应用3D打印技术辅助识别昆虫脑解剖结构

【目的】明确烟芽夜蛾Heliothis virescens雄成虫脑的结构,构建和打印脑的三维模型,并将该技术扩展应用到黑腹果蝇Drosophila melanogaster、西方蜜蜂Apis mellifera和沙漠蝗Schistocerca gregaria上,制作这些模式昆虫脑的3D打印模型。【方法】首先采用突触蛋白抗体免疫组织化学染色标记的方法研究烟芽夜蛾雄成虫脑的结构,利用激光扫描共聚焦显微镜获取脑解剖结构图像,利用图形分析软件创建三维脑模型,并利用3D打印技术进行三维图像打印。【结果】鉴定出烟芽夜蛾雄成虫脑及颚神经节、触角叶、视叶、前视结节、中央体和蕈形体等主要神经髓结构,并构建了三维数字化模型。首次成功应用3D打印技术打印了烟芽夜蛾雄成虫脑的三维数字化模型,获得实体模型。并将该技术进一步应用到黑腹果蝇、西方蜜蜂和沙漠蝗,获得了这些昆虫的脑实体模型。基于脑模型,对这些昆虫的味觉中枢、嗅觉中枢、视觉中枢和学习及记忆中枢等神经髓结构做了系统比较。【结论】3D打印模型为脑解剖结构的观察提供了新形式,并提供了便利工具。3D打印所得脑实体模型大小适中,可以放在手中,任意旋转,从不同角度观察昆虫脑不规则结构的形态、位置和空间关系,也便于比较不同昆虫脑结构异同,加深对昆虫脑结构和功能及其演化的认识。     

茶尺蠖幼虫脑的解剖结构

【目的】明确茶尺蠖Ectropis obliqua Prout 5龄幼虫脑解剖结构,并分析和构建幼虫脑以及脑内部各神经髓结构的三维结构模型。【方法】采用免疫组织化学方法,利用突触蛋白抗体,染色标记脑内神经突触,定位突触联系密集分布的区域,获得脑内部神经髓的结构。利用激光共聚焦显微镜获取脑扫描图像,然后利用三维图像分析软件AMIRA进行图像分析,构建脑的三维结构模型,并计算脑以及脑内各神经髓结构的体积。【结果】突触蛋白抗体染色显示,茶尺蠖5龄幼虫脑内具有很多神经突触联系密集分布的区域,这些不同区域即为脑的不同神经髓结构。茶尺蠖幼虫脑主要包括前脑、中脑和后脑3个组成部分。其中前脑最大,包括成对的视叶、蕈形体、前脑桥和侧副叶以及不成对的中央体。视叶位于前脑的两侧后端。蕈形体位于脑半球正中间位置。侧副叶在中央体的下前方两侧。中央体在脑的正中心。前脑桥在中央体的上方后侧。除这些形态结构明显的神经髓区域外,前脑还包括大量内部边界不明显的神经髓区域,位于前脑左右两侧以及背侧和腹侧,这些区域被总称为中间脑,占整个脑神经髓的66%。触角叶为中脑的主要组成部分,在脑的下部最前端,为一对球状结构。后脑在脑的腹侧和触角叶下方,即围咽神经索进入脑的入口处。【结论】构建了茶尺蠖5龄幼虫脑以及各神经髓结构三维模型,分析了脑内各个神经髓之间的空间位置关系,明确了各神经髓的体积。茶尺蠖幼虫脑体积小而且结构简单的特征与其幼期视觉、嗅觉等感觉器官不发达、活动能力弱、行为简单的生物学习性相对应。     

