暗褐蝈螽与优雅蝈螽精子超微结构的比较研究(直翅目,螽斯科)

研究了暗褐蝈螽Gampsocleis sedakovii(Fischer von Waldheim)和优雅蝈螽G.gratiosa Brunner von Wattenwyl精子的超微结构。这两种蝈螽精子头部的顶体复合体由顶体外层、顶体本体和顶体组成,顶体复合体位于细胞核前端,并包裹部分细胞核;颈部具5纵层细胞器;尾部鞭毛轴丝为典型的9+9+2型,线粒体衍生体部分晶状化。暗褐蝈螽精子较短,顶体复合体夹角较大,精子鞭毛横切面直径稍大;优雅蝈螽精子稍长,顶体复合体夹角较小,精子鞭毛横切面直径较小,两种精子超微结构差异不显著,其生殖隔离机制有待进一步研究。     

优雅蝈螽与暗褐蝈螽精子束的显微观察

本文应用微分干涉相衬法对优雅蝈螽Ｇampsocleis gratiosa Brunner von Wattenwyl和暗褐蝈螽G. sedakovii （Fischer von Waldheim） 雄性精巢管基部、输精管、贮精囊和精包,及雌性受精囊中精子束的形态变化进行了观察,对探讨螽斯近缘种的生殖隔离机制和生殖生物学具有重要意义.结果表明：这两种蝈螽的精子束通过精包转移到雌性受精囊后,精子束的形态发生了显著变化.精巢管基部的精子为游离的单个精子;输精管、贮精囊和精包中精子成束排列形成较分散的精子束,精子束头部包裹有粘液帽;雌性受精囊中的精子束的精子呈羽状排列,精子的头部汇集在中央轴上.两种蝈螽精子束形态差异不显著.     

乌苏里蝈螽和优雅蝈螽雄性鸣声结构的比较研究

应用计算机技术分析了内蒙古草原乌苏里蝈螽Gampsocleis ussuriensis和优雅蝈螽Gampsocleis gratiosa2种螽斯雄性的鸣声结构。乌苏里蝈螽鸣声较复杂,一次鸣叫持续时间6~27s(平均12.5s),每个脉冲组由两类脉冲组组分构成,第1类脉冲组组分为振幅较大的脉冲组,脉冲组持续时间0.0065s,由6~8个脉冲串组成,每个脉冲串持续时间为0.00047s,间隔时间为0.00027s,每个脉冲串含5~10个单脉冲,脉冲串持续时间、间隔时间较短;第2类脉冲组组分为振幅较小的脉冲组,脉冲组持续时间0.0191s,约含有15个左右脉冲串,持续时间为0.00041s,间隔时间约为0.00127s,每个脉冲串含有3~5个单脉冲,脉冲串持续时间、间隔时间也较短,主能峰频率为7.98kHz。优雅蝈螽鸣声则较规则,一次鸣叫持续时间4~232s(平均41.7s)。每个脉冲组也由两类脉冲组组分构成,第1类脉冲组组分振幅较大且逐步增强,脉冲组持续时间0.119s,由10个振幅较大的脉冲串组成,每个脉冲串持续时间为0.00576s,间隔时间为0.005s,每个脉冲串含18~25个单脉冲;第2类脉冲组组分为振幅较小的脉冲组,脉冲组持续时间0.07s,约含有25个左右脉冲串,脉冲串振幅较小,含有6~9个单脉冲,主能峰频率为6.87kHz,次能峰频率为3.25kHz。结果表明乌苏里蝈螽与优雅蝈螽雄性鸣声既有相似的共同特征,同时存在较显著差异。     

优雅蝈螽雄性附腺结构与分泌蛋白特性

通过组织切片和SDS-PAGE方法,研究优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl雄性附腺的结构及分泌蛋白的特性。结果表明,优雅蝈螽雄性附腺由3类腺管组成:乳白长腺管、透明腺管和乳白短腺管,腺管的管壁组织结构相似,从内到外依次为单层上皮细胞、基膜、肌肉层,不同腺管管腔分泌物H-E染色后呈现不同颜色。SDS-PAGE分析各种腺管的分泌蛋白具有特异性。     

优雅蝈螽VASA蛋白cDNA序列的克隆与生物信息学分析

Vasa基因属于DEAD-box家族,其功能主要是特定mRNA的翻译调控。在许多动物中,它都是生殖系细胞发育所必须,对生殖干细胞分化具有重要作用。为探究vasa基因在半变态类昆虫生殖系细胞发育中的作用,本研究首先从基于Illumina高通量测序平台测得的优雅蝈螽Gampsocleis gratiosa成体转录组数据中筛选出一段长度为1215 bp的vasa基因片段,进而设计引物并利用RT-PCR和RACE技术获得其c DNA序列全长,最后利用生物信息学技术进行分析。结果显示:优雅蝈螽vasa基因的c DNA序列全长3359 bp,其中,5'端非编码区82bp,3'端非编码区1306 bp,开放阅读框1971 bp编码656个氨基酸,理论蛋白相对分子量(Mw)72.3 k Da,等电点(p I)5.48。通过与Gen Bank数据库中收录的其他VASA蛋白序列比对,发现优雅蝈螽VASA蛋白具有DEAD-box蛋白家族所共有的9个保守基序,Ax TGo GKT(I)、PTRELA(Ia)、TPGR(Ib)、DEAD(Ⅱ)、SAT(Ⅲ)、LVFVE(Ⅳ)、TDVu ARGID(Ⅴ)、HRIGRTGR(Ⅵ)和Gacc Poh1Q(Q),其中,Gacc Poh1Q(Q)的第3个氨基酸残基存在显著变化,建议将Gacc Poh1Q(Q)修改为Gaxc Poh1Q(Q)。此外,优雅蝈螽VASA蛋白的N端还具有10个RG和2个RGG重复序列、起始及终止密码子附近的色氨酸(W)、C末端的7个氨基酸残基中有4个为酸性氨基酸残基(E),表明其具有ATP依赖的RNA解旋酶活性。基于氨基酸序列聚类结果显示:优雅蝈螽位于六足动物分枝末梢,与双斑蟋Gryllus bimaculatus的亲缘关系最近,这与二者的分类学地位相符。本研究表明基于短读长二代测序平台获得的转录组数据可以很好地服务于功能基因研究,所获得的优雅蝈螽vasa基因c DNA全长对于进一步深入研究VASA蛋白在半变态类昆虫生殖系细胞发育研究具有重要意义。     

优雅蝈螽精子形成过程中F-肌动蛋白的动态变化

为阐明F-肌动蛋白在优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl精子形成过程中的动态变化, 本研究利用微分干涉相衬技术和免疫荧光技术首次对优雅蝈螽精子形成过程中的F-肌动蛋白进行了细胞定位, 利用透射电镜技术从超微水平观察了优雅蝈螽精子顶体复合体的结构。结果显示： 精子形成早期, F-肌动蛋白富集于亚顶体区域, 形态由“球状”转变为“棒锥状”; 精子形成中期, F-肌动蛋白呈“倒Y型”分布于亚顶体区域和细胞核前端两侧; 精子形成后期, 亚顶体区域的F 肌动蛋白解聚消失, F-肌动蛋白呈“箭头状”, 仅分布于顶体复合体扩张的两翼中。F-肌动蛋白动态变化伴随着细胞核和精子头部的形态改变, F-肌动蛋白的动态装配在精子顶体复合体形态构建和细胞核的形变中起着重要的作用。本研究还发现未成熟的精子尾部有一些富含F-肌动蛋白的细胞质微滴, 与精子形成过程中多余细胞质和细胞器的外排有关。F-肌动蛋白的动态变化研究为进一步阐明细胞骨架蛋白在昆虫精子形成过程中的功能和作用机制奠定了基础。     

三种螽斯的性选择行为比较研究

本文通过设计Y形迷宫实验,对螽斯属的优雅蝈螽Gampsocleis gratiosa、暗褐蝈螽Gampsocleis sedakovii obscura和鸣螽属的鼓翅鸣螽Uvarovites inflatus雌虫的性选择行为和交配行为进行了观察研究。结果表明:3种螽斯的交配行为谱一致,但交配时间上有差别;3种螽斯雌虫趋于选择体重较大的雄虫交配;体重和精包重之间呈现较复杂的相关性,但均以三次函数的拟合度(R2)最大;雄虫精包重占体重均在10.0%以上,分别为优雅蝈螽11.1%、暗褐蝈螽12.5%、鼓翅鸣螽14.9%。     

优雅蝈螽精子形成过程中核的形态结构变化观察（英文）

【目的】螽斯精子结构复杂,具有特征性的箭头状顶体,是研究昆虫精子形成的理想材料。为了研究螽斯精子形成过程中的动态变化机制,特别是细胞核的凝集机制和箭头状顶体的发生机制,本研究对优雅蝈螽Gampsocleis gratiosa精细胞和精子的细胞核进行了观察。【方法】选择发育良好的优雅蝈螽成虫精巢为研究材料,利用透射电镜技术、普通光学显微镜和荧光显微镜技术,制作光镜切片和电镜切片进行观察。【结果】根据其形态结构变化特征,将优雅蝈螽精子形成过程中的细胞核分为4个阶段：圆形核、叶形核、柱状核和成熟阶段。我们还通过常规HE染色,结合DNA特异性荧光探针DAPI,证明了圆形核时期,精细胞内具有两个明显的球状结构,一个为细胞核,另一个是原顶体;精子成熟阶段,精子尾部排出的细胞质微滴中含有DNA。【结论】优雅蝈螽精子形成过程中,精细胞的细胞核经历了显著的形态变化,精细胞核的形态变化与细胞骨架微管相关,细胞核塑形伴随着染色质的重组。本研究为进一步阐明直翅目昆虫精子形成的分子机制奠定了基础。     

