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.     

家蚕幼虫5龄期中肠蛋白质组学

利用双向电泳技术对家蚕幼虫5龄期第2天、第5天和第7天的中肠蛋白质进行分离, 并利用ImageMaster 2D Platinum对所分离得到的蛋白图谱进行比较, 并对一些蛋白质斑点进行了质谱鉴定。研究发现, 家蚕中肠蛋白质具有区别于家蚕其他组织的明显特征: 蛋白质大多分布在PI值4-8、分子量20~70 kD的区域, 且分布不均匀, 主要集中在酸性一侧, 这一特点在家蚕5龄期第7天的图谱尤为明显。5龄期家蚕第2天的蛋白质斑点数目为869个, 而到第5天增加到966个, 新增蛋白数目97个, 进一步观察发现增加的蛋白主要分布在PI值6-9, 分子量20~40 kD区间; 随着进入幼虫成熟期, 蛋白质斑点数目明显减少, 第7天斑点数仅为420个, 比第5天减少了56.5%。这些结果说明家蚕中肠组织蛋白质组成在5龄早、中、晚期经历了较大变化, 暗示这可能与中肠的功能相适应。从MALDI-TOF-MS鉴定的斑点发现了构成家蚕中肠组织的一些新的部分结构蛋白和一些可能与消化、吸收相关的蛋白, 还发现一些能够抵御外界微生物入侵的相关蛋白。这些结果为进一步认识家蚕中肠提供了重要的理论基础。     

Possible Alteration of Female Specific Protein by Estradiol-17β in Silkworm,Bombyx mori L

In our attempt to understand the biological significance of natural occurrence of estradiol-17β (E₂, a vertebrate female sex steroid) in silkworms effect of exogenous estradiol-17β injection on the appearance of female-specific proteins both in pupal hemolymph and ovarian extracts was undertaken. A single injection of 2 μg of E₂ per g of body weight on the 3rd day in the 5th instar female silkworm (Bombyx mori L. race Nistari) larvae revealed more accumulation of both 170- and 43 kDa proteins from the day 3 to 7 of pupal life with a peak on the day 7 in comparison to the untreated (control) hemolymph. On the contrary, the ovarian extracts revealed highest accumulation of both the proteins in female silkworm pupae on the day 4 and thereafter, declined gradually to level up with the control on the day 8. The fertilized egg extracts irrespective of control and E₂-treated silkworms failed to establish quantitative differences of the proteins in question. Thus the metabolic role of E₂ on the female-specific proteins in silkworm could not be ruled out.     

DFMO feeding lowers polyamine levels and causes developmental defects in the silkworm Bombyx mori

The domesticated silkworm Bombyx mori is an economically important insect that produces large quantities of silk during its 5th instar larval stage. Polyamines are important regulators of growth and have been shown to affect silk production, however their role in larval development is not completely understood. L-ornithine decarboxylase (ODC), a key regulatory enzyme in the polyamine biosynthetic pathway catalyzes the conversion of ornithine to putrescine, which is further broken down to spermidine and spermine. In this study, we set out to understand the role of ODC on the growth and development of silkworm larvae. We fed 5th instar larvae with α-difluoromethylornithine (DFMO), an ODC inhibitor and studied its impact on larval silk glands. Feeding DFMO did not alter the expression of L-ODC but led to a significant reduction in putrescine and spermidine levels. Furthermore, reduced cellular levels of polyamine led to increased oxidative stress and decreased cell viability. Subsequently, this resulted in several developmental defects at the pupal and moth stages. These findings highlight the importance of ODC in the growth and development of B. mori larvae.     

The effect of larval nutrition on egg production in Rhodnius prolixus

Rhodnius prolixus adults which had fed to repletion at each larval instar were frequently autogenous, while those allowed to take only partial blood meals at the 3rd, 4th and 5th larval instars were always anautogenous.Bugs fed to repletion at each larval instar had large abdominal fat bodies throughout adult life. In such bugs, the quantity of blood associated with the production of one egg was small and constant during the course of several blood meals.Bugs fed partial blood meals as larvae had small abdominal fat bodies following ecdysis to the adult; the quantity of blood associated with the production of one egg was large following the first adult feed because some of the blood meal was used to increase the size of the abdominal fat bodies.     

Haemolymph levels of juvenile hormone,ecdysteroids and protein during the last two larval instars of Locusta migratoria

The radioimmunoassay of ecdysteroids and juvenile hormone has enabled us to relate hormone levels to haemolymph protein concentrations and weight increase during the 4th and 5th instar of the migratory locust. The two hormones are never present in high concentrations in the blood simultaneously. The levels of ecdysteroids are high on the 5th day during the 4th larval stage: they show a small peak on the 3rd day, and then a large peak on the 8th day during the 5th instar. JHI-immunoreactive substances are high during the first 4 days of the 4th instar, and during the first 5 hr during the 5th instar. Protein concentrations in the haemolymph begin to rise when ecdysteroid levels increase during stage 4, and immediately after the small peak (on day 3) in the 4th stage larva. The rise in protein levels is correlated with an increase in weight.     