绿盲蝽腹神经节的解剖结构

【目的】揭示绿盲蝽Apolygus lucorum腹神经节的组成结构。【方法】采用免疫组织化学染色方法,利用突触蛋白抗体对绿盲蝽成虫的腹神经节进行免疫标记,激光共聚焦扫描显微镜扫描照相获得原始数据,用图像分析软件进行标记,构建三维结构模型。【结果】绿盲蝽成虫腹神经节位于腹神经索的末端,与其前方的后胸神经节和中胸神经节紧密融合,形成后部神经节。与脑和胸神经节类似,腹神经节由周围的细胞体和内部的神经髓构成。腹神经节的神经纤维束主要包括位于腹侧的两条纵向神经连索和向两侧发出的9束神经纤维。9束神经纤维连接着9个神经原节,即富含突触联系的神经髓。这些神经原节紧密融合,无明显的边界,最后两节形成膨大的末端腹神经节。两侧的神经原节由横向的神经连锁连接起来。腹神经节外周的细胞体数量较多,排列紧密,大小一致,仅在前端背侧中间和后端腹侧中间位置分别有2个和5个体积较大的细胞体。【结论】本研究结果明确了绿盲蝽腹神经节的结构,为进一步研究昆虫的行为调控及神经系统发育和演化奠定一定的形态学基础。     

棉铃虫幼虫脑和咽下神经节的三维结构构建

【目的】解剖棉铃虫Helicoverpa armigera (Hübner) 5龄幼虫脑和咽下神经节及其内部神经髓形态结构,并分析和构建幼虫脑和咽下神经节以及各神经髓的三维结构模型。【方法】采用免疫组织化学方法解剖脑和咽下神经节的内部神经髓结构,利用激光共聚焦显微镜获取脑和咽下神经节扫描图像,然后利用AMIRA 三维图像分析软件进行图像分析,从而构建脑和咽下神经节的三维结构模型,并测量脑和咽下神经节以及内部各神经髓的体积,并分析了相对比例。【结果】 棉铃虫5龄幼虫脑和咽下神经节由围咽神经索连接在一起。脑主要由前脑、中脑和后脑3部分组成。前脑内包括视叶、蕈形体和中央体等形态结构较明显的神经髓。此外,前脑还包括其他位于脑的左右两侧以及背侧和腹侧大量神经髓区域,约占脑总神经髓的59.65%。这些神经髓区域边界不明显。中脑主要包括1对触角叶;后脑位于脑的腹侧和触角叶的下方,体积较小。咽下神经节由3个神经节融合构成,从前到后分别为上颚神经节、下颚神经节和下唇神经节,由于融合的紧密程度高,3个神经节间的边界不明显。【结论】阐明了棉铃虫5龄幼虫脑和咽下神经节的神经髓形态结构,构建了脑和咽下神经节以及内部神经髓的三维结构模型。三维模型可以任意旋转,能从任何角度观察脑、咽下神经节和内部不同神经髓的结构及其它们之间的空间关系。本研究结果对研究棉铃虫脑和咽下神经节信息接收、处理及调控行为的机制奠定了解剖学基础。     

绿盲蝽中枢神经系统的解剖结构

【目的】阐述绿盲蝽Apolygus lucorum中枢神经系统的组成,辨识各组成部分的神经节解剖结构及其形态,计算中枢神经系统各神经节结构体积大小、解析其空间分布关系以及连接模式。【方法】采用免疫组织化学方法,使用突触蛋白抗体对绿盲蝽中枢神经系统神经髓进行染色标记,利用共聚焦激光扫描显微镜获取中枢神经系统各结构数码图像,使用三维图像分析软件对绿盲蝽中枢神经系统进行分析,并构建三维模型。【结果】绿盲蝽中枢神经系统从前往后分别由脑神经节、咽下神经节、前胸神经节和后部神经节组成。脑、咽下神经节和前胸神经节3个神经节融合在一块,形成脑-咽下神经节-前胸神经节复合体,并通过长的神经连索与后部神经节相连,从外观上看似由2个大的神经节构成,这种神经节愈合形式尚未在昆虫中发现过。前胸神经节与后部神经节分离,二者由长的神经连索连接起来。除前胸神经节由单独的神经原节构成外,其他3个神经节又由多个神经原节融合而成。脑包括前脑、中脑和后脑3部分。咽下神经节包括上颚神经节、下颚神经节和下唇神经节。后部神经节包括中胸、后胸和腹部神经节3部分。【结论】明确了绿盲蝽中枢神经系统的神经节构成,发现了绿盲蝽中枢神经系统各神经节的高度融合特性。该项研究结果为研究绿盲蝽中枢神经系统的发育、重塑和系统演化奠定了形态学基础,为研究中枢神经元形态、分布以及其对昆虫生理和行为的功能调控机制提供了结构框架。     

5-羟色胺在粘虫视叶中的分布

【目的】解剖分析粘虫Mythimna separata成虫视叶的结构,研究5-羟色胺(5-hydroxytryptamine,5-HT)在视叶中的分布。【方法】采用组织包埋切片技术和免疫组织化学方法,使用突触蛋白抗体标记粘虫成虫视叶内神经髓结构,用抗5-HT血清标记5-HT;利用激光共聚焦扫描显微镜照相获取数码图像,利用图形分析软件识别神经髓结构及进行细胞体群分组与计数。【结果】粘虫成虫的视叶由视神经节层、视髓、副视髓、视小叶和视小叶板5个神经髓结构组成。免疫染色显示粘虫视叶内具有5-HT,每个视叶内约具有40个5-HT免疫标记的细胞体,这些细胞体分为3个细胞体群,位于视髓前方或腹面的内侧。视叶内所有的神经髓结构均含有5-HT免疫标记神经纤维。视神经节层的5-HT免疫标记神经纤维来自视叶的切向神经元,视髓的5-HT免疫标记神经纤维主要来自视叶的切向神经元、远心神经元和无长突神经元。视髓呈明显分层现象,主要分为3层,其中中间一层5-HT免疫标记神经纤维较密集。副视髓存在少量的5-HT免疫标记神经纤维。视小叶和视小叶板中的5-HT免疫标记神经纤维来自远心神经元,分为明显的2层,其中有少量神经纤维投射至视髓,将视小叶、视小叶板和视髓连接起来。【结论】粘虫视叶中广泛分布着5-HT免疫标记神经纤维。该研究结果为进一步研究5-HT在视觉机制中的作用奠定一定的解剖学基础。     

昆虫脑神经髓结构图谱构建方法综述

脑控制和调节所有动物的行为,昆虫也不例外,构建脑神经髓结构图谱有利于阐明其对行为调控的神经机制。目前,除一些模式昆虫的脑图谱被构建外,大多数昆虫仅针对少数易识别的神经髓(如视叶、触角叶和蕈形体等)进行了三维重建,而对脑内大部分区域还未描述,这与其结构的复杂性有关。随着共聚焦显微成像和计算机三维重建技术的发展,人们有机会获取全脑的结构信息,并构建出三维可视化的图谱,这为研究全脑神经髓的功能提供了重要平台。特别是果蝇全脑神经髓的系统命名法的建立,极大的推动了昆虫脑神经髓结构的研究进展。本文对昆虫脑的结构组成、免疫染色方法、标准脑构建及脑神经髓命名等方面进行综述,提出在构建脑神经髓图谱过程中需要注意的问题及解决办法,这将为推动国内昆虫脑神经髓图谱的构建提供参考。     

桔小实蝇视叶结构特征及5-羟色胺能神经元的分布

桔小实蝇是重要的果蔬害虫,它对不同颜色的光表现出不同的趋性。为了明确其视觉感受的结构基础,本研究采用免疫组织化学染色技术结合激光共聚焦成像分析了桔小实蝇成虫视叶内神经髓结构组成和体积大小,并利用5-羟色胺(5-hydroxytryptamine,5-HT)抗体标记了视叶内5-羟色胺能神经元,研究了其在视叶内的分布特征及细胞体数量。结果表明,桔小实蝇成虫的视叶由视神经节层、视髓、副视髓、视小叶和视小叶板5个神经髓结构组成,其中雌成虫的视髓相对体积极显著的大于雄虫的视髓相对体积。桔小实蝇每个视叶中包含12个5-HT能神经元细胞体,位于视髓的腹内侧,副视髓的前方。视叶5个神经髓区均含5-HT能神经纤维,但它们的神经纤维来自不同的神经元。对视叶神经髓结构及5-HT能神经元分布特征的研究将为未来构建桔小实蝇视觉神经通路和阐明5-HT对视觉感受的调控机制奠定解剖学基础。     

