Changes in external morphology and integument ultrastructure of the 5th instar larvae of Lymantria dispar ( Lepidoptera: Lymantridae) treated by 20-hydroxyecdysone

为了探明20-羟基蜕皮甾酮对昆虫蜕皮过程中体壁的表皮层、皮细胞及其细胞器的具体影响过程,本研究利用透射电镜技术研究了20-羟基蜕皮甾酮对舞毒蛾Lymantria dispar( Linnaeus)5龄幼虫体壁超微结构的变化.结果表明,用高浓度20-羟基蜕皮甾酮溶液浸过的白桦叶片饲喂幼虫,处理6h,摄人约400 μg 20-羟基蜕皮甾酮后,幼虫停止取食;处理12h时表皮细胞顶膜上的微绒毛减少,在皮细胞与旧表皮之间形成蜕皮间隙,旧头壳从幼虫头部脱离;处理24 h时蜕皮间隙继续增大,旧表皮与皮细胞进一步分离,新表皮质层开始形成;处理36 h时皮细胞顶膜形成较短的微绒毛,胞质区域出现数量较多的电子疏松泡,新表皮由上表皮、外表皮及8层左右内表皮片层组成;处理48 h时顶膜与内表皮界限模糊,内表皮继续合成至16层左右;72h时细胞内出现大面积电子疏松泡,内表皮合成至20层左右.处理96 h时,与对照组相比,皮细胞细胞器较少,核仁周围出现小部分空白区域,胞质区域内含物减少;虫体发黑缩小,即将死亡;内表皮层数仍旧保持20层左右.对照组幼虫6 -96 h虫体活跃,正常取食,外部观察及透射电镜结果均未显现蜕皮现象;表皮层由上表皮、外表皮及内表皮组成;皮细胞顶膜微绒毛密度高;表皮细胞分泌活动旺盛,胞质区域细胞界限明显,内含物丰富;细胞器典型而且活跃;内表皮片层随时间不断增加至50层左右.结果提示,外源20-羟基蜕皮甾酮能够导致舞毒蛾5龄幼虫的致死性蜕皮.     

新型非甾醇蜕皮激素类杀虫剂对棉铃虫幼虫蜕皮的影响

用透射电镜技术研究了新型非甾醇蜕皮激素类杀虫剂W200013对棉铃虫4龄幼虫蜕皮的影响。结果表明W200013使棉铃虫产生早熟、致死的蜕皮。中毒试虫6 h表现出内表皮层沉积加快,皮细胞中粗面型内质网大量增加,糖原颗粒减少;12 h蜕皮间隙开始形成,细胞质凝集;24 h新、旧表皮同时存在,皮细胞空泡化严重;36 h新上表皮覆盖于仅沉积几层的新原表皮上,旧表皮仍然保持,皮细胞呈恶化、降解状态,宏观上虫体不表现出蜕皮行为而死亡。而且比较研究可见W200013在中毒症状、生物测定结果、超微结构水平与RH-5992具有相似的作用。     

非甾醇类蜕皮激素竞争物(RH-2485)对棉铃虫六龄幼虫部分组织蛋白质表达和体壁结构的作用

棉铃虫六龄幼虫经非甾醇类蜕皮激素竞争物（RH－2485）饲喂处理后,取部分组织做酶／蛋白检测以及切片光镜观察。变性聚丙烯酰胺凝胶电泳显示,处理24和48h幼虫的脂肪体和体壁的一种主要蛋白质表达量降低;非变性聚丙烯酰胺凝胶电泳酯酶染色显示,处理48h后血淋巴和中肠有新的酯酶同功酶表达;处理24h幼虫的体壁组织切片出现蜕皮特征（中表皮＋内表皮囊泡化、上表皮＋外表皮与真皮细胞层分离）,以及异常蜕皮（真皮细胞层变薄）。这些结果深化了RH－2485杀虫机制的认识。     