Changes in the activity of dopa quinone imine conversion factor during the development of Bombyx mori

The activity of DOPA quinone imine conversion factor (QICF) in tissues at different developmental stages of the silkworm, Bombyx mori, was determined. QICF activity was detected in all developmental stages from egg to pupa although the activities, other than in fifth instar larvae, were quite low. Activity in whole larvae peaked one day before the onset of larval-pupal development and declined to a low level shortly before ecdysis. In whole pupae, maximal QICF activity was obtained 1 h after pupation. The activity in larval cuticles was elevated on the last day of the fourth instar and again between days 4 and 8 of the fifth instar, decreasing to very low levels before pupal ecdysis. QICF was detectable in pupal cuticles with most of the pupal activity found in homogenates of mid and hind guts. A major part of the total larval QICF activity was found in hemolymph. Activity in hemolymph varied in a different manner from that in cuticles, with markedly raised levels immediately before pupal ecdysis when the cuticular activity had declined. It is postulated that QICF in cuticles plays some role in wound healing and/or sclerotizatio,, while QICF in hemolymph participates in melanization in the humoral immune system.     

Physiological aspects of nm-g mutant: An ecdysteroid-deficient mutant of the silkworm,Bombyx mori

We examined physiological and endocrinological properties of the nm-g mutant of the silkworm, whose development was arrested at 1st or 2nd larval instar. The larva of the nm-g homozygote continued to feed and grow past the stage when a normal larva moulted to the next instar. The nm-g larva attained more than twice the body weight of a normal moulting larva, but remained in the same instar with no sign of the moulting until it finally died. The ecdysteroid concentration in the haemolymph of the nm-g larva was reduced and an increase in the titre, which occurred before moulting in the normal larva, was not observed. Injection of 20-hydroxyecdysone induced the nm-g larva to moult and ecdyse to the next larval instar. From these results, we conclude that nm-g is an ecdysteroid-deficient mutant.     

Involvement of adipokinetic hormone in the homeostatic control of hemolymph trehalose concentration in the larvae of Bombyx mori

Prior to wandering, 5th instar larvae of the silkworm, Bombyx mori, maintain constant hemolymph titers of trehalose. Head ligation of day 3, 5th instar larvae significantly decreased the hemolymph trehalose concentrations, but the concentrations did not decrease in starved larvae. After being diluted by replacement of larval hemolymph with insect Ringer's solution, the trehalose concentrations recovered the initial levels in 90 min in the non-ligated larvae, while they were not restored in 90 min in the neck-ligated larvae. These results suggest that a head factor(s) with hypertrehalosemic activity is involved in the homeostatic control of hemolymph trehalose concentration. When adipokinetic hormone (AKH) was injected into neck-ligated larvae, the trehalose concentrations increased in 2 h and decreased thereafter. Repeated injections of AKH every 4 h maintained the concentrations for 12 h. These findings suggest that AKH induces a hypertrehalosemic response and is involved in the homeostasis of hemolymph trehalose concentration in the larval feeding period.     

Ecdysteroid as a mediator in the regulation of silk protein synthesis and its influence on silkworm (Bom., Lepidoptera) genome

Abstract:  Ecdysteroid is one of the two most important insect hormones, which controls growth and developmental activities of insect. In silkworm, Bombyx mori L., basically ecdysteroid induces molting and metamorphosis in larvae and exogenous administration hastens the silk synthetic activity and cocoon spinning process but it strictly depends on the time of administration. In the present study, we administered phytogenous ecdysteroid to the 48-h-old 5th instar silkworm, B. mori at a single dose of 2  μ g per larva to study its effects on the larval growth, cocoon and silk variables. The possible role of ecdysteroid in altering the developmental sequence of silkworm and thereby its effect on cocoon and silk production at molecular level have been tried to get elucidated. The genomic DNA was isolated from the posterior silk gland on day 5 and 7 of the 5th instar from the ecdysteroid treated and the control larvae and was randomly amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of ecdysteroid in regulating the silk gene. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of ecdysteroid in the silk biosynthetic process with its physiological and molecular implications was discussed.     

