Macromolecular synthesis in wing discs of Spodoptera mauritia Boisd: effects of a juvenile hormone analogue

Last instar larvae of S. mauritia treated topically on day 0, day 1, day 2 and day 3 with a daily (dose of 25 microg juvenile hormone analogue (JHA) moulted into supernumerary larvae. The imaginal discs of the supernumerary larvae especially those of mouthparts and thoracic appendages showed pupal characteristics. However the wing discs, which showed only partial differentiation, were uneverted and highly tanned. In an effort to provide an explanation to this anomaly the RNA, DNA and protein profile in the wing discs of supernumerary larvae were studied. Quantitative analysis of DNA, RNA and protein showed a considerable increase in the amount of DNA and protein and a decline in RNA level. SDS-PAGE analysis of wing disc proteins of JHA treated larvae showed a reduction in the expression of many major proteins that were predominant in the wing discs of control larvae. The results suggest that JHA induced inactivation of genes involved in the synthesis of proteins needed for evagination process may be responsible for the formation of uneverted, partially differentiated pupal wing discs in supernumerary larvae.     

低致死剂量氯虫苯甲酰胺对沟金针虫食物利用和相关生理生化指标的剂量和时间效应

【目的】明确氯虫苯甲酰胺对沟金针虫Pleonomus canaliculatus亚致死效应的生理生化机制,阐明氯虫苯甲酰胺低致死剂量对沟金针虫食物利用、能量物质含量以及体内消化酶、保护酶和解毒酶活力的影响。【方法】室内采用土壤混药法测定氯虫苯甲酰胺对沟金针虫3龄幼虫毒力,并测定了氯虫苯甲酰胺LC₁₀, LC₂₅和LC₄₀低致死剂量对沟金针虫3龄幼虫营养指标和体内能量物质含量的影响;采用酶动力学法检测了氯虫苯甲酰胺低致死剂量处理1, 6, 12, 24, 48和72 h后沟金针虫3龄幼虫体内消化酶(蛋白酶、α-淀粉酶、脂肪酶、海藻糖酶)、保护酶(CAT, POD和SOD)以及解毒酶(CarE, MFO和GST)活力的动态变化。【结果】氯虫苯甲酰胺对沟金针虫3龄幼虫有较高毒力,其LC₅₀值为1.2397 mg/kg。LC₁₀和LC₄₀剂量氯虫苯甲酰胺处理沟金针虫3龄幼虫后,平均相对生长率(MRGR)和近似消化率(AD)显著降低,严重干扰其对食物的利用;LC₁₀, LC₂₅和LC₄₀剂量处理后沟金针虫3龄幼虫体内主要的能量物质(蛋白质、脂质、碳水化合物、海藻糖)含量和消化酶活力均明显降低,而解毒酶和保护酶活力显著增加,最终延缓其生长发育。【结论】氯虫苯甲酰胺对沟金针虫幼虫具有很高的杀虫活性,低致死剂量氯虫苯甲酰胺处理沟金针虫幼虫后,通过抑制消化酶活性,使其对食物的利用能力降低和生长发育延缓,以及诱导解毒酶和保护酶活性来阻止外界毒物侵害。研究结果为阐明氯虫苯甲酰胺对沟金针虫的亚致死效应机制及作用机理提供了一定的理论基础。     

饲喂四环素和链霉素对贡嘎蝠蛾幼虫生长和肠道消化酶的影响

以抗生素(四环素和链霉素)处理的食物饲喂贡嘎蝠蛾Hepjalus gonggaensis Fu & Huang,sd.nov.4龄幼虫1个月后,肠道主要消化酶的种类和酶活都发生了显著变化。经测定知,淀粉酶和蛋白酶是4龄幼虫主要的消化酶,其次为海藻糖酶、麦芽糖酶和纤维素酶。经抗生素处理的食物饲喂后,与正常对照相比,抗生素处理的幼虫体重增长百分比显著降低(P<0.01),所测的消化酶酶活比对照也显著降低(P<0.01)。另外,肠道内原有的半纤维素酶和纤维素酶在抗生素处理后没有检测出酶活。据此认为,抗生素可能对幼虫生长有毒害作用。其原因可能是抗生素长期使用导致肠道菌群紊乱,消化生理发生改变,从而导致一些消化酶活性下降甚至完全失去活性。     

Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson

During larval diapause lasting 9 months from September to May, trehalase activity in the midgut of the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae) was low from December to April, followed by a fourfold increase in May that remained high during the pupal stage in July. An application of juvenile hormone analog (JHA) produced increases in the ecdysteroid titer, while trehalase activity was increased by both JHA and 20-hydroxyecdysone (20E) injection. The trehalase activity in the midgut of diapausing larvae was doubled by incubating the midgut with 20E for 48h. During diapause as well as after JHA application, expression of two ecdysone receptor isoform genes (EcR-A and EcR-B1) in the midgut increased simultaneously with the increase in hemolymph ecdysteroid titer, followed by an increase in trehalase activity. The hemolymph of diapausing larvae contained a trehalase inhibitor and inhibitory activity was high during diapause. After 20E injection, trehalase inhibition decreased as midgut trehalase activity increased. Taken together, at least two factors may participate in the change in midgut trehalase activity: increase in trehalase activity and decrease in trehalase inhibitor activity, both of which may be induced by 20E.     

转Bt基因抗虫棉对棉铃虫拒食作用及其机理研究

转Bt基因抗虫棉对 3 ,4,5龄棉铃虫幼虫的抗性表现形式为拒食作用 ,且随幼虫龄期的增加拒食作用明显降低 ,其中对 5龄幼虫的拒食作用很低。取食Bt棉后 ,3 ,4,5龄棉铃虫幼虫中肠消化酶比活力均较对照有所减退 ,且随幼虫龄期的增加减退率明显降低 ,其中对 5龄幼虫减退率最低。由此 ,解释了Bt棉对 3龄及 3龄以上棉铃虫幼虫抗性表现形式、抗性随不同幼虫龄期的差异性 ,及其抗性差异性的消化机理。     

外源激素处理后家蚕吡哆醛激酶和磷酸吡哆醇氧化酶转录水平的变化

【目的】研究家蚕 Bombyx mori 经蜕皮激素(20-hydroxyecdysone, 20-E)和保幼激素类似物(juvenile hormone analogue, JHA)处理后引起吡哆醛激酶(pyridoxal kinase, PLK)和磷酸吡哆醇氧化酶(pyridoxine-5′-phosphate oxidase, PNPO)的转录水平变化,为进一步研究激素对蚕体营养代谢等工作奠定基础。【方法】以20-E和JHA分别喂食不同发育时期(5龄第1, 3和5天)的家蚕幼虫,以喂食蒸馏水的家蚕为对照,采用实时荧光定量PCR(real-time quantitative PCR)方法在处理后24 和48 h对各组幼虫后部丝腺中PLP合成酶PLK和PNPO的转录水平进行分析。【结果】5龄第1天幼虫经20-E处理24和48 h后,PLK和PNPO的转录水平出现上调且与对照的差异达到极显著 (P＜0.01);5龄第3天幼虫经20-E处理,PLK的转录水平在48 h出现下调且与对照的差异达到显著(P＜0.05),PNPO的转录水平在24 和48 h均出现上调且与对照的差异达到极显著 (P＜0.01);5龄第5天幼虫经20-E处理后PLK和PNPO的转录水平无变化。5龄第1天幼虫经JHA处理后PLK和PNPO的转录水平未受到影响;5龄第3天幼虫经JHA处理后,PLK的转录水平在48 h出现显著下调且与对照的差异达到显著(P＜0.05),PNPO的转录水平在24和48 h后均出现显著下调且与对照的差异达到极显著(P＜0.05);5龄第5天幼虫经JHA处理24和48 h后,PLK和PNPO的转录水平出现下调且与对照的差异达到极显著 (P＜0.01)。【结论】20-E和JHA显著影响家蚕5龄幼虫PLK和PNPO的转录水平,20-E提高5龄前期家蚕PLK和PNPO的转录水平,JHA降低5龄后期它们的转录水平,为深入研究激素对VB₆的调控奠定基础。     

Ambient temperature‐mediated enzymic activities and intestinal microflora in Lymantria dispar larvae

To understand how ambient temperature affect the gypsy moth larvae, and provide a theoretical basis for pest control in different environments. Fourth instar gypsy moth larvae were incubating for 3 hr at 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃, respectively. Afterward, digestive and antioxidant enzyme activities, total antioxidant capacity, and intestinal microflora community were analyzed to reveal how the caterpillars respond to ambient temperature stress. Results showed that both digestive and antioxidant enzymes were regulated by the ambient temperature. The optimum incubation temperatures of protease, amylase, trehalase, and lipase in gypsy moth larvae were 30℃, 25℃, and 20℃, respectively. When the incubation temperature was deviated optimum temperatures, digestive enzyme activities would be downregulated depending on the extent of temperature stress. In addition, glutathione S‐transferase, peroxidase, catalase, and polyphenol oxidase would be activated under a sufferable temperature stress, but superoxide dismutase and carboxylesterase (CarE) would be inhibited. In addition, results showed that the top two abundant phyla were Proteobacteria and Firmicutes. The phylum Firmicutes abundance was decreased and phylum Proteobacteria abundance was increased by ambient temperature stress. Moreover, it suggested that gypsy moth caterpillars at different ambient temperature mainly differed from each other by Escherichia‐Shigella and Bifidobacterium in control, Acinetobacter in T15, and Lactobacillus in T40, respectively.     