中华绒螯蟹蜕皮过程中体壁结构和主要成分的变化

采用组织化学和原子吸收分析等方法, 研究了中华绒螯蟹蜕皮过程中体壁结构和主要成分的变化。结果显示: 中华绒螯蟹体壁分为上表皮、外表皮、内表皮和膜层, 糖类物质各层均有分布, 胶原纤维分布在除上表皮外的其他各层。在蜕皮前, 糖类、胶原纤维都被重吸收, 体壁上表皮和外表皮在蜕皮前形成, 内表皮和膜层在蜕皮后形成。体壁粗蛋白含量在蜕皮前期(D1-D3-4期)降低(P0.05), 蜕皮后A-B期含量极高(P0.05)。几丁质含量在蜕皮过程中变化不显著(P0.05), 只是在蜕皮前稍有上升。Ca2+和Mg2+含量在蜕皮前D1期显著低于蜕皮间期和蜕皮前其他时期(P0.05), 而蜕皮后A-B期降到最低(P0.05), 蜕下的甲壳中则含有较多的Ca2+和Mg2+ (P0.05)。Cu2+和Zn2+含量除蜕皮后A-B期升高外(P0.05), 其余时期变化不明显(P0.05)。这些研究结果表明, 中华绒螯蟹体壁结构和成分变化与蜕皮周期密切相关。       

黄粉虫幼虫体壁硬化过程中酚氧化酶活性的变化

为研究酚氧化酶(PO)在昆虫蜕皮过程中的功能和作用, 采用微量测定法研究了黄粉虫Tenebrio molitor体壁硬化过程中血淋巴和表皮中的PO活性变化。结果表明：初蜕皮幼虫血淋巴中PO活性较高, 但随着体壁的不断黑化与硬化, 其活性呈现下降趋势, 在3～4 h内达到最低点, 而后PO活性逐渐上升, 7 h左右活性上升至最高, 并接近于正常幼虫的水平;在刚蜕完皮后的1 h内, 体壁中 PO活性基本无变化, 但随后即开始下降, 3 h左右降到最低点, 然后开始回升, 6~7 h左右恢复到正常水平, 并趋于稳定;以L-DOPA为底物, 通过双倒数曲线作图法求得黄粉虫血淋巴PO的Km＝1.176 mmol/L, 体壁PO的Km＝0.881 mmol/L, 表明体壁PO与底物L-DOPA的亲和力要高于血淋巴PO。研究表明两种来源的酚氧化酶均参与了黄粉虫幼虫的体壁硬化过程, 但在作用方式及与底物的亲和力方面存在差异。     

保幼激素类似物及蜕皮甾类对亚洲玉米螟幼虫酚氧化酶活性的影响

分别用1 μg/头、0.1 μg/头和0.01 μg/头浓度的保幼激素类似物methoprene（蒙五一五）体外处理亚洲玉米螟5龄幼虫,测定幼虫体壁组织、血清和血细胞溶离物中酚氧化酶的活性。结果表明： 1 μg/头 methoprene处理组和0.1 μg/头处理组幼虫体壁组织中酚氧化酶活性与对照组相比有显著提高（P<0.01）,血清和血细胞溶离物中酚氧化酶活性也显著上升（P<0.01）。将含有20-羟基蜕皮酮的人工饲料饲喂亚洲玉米螟5龄幼虫,处理组幼虫体壁组织的酚氧化酶活性下降(P<0.05),血清和血细胞溶离物中的酚氧化酶活性均低于对照组 （P<0.01）。这些结果表明methoprene可以诱导亚洲玉米螟5龄幼虫体内酚氧化酶活性的上升,而20-羟基蜕皮酮则抑制了酚氧化酶的活性。     

华麻花头根中的蜕皮甾酮类成分

从华麻花头(Serratula chinensis S．Moore)根中分得7种蜕皮甾酮类化合物,经光谱分析和化学方法,分别鉴定为：20-羟基蜕皮松(1),podecdysone C(2),3-氧-乙酰基-20-羟基蜕皮松(3),20-羟基蜕皮松-20,22．-缩丁醛(4),shidasterone(5),atrotosterone C(6)和carthamosterone(7),其中20-羟基蜕皮松-20,22．缩丁醛为一新的化合物。     

平卧川牛漆甾酮对小菜蛾生长发育的影响(英文)

