Swallowing motor pattern triggered and modified by sucrose stimulation in the larvae of the silkworm, Bombyx mori

To determine the contribution of sucrose signals to swallowing motor patterns, a series of behavioral, morphological and electrophysiological experiments were carried out in the larvae of the silkworm, Bombyx mori. The larvae ingested a droplet of sucrose solution applied to the mouth. The rate of ingestion was increased for higher sucrose concentrations. The swallowing movements were produced by a cibarial pump system that consisted of a circular compressor and pairs of dilators. The circular compressor was innervated by at least two dorsal motor neurons with the somata in the frontal ganglion. One of these neurons with arborized in both the frontal ganglion and the tritocerebrum of the brain. Both extra- and intracellular recording from the compressor showed that the rhythmic motor patterns were modified by different concentration of sucrose. A higher concentration of sucrose lengthened the duration of a burst or caused more excitatory junction potentials (EJPs) in the compressor, resulting in stronger swallowing contractions. Transection of both frontal connectives deleted the sucrose response, but spontaneous rhythmic motor patterns remained in the compressor, suggesting that the motor rhythm could be generated in the frontal ganglion, and triggered and/or modified by sucrose signals processed through the tritocerebrum of the brain.     

Effects of diet-deprivation and physical stimulation on the feeding behaviour of the larvae of the silkworm, Bombyx mori

Continuous observations of larvae of the silkworm, Bombyx mori, revealed that feeding occurred at regular intervals throughout larval development. To investigate possible factors influencing meal-timing, the behaviours of diet-deprived Bombyx larvae were also analysed. Diet-deprivation resulted in longer durations of the first meals after diet replacement, but did not affect feeding patterns. Furthermore, long-term diet-deprivation promoted wandering behaviour and a consequent delay in feeding after diet replacement. Under diet-deprivation conditions, meal-starts appeared to be inducible by defecation and physical stimulation. However, stimulation-induced meal-starts were dependent on the time elapsed since the larvae's previous meals. Provided that more than 1h had elapsed since their previous meals, larvae could be induced to feed by defecation and tapping. At less than 1h post-meal, larvae were less likely to begin feeding after defecation or physical stimulation. Activated locomotions such as wandering and feeding were observed in the long-term diet-deprived larvae only after diet blocks were replaced, while long-term diet-deprived larvae did not show activated locomotion during the absence of diet blocks. Collectively, these data suggest that a combination of elevated locomotion activity and the presence of diet may be necessary for the initiation of feeding in diet-deprived larvae.     

家蚕耐氟性差异的细胞化学研究

对不同蚕品种的耐氟性、ACPase的氟敏感性、蚕品种耐氟性机理的研究表明,在供试蚕品种中以浙农1号的耐氟性最强,杭 8的耐氟性最弱;家蚕Bombyx mori血淋巴ACPase活性与蚕品种的耐氟性无明显关系;氟对蚕的血淋巴和中肠组织细胞的ACPase活性都有抑制作用,并随着氟添食浓度的增加ACPase活性降低,但超过一定浓度的氟添食,血淋巴ACPase活性反而有一个回升的过程,这个转折点出现可能的浓度及回升的幅度与蚕品种的耐氟性有关;细胞化学研究发现此转折点的出现是由于高浓度氟引起细胞结构的破坏而导致蚕体组织细胞内的ACPase大量向血腔释放的结果;氟敏感性蚕品种杭 8在很低氟量添食即可引起中肠组织细胞的ACPase大量向血腔释放,使血淋巴中的ACPase活性大幅度上升,随后ACPase活性受到完全的抑制;耐氟性较强的蚕品种浙农1号则在较高的氟含量添食时才向血腔释放ACPase,且血淋巴中ACPase增高的幅度小,在很高的氟量添食时全面抑制中肠ACPase活性。氟对不同品种ACPase活性影响的差异被认为是家蚕品种耐氟性差异机理之一。     