Regulation of soluble and membrane-bound trehalase activity and expression of the enzyme in the larval midgut of the bamboo borer Omphisa fuscidentalis

Diapausing larvae of Omphisa fuscidentalis contain soluble and membrane-bound trehalase in the midgut. Soluble trehalase activity accounts for three-fourths of the total trehalase activity in midgut homogenates. The exposure of diapausing larvae to juvenile hormone analog (JHA) induced pupation, accompanied by an increase in soluble trehalase activity at the beginning of the prepupal period. Injection of 20-hydroxyecdysone (20E) increased the level of soluble trehalase activity 5 days postinjection in a dose-dependent manner. In contrast, no increase in membrane-bound trehalase activity was observed under the same conditions. We cloned the cDNAs that encode the soluble and membrane-bound forms of trehalase in O. fuscidentalis trehalase-1 (OfTreh-1) and trehalase-2 (OfTreh-2), respectively. Treh-1 encodes a 581-aa protein while Treh-2 encodes a 648-aa protein with one putative transmembrane domain near the C-terminus. The mRNA expression level of Treh-1 was 27-fold higher than that of Treh-2 in diapausing larval midgut. Following the exposure of diapausing larvae to JHA, Treh-1 mRNA expression increased gradually until the prepupal period whereupon it increased dramatically; in contrast, the mRNA expression of Treh-2 remained at its initial level. Similarly, 20E upregulated Treh-1 expression but had no effect on Treh-2 expression. Taken together, these results suggest that an increase in the soluble trehalase activity at pupation is caused by upregulation of Treh-1 gene. Moreover, membrane-bound trehalase does not appear to be involved in the dynamic changes in the hemolymph trehalose concentration that occur during the larval-pupal transformation.     

Death commitment in the anterior silk gland of the silkworm, Bombyx mori

The insect steroid hormone, 20-hydroxyecdysone (20E) triggers the programmed cell death (PCD) of the anterior silk glands (ASGs) of the silkworm, Bombyx mori. We tried to determine the time of commitment to die (death commitment) by examining ASG responses to 20E and juvenile hormone analogue (JHA) in vivo as well as in vitro. The ASGs obtained late on day 6 of the fifth instar completed PCD when cultured with 20E, while the ASGs obtained on day 4 and cultured with 20E did not undergo PCD. The ASGs became competent to respond to 20E at mid-day 5. The ASGs with responsiveness to 20E were not sensitive to JHA, indicating that the ASGs were committed to die before becoming capable of responding to 20E. Topical application of JHA on day 4 suppressed 20E-induced PCD, but that on day 5 failed to do so, indicating that the death commitment might occur between day 4 and 5. We also determined the time of death commitment after allatectomy of the fourth instar larvae, a procedure that induced the precocious PCD. Timed application of JHA and culture of ASGs with 20E in the presence of JHA showed that the ASGs had lost their sensitivity to JHA between 72 and 96 h after allatectomy, i.e. 24-48 h before precocious gut purge in the allatectomized larvae. This result is similar to that obtained in the fifth instar. We conclude that the cellular commitment to die takes place one day before the ASGs become competent to respond to 20E.     

Age-dependent responses of last instar larvae of Spodoptera mauritia to precocene II

ABSTRACT. Last instar larvae of Spodoptera mauritia Boisd. (Lepidoptera: Noctuidae) of various ages were treated with a single dose of 80 μg or 160 μg of precocene II (PII) and the effects of larval—pupal transformation were studied. Final instar (sixth) larvae treated on day 0, 1 or 2 with 80 μg P II showed a high mortality; P II treatment during subsequent days of this stadium showed a diminishing tendency for mortality. P II prolonged the larval—pupal period in all larvae but the effect was somewhat reduced in the mid-stadium.
Treatments of 160μg P II to 0-day-old, 1-day-old, 2-day-old and 3-day-old larvae were highly toxic. Treatments of 4-day-old and 5-day-old larvae with 160 μg P II delayed pupation by 1 day, but in addition the larvae often moulted into larval—pupal intermediates. Co-application of 50μg juvenile hormone analogue (ZR 512) with 160 μg P II restored both normal morphogenesis and a normally-timed moult.     