Changes in calmodulin levels during development of the silkworm,Bombyx mori

Calmodulin levels were measured in various tissues during the larval-adult development of the silkworm, Bombyx mori. In the larval period, calmodulin levels in fat body, midgut and testis were in a range of 0.3–1.7 μg/mg protein and remained almost constant during larval growth. The silk gland contained a relatively high (0.2 μg/mg protein) level of calmodulin early in the fifth instar which gradually decreased during maturation of the larva. At pupation, testis calmodulin dropped from 1.5 to 1.7 μg/mg protein to about 1 μg/mg, and remained constant thereafter. The most striking change occurred in fat body calmodulin which fell from 0.5 to 0.6 μg/mg in the larval stage to 0.01–0.03 μg/mg during pupal-adult metamorphosis. Midgut calmodulin levels were unchanged at pupation and remained constant during pupal-adult development.When expressed on per g wet weight basis, calmodulin levels in silkworm tissues were comparable to mammalian tissue levels. However, only 2–4% of the total calmodulin in silkworm tissues was in a membrane-bound form compared to 20–60% for membrane-bound calmodulin in mammals.     

Brain-independent development in the moth Sesamia nonagrioides

The caterpillars of Sesamia nonagrioides developing under long-day (LD) photoperiod pupate in the 5th or 6th instar whereas under short day (SD) conditions they enter diapause and undergo several extra larval molts. The diapause is terminated within 1-3 instars upon transfer of SD larvae to the LD conditions. Brain removal from the 6th instar larvae promotes pupation followed by imaginal development; however, one third of the SD larvae and 12% of the LD larvae debrained at the start of the instar first undergo 1-2 larval molts. The incidence of larval molts is enhanced by the brain implants. Exclusively pupal molts occur in the LD larvae debrained late in the 6th instar. Decapitation elicits pupation in both LD and SD larvae, except for some of the 4th and 5th and rarely 6th instar that are induced to a fast larval molt. The pupation of decapitated larvae is reverted to a larval molt by application of a juvenile hormone (JH) agonist. No molts occur in abdomens isolated from the head and thorax prior to the wandering stage. Abdomens isolated later undergo a larval (SD insects) or a pupal (LD insects) molt. Taken together the data reveal that in S. nonagrioides (1) several larval molts followed by a pupal and imaginal molt can occur without brain; (2) an unknown head factor outside the brain is needed for the pupal-adult molt; (3) brain exerts both stimulatory and inhibitory effect on the corpora allata (CA); (4) larval molts induced in CA absence suggest considerable JH persistence.     

电穿孔法可用于家蚕基因功能分析(英文)

【目的】明确基于电穿孔的基因功能分析方法在家蚕Bombyx mori活体内的应用实效。【方法】针对调控家蚕幼虫体表斑纹黑色素合成的靶基因Wnt1 (Wingless),人工合成特异性siRNA,向4龄第3天家蚕幼虫注射Wnt1 siRNA并进行电穿孔作为处理组(ERFA-RNAi),以注射Wnt1 siRNA但未进行电穿孔的幼虫作为阴性对照组(negative control, NC),解剖5龄幼虫斑纹区的表皮,用实时定量RT-PCR测定表皮中Wnt1的相对表达量,验证电穿孔介导的RNAi效果。带有增强型绿色荧光蛋白报告基因EGFP和红色荧光蛋白报告基因DsRed2表达元件的转座子载体pPIG-A3GR,通过电穿孔导入家蚕2龄幼虫;正常饲养72 h后,用荧光体视显微镜观察幼虫中EGFP和DsRed2的表达,验证当世代家蚕的嵌合体转基因。【结果】家蚕4龄第3天幼虫中导入Wnt1的特异性siRNA后,5龄幼虫体表特定部位斑纹的形成明显受到抑制,实时定量RT PCR分析表明5龄幼虫表皮中Wnt1的表达水平显著降低。嵌合体转基因家蚕的阳性率达56.60%,并且两个荧光报告基因EGFP和DsRed2在幼虫、蛹及成虫期持续表达。【结论】电穿孔技术可快捷、高效地向家蚕活体内导入外源RNA或DNA,是家蚕乃至其他昆虫基因功能解析的有效方法。     

家蚕幼虫血细胞密度变化成因及血细胞密度与耐高温性的关系

[目的]血细胞是昆虫血淋巴免疫的主导者.调查家蚕Bombyx mori幼虫血细胞密度变化和成因、血细胞密度与家蚕抗性的关系,是研究家蚕血细胞相关的免疫调控和抗性育种的重要组成.[方法]用细胞计数板统计家蚕品种大造不同龄期(4龄第1-4天、5龄第1-8天和上蔟期)幼虫10 μL血淋巴中的血细胞数目并计算血细胞密度,利用I...     