家蚕味觉电生理反应的个体差异

为探讨家蚕Bombyx mori人工饲料饲养发育不齐的生理原因,从同一蚕品种中挑选出对人工饲料摄食性不同的个体,用电生理学方法测定了下颚瘤状体味觉感器对4种代表性物质（蔗糖、肌醇、大豆粉提取物和柠檬酸）的电生理反应。结果表明,栓锥感器Ss-Ⅰ对蔗糖等促食物质的反应以及栓锥感器Ss-Ⅱ对大豆粉提取物等阻食物质的反应,均存在明显的个体差异。在临界浓度下,低摄食性个体的放电脉冲频率显著高于高摄食性个体。说明低摄食性蚕的味觉反应比高摄食性蚕敏感。     

Morphological and electrophysiological characteristics of the epipharyngeal sensilla of the silkworm, Bombyx mori

One pair of gustatory sensilla was found on the epipharynx ofBombyx mori larvae, and some morphological and electrophysiological characteristics of the epipharyngeal sensilla were investigated. They are sensilla coeloconica composed of a small papilla with a pore at the tip and a swelling of cuticle encircling the papilla. Three bipolar neurons innervate each sensillum. One neuron is an inositol receptor which responds to inositol only. Another cell responds with action potentials of relatively large amplitude to some feeding deterrent substances, such as strychnine nitrate. The thresholds of these cells for inositol and strychnine nitrate are approximately 10⁻⁴ M and 10⁻⁷ M, respectively. At least two kinds of spikes can be observed when these sensilla are stimulated with some salts and acids. Dose-response relationships and time courses of responses to inositol and strychnine nitrate were also examined in this study.     

Recycling of urea associated with the host plant urease in the silkworm larvae,Bombyx mori

Urea concentration and urease activity in the midgut content were compared between larvae of the silkworm, Bombyx mori fed an artificial diet and those fed fresh mulberry leaves. A considerable amount of urea was found in the midgut content of the both larvae, however it was significantly lower in the larvae fed fresh mulberry leaves than in the larvae fed the artificial diet; average urea concentrations in the midgut content of the larvae fed fresh mulberry leaves and the artificial diet were 2.9 and 4.6 &mgr;mol/g, respectively. Urea in the midgut content seems to be secreted from the insect itself since the amount of urea in both diets were negligibly small. Urease activity was detected only in the midgut content of the larvae fed fresh mulberry leaves but not in other tissues of the larvae. On the other hand, no urease activity was detected in the midgut content of the larvae fed the artificial diet. Subsequently, to elucidate the role of mulberry leaf urease in the midgut lumen, larvae that had been reared on the artificial diet were switched to fresh mulberry leaves. The diet switch caused a rapid decrease in urea concentration in the midgut content and an increase in ammonia concentration in the midgut content, suggesting that secreted urea could be hydrolyzed to ammonia by mulberry leaf urease in the midgut lumen. Furthermore, to investigate the physiological significance of mulberry leaf urease on urea metabolism of the silkworm, (15)N-urea was injected into the hemocoel, and after 12 h the larvae were dissected for (15)N analysis. A considerable amount of (15)N was found to be incorporated into the silk-protein of the larvae fed fresh mulberry leaves, but there was little incorporation of (15)N into the silk-protein of the larvae fed the artificial diet. These data indicate that urea is converted into ammonia by the action of mulberry leaf urease in the midgut lumen and used as a nitrogen source in larvae fed mulberry leaves.     

A novel sucrose hydrolase from the bombycoid silkworms Bombyx mori,Trilocha varians,and Samia cynthia ricini with a substrate specificity for sucrose

Although membrane-associated sucrase activity has been detected in the midgut of various lepidopteran species, it has not yet been identified and characterized at the molecular level. In the present study, we identified a novel sucrose hydrolase (SUH) gene from the following three bombycoid silkworms: Bombyx mori, Trilocha varians, and Samia cynthia ricini and named them BmSuh, TvSuh, and ScSuh, respectively. The EST dataset showed that BmSuh is one of the major glycoside hydrolase genes in the larval midgut of B. mori. These genes were almost exclusively expressed in the larval midgut in all three species, mainly at the feeding stage. SUHs are classified into the glycoside hydrolase family 13 and show significant homology to insect maltases. Enzymatic assays revealed that recombinant SUHs were distinct from conventional maltases and exhibited substrate specificity for sucrose. The recombinant BmSUH was less sensitive to sugar-mimic alkaloids than TvSUH and ScSUH, which may explain the reason why the sucrase activity in the B. mori midgut was less affected by the sugar-mimic alkaloids derived from mulberry.     

