 揭示其信息化学物质利用和分配模式奠定基础。     

伪鞘榕小蜂雌雄成虫触角感器的超微形态、分布及适生意义

伪鞘榕小蜂Sycoscapter trifemmensis是一种寄生于鸡嗉子榕间花期榕果的专性寄生蜂,雌雄两性繁殖策略分化明显,为更好理解和诠释雌蜂寄主定位和雄蜂配偶识别机制,有必要对两性的触角感器进行观察。运用环境扫描电镜观察,对比和探讨了伪鞘榕小蜂雌雄成虫的触角和触角感器类型、分布、数量及其生态适应性。结果表明:雌蜂触角鞭节由11鞭小节组成,总长817.82±33.23μm,分布有毛形感器、刺形感器(类型1)、锥形感器(类型1)、多孔板形感器(类型1)、栓锥形乳突状感器5类5种;雄蜂触角鞭节仅由6鞭小节组成,全长为雌蜂的1/3,且各节有明显的缩短和增粗特征,着生感器包括毛形感器、刺形感器(类型2和类型3)、锥形感器(类型1和类型2)、多孔板形感器(类型2)、腔锥形感器5类7种。雌蜂触角感器的数量与分布显著高于雄蜂,且同类型感器在雌蜂上具有明显的延伸、增粗、分支的特征,以板形感器和锥形感器最为突出。伪鞘榕小蜂雌雄成虫的触角及其感器有明显的性二型,特别是与化学信息识别相关的感器,反映了雌雄蜂在不同生态环境和繁殖压力下的形态分化、行为策略和生态适应。