利用单感器记录与神经元示踪结合对棉铃虫主要性信息素感器内神经元投射的鉴定

【目的】鉴定雄性棉铃虫Helicoverpa armigera成虫触角性信息素感器嗅觉受体神经元的功能、形态及中枢投射路径。【方法】利用单感器记录技术记录棉铃虫嗅觉受体神经元对性信息素的反应,同时采用荧光染料作为示踪剂染色标记嗅觉受体神经元;使用免疫组织化学方法处理相应的脑组织,标记脑内触角叶的神经纤维球结构;用激光扫描共聚焦显微镜获取图像数据,使用图形软件ZEN和Amira 4.1.1进行三维结构重建。【结果】记录到雄性棉铃虫成虫触角上长毛形感器对主要性信息素成分Z11-16∶Ald产生明显的电生理反应,并成功染色标记了该感器内的嗅觉受体神经元。染色标记显示该感器内具有两个嗅觉受体神经元,其轴突通过触角神经分别投射触角叶内的云状体神经纤维球和普通神经纤维球。【结论】单感器记录与神经元示踪两技术结合能够用于鉴定昆虫触角嗅觉受体神经元的功能、形态和投射至神经纤维球的路径。与赖氨酸钴方法比较,使用荧光染料法进行神经元示踪,操作更简便,且易于进行三维空间分析,为调查棉铃虫其他嗅觉神经元的投射路径以明确外周气味受体感受与中枢系统的联系提供了有力技术支持。     

刺螫库蠓成虫内部器官系统组织学观察

【目的】刺螫库蠓Culicoides punctatus是一种重要媒介蠓虫,是施马伦贝格病毒(Schmallenberg virus, SBV)的主要传播载体。通过石蜡切片和苏木素 伊红染色技术观察刺螫库蠓成虫内部器官系统的组织结构。【方法】利用网扫法、灯诱法采集刺螫库蠓成虫,除足和翅外置于Duboscq-Brasil固定液中常温固定,然后经过逐级脱水、二甲苯透明、石蜡包埋、连续切片以及苏木素-伊红染色等过程制成玻片,利用光学显微镜进行观察和拍照。【结果】刺螫库蠓成虫消化系统不存在性别差异,由消化道及唾液腺组成。中枢神经系统由脑和腹神经索组成。脑可划分为前脑、中脑和后脑3个功能区。复眼和触角是刺螫库蠓主要的感觉器官。腹神经索可分为咽下神经节、胸神经节和腹神经节。呼吸系统主要由气管组成,气管遍布全身,无肺组织,胸部具有2对气门,分别位于中胸和后胸;腹支囊组织呈泡状,着色不明显。生殖系统包括内生殖器官和外生殖器官,其中雌性内生殖器官包括卵巢、输卵管、受精囊及生殖附腺,雄性内生殖器官包括精巢、输精管、射精管及生殖附腺。【结论】本研究明确了刺螫库蠓成虫的消化系统、神经系统、呼吸系统、生殖系统以及感觉器官的结构特征,为库蠓的发育研究提供了更为直接、准确的证据,有助于提高蠓媒监测、预报及防制的全面性和精确性。     

Insect myotropic peptides: differential distribution of locustatachykinin- and leucokinin-like immunoreactive neurons in the locust brain

Locustatachykinin I is one of four closely related myotropic neuropeptides isolated from brain and corpora-cardiaca complexes of the locust Locusta migratoria. Antiserum was raised against locustatachykinin I for use in immunocytochemistry. It was found that the antiserum recognizes also locustatachykinin II and hence probably also the other two locustatachykinins due to their similarities in primary structure. Locustatachykinin-like immunoreactive (LomTK-LI) neurons were mapped in the brain of the locust, L. migratoria. A total of approximately 800 Lom TK-LI neurons were found with cell bodies distributed in the proto-, deutoand tritocerebrum, in the optic lobes and in the frontal ganglion. Processes of these neurons innervate most of the synaptic neuropils of the brain and optic lobes, as well as the frontal ganglion and hypocerebral ganglion. The widespread distribution of LomTK-LI neurons in the locust brain indicates an important role of the locustatachykinins in signal transfer or regulation thereof. As a comparison neurons were mapped with an antiserum against the cockroach myotropic peptide leucokinin I. This antiserum, which probably recognizes the native peptide locustakinin, labels a population of about 140 neurons distinct from the LomTK-LI neurons (no colocalized immunoreactivity). These neurons have cell bodics that are distributed in the proto- and tritocerebrum and in the optic lobe. The processes of the leucokinin-like immunoreactive (LK-LI) neurons do not invade as large areas in neuropil as the Lom TK-LI neurons do and some neuropils, e.g. the mushroom bodies, totally lack innervation by LK-LI fibers. In some regions, however, the processes of the Lom TK-LI and LK-LI neurons are superimposed: most notably in the central body and optic lobes. A functinal relation between the two types of neuropeptide in the locust brain can, however, not be inferred from the present findings.     

Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster

Summary The larval and early pupal development of the optic lobes in Drosophila is described qualitatively and quantitatively using [³H]thymidine autoradiography on 2-m plastic sections. The optic lobes develop from 30–40 precursor cells present in each hemisphere of the freshly hatched larva. During the first and second larval instars, these cells develop to neuroblasts arranged in two epithelial optic anlagen. In the third larval instar and in the early pupa these neuroblasts generate the cells of the imaginal optic lobes at discrete proliferation zones, which can be correlated with individual visual neuropils.The different neuropils as well as the repetitive elements of each neuropil are generated in a defined temporal sequence. Cells of the medulla are the first to become postmitotic with the onset of the third larval instar, followed by cells of the lobula complex and finally of the lamina at about the middle of the third instar. The elements of each neuropil connected to the most posterior part of the retina are generated first, elements corresponding to the most anterior retina are generated last.The proliferation pattern of neuroblasts into ganglion mother cells and ganglion cells is likely to include equal as well as unequal divisions of neuroblasts, followed by one or two generations of ganglion mother cells. For the lamina the proliferation pattern and its temporal coordination with the differentiation of the retina are shown.     

Neurobiology of the scallop. II. structure of the parietovisceral ganglion lateral lobes in relation to afferent projections from the mantle eyes

The lateral lobes of the scallop parietovisceral ganglion have been examined morphologically with respect to their functional role as optic lobes. The gross morphology of the lateral lobe and projections of optic nerve fibers within it were investigated by 1) supravital methylene blue staining, and 2) autoradiography using tritiated proline injected intraocularly for incorporation and transport by the optic fibers. Ultrastruc‐turally, the lateral lobe was examined using standard electron microscopic techniques. The lateral lobe is composed of a cortical rind of cells, 8–15 μm in diameter at the ventral surface and 15–20 μm in diameter at the ventral surface, surrounding a central neuropil. The neuropil contains three distinct regions: 1) the glomerular neuropil, a series of densely staining spherical subunits associated with the eyes and pallial nerves, 2) the subcellular neuropil, a synaptic region adjacent to the ventral cell layer also having a visual function, and 3) the subglomerular neuropil, the remaining, rather unspecialized neuropil of the lateral lobe. Synaptic profiles with symmetrical membrane thickenings, a 32 nm synaptic cleft, and three types of vesicles are seen throughout the neuropil, although the density of synapses is greater in the glomerular region. Clear, dense core and neurosecretory vesicles are seen individually or as mixed populations in the presynaptic terminals. Autoradiographic experiments have revealed that optic fibers enter the lateral lobe and project directly to the subcellular neuropil where they synapse with cells located on the ventral surface of the lateral lobe cells. These cells in turn form the dense glomerular structures previously identified as visual association centers and send efferent fibers into the pallial nerves. The projection of optic fibers to the ventral surface of the lobe is consistent with previous electrophysiological recordings of visual activity at this site.     

Insect optic lobe neurons identifiable with monoclonal antibodies to GABA

Five monoclonal antibodies against GABA were tested on glutaraldehyde fixed sections of optic lobes of three insect species, blowflies, houseflies and worker bees. The specificity of these antibodies was analyzed in several tests and compared with commercially available anti-GABA antiserum. A very large number of GABA-like immunoreactive neurons innervate all the neuropil regions of these optic lobes. Immunoreactive processes are found in different layers of the neuropils. The immunoreactive neurons are amacrines and columnar or noncolumnar neurons connecting the optic lobe neuropils. In addition some large immunoreactive neurons connect the optic lobes with centers of the brain. Some neuron types could be matched with neurons previously identified with other methods. The connections of a few of these neuron types are partly known from electron microscopy or electrophysiology and a possible role of GABA in certain neural circuits can be discussed.     