Effects of juvenile hormone on the secretion of prothoracicotropic hormone in the last- and penultimate-instar larvae of the silkworm Bombyx mori

The effect of juvenile hormone (JH) on the secretion of the prothoracicotropic hormone (PTTH) was investigated, by examining the changes in hemolymph PTTH titer after the topical application of JH-I on the larvae of the silkworm, Bombyx mori. The titer of PTTH was determined by the time-resolved fluoroimmunoassay. JH-I application at very early stages of development in the fifth (last) instar resulted in a significant increase in the PTTH titer, but this effect became less evident thereafter. After the onset of wandering (day 6 of the fifth instar), JH-I did not affect the hemolymph PTTH titer. JH-I application on day 5 resulted in the delay of spinneret pigmentation on day 6, which is induced by an increase in the ecdysteroid titer on day 5 and is the first visible indication of larval-pupal transformation. However, the JH-I application did not suppress the increase in either PTTH or ecdysteroid titer on day 5, suggesting that JH-I acts on the spinneret to inhibit the response of the tissue to ecdysteroids. JH-I also exhibited a PTTH titer-elevating effect in the fourth instar. These results suggest that JH has a role as a potent stimulator of PTTH secretion in both the penultimate and last instar of the silkworm.     

Characterization of a ligand-gated cation channel based on an inositol receptor in the silkworm,Bombyx mori

Insect herbivores recognize non-volatile compounds in plants to direct their feeding behavior. Gustatory receptors (Gr) appear to be required for nutrient recognition by gustatory organs in the mouthparts of insects. Gr10 is expressed in Bombyx mori (BmGr10) mouthparts such as maxillary galea, maxillary palp, and labrum. BmGr10 is predicted to function in sugar recognition; however, the precise biochemical function remains obscure. Larvae of B. mori are monophagous feeders able to find and feed on mulberry leaves. Soluble mulberry leaf extract contains sucrose, glucose, fructose, and myo-inositol. In this study, we identified BmGr10 as an inositol receptor using electrophysiological analysis with the Xenopus oocyte expression system and Ca²⁺ imaging techniques using mammalian cells. These results demonstrated that Xenopus oocytes or HEK293T cells expressing BmGr10 specifically respond to myo-inositol and epi-inositol but do not respond to any mono-, di-, or tri-saccharides or to some sugar alcohols. These inositols caused Ca²⁺ and Na⁺ influxes into the cytoplasm independently of a G protein-mediated signaling cascade, indicating that BmGr10 is a ligand-gated cation channel. Overall, BmGr10 plays an important role in the myo-inositol recognition required for B. mori larval feeding behavior.     

摄入异烟肼对家蚕幼虫体内谷胱甘肽氧化还原循环和谷胱甘肽S-转移酶活性的影响

为了建立家蚕Bombyx mori的药物筛选和毒性评价模型, 以剂量为2 000 mg/kg的抗结核模药异烟肼饲喂家蚕5龄第3天幼虫后检测其中肠和脂肪体的抗氧化解毒相关代谢的变化。结果表明： 雌蚕中肠组织中, 总谷胱甘肽(GSH+2GSSG)、 还原型谷胱甘肽(reduced glutathione, GSH)和氧化型谷胱甘肽(oxidized glutathione, GSSG)含量均呈现迅速上升再缓慢下降趋势; 谷胱甘肽S 转移酶(glutathione S-transferase, GST)活性升高到较大值后逐渐降低; GSH/GSSG的比值下降表明, 在72 min后中肠组织向氧化态转移。脂肪体组织中, 总谷胱甘肽、 GSH和GSSG含量变化均呈现迅速下降再迅速上升的趋势; GST活性达到最大值后逐渐降低后趋于平稳; GSH/GSSG比值升高表明, 在72 min后脂肪体组织向还原态转移。无论雌蚕还是雄蚕, 总谷胱甘肽、 GSH和GSSG含量以及GST活性均是脂肪体高于中肠。雌蚕的总谷胱甘肽含量、 GSH和GSSG含量高于雄蚕, 但雄蚕的GST活性高于雌性。结果说明, 摄入异烟肼引起了家蚕幼虫体内谷胱甘肽氧化还原状态的改变和酶活性的变化, 在这个过程中脂肪体起主要解毒代谢作用。     

Effects of Diflubenzuron on the Development of Bombyx mori (Lepidoptera: Bombycidae)

ABSTRACT This study was conducted to determine the effects of diflubenzuron (DFB) on the growth and development of the silkworm, Bombyx mori (L.). The DFB treatment extended abnormally the larval duration and affected negatively on larval spinning of the 5th instar. All the larvae treated with high DFB concentration (2.5 × 10^-1 and 2.5x 10^-1μg/μl) lost their spinning capability and finally died, whereas 62% of larvae treated with low DFB concentration (2.5 × 10^-1μg/μl)) spinned cocoon. The larval weights depended sensitively on the DFB treatment period rather than on the DFB concentration. The DFB treatment decreased the larval maturity less than 6% without regard to the concentration and treatment period. All the larva, when treated with DFB before the 5th days of the 5th instar, were not matured.     

家蚕组织蛋白酶基因家族的鉴定及表达特征分析

家蚕是鳞翅目完全变态昆虫,在其变态过程中伴随着巨大的形态变化,包括旧组织的解离和新组织的形成,在这过程中有多种组织蛋白酶参与。组织蛋白酶是一类细胞内蛋白酶,广泛存在于各个物种中,包括组织蛋白酶B、H、L等几个亚家族。对家蚕组织蛋白酶的研究将有利于阐明家蚕变态发育的详细过程。通过对家蚕基因组数据库进行筛选,共在家蚕中鉴定到13种组织蛋白酶,并对这13种组织蛋白酶的基本信息和表达模式进行了分析。另外,利用家蚕基因芯片数据和荧光定量PCR分析,鉴定编号为BGIBMGA004622的基因为卵巢特异表达的组织蛋白酶L亚家族基因。该基因全长1 209 bp,编码402个氨基酸。经过序列分析,该酶与其他物种的组织蛋白酶L具有较高的同源性,其活性位点高度保守,且与鳞翅目的组织蛋白酶L在进化上聚为一支。同时,对该基因进行克隆并原核表达,结果显示重组蛋白以包涵体的形式表达。定量PCR结果显示,该酶在蛹发育初期表达量逐渐升高,至蛹3 d达到最高值,推测其可能参与卵巢与卵母细胞的发育过程。     

Steroidal regulation of hydrolyzing activity of the dietary carbohydrates in the silkworm, Bombyx mori

Blood sugar is an essential energy source for growth and development and is maintained at a constant level through precise regulation of formation and utilization. Sugars are produced from dietary carbohydrates by enzymatic hydrolysis in the digestive tract, which are under the homeostatic control of paracrine and prandial mechanisms in mammals. Here, we show that dietary carbohydrates hydrolyzing activity of the digestive tract is developmentally regulated by the steroid hormone ecdysone in the silkworm, Bombyx mori. The dietary carbohydrates hydrolyzing activity remained high throughout the last larval period and then decreased to negligible levels until the pupal period. However, dietary carbohydrates digestive activities were constitutively high when the steroidogenic organ, prothoracic glands were ablated. The prothoracic glands produced and released a large amount of ecdysone at the end of the larval period, suggesting that ecdysone is responsible for the decrease in dietary carbohydrates hydrolyzing activity. In fact, ecdysone decreased the activity to negligible levels in silkworms lacking the prothoracic glands. The present results indicate that the dietary carbohydrates hydrolyzing activity is regulated by ecdysone and that an increase in ecdysone titer decreases that activity at the end of the larval period, suggesting that ecdysone is essential for metabolic coordination during development.     

Analysis of ESTs and gene expression patterns of the posterior silkgland in the fifth instar larvae of silkworm, Bombyx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without homology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.     

