Developmental changes in midgut trehalase activity and its localization in the silkworm, Bombyx mori

The changes in trehalase activity and its localization in the midgut of the silkworm, Bombyx mori, were studied during larval-pupal-adult development. Trehalase activity in larval midgut epithelium increased with the larval growth, reached a maximum level at the middle of the fifth instar, and then decreased gradually. Trehalase activity in larval midgut was found in the epithelial tissue but not in the digestive juice or the midgut contents.The trehalase activity in the whole midgut started to rise at the onset of spinning and increased abruptly at larval-pupal ecdysis to reach an extremely high level 3 days later. This high activity was maintained throughout the subsequent pharate adult development and dropped suddenly at emergence. The midgut trehalase activity during pupal-adult development was mainly found in the midgut contents but scarcely any in the epithelium.Subcellular distribution of midgut trehalase depended upon larval-pupal-adult development. The activity was concentrated in a precipitate fraction of the epithelium until the middle of the fifth instar. During larval-pupal development, however, the activity increased in the soluble fraction with a concomitant decrease in the precipitate fraction. Almost all the trehalase activity in pupal and pharate adult midgut was recovered in the soluble fraction of the midgut contents. The data are discussed from a viewpoint of the histolysis.     

Toxic impact of ingested Jatropherol-I on selected enzymatic activities and the ultrastructure of midgut cells in silkworm, Bombyx mori L.

Abstract:  Jatropherol-I, a phorbol-type diterpene from Jatropha curcas seeds was found highly toxic to third instars silkworm larvae after ingestion with LC₅₀ values 0.5793, 0.2197 and 0.1578 mg/ml at 48, 72 and 120 h respectively. The acute toxicity was associated with changes in activities of several midgut enzymes and pathological changes in midgut epithelial cells. Jatropherol-I caused various fluctuations in activities of different midgut enzymes in third instars larvae of silkworm. Compared with controls, both esterase and carboxyliesterase showed significantly depressed activities at 3 h ( t -test, P < 0.05) and increased activities at 24 h and rapidly decreased activities at 48 h after ingestion of 0.125 and 0.25 mg/ml of Jatropherol-I. Jatropherol-I induced two (E4 and E5) and depressed one (E2) of midgut esterase isozymes analysed 3 h after ingestion. There was no significantly different glutathione S-transferase activity between most of treated silkworm and control ( t -test, P > 0.05). Activities of acetylcholinesterase fluctuated weakly in treated silkworm in 48 h. Jatropherol-I induced a gradual decline in midgut protease activities in silkworm with an obvious dose- and time-dependent effect. Jatropherol-I also caused pathological changes in midgut cells, especially in their endoplasmic reticulum. They were noted slight dilatation on 12 h exposure, while extreme dilatation, vesiculations and shedding of ribosome from membrane were observed in endoplasmic reticulum after a longer time exposure. Other pathological changes in microvilli, lysosome, mitochondria and chromatin were also observed in midgut cell of treated silkworm.     

Expression analysis of chlorophyllid a binding protein a secretory,red fluorescence protein in the midgut of silkworm,Bombyx mori

Chlorophyllid a binding protein （ chbp） was recently characterized by its ability to bind the prosthetic group of chlorophylls and little information is known regarding its expression. In the present study, we found that chpb was expressed highly and exclusively in the midgut of silkworm, Bombyx mori. The expression level of chbp was very high in the newly molted fifth instar larvae followed by gradual decline in the same instar. Our results demonstrated that CHBP was a secretory protein and located mainly in the apical of midgut epithelial cells. Real-time polymerase chain reaction analysis results showed that chpb highly expressed in the anterior midgut, threefold and sixfold higher compared with that of the middle midgut and posterior midgut, respectively, and chpb expression declined in darkness. In addition, the expression of chbp was affected by high-dose virus or bacterium infection.     