Insect optic lobe neurons identifiable with monoclonal antibodies to GABA

Summary Five monoclonal antibodies aginst GABA were tested on glutaraldehyde fixed sections of optic lobes of three insect species, blowflies, houseflies and worker bees. The specificity of these antibodies was analyzed in several tests and compared with commercially available anti-GABA antiserum.A very large number of GABA-like immunoreactive neurons inncrvate all the neuropil regions of these optic lobes. Immunoreactive processes are found in different layers of the neuropils. The immunoreactive neurons are amacrines and columnar or noncolumnar neurons connecting the optic lobe neuropils. In addition some large immunoreactive neurons connect the optic lobes with centers of the brain.Some neuron types could be matched with neurons previously identified with other methods. The connections of a few of these neuron types are partly known from electron microscopy or electrophysiology and a possible role of GABA in certain neural circuits can be discussed.     

Brain organization and the origin of insects: an assessment

Within the Arthropoda, morphologies of neurons, the organization of neurons within neuropils and the occurrence of neuropils can be highly conserved and provide robust characters for phylogenetic analyses. The present paper reviews some features of insect and crustacean brains that speak against an entomostracan origin of the insects, contrary to received opinion. Neural organization in brain centres, comprising olfactory pathways, optic lobes and a central neuropil that is thought to play a cardinal role in multi-joint movement, support affinities between insects and malacostracan crustaceans.     

烟夜蛾性肽受体基因的克隆与表达模式分析

【目的】克隆烟夜蛾Helicoverpa assulta (Guenée)性肽受体基因并分析其表达模式, 为深入研究性肽与交配后反应的关系奠定基础。【方法】采用RT-PCR方法, 从烟夜蛾雌蛾性信息素腺体中得到性肽受体基因cDNA全序列。利用荧光定量PCR方法, 分析该基因的表达模式。【结果】序列分析结果显示, 烟夜蛾性肽受体基因cDNA全长2 048 bp, 命名为HassSPR（GenBank登录号: AFH53182.1）。该基因的开放阅读框长1 275 bp, 编码424个氨基酸残基, 序列中含有7个跨膜域结构, 预测分子量和等电点分别为48.6 kDa和9.25。序列比对分析表明, HassSPR与近缘种棉铃虫H. armigera和其他蛾类性肽受体的氨基酸序列一致性分别达98.35%和超过84%, 与已经报道的其他昆虫的性肽受体的氨基酸序列一致性也在64%以上。不同组织表达分析表明, HassSPR在测定的1日龄雌蛾不同组织中均有表达, 以在脑中的表达量最高。时序表达分析表明, 在羽化前1 天至羽化后6日龄雌蛾的信息素腺体中均有表达, 以3日龄表达量最高。雌蛾交配后, HassSPR在性信息素腺体和脑中的表达量显著上调, 而在交配囊和卵巢中的表达量显著下调。【结论】从烟夜蛾雌蛾性信息素腺体中克隆得到性肽受体基因HassSPR, 其表达模式提示该基因的表达水平与雌蛾的生殖生理和生殖行为有关。     

Division of labor in the hyperdiverse ant genus Pheidole is associated with distinct subcaste- and age-related patterns of worker brain organization

The evolutionary success of ants and other social insects is considered to be intrinsically linked to division of labor among workers. The role of the brains of individual ants in generating division of labor, however, is poorly understood, as is the degree to which interspecific variation in worker social phenotypes is underscored by functional neurobiological differentiation. Here we demonstrate that dimorphic minor and major workers of different ages from three ecotypical species of the hyperdiverse ant genus Pheidole have distinct patterns of neuropil size variation. Brain subregions involved in sensory input (optic and antennal lobes), sensory integration, learning and memory (mushroom bodies), and motor functions (central body and subesophageal ganglion) vary significantly in relative size, reflecting differential investment in neuropils that likely regulate subcaste- and age-correlated task performance. Worker groups differ in brain size and display patterns of altered isometric and allometric subregion scaling that affect brain architecture independently of brain size variation. In particular, mushroom body size was positively correlated with task plasticity in the context of both age- and subcaste-related polyethism, providing strong, novel support that greater investment in this neuropil increases behavioral flexibility. Our findings reveal striking levels of developmental plasticity and evolutionary flexibility in Pheidole worker neuroanatomy, supporting the hypothesis that mosaic alterations of brain composition contribute to adaptive colony structure and interspecific variation in social organization.