The title of the article: effects on growth and digestive enzyme activities of the <Emphasis Type="Italic">Hepialus gonggaensis</Emphasis> larvae caused by introducing probiotics

A bacteria strain Hg4-03 of Carnobacterium sp., isolated from the intestine of Hepialus gonggaensis larvae, was fed back to the fourth instars larvae as probiotics to evaluate its impact on the growth performance and digestive enzymes. The larvae were reared in the lab with a natural diet treated with different concentrations of bacterial fermentation and heating killed bacteria, respectively. Compared with the control group, results showed that the growth rates significantly increased and the insect mortality rate decreased significantly after feeding with live probiotics. Meanwhile, the activities of protease, total amylase and trehalase rose significantly in intestinal fluid of the group fed with live probiotics compared with the control treatment. These findings demonstrated that the intestinal bacteria Hg4-03 play an important role for the growth of H. gonggaensis larvae. The bacteria community can improve the growth of H. gonggaensis larvae, indicating that intestinal bacteria may probably be one of the most important factors impacting H. gonggaensis larvae reared in control conditions.     

Modulation of digestive enzyme activities during ontogeny of Labeo rohita larvae fed ascorbic acid enriched zooplankton

The effect of supplementation of ascorbic acid through enriched zooplankton [10%, 20% and 30% ascorbyl palmitate (AP) inclusion in diet of zooplankton] on different digestive enzyme activities during ontogeny of Labeo rohita larvae was studied from 4 day to 15 day post hatch. Ascorbic acid (AA) content in different groups of unenriched (8.6+/-0.71) and enriched zooplankton were, 750+/-29.3, 1409.1+/-45.5, 2009.21+/-199.2 mug/g respectively on dry matter basis with differences (P<0.05) between the treatments. A difference (P<0.05) was found in tissue AA level in different dietary groups. Low amylase, protease, lipase and alkaline phosphatase activities were present in rohu larvae from the mouth opening stage which showed increasing trend with the age of larvae and increasing dietary AA content. A clear dose-dependent modulation of digestive enzyme activities in response to 10%, 20% and 30% AP enriched zooplankton feeding was evidenced from positive correlations between dietary AA content with magnitude of elevation of enzyme activity in different groups. There were 57, 55, 29.2 and 2 fold increases in amylase activity; 7.35, 7.02, 4.43 and 2.73 fold increases in protease activity; 45.636, 41.50, 19.83 and 13.69 fold increases in lipase activity and 6, 5, 3, and 2 fold increases in alkaline phosphatase activity observed in the 15th day post hatch larvae fed 20%, 30%, 10%AP enriched and normal zooplankton respectively, than 4-day post hatch larvae of the respective groups. Enzyme activities were also positively correlated with specific growth rates of wet weight of rohu larvae at the 15th day post hatch. Increased AA might have played an important role in advancing morphological transformation of the digestive tract, protecting gastric mucosa and accelerating growth by the process of tissue formation, which necessitated the requirement of more nutrient thereby, increasing digestive enzyme activity. The regulatory role of AA in the modulation of different digestive enzymes activity and its physiological consequences of nutrient digestibility and utilization during ontogenesis could be extrapolated for better nutrient management of the larvae.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

亚致死剂量阿维菌素对家蚕食物利用及中肠消化酶活性的影响

采用质量法和酶活力测定法研究了阿维菌素3种亚致死剂量（LC₅、LC₁₀和LC₂₀）处理对家蚕5 龄幼虫的食物利用及中肠3种消化酶活性的影响.结果表明：亚致死剂量阿维菌素处理对家蚕5龄幼虫的生长和食物利用有明显的抑制作用,体质量及其增加量、相对生长率均显著低于对照,同时伴随着取食量、相对取食量和排粪量减少,食物利用率和食物转化率显著降低,而近似消化率升高.阿维菌素处理的家蚕,其中肠淀粉酶和蔗糖酶活性前期受到抑制且持续时间较长,但随着幼虫的生长发育,逐渐恢复正常或高于对照;而海藻糖酶活性前期受抑制时间较短,随后活性迅速增强,并维持在与对照同一水平或高于对照.这些变化说明亚致死剂量阿维菌素对家蚕仍有一定的毒性,其程度随剂量的提高而增强,可导致家蚕消化系统紊乱,进而影响其生长发育和对食物的利用.     