Neuroendocrine regulation of corpus allatum activity in Manduca sexta: The endocrine basis for starvation-induced supernumerary larval moult

When newly-ecdysed 5th instar larvae of Manduca sexta were starved for 3 days and thereafter fed on standard diet the majority (90%) of the surviving larvae moulted into 6th instars. Allatectomy prior to starvation abolished the supernumerary moult, while denervation of the corpora allata (CA) had no effect.Cautery of medial neurosecretory cells, but not of the lateral cells, prevented supernumerary moulting and pupation ensued. Transplantation of brains from young 5th instar donors into larvae, whose medial neurosecretory cells were cauterized prior to starvation, restored the extra larval moult. Neither CA nor corpora cardiaca (CC) could be substituted for the medial neurosecretory cells.For induction of the supernumerary moult the medial neurosecretory cells are required only until day 1 after refeeding whereas the CA are required until day 3 after refeeding. Allatectomy on day 3 after refeeding resulted in the production of black 6th instar larvae.We conclude that starvation-induced supernumerary moulting is due to activation of the CA by allatotropin produced by medial neurosecretory cells in the brain. The anteromedial cells (group II) appear to be the source of allatotropin.     

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.     

Effects of applaud on the growth of silkworm (Lepidoptera: Bombycidae)

An experiment was carried out to evaluate the effect of the insecticide Applaud (buprofezin 25% WP) on the silkworm Bombyx mori (L.). This insecticide belongs to the class of insect growth regulators (IGR). The larvae were fed on leaves treated with 3 different concentrations (0.5, 1, 2 g/liter) of Applaud on the 1st d of each instar. Analysis of data with the Tukey-Kramer test at 1% significant level revealed that mortality and larval duration did not differ among the treatments. On the contrary, the larval weight, which was estimated just before mounting (procedure during which the mature larva climbing on a branch or other material to spin the cocoon), differed among the treatments. Also, cocoon weight, shell weight, and cocoon sericin and fibroin content were different among the treatments, except the shell cocoon ratio. Maximum weight was observed in the controls and minimum in the last instar treatments. Our data suggest that supplementation of Applaud through food to larvae does not affect their mortality rate. On the contrary, it affects larval growth and cocoon parameters.     

中华花翅跳小蜂生物学特性和种群动态的研究

中华花翅跳小蜂Microterys sinicus Jiang是白蜡虫成虫产卵期的主要天敌。该蜂成虫喜在自然光下活动,取食和交配都在寄主树上进行。中华花翅跳小蜂每雌平均怀卵量34.39粒,产卵前期2～3天。产卵量、产卵率和产卵天数与温度密切相关,27℃时产卵量和产卵率最高。30℃时产卵天数最少。21～27℃卵孵化率达80%以上。幼虫共5龄。每头跳小蜂幼虫平均取食蜡虫卵数分别为：1龄14.6粒,2龄29.8粒,3龄69.4粒,4龄126.9粒。5龄幼虫不取食。全代发育起点温度和有效积温分别为11.84℃和574.32日·度。补充营养能显著延长成虫寿命和产卵天数,提高雌虫产卵量。中华花翅跳小蜂成虫在田间有两个高峰期,分别发生在4月上中旬和5月中下旬。每粒虫囊内跳小蜂幼虫数与白蜡虫健卵百分率呈负指数关系,拟合方程为：Y=96.0829Exp(-0.1872x)。     

基于双元转基因CRISPR/Cas9系统的家蚕pax3基因功能分析

【目的】本研究旨在以家蚕Bombyx mori为研究模型探索pax3基因在鳞翅目昆虫中的生物学功能。【方法】利用PCR扩增验证家蚕Bmpax3外显子序列;利用qRT-PCR检测Bmpax3在5龄第3天家蚕幼虫头、表皮、脂肪体、中肠、马氏管、前部丝腺、中部丝腺、后部丝腺和生殖腺(包括精巢和卵巢)中的表达谱;利用双元转基因CRISPR/Cas9系统构建Bmpax3敲除突变体,分析Bmpax3突变对家蚕幼虫存活、体节分化及性别差异的影响。【结果】Bmpax3在家蚕5龄第3天幼虫头、中肠和丝腺中均有表达,其中在前部丝腺表达量最高。Bmpax3突变体的卵孵化率约为90%,但约有80%的突变体在1龄幼虫期死亡,有将近10%的突变个体能幸存并发育到成虫阶段,并且存活成虫数存在性别差异,雄性显著多于雌性。在幸存的成虫中,约有将近1/2的个体腹部末端体节分节异常,表皮条纹混乱,腹节腹板部分缺失,生殖器官及其周围的其他辅助器官出现发育缺陷。【结论】Bmpax3发生突变后会对家蚕的生存及形态发育产生较大的影响,提示Bmpax3可能参与了家蚕的生长发育过程。     

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.