乐果对家蚕生殖腺及氧化应激反应的影响

【目的】农药施加不当导致环境污染严重,已成为蚕桑业发展面临的紧迫问题。本研究旨在评估大田喷洒有机磷农药乐果污染间作桑园可能导致对家蚕Bombyx mori正常发育的影响,以分析乐果对家蚕生殖腺的毒理损伤影响。【方法】以文献报道的乐果对家蚕幼虫的LD₅₀为400 mg/L作为参考依据,给家蚕5龄幼虫添食不同浓度（0, 50, 100, 200和400 mg/L）乐果溶液浸叶处理的桑叶,测定乐果处理后5龄幼虫茧质和体重,雌、雄幼虫生殖腺中乐果残留含量和H2O2含量,抗氧化酶（SOD和CAT）活性及其mRNA表达。【结果】结果显示,乐果处理后家蚕5龄幼虫茧质和体重以及雌、雄幼虫生殖腺中乐果残留含量均具有浓度 效应关系。100~200 mg/L乐果浓度添食下,雌雄幼虫生殖腺中H₂O₂含量与对照（清水）相比均显著上升。随着添食浓度的增加,SOD和CAT活性都出现先降低后升高的趋势,但均低于对照组。sod和cat的mRNA表达水平与相应的酶活性具有正相关性。通过HE染色以及生精囊的体外培养,观察到在乐果处理后雌、雄幼虫生殖腺的形态结构发生变化,表现为形态畸形,细胞内空泡增大,生殖细胞数目相对减少,且随浓度的增加,生殖腺损伤越严重。【结论】本研究结果说明,采用乐果对家蚕的LD₅₀以下浓度溶液浸叶添食于5龄幼虫,对家蚕生殖腺发育有毒理损伤的影响,这为从蚕业生产角度全面禁止大田使用乐果和加强农业生产区划管理提供了依据。     

Larval feeding status regulates the transcript levels of genes encoding PTTH and allatoregulatory peptides in silkworm Bombyx mori

In Bombyx mori, two dorsolateral neurosecretory cells (NSCs) in each of the two brain lobes have been identified as prothoracicotropic hormone (PTTH) producing cells. This neuropeptide in insects stimulates the prothoracic gland for the synthesis and release of ecdysone, responsible for the molting events. Allatotropin (AT) and allatostatin (AST) are allatoregulatory neuropeptides that regulate juvenile hormone biosynthesis. Here, by using RT-qPCR, we showed that in B. mori, nutritional stress modulates the mRNA expression of AT and AST-C (allatostain type C) in the central nervous system consisting of the brain lobes and all the associated ganglia. Using whole-mount in situ hybridization, we showed that the feeding status of Bombyx larvae also influences the expression of PTTH in the NSCs of the brain. Food deprivation significantly decreased the mRNA expression levels of PTTH in larvae at active or terminal growth period. Further, we showed that insulin modulates the expression level of PTTH. However, its action was dependent on the feeding status of the larvae. At feeding, the insulin decreased the PTTH expression level, while at food deprivation, the insulin increased the PTTH expression level. The data thus indicates that larval feeding status plays an important role in altering the mRNA expression levels of allatoregulatory peptide genes and PTTH.     

家蚕免疫稳态调控分子的鉴定和表达模式分析

昆虫免疫稳态的维持有赖于准确地激活和有效地抑制Toll或IMD信号通路中的关键转录因子-- Dorsal/Dif或Relish。在果蝇等昆虫中, 已报道了多种降低转录因子稳定性和活性的免疫稳态调控分子, 突变或敲除这类分子导致免疫系统的过度激活。对家蚕Bombyx mori免疫信号通路的研究中, 至今为止尚无对这类分子的探索。本研究通过比较基因组学, 在家蚕基因组中鉴定了多个可能参与免疫稳态调控的分子, 包括Wnt家族成员、 Ubc9、 FAF和POSH等; 并通过检测家蚕被微生物感染后这些分子在多种免疫器官中的诱导表达模式, 发现这些分子的表达水平在微生物感染后普遍呈下降趋势, 虽然在某些组织中表达量有明显的升高（>1.5倍）, 但此高表达水平均不能维持且迅速下降; 而且免疫稳态调控分子和受其调控的信号通路的对应关系在不同组织中表现出差异。本研究是首次对家蚕免疫稳态调控分子的报道, 为深入研究家蚕免疫负调控的分子机制提供了参考。     

Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.