保幼激素类似物对斜纹夜蛾核多角体病毒增殖的影响

分别用5×10⁶,1×10⁷,5×10⁷,1×10⁸ PIBs/mL 4种浓度的斜纹夜蛾核多角体病毒(SpltNPV)感染斜纹夜蛾末龄幼虫（第6龄）,并于同龄期以10μg/头的保幼激素类似物(JHA) methoprene分别对各感染组进行点滴处理,以研究JHA对SpltNPV增殖的影响。研究表明,与对照组相比各不同浓度处理组病毒总产量分别提高227.06%、128.71%、52.62%、33.15%,平均单头病毒含量分别提高49.15%、48.40%、36.40%和31.11%,病毒感染死亡率分别提高119.21%、52.72%、12.64%和1.12%。其中以1×10.7 PIBs/mL感染再经JHA处理的病毒总产量和平均单头病毒含量最高,分别为1 701.8×10⁸PIBs和60.1×10⁸ PIBs。各处理组的病毒总产量和平均单头病毒含量均显著高于其对照组。在此基础上,进一步研究了JHA对染毒与未染毒宿主消化生理的影响。结果表明,JHA处理不仅延长了6龄幼虫的寿命,增加了其取食量,而且还显著提高了幼虫的食物转化率和病毒产量。     

Age-specific digestion of Tenebrio molitor (Coleoptera: Tenebrionidae) and inhibition of proteolytic and amylolytic activity by plant proteinaceous seed extracts

Tenebrio molitor L. (Coleoptera: Tenebrionidae), is an international and serious pest of stored products. So far nothing is known about the activity for each growth stage digestive enzyme regarding this insect species. Thus, the aim of the current study was to get in depth analysis of the stage specific digestion and to investigate the effect of cereal (wheat cultivars including MV17, Aflak, Sivand, Saymon, and Zare) and legume (bean) seed extracts on the two main digestive enzymes i.e. α-amylases and proteases. Therefore, gut enzymes were extracted using distilled water and wheat cultivars and bean seed proteinaceous compounds were extracted using 0.1 M NaCl. Results showed that a steady state increase in the number and amount of digestive enzyme activities from first to fourth instar larvae was seen in both enzyme and in gel assays. In the first instar larvae (L1) only one band of α-amylase activity was seen (A1), whereas in the second (L2), third (L3), fourth (L4) and fifth (L5) instar larvae as well as in the adult (A) more than one amylase band (up to 4 isoenzymes) was seen. The same pattern was observed for α and β glucosidases and proteases. Probit analysis showed that bean and MV17 inhibited the amylase activity with an I₅₀ of 9.73 and 7.4 μg, respectively. The same cultivar seed extract inhibited protease activity with I₅₀s of 11.54 and 6.5 μg proteins. It is concluded that proteinaceous extract of cereals and bean seeds have a strong potential to be used in this pest management.     

保幼激素类似物对家蚕丝腺与脂肪体蛋白质合成的调节作用

应用示踪原子法,研究了家蚕Bombyx mori 5龄幼虫丝腺与脂肪体细胞内蛋白质合成的变化规律及保幼激素类似物(JHA738)的调节作用。从5龄初到龄末,家蚕丝腺细胞内蛋白质合成持续升高,5龄中期、后期的蛋白质合成活性分别是前期的1.60倍和2.86倍;全龄出现2个合成高峰,一个是在5龄72h,为细胞固有蛋白质合成高峰,另一个是在5龄192h,为丝蛋白合成高峰。脂肪体细胞内蛋白质合成作用呈现脉冲式的变化。在5龄前期和中期用JHA处理家蚕(剂量为4μg/条),对丝腺细胞固有蛋白质合成和脂肪体细胞蛋白质合成均表现出抑制作用,而对丝蛋白合成则表现出促进作用。本实验结果为进一步阐明JHA增丝机理提供了直接证据。     

The effects of juvenile hormone analogues on the embryogenesis of Drosophila melanogaster

Two juvenile hormone analogues (JHA), 6,7-Epoxy-3-methyl-7-ethyl-1-(3,4-(methyl-enedioxy)phenoxy)-2-cis/trans-octene (Hoffman-LaRoche RO 20-3600) and 6,7-Epoxy-3,7-dimethyl-1-(p-ethyphenoxy)-2-cis/trans-octene (Stauffer R-20458), produced embryonic and some first larval instar lethality when topically applied to freshly laid Drosophila eggs at concentrations above 0·1μg RO-20-3600/μl acetone or 1·0 μg R-20458/μl acetone. With progressively later JHA treatments during embryonic development, lethality decreased while the proportion of larvae dying at the time of hatching increased. No delayed postembryonic lethality was observed after the first larval instar. JHA applications to freshly laid eggs produced abnormal head and tracheal formation in some embryos treated with RO-20-3600 and caused developmental arrest during the beginning of posterior gut formation in most eggs treated with R-20458. Treatment with either JHA after the beginning of blastoderm formation resulted in normal looking first instar larvae which failed to hatch.