平卧川牛漆甾酮是紫背金盘中的主要植物蜕皮甾类 ,在筋骨草属植物中普遍存在。试验结果表明 ,用 5 0mg L平卧川牛漆甾酮处理小菜蛾卵 ,其孵化受到抑制 ,总孵化率为 94.7% ,显著低于对照的 1 0 0 %孵化。平卧川牛漆甾酮对幼虫具有弱的毒杀活性和良好的拒食活性 ,且与浓度相关。小菜蛾的生长发育也受平卧川牛漆甾酮的影响。用高于 5 0mg L浓度处理幼虫 ,其生长受抑及蛾的产卵量下降 ,而较低浓度处理则有利于幼虫生长 ,卵量提高。这种双重作用可能是由于平卧川牛漆甾酮的激素活性和拒食活性的作用结果 ,后者导致昆虫营养不良。处理幼虫后 ,试虫的化蛹和羽化受阻。小菜蛾对平卧川牛漆甾酮比 2 0 羟基蜕皮酮更敏感。     

小地老虎及粘虫幼虫对灭幼脲表现自然耐药力的体壁结构和生化基础

小地老虎Agrotis ypsilon rottem.幼虫对灭幼脲具有一定的自然耐药力。本文以粘虫Mythimna separata(Walker)作为敏感性虫种与之进行比较,实验结果表明,灭幼脲对两种试虫的室内毒力相差4倍左右,引起差异的原因,在体壁结构方面主要在于:(1)小地老虎幼虫的表皮层较粘虫的厚4.2倍左右;(2)上表皮不是匀质结构,依靠少数蜡道与体表沟通;(3)几丁质片层内的孔道数较少,仅及粘虫的1/4。由此构成了表皮对疏水性的灭幼脲表现抗穿透的性能。小地老虎幼虫体壁还含有较强的生化防卫体系,灭幼脲对多功能氧化酶、芳基酰胺酶有明显激活效应,这两种酶都是灭幼脲的降解酶。由此认为,小地老虎幼虫对灭幼脲所表现的自然耐药力,是由体壁的抗穿透性能以及由灭幼脲所激活的适应酶所造成。     

20-羟基蜕皮甾酮对大鼠全脑缺血再灌注后海马神经元损伤和炎症反应的影响

目的:观察20-羟基蜕皮甾酮对全脑缺血再灌注后SD大鼠海马神经元和认知功能的保护作用,并探讨其相关机制。方法:采用四血管闭塞法建立SD大鼠全脑缺血再灌注模型,脑电图和脑组织Nissl染色评估模型的可靠性。将实验动物分为假手术组,缺血再灌注组和缺血再灌注+20-羟基蜕皮甾酮组。TUNEL染色观察海马神经元凋亡,Morris水迷宫实验评价大鼠的认知功能,酶联免疫法测定缺血再灌注后3-24小时大鼠血清中白细胞介素-1β(IL-1β)和肿瘤坏死因子α(TNF-α)的浓度。结果:全脑缺血再灌注后大鼠海马神经元凋亡率从4.50±1.90%上升至72.90±8.40%(p0.01),给予20和40 mg/kg 20-羟基蜕皮甾酮干预,大鼠海马神经元凋亡率分别下降至51.40±8.60%(p0.05)和42.70±6.80%(p0.01)。与假手术组相比,全脑缺血再灌注后大鼠在Morris水迷宫定位航行试验中逃避潜伏期明显延长(p0.01),在空间探索试验中目标象限停留时间和穿越目标象限次数明显减少(p0.01),而20-羟基蜕皮甾酮显著抑制上述变化,改善大鼠的认知功能。缺血再灌注后3-24小时,大鼠血清中IL-1β和TNFα浓度较假手术组显著升高,20-羟基蜕皮甾酮能抑制上述各时间点大鼠血清中IL-1β和TNFα浓度的升高。结论:20-羟基蜕皮甾酮对全脑缺血再灌注后大鼠海马神经元和认知功能有显著保护作用,抑制缺血再灌注后的炎症反应是其保护机制之一。     

Fine structural survey of old cuticle degradation during pre-ecdysis in two European Atlantic crabs

Light and transmission electron microscopy were used to monitor changes due to the degradation of the old exoskeleton and related events in the sclerites, articular membranes, and gills of two decapod crustaceans (Carcinus maenas and Macropipus puber) during pre-ecdysis. In both sclerites and articular membranes, degradation follows a similar general pattern in both crab species, while the gill cuticle appears unaltered. In early pre-ecdysis (D(0)), the degradation of the old cuticle starts with the secretion of ecdysial droplets by the epidermis. Apolysis, occurring at stage D(1)', is re-defined as an event, not necessarily morphologically observable, consisting in the loss of adherence between the epidermis and the old cuticle during early pre-ecdysis of arthropods. At the stage D(1)', the moulding of the epidermal cell surface occurs in preparation to the deposition of the new cuticle and causes the opening of the ecdysial cleft. In the principal layer of sclerites, degradation of the chitin-protein microfibres should precede mineral dissolution. In contrast to the other degraded cuticle layers, the membranous layer of sclerites and the innermost endocuticular lamellae of articular membranes are transformed into a digestion-resistant fibrous network resembling the ecdysial membrane of insects.     

The formation of the ecdysial droplets and the ecdysial membrane in an insect

Insect cuticle forms as a result of overlapping sequences of two kinds of process, those involving vesicles of the Golgi complex, and those related to transport through and/or assembly at the apical plasma membrane. The ecdysial droplets are the last layer of old cuticle to be deposited before ecdysis and form from the contents of secretory vesicles from Golgi complexes. Ecdysial droplets and secretory vesicles both stain with PTA and react with silver hexamine after oxidation with periodic acid. The vesicles discharge in localized apical areas devoid of microvilli where they accumulate as droplets measuring about 3 [ x 1 [. The. droplets span the last few lamellae of the endocuticle which becomes the ecdysial membrane. They dissolve to leave the ecdysial membrane full of holes at the time that the rest of the old cuticle is digested.     

Ultrastructural Effects of a Non-Steroidal Ecdysone Agonist,RH-5992, on the Sixth Instar Larva of the Spruce Budworm,Choristoneura fumiferana

Force feeding of RH-5992 (Tebufenozide), a non-steroidal ecdysone agonist to newly moulted sixth instar larvae of the spruce budworm, Choristoneura fumiferana, (Lepidoptera: Tortricidae) initiates a precocious, incomplete moult. Within 6h post treatment (pt) the larva stops feeding and remains quiescent. Around 12hpt, the head capsule slips partially revealing an untanned new head capsule that appears wrinkled and poorly formed. By 24hrpt, the head capsule slippage is pronounced and there is a mid-dorsal split of the old cuticle in the thoracic region but there is no ecdysis. The larva remains moribund in this state and ultimately dies of starvation and desiccation. The temporal sequence of the external and internal changes of the integument were studied using both scanning and transmission electron microscopy. Within 3hpt, there is hypertrophy of the Golgi complex indicating synthetic activity and soon after, large, putative ecdysial droplets are seen. Within 24h, a new cuticle that lacks the endocuticular lamellae is formed. The formation of the various cuticular components, the degradation of the old cuticle and changes in the organelles of the epidermal cells of the mesothoracic tergite are described. The difference between the natural moult and the one induced by RH-5992 are explained on the basis of molecular events that take place during the moulting cycle. The persistence of this ecdysone agonist in the tissues permits the expression of all the genes that are up-regulated by the presence of the natural hormone but those that are turned on in the absence of the hormone are not expressed.     

Penetration of the cuticular layers of elaterid larvae (Coleoptera) by the fungus Metarrhizium anisopliae, and notes on a bacterial invasion

The penetrant hyphae of Metarrhizium anisopliae in the exuvial cuticle of a molting wireworm can form secondary appressoria on the developing new cuticle. From these a new penetrant fungal apparatus can develop through the new cuticle toward the body cavity. The penetrant fungal apparatus in the ecdysial space of the host does not appear to be affected by the histolytic enzymes in the wireworm molting fluid. A mucoidlike substance that envelopes the fungus in the ecdysial space may be, in part, the protective mechanism involved. Bacteria from the soil often invade the ecdysial space of molting wireworms that have difficulty in shedding their exuvia. Pseudomonas aeruginosa can histolyze the proteinaceous exocuticle of the exuvium, the ecdysial membrane, and the dense inner epicuticle of the new cuticle, but not the epicuticle of the exuvium, when it invades the ecdysial space of a molting wireworm.     

Fine structure and development of the silver and golden cuticle in butterfly pupae

Pupae of the butterflies Danaus chrysippus and Helioconius charitonius display characteristic patterns of golden spots, while the pupae of the genera Euploea and Amauris exhibit metallic lustre over most of their surface; E. core and midamus more golden, A. ochlea and niavius more silvery. The absolute reflectance exceeds 80% at wavelengths longer than 550 nm, but drops more or less steeply at shorter wavelengths (shown by microspectrophotometry for E. core and A. ochlea; in all species this effect is caused by constructive interference of the incident light at Multiple Endocuticular Thin Alternating Layers (METAL cuticle). Dense, cuticular D layers alternate with clear, watery C layers and form over 200 double layers. The thickness of the D layers is fairly constant throughout the stack, whereas the C layers systematically increase and decrease in thickness, thus causing the broad bandwidth of the reflector. Connecting filaments, traversing the C layers in zig zag course, probably secure the mechanical stability of the arrangement. After drying, the C layers have vanished and the lustre is lost; the cuticle is now perfectly transparent, except for D. chrysippus, where it is partly transparent and partly yellow. The metallic reflectance develops between 20 and 30 hr after pupal ecdysis, starting with blue colours which change via green to gold or silver. About half a day before emergence of the imago, the reflection fades again via the opposite colour sequence. Coincident with these colour changes, the METAL cuticle is being deposited and decomposed, respectively. The deposition zone immediately above the apical epidermal microvilli consists of about three helicoidal lamellae as in normal, non-reflecting cuticle. The METAL cuticle is formed abruptly at the outer border of the deposition zone, possibly during condensation of the cuticular microfibres. The periodicity it is suggested is controlled either directly by the epidermal cells or indirectly via appropriate self-assembling processes.     

Molting and cuticle deposition in the subterranean trichoniscid Titanethes albus (Crustacea, Isopoda)

Terrestrial isopods are a suitable group for the study of cuticle synthesis and calcium dynamics because they molt frequently and have evolved means to store calcium during molt. Little data is currently available on molting in Synocheta and subterranean isopods. We studied the molting dynamics in the subterranean trichoniscid Titanethes albus under laboratory conditions and performed a microscopic investigation of sternal CaCO(3) deposits and the tergal epithelium during molt in this species. In accordance with its lower metabolic rate, molting in the laboratory is roughly 2-3 times less frequent in Titanethes albus than would be expected for an epigean isopod under similar conditions. Animals assumed characteristic postures following the molt of each body half and did not consume the posterior exuviae after posterior molt. The structure of sternal calcium deposits and the ultrastructural characteristics of the epidermis during cuticle formation in Titanethes albus are similar to those described in representatives of Ligiidae. During the deposition of the exocuticle, the apical plasma membrane of epidermal cells forms finger-like extensions and numerous invaginations. In the ecdysial space of individuals in late premolt we observed cellular extensions surrounded by bundles of tubules.     

Attachment and germination of Entomophaga maimaiga conidia on host and non-host larval cuticle