细菌感染后家蚕幼虫中肠差异表达基因的鉴定（英文）

作为遭遇各种微生物的前线, 昆虫的消化道在免疫反应中起到重要的作用。为了研究家蚕Bombyx mori肠道免疫反应, 我们利用基于ACP (annealing control primer)的反转录PCR技术, 从中肠组织中鉴定得到18个在经口器感染绿脓杆菌Pseudomonas aeruginosa 和金黄色葡萄球菌Staphylococcus aureus后的差异表达基因,并利用荧光定量PCR分析了其中4个基因在感染后24 h内的动态变化。结果表明： 肽聚糖识别蛋白-L1（PGRP-L1)和一个丝氨酸蛋白酶前体基因特异性地受绿脓杆菌感染后上调; 30kP蛋白酶A基因受绿脓杆菌和金黄色葡萄球菌2种细菌感染后上调。我们的研究鉴定了经口器感染后家蚕中肠中参与入侵细菌识别和免疫信号途径的基因, 可为对这些基因进一步的功能研究提供线索。     

Induction of non-diapause eggs in Bombyx mori by a trehalase inhibitor

The effect of validoxylamine A (VAA), a potent and specific trehalase inhibitor, on the induction of non-diapause in Bombyx mori was examined. The VAA induced non-diapause eggs and prevented the glucogen accumulation in the eggs. Trehalase activity of the pupal ovary was effectively inhibited by the VAA injection.     

家蚕中肠组织抗核型多角体病毒病的相关蛋白分析

家蚕中肠上皮是病毒经口侵入遇到的第一个组织。昆虫幼虫抵御杆状病毒的感染,可通过选择性的使感染的中肠上皮细胞发生调亡并在释放病毒粒子进入血淋巴之前使感染的细胞从中肠脱落。为研究家蚕抗核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)病的机制,通过对BmNPV高度抗性和高度敏感性的家蚕品系杂交和回交构建了近等基因系。本文对家蚕高抗,敏感及近等基因系5龄起蚕中肠组织的蛋白质表达谱进行了二维电泳 (two-dimensional gel electrophoresis,2-DE) 分析,并利用基质辅助激光解吸电离飞行时间 (matrix-assisted laser desorption/ionization-time of flight, MALDI-TOF) 质谱对差异蛋白进行鉴定。结果发现了5个差异表达的蛋白。推测这些蛋白可能与家蚕中肠对BmNPV的抗性或感性有关。     

Absorption of horseradish peroxidase in Bombyx mori larval midgut

Increasing experimental evidence indicates that ingested proteins can in part reach the haemocoel undegraded, but information on the mechanisms involved in protein transport across the insect gut is very limited, in spite of the implications that this may have on the development of novel delivery strategies of insecticide proteins targeting haemocoelic receptors. Here we contribute to this field of study, by focusing on horseradish peroxidase (HRP) transport through Bombyx mori larval midgut, isolated and perfused in vitro. The protein crossed the intestinal barrier in a time-dependent manner and the influx was linearly related to time between 30 and 90 min of incubation. HRP absorption was strongly affected by temperature and inhibition of cell metabolism: protein influx at 4 degrees C was reduced to 27% of that measured at 25 degrees C and was similarly inhibited by the metabolic inhibitor DNP. Transmission electron microscopy analysis of midgut columnar cells exposed to HRP showed the presence of the protein both in vesicular structures inside the cytoplasm and in the space between two adjacent absorptive cells, indicating the occurrence of both a transcellular and a paracellular permeation route. The analysis of HRP influx as a function of increasing protein concentration in the lumen supported this morphological indication. The J(max) relative to the HRP transcellular transport component was 121+/-24 pmol/cm(2)/h and the K(d) of the passage through the paracellular route was 1.9+/-0.3 microl/cm(2)/h. The paracellular electrical resistance decreased in midguts exposed to HRP, indicating that its passage through this pathway was likely due to an alteration exerted on the junctional complex by the protein itself. The role of the cytoskeleton in HRP transport was investigated by assessing the impact of drugs affecting microtubules and actin filaments.     

Membrane anchors of alkaline phosphatase and trehalase associated with the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori

The larval midgut epithelial cell of the silkworm, Bombyx mori, has two forms of alkaline phosphatase and trehalase, soluble and membrane-bound. Alkaline phosphatase and trehalase of the latter form are found in the brush border membrane and the basolateral membrane, respectively. In this work we studied the membrane anchors of these membrane-bound enzymes. Alkaline phosphatase was solubilized by phosphatidyl-inositol-specific phospholipase C, but not by papain. Conversely, trehalase was released from the membrane by papain, but not by phosphatidylinositol-specific phospholipase C. Both enzymes were solubilized in an amphiphilic form with 0.5% Triton X-100 plus 0.5% sodium deoxycholate (pH 7.0). The detergent-solubilized alkaline phosphatase and trehalase were converted to hydrophilