20-羟基蜕皮甾酮处理后舞毒蛾外部形态和幼虫体壁超微结构的变化

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6 h, 摄入约400 μg 20-羟基蜕皮甾酮后, 幼虫停止取食; 处理12 h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; 72 h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。     

Metabolism of poplar salicinoids by the generalist herbivore Lymantria dispar (Lepidoptera)

The survival of insect herbivores on chemically defended plants may often depend on their ability to metabolize these defense compounds. However, only little knowledge is available on how insects actually process most plant defense compounds. We investigated the metabolism of salicinoids, a major group of phenolic glycosides in poplar and willow species, by a generalist herbivore, the gypsy moth (Lymantria dispar). Seven salicinoid metabolites identified in gypsy moth caterpillar feces were mostly conjugates with glucose, cysteine or glycine. Two of the glucosides were phosphorylated, a feature not previously reported for insect metabolites of plant defense compounds. The origins of these metabolites were traced to specific moieties of three major poplar salicinoids ingested, salicin, salicortin and tremulacin. Based on the observed metabolite patterns we were able to deduce the initial steps of salicinoid breakdown in L. dispar guts, which involves cleavage of ester bonds. The conjugated molecules were effectively eliminated within 24 h after ingestion. Some of the initial breakdown products (salicin and catechol) demonstrated negative effects on insect growth and survival in bioassays on artificial diets. Gypsy moth caterpillars with prior feeding experience on salicinoid-containing poplar foliage converted salicinoids to the identified metabolites more efficiently than caterpillars pre-fed an artificial diet. The majority of the metabolites we identified were also produced by other common poplar-feeding insects. The conversion of plant defenses like salicinoids to a variety of water-soluble sugar, phosphate and amino acid conjugates and their subsequent excretion fits the general detoxification strategy found in insect herbivores and other animals.     

Inheritance of female flight in Lymantria dispar (Lepidoptera: Lymantriidae)

A clinal female flight polymorphism exists in the gypsy moth, Lymantria dispar L., where female flight diminishes from east to west across Eurasia. A Russian population where females are capable of sustained ascending flight and a North American population with females incapable of flight were crossed: parentals, reciprocal F(1) hybrids, double reciprocal F(2) hybrids, and all possible backcrosses to both the parental lines were compared. Heritabilities were estimated using a threshold model, female offspring on female parent regressions, and joint-scaling analyses. Heritability of female flight capability measured using a free flight test was at least 0.60, and variation in wing size, muscle strength, and flight behaviors contributed to the flight polymorphism. Relative wing size varied continuously and had a heritability of 0.70. Environmental variation accounted for >90% of the variation in female preflight weight and relative flight muscle strength, as estimated by an inverted female's ability to right herself. Preflight walking behavior and early deposition of eggs were each inherited through a single gene with two co-dominant alleles. There was no evidence for sex-linkage or maternal effects in female flight capability or associated traits. Continued vigilance to exclude and eradicate introductions of strains capable of female flight in North America is warranted even in areas where no females fly, because some of the alleles needed for full flight capability may not be present in the North American populations, and some flight capability is maintained in the hybrids that could increase the rate of spread of L. dispar.     

Diel pattern in the structure and function of the gut microbial community in Lymantria dispar asiatica (Lepidoptera: Lymantriidae) larvae

In the present study, diel pattern in gut microbial communities in insects were evaluated. Lymantria dispar asiatica fourth instar larvae (72 ± 2 hr after molting) at noon (LdD) and midnight (LdN) were used for a comparative analysis of the gut microbial community. Ten bacterial operational taxonomic units (OTUs) were shared between LdD and LdN samples. One bacterial OTU was specific to LdD. The dominant gut microbes were OTU72 in LdD and OTU75 in LdN. A linear discriminant analysis effect size cladogram suggested that ten bacterial OTUs maintain significant differences in relative abundances between LdD and LdN. These results agreed with the discrete ellipses between LdD and LdN in principal coordinates analysis plots. Additionally, using phylogenetic investigation of communities by reconstruction of unobserved states, the gut microbial community was assigned to 23 functional terms, among which 22 exhibited significant differences between LdD and LdN. To conclude, the present study documented a diel pattern in the gut microbial community of L. dispar asiatica larvae.     

Identification of 20-hydroxyecdysone and ecdysone from the pupa of the gypsy moth,Lymantria dispar

Normal and reverse-phase high-performance liquid chromatography in conjunction with radioimmunoassay and mass spectrometry were used to identify the free and conjugated ecdysteroids (after enzymatic hydrolysis) from day-4 pupae of the gypsy moth, Lymantria dispar L. Seven ecdysteroids were searched for, but only 20-hydroxyecdysone (964 ng/g fresh weight) and ecdysone (367 ng/g fresh weight) were detected. Analysis of conjugated ecdysteriods after liberation by hydrolysis also indicated the presence of 20-hydroxyecdysone (21.6 ng/g fresh weight) and ecdysone (2.4 ng/g fresh weight). Neither 26-hydroxyecdysone nor the 3α-epimers of 20-hydroxyecdysone or ecdysone were detected.     

舞毒蛾末龄幼虫部分口器的扫描电镜观察

利用扫描电子显微镜对亚洲型舞毒蛾末龄幼虫口器的下颚和下唇的形态结构特征进行了观察。结果表明：在下颚和下唇须上共存在5种感器和1种角质齿,即毛形感器、刺形感器、锥形感器、栓锥形感器、钟形感器和刺状角质齿,其中毛形感器最发达。     

Inhibition of juvenile hormone esterase activity in Lymantria dispar (Lepidoptera, Lymantriidae) larvae parasitized by Glyptapanteles liparidis (Hymenoptera, Braconidae)

Glyptapanteles liparidis is a gregarious, polydnavirus (PDV)-carrying braconid wasp that parasitizes larval stages of Lymantria dispar. In previous studies we showed that parasitized hosts dramatically increase juvenile hormone (JH) titers, whereas JH degradation is significantly inhibited in the hemolymph. Here we (i) quantified the effects of parasitism on JH esterase (JHE) activity in hemolymph and fat body of penultimate and final instars of L. dispar hosts and (ii) assessed the relative contribution of individual and combined wasp factors (PDV/venom, teratocytes, and wasp larvae) to the inhibition of host JHE activity. The effects of PDV/venom was investigated through the use of gamma-irradiated wasps, which lay non-viable eggs (leading to pseudoparasitization), while the effects of teratocytes and wasp larvae were examined by injection or insertion of these two components in either control or pseudoparasitized L. dispar larvae. Parasitism strongly suppressed host JHE activity in both hemolymph and fat body irrespective of whether the host was parasitized early (premolt-third instar) or late (mid-fourth instar). Down-regulation of JHE activity is primarily due to the injection of PDV/venom at the time of oviposition, with only very small additive effects of teratocytes and wasp larvae under certain experimental conditions. We compare the results with those reported earlier for L. dispar larvae parasitized by G. liparidis and discuss the possible role of JH alterations in host development disruption.     

TAT-Cloan-DH-EGFP融合蛋白的原核表达及其在舞毒蛾幼虫体内的跨膜转导

【目的】为了探究滞育激素经昆虫取食的生物效应, 需设计一种能够加速跨膜运输的融合蛋白, 克服经昆虫消化道的降解作用。【方法】本研究以分月扇舟蛾Clostera anastomosis滞育激素基因Cloan-DH和TAT穿膜肽编码序列为基础, 构建TAT-Cloan-DH融合基因; 然后以增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）为标签, 构建在pET-22b载体中。在0.2 mmol/L IPTG诱导下, 在大肠杆菌Escherichia coli BL21 Rosetta(DE3)中表达出TAT-Cloan-DH-EGFP融合蛋白。 再分别将表达有TAT-Cloan-DH-EGFP和EGFP融合蛋白的大肠杆菌BL21菌体拌入人工饲料,经舞毒蛾Lymantria dispar幼虫持续取食0, 8, 24, 48, 72和90 h后, 随机挑选试虫进行组织固定并制作石蜡切片, 选取中肠所在体段切片进行组织荧光分析。【结果】37℃ 0.2 mmol/L IPTG诱导条件下, 表达的TAT-Cloan-DH-EGFP融合蛋白的分子量为61 kD, 主要以包涵体形式表达, 融合蛋白约占细胞总蛋白含量的18.1%; 随着试虫取食含TAT-Cloan-DH-EGFP融合蛋白饲料的增加, 虫体组织的绿色荧光强度逐渐增加, 取食72 h后, 组织荧光强度达到最高。【结论】实验结果说明融合蛋白TAT-Cloan-DH-EGFP可以通过消化道横向跨膜运输, 到达虫体其他组织, 且该过程与蛋白的摄入量成比例。     

World distribution of female flight and genetic variation in Lymantria dispar (Lepidoptera: Lymantriidae)

Female gypsy moths, Lymantria dispar L., from 46 geographic strains were evaluated for flight capability and related traits. Males from 31 of the same strains were evaluated for genetic diversity using two polymorphic cytochrome oxidase I mitochondrial DNA restriction sites, the nuclear FS1 marker, and four microsatellite loci. Females capable of strong directed flight were found in strains that originated from Asia, Siberia, and the northeastern parts of Europe, but flight capability was not fixed in most strains. No flight-capable females were found in strains from the United States or southern and western Europe. Wing size and musculature were shown to correlate with flight capability and potentially could be used in predicting female flight capability. The mtDNA haplotypes broadly separated the gypsy moth strains into three groups: North American, European/Siberian, and Asian. Specific microsatellite or FS1 alleles were only fixed in a few strains, and there was a gradual increase in the frequency of alleles dominant in Asia at both the nuclear and microsatellite loci moving geographically from west to east. When all the genetic marker information was used, 94% of the individuals were accurately assigned to their broad geographic group of origin (North American, European, Siberian, and Asian), but female flight capability could not be predicted accurately. This suggests that gene flow or barriers to it are important in determining the current distribution of flight-capable females and shows the need for added markers when trying to predict female flight capability in introduced populations, especially when a European origin is suspected.     

Fine structure of the galeal styloconic sensilla of larval Lymantria dispar (Lepidoptera: Lymantriidae)

Lepidopteran larvae possess two pairs of styloconic sensilla located on the maxillary galea. These sensilla, namely the lateral and medial styloconic sensilla, are each comprised of a smaller cone, which is inserted into a style. They are thought to play an important role in host-plant selection and are the main organs involved in feeding. Ultrastructural examination of these sensilla of fifth instar Lymantria dispar (L.) larvae reveal that they are each approximately 70 um in length and 30 um in width. Each sensillum consists of a single sensory peg inserted into the socket of a large style. Each peg bears a slightly subapical terminal pore averaging 317 nm in lateral and 179 nm in medial sensilla. Each sensillum houses five bipolar neurons. The proximal dendritic segment of each neuron gives rise to an unbranched distal dendritic segment. Four of these dendrites terminate near the tip of the sensillum below the pore and bear ultrastructural features consistent with contact chemosensilla. The fifth distal dendrite terminates near the base of the peg and bears ultrastructural features consistent with mechanosensilla. Thus, these sensilla each bear a bimodal chemo-mechanosensory function. The distal dendrites lie within the dendritic channel and are enclosed by a dendritic sheath. The intermediate and outer sheath cells enclose a large sensillar sinus, whereas the smaller ciliary sinus is enclosed by the inner cell. The neurons are ensheathed successively by the inner, intermediate, and outer sheath cells.     

Inhibitory effects of tebufenozide on dopa decarboxylase and tyrosine hydroxylase in 5th instar larvae of Spodoptera exigua (Hiibner) ( Lepidoptera: Noctuidae)

虫酰肼模拟昆虫蜕皮激素的作用干扰新表皮的形成.为了探讨虫酰肼对昆虫新表皮形成的影响是否与抑制表皮形成相关酶的活性有关,本研究应用高效液相色谱-荧光检测法(HPLC-RP),测定了甜菜夜蛾Spodoptera exigua5龄幼虫用虫酰肼处理不同时间(24,48和72 h)后多巴脱羧酶和酪氨酸羟化酶的活性.结果表明:用LCu( 28.41 μmol/L)和LC33 (85.23 μmol/L)两个亚致死剂量的虫酰肼处理5龄幼虫后,多巴脱羧酶和酪氨酸羟化酶的活性均受到明显抑制,高浓度的抑制作用大于低浓度的抑制作用.随着处理时间的延长,同一剂量的抑制作用逐渐增强.进一步测定虫酰肼处理24,48和72 h后5龄幼虫血淋巴、脂肪体、中肠、表皮和头部的多巴脱羧酶和酪氨酸羟化酶的活性,可看出虫酰肼对幼虫不同组织的多巴脱羧酶和酪氨酸羟化酶的活性也具有相似的抑制作用.结果提示,虫酰肼对甜菜夜蛾幼虫多巴脱羧酶和酪氨酸羟化酶活性具有明显抑制作用,幼虫新表皮形成受阻可能与虫酰肼抑制多巴脱羧酶和酪氨酸羟化酶的活性有关.     

Food utilization values of gypsy moth Lymantria dispar (Lepidoptera: Lymantriidae) larvae infected with the microsporidium Vairimorpha sp. (Microsporidia: Burenellidae)

Infection of the gypsy moth, Lymantria dispar, with the microsporidium Vairimorpha sp. strongly influences the development of the host in ways typical of many species of terrestrial entomopathogenic Microsporidia; growth is reduced while development time is extended in infected insects. The appearance of the different stages of the parasite in the host relative to the elapsed time after oral infection, as well as the influence of the parasite proliferation on food utilization of the host, were examined. At 3 days postinfection, midgut muscle cells were infected with primary spores, and the fat body tissues contained meronts, sporonts, and primary spores. Many more fat body cells contained vegetative stages and primary spores at 4 and 5 days postinfection, and diplokaryotic spores and immature octospores were also present. Approximate digestibility of infected larvae increased during this time period, whereas the conversion of ingested and digested food to body substance decreased. The relative growth rate of infected and uninfected groups did not differ significantly between 4 and 5 days postinfection, although the relative consumption rate in infected L. dispar larvae was higher. Between 8 and 10 days postinfection, the relative growth rate of uninfected larvae increased. The infected group did not demonstrate this increase at a time period characterized by maturation of diplokaryotic spores and octospores in larval fat body tissues. Total body weight of uninfected larvae remained higher than that of infected larvae after 8 days postinfection.     

The effect of varying alkaloid concentrations on the feeding behavior of gypsy moth larvae, Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Nine alkaloids (acridine, aristolochic acid, atropine, berberine, caffeine, nicotine, scopolamine, sparteine, and strychnine) were evaluated as feeding deterrents for gypsy moth larvae (Lymantria dispar (L.); Lepidoptera: Lymantriidae). Our aim was to determine and compare the taste threshold concentrations, as well as the ED₅₀ values, of the nine alkaloids to determine their potency as feeding deterrents. The alkaloids were applied to disks cut from red oak leaves (Quercus rubra) (L.), a plant species highly favored by larvae of this polyphagous insect species. We used two-choice feeding bioassays to test a broad range of biologically relevant alkaloid concentrations spanning five logarithmic steps. We observed increasing feeding deterrent responses for all the alkaloids tested and found that the alkaloids tested exhibited different deterrency threshold concentrations ranging from 0.1 to 10 mM. In conclusion, it appears that this generalist insect species bears a relatively high sensitivity to these alkaloids, which confirms behavioral observations that it avoids foliage containing alkaloids. Berberine and aristolochic acid were found to have the lowest ED₅₀ values and were the most potent antifeedants. Handling Editor: Joseph Dickens.     

柚木野螟成虫复眼外部形态和超微结构观察

【目的】柚木野螟Eutectona machaeralis主要取食危害珍贵树种柚木。本研究旨在观察研究柚木野螟成虫复眼的形态、组织结构和超微结构,分析其复眼结构特征,为更好了解该物种复杂的视觉行为与感光、趋光机制的关系奠定基础。【方法】运用光学显微镜以及扫描和透射电子显微镜技术观察了柚木野螟成虫复眼的形态、组织结构和超微结构。【结果】柚木野螟成虫复眼着生于头部触角基部,呈椭球形,属对称性复眼。雌、雄成虫复眼分别有2 300~2 755和1 950~2 316个小眼。小眼呈正六边形,表面密被角膜乳突,间隙偶有感觉毛。每个小眼由1个角膜、4个晶锥细胞、1对初级色素细胞,6个次级色素细胞、不同水平面分布的12个视网膜细胞和基膜等组成。沿小眼纵轴11个视网膜细胞的向心侧细胞膜特化成细丝状微绒毛,形成放射状排列的视小杆,组合呈融合型视杆;第12个视网膜细胞位于小眼基部。基膜上方,视网膜细胞和次级色素细胞末端膨大,以轴突形式穿过基底膜。【结论】柚木野螟复眼为典型的重叠像眼,雌、雄成虫小眼排列方式及内部结构无明显差异,但雌、雄虫小眼数量和大小具有明显的性二型现象。     

虫酰肼对甜菜夜蛾多巴脱羧酶和酪氨酸羟化酶的抑制作用

虫酰肼模拟昆虫蜕皮激素的作用干扰新表皮的形成。为了探讨虫酰肼对昆虫新表皮形成的影响是否与抑制表皮形成相关酶的活性有关, 本研究应用高效液相色谱-荧光检测法（HPLC-RP）, 测定了甜菜夜蛾Spodoptera exigua 5龄幼虫用虫酰肼处理不同时间（24, 48和72 h）后多巴脱羧酶和酪氨酸羟化酶的活性。结果表明： 用LC₁₁ （28.41 μmol/L）和LC₃₃ （85.23 μmol/L）两个亚致死剂量的虫酰肼处理5龄幼虫后, 多巴脱羧酶和酪氨酸羟化酶的活性均受到明显抑制, 高浓度的抑制作用大于低浓度的抑制作用。随着处理时间的延长, 同一剂量的抑制作用逐渐增强。进一步测定虫酰肼处理24, 48和72 h后5龄幼虫血淋巴、 脂肪体、 中肠、 表皮和头部的多巴脱羧酶和酪氨酸羟化酶的活性, 可看出虫酰肼对幼虫不同组织的多巴脱羧酶和酪氨酸羟化酶的活性也具有相似的抑制作用。结果提示, 虫酰肼对甜菜夜蛾幼虫多巴脱羧酶和酪氨酸羟化酶活性具有明显抑制作用, 幼虫新表皮形成受阻可能与虫酰肼抑制多巴脱羧酶和酪氨酸羟化酶的活性有关。     

Characterisation of three Alphabaculovirus isolates from the gypsy moth,Lymantria dispar dispar (Lepidoptera: Erebidae), in Turkey

In this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170?kb, respectively. Insecticidal activities of each isolate were tested on L. d. dispar larvae using four different viral concentrations between 10³ and 10⁶?OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13–53%, 47–100% and 46–93%, respectively. The LC₅₀ and LC₉₅ values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC₅₀ value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L. d. dispar larvae.     

Larvicidal and antifeedant activity of some plant-derived compounds to Lymantria dispar L. (Lepidoptera: Limantriidae)

Ethanol solutions of essential oil of Ocimum basilicum and its main component, linalool (both isomer forms), all in three concentrations, as well as botanical standard Bioneem (0.5%), were tested for their toxicity and antifeedant activity against the second instar gypsy moth larvae in the laboratory bioassay. The essential oil of O. basilicum was subjected to gas chromatography analysis, and totally 37 compounds were detected, of which linalool was predominantly present. All tested solutions showed low to moderate larvicidal effect in both residual toxicity test and in chronic larval mortality bioassay. Chronic mortality tests showed that obtained mortality was a consequence of starving rather than ingestion of treated leaves. However, antifeedant index achieved by application of tested solutions in feeding choice assay was remarkable. Foliar application of all tested compounds deterred feeding by L2 in the same percent as Bioneem. Antifeedant index was relatively high at all tested treatments (85-94%); moreover, the larval desensitization to repelling volatiles has not occurred after five days of observation. Low toxic and high antifeedant properties make these plant-derived compounds suitable for incorporation in integrated pest management programs, especially in urban environments.     

Larval integument and its differentiations in Micropterix calthella L. (Lepidoptera : Micropterigidae): Anatomy and ultrastructure

The outer layer of the integument of the larva of Micropterix calthella (Lepidoptera : Micropterigidae) shows numerous modifications shaped like crests, nipples, and cones. Sections made at the level of these modifications or structures show that internal and external layers of the exocuticle are separate and form intracuticular cavities filled with a fluid. Every segment on the thorax and abdomen bears 3 disc-shaped structures, one dorsally and 2 laterally. These discs correspond to muscle attachments. Leaf-shaped structures, measuring up to 180 μm high in the last-instar larva, are located along longitudinal lines on all segments. Despite their great anatomical diversity, these structures have the same ultrastructural organization. Although larvae appear to lack spiracles, they have an internal tracheal system, which does not seem to be linked with the intracuticular cavities.     

Conversion of ecdysone and 20-hydroxyecdysone into 3-dehydroecdysteroids is a major pathway in third instar Drosophila melanogaster larvae

Ecdysone and 20-hydroxyecdysone metabolism was investigated in third instar Drosophila larvae both in vivo by injecting radiolabelled ecdysteroids and in vitro by incubating various tissues with labelled ecdysteroids.Ecdysone metabolism proceeds through different pathways: (1) C-20 hydroxylation; (2) C-26 hydroxylation and C-26 oxidation leading to the formation of 26-hydroxyecdysteroids (26-hydroxyecdysone and 20,26-dihydroxyecdysone) and acidic compounds (ecdysonoic acid and 20-hydroxyecdysonoic acid); C-3 oxidation and C-3 epimerization then conjugation leading to the formation of 3-dehydrocompounds (3-dehydroecdysone and 3-dehydro-20-hydroxyecdysone), 3-epimers (3-epiecdysone and 3-epi-20-hydroxyecdysone) and conjugates (only one conjugate was tentatively characterized as 3-epi-20-hydroxyecdysone-3-phosphate). 3-Dehydrocompounds are the major metabolites formed in third instar Drosophila larvae and C-3 oxidation occurs in various tissues. Experiments using tritiated cholesterol provided evidence that 3-dehydroecdysone and 3-dehydro-20-hydroxyecdysone are true endogenous ecdysteroids in Drosophila larvae.