The lepidopteran-specific fungal pathogen Entomophaga maimaiga is highly virulent against Lymantria dispar (gypsy moth) larvae, and other members of the family Lymantriidae. Numerous species in the subfamily Cuculliinae (Family Noctuidae) are not susceptible to E. maimaiga due to the inability of this fungus to penetrate the larval cuticle. Conidial attachment and germination were compared among five cuculliine species and L. dispar using bioassays and scanning electron microscopy. Although conidia were showered evenly across larvae during bioassays, on L. dispar conidia were most abundant on segments, where they adhered well to the cuticle and germinated at high percentages. Conidia on cuculliine cuticles were predominantly found in large, loose aggregations in intersegmental areas. Few conidia on cuculliine cuticle germinated and scanning electron microscopy revealed a thick film of mucous enveloping conidia. We hypothesize that the conidia on cuculliines become coated by this film and were only loosely attached to the larval cuticle. No such film was seen on L. dispar larvae where individual conidia appeared well attached. On L. dispar larvae many conidia also adhered to setae. To determine if hydrophobicity affected the ability of E. maimaiga conidia to attach and germinate on a substrate, a goniometer was used to determine relative hydrophobicity of larval cuticles. L. dispar cuticle was more hydrophobic than cuculliine cuticle, suggesting that a high level of hydrophobicity could be a required characteristic for hosts. Cuticles from four cuculliine species and L. dispar were sequentially extracted using hexane, chloroform, and methanol. Conidia were showered onto glass slides coated with the different extracts and germination was quantified. Methanol extracts of cuculliine cuticle consistently decreased germination, compared to all extracts of L. dispar cuticle. For all L. dispar extracts, the majority of conidia produced germ tubes, which is a normal prerequisite for cuticular penetration. For the cuculliines, conidia exposed to hexane and chloroform extracts produced secondary conidia as did all controls, but the conidia exposed to cuculliine methanol extracts that germinated produced germ tubes. These studies demonstrated that a range of factors act in concert to prevent E. maimaiga infection of the cuculliine species investigated.     

Effects of a fat body extract on larval midgut cells and growth of lepidoptera

Treatment with fat body extract (FBX) from pupae of the tobacco hornworm, Manduca sexta, caused mortality in larvae of two pest lepidopterans, the gypsy moth, Lymantria dispar, and the cotton leafworm, Spodoptera littoralis. In FBX-treated larvae, the feeding rate was depressed, causing reduced weight gain and then larval death. Their midgut showed formation of multicellular layers of midgut epidermis, indicating stem-cell hyperplasia. Hence, the integument of FBX-treated larvae had a double cuticle, indicating induction of premature molting. But radioimmunoassay measurements confirmed that the amount of ecdysteroids in FBX was too low to be responsible for the molt-inducing effects observed after treatment with FBX. With midgut stem cell cultures in vitro, addition of FBX to the culture medium stimulated cell proliferation and differentiation in a concentration-dependent manner. This effect was compared with those of insect molting hormones, ecdysone and 20-hydroxyecdysone; an ecdysteroid agonist, RH-2485; and a purified protein from FBX (multiplication factor). This article describes the mode of action of FBX and possible interplay between fat body factor(s) and insect hormones in the development and metamorphosis of the insect midgut.     

Exoskeletal cuticle differentiation during intramarsupial development of Porcellio scaber (Crustacea: Isopoda)

Exoskeletal crustacean cuticle is a calcified apical extracellular matrix of epidermal cells, illustrating the chitin-based organic scaffold for biomineralization. Studies of cuticle formation during molting reveal significant dynamics and complexity of the assembly processes, while cuticle formation during embryogenesis is poorly investigated. This study reveals in the terrestrial isopod Porcellio scaber, the ultrastructural organization of the differentiating precuticular matrices and exoskeletal cuticles during embryonic and larval intramarsupial development. The composition of the epidermal matrices was obtained by WGA lectin labelling and EDXS analysis. At least two precuticular matrices, consisting of loosely arranged material with overlying electron dense lamina, are secreted by the epidermis in the mid-stage embryo. The prehatching embryo is the earliest developmental stage with a cuticular matrix consisting of an epicuticle and a procuticle, displaying WGA binding and forming cuticular scales. In newly hatched marsupial larva manca, a new cuticle is formed and calcium sequestration in the cuticle is evident. Progression of larval development leads to the cuticle thickening, structural differentiation of cuticular layers and prominent cuticle calcification. Morphological characteristics of exoskeleton renewal in marsupial manca are described. Elaborated cuticle in marsupial larvae indicates the importance of the exoskeleton in protection and support of the larval body in the marsupium and during the release of larvae in the external environment.