优雅蝈螽雄性附腺的超微结构及其腺管提取物对精子束的作用

为阐明优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl雄性附腺的结构与功能的关系, 本文利用透射电镜(transmission electron microscope, TEM)技术研究了优雅蝈螽雄性附腺的超微结构, 利用微分干涉相差显微镜(differential interference contrast microscope, DIC)技术并结合雄性附腺匀浆提取物与精子束在体外的短暂培养, 研究了优雅蝈螽雄性附腺对精子束的作用。结果表明： 优雅蝈螽雄性附腺3类腺管组织结构相似, 腺管管壁为单层上皮细胞, 缺少内表皮, 说明其来源于中胚层。上皮细胞富含粗面内质网、 线粒体、 高尔基体, 具有分泌细胞的特点。腺管管腔中分泌物有4种形态, 即电子透明的物质、 电子致密的颗粒物质、 细纤维状物质以及绒球状物质。上皮细胞的分泌方式主要有2种, 即顶质分泌和局部分泌。乳白短腺管的匀浆提取物参与了帽状精子束解聚的过程, 乳白长腺管和透明腺管的匀浆提取物有维持精子束活性的作用。本研究结果为进一步阐明螽斯雄性附腺的生理功能奠定了基础。     

优雅蝈螽转录组和基因组浅层测序结果中SSR位点鉴定、特征及分布规律

优雅蝈螽Gampsocleis gratiosa是一种在我国具有悠久人工饲养历史的鸣虫。简单重复序列(Simple sequence repeats,SSR)非常适用于种群遗传研究。本文运用MISA软件分别在优雅蝈螽转录组和基因组浅层测序结果中发现15 042和40 611个SSR位点。转录组中,双碱基型最为丰富5 444个(36.19%),其次为单碱基型4 785个(31.81%)和三碱基型4 273个(28.41%),而四碱基型514(5.83%)和五碱基型26(0.33%)极少,没有发现六碱基型。基因组浅层测序结果中,以三碱基型(38.89%)和四碱基型(38.43%)最为丰富,单碱基型(10.34%)和双碱基型(11.72%)次之,五碱基型(0.54%)和六碱基型(0.08%)最少。基因组浅层测序结果中SSR位点的密度为876.20个/Mb,约为转录组143.06个/Mb的6倍。利用primer 3_2.2.3搜索发现优雅蝈螽转录组和基因组浅层测序结果中侧翼序列满足设计引物条件的完整型SSR位点数分别为9 969个(70.18%)和21 437个(67.75%)。本研究加深了对优雅蝈螽基因组的认识和了解,并为下一步大量开发和筛选高质量微卫星标记提供数据支持。     

The complete mitogenome of the Chinese bush cricket, Gampsocleis gratiosa （Orthoptera： Tettigonioidea）

The complete mitochondrial genome (mitogenome) of Gampsocleis gratiosa was determined. The 15, 929 bp in the size of G. gratiosa mitogenome contains a typical gene content, base composition, and codon usage found in metazoan. All 13 protein coding genes (PCGs) of the G. gratiosa mitogenome start with a typical ATN codon. The usual termination codons (TAA and TAG) were found from 10 PCGs. However, the atp6, nad4, and nad5 had incomplete termination codon (T). The anticodons of all tRNAs are identical to those observed in Drosophila yakuba and Locusta migratoria, and can be folded in the form of a typical clover leaf structure except for trnS (AGN). The secondary structure of trnS (AGN) was drawn according with the Steinberg-Cedergren tertiary structure. The A T content (67.4%) of the A T-rich region is relatively lower among the mitogenome regions, in contrast, it usually contains the highest A T content for most insects. Two isolated sequence repeat regions (202 bp) were found in the A T-rich region with mapping and secondary structure information.     

Effects of mating status on copulation investment by male bushcricket Gampsocleis gratiosa (Tettigoniidae, Orthoptera)

In many insect species, male will donate prey, se-cretion, and spermatophore as “nuptial gift” or “court- ship feeding” to females prior to, after or during the copulation[1,2]. The nuptial gifts represented great re-productive cost. In bushcrickets, …     

Mate sampling by female barking treefrogs (Hyla gratiosa)

Despite intense interest in mate choice, relatively little isknown about how individuals sample prospective mates. Indeed,a key issue is whether females sample males or simply matewith the first male encountered. We investigated mate samplingby female barking treefrogs (Hyla gratiosa). Females choosingmates in natural choruses did not move between males but insteadmated with the first male they approached closely. Most femalesmated with the male closest to them at the start of their mate-choiceprocess, and females were more likely to mate with the closest male when the distance to other males was large. These observationsare consistent with the hypothesis that females do not samplepotential mates but instead mate with the first male they distinguishfrom the rest of the chorus. To test this initial detectionhypothesis, we conducted a playback experiment in which weoffered females a choice between two calls, one of which was detectable above the background chorus sound at the female'srelease point, and one of which became detectable only as femalesmoved toward the initially detectable call. Females did notprefer the initially detectable call, thus ruling out the initialdetection hypothesis and implicating sampling of potentialmates by females. Based on the behavior of females in natural choruses, we hypothesize that females approach the chorus, moveto locations where they are able to detect the calls of severalmales simultaneously, and choose a mate from among these malesat some distance from the males. Such simultaneous samplingmay be common in lekking and chorusing species, which havebeen the subjects of many studies of sexual selection.     

Tribological Behavior of Gampsocleis Gratiosa Foot Pad Against Vertical Flat Surfaces

Some tribological behavior between mature Gampsocleis gratiosa foot pads and vertical flats of different materials were studied in this work. stereomicroscope （SMS） and scanning electron microscope （SEM） were used to measure the morphology of the Gampsocleis gratiosa foot pads. An atomic force microscope （AFM） was used to measure the morphologies of the surfaces of glass and a wall doped with calcium carbonate material. The attaching behavior of Gampsocleis gratiosa feet on the two vertical surfaces was observed. The attaching force （perpendicular to the vertical surface） and the static frictional force （along the direction of gravitation） of Gampsocleis gratiosa foot pads on a vertical glass were measured. It was shown that the average attaching force is 50.59 mN and the static frictional force is 259.10 mN. The physical models of the attaching interface between Gampsocleis gratiosa foot pads and the two vertical surfaces were proposed. It was observed that the foot pads are smooth in macroscale; however, the pad surface is composed by approximate hexagonal units with sizes of 3 μm to 7 μm in microscale; the adjacent units are separated by nanoscale grooves. The Observations showed that the Gampsocleis gratiosa can not climb the vertical calcium carbonate wall; in contrast, they can easily climb the vertical glass surface. Based on the features of the geometrical morphologies of the foot pads and the glass surface, we speculate that the attaching force and strong static frictional force are attributed to the interinlays between the deformable Gampsocleis gratiosa foot pads and the nanoscale sharp tips of the glass surface.     

Effects of mating status on copulation investment by male bushcricket Gampsocleis gratiosa (Tettigoniidae, Orthoptera)

Male’s copulation investment, including spermatophore and sperm investment were very high in the Chinese bushcricket Gampsocleis gratiosa. The effects of mating status of both males and females on male’s copulation investment were examined in this study. The fresh weight of spermatophylax increased positively with the weight of males’ body. This indicated that the nutritional investment during copulation depended on male’s quality. Spermatophore investment showed insignificant differences in every copulation protocols. This finding supported the paternal investment hypothesis, that is, males contributed to their offspring with little attention to their partners. Sperm releasing per ejaculation varied significantly among the trials. Males decreased 54.19% sperm in second mating than in its first mating, demonsrated that males regarded the first mating highly, and were more prudent in subsequent mating. These males’ strategies may contribute to the viability of the offspring.     

迷卡斗蟋和短翅鸣螽的行为谱及交配行为

研究了迷卡斗蟋和短翅鸣螽的行为模式及迷卡斗蟋鸣叫的时间分布。结果表明迷卡斗蟋雄虫单独存在时 ,在一昼夜内其鸣叫的持续时间为 668.38± 1 0 9.86分钟 ( 5 ) ,引入雌虫后鸣叫累计时间减少为 1 7.3± 6.7分钟 ( 5 ) ,并产生音调低沉的求偶声。在昼夜鸣叫活动型式中 ,雄虫单独存在时的鸣叫活动集中于夜间 ;引入雌虫后 ,占区鸣叫、求偶鸣唱和交配行为多集中于白天。根据上述结果提出了机会 -风险平衡假说来解释此现象 ,即白天交配有被捕食的危险 ,雄蟋短期内重复交配以保证精液进入雌蟋体内 ,从而保证了交配的成功 ,补偿了雄蟋所冒风险。短翅鸣螽雄性产生较大的精包 ,当雌体取食精包时 ,精液进入雌体内以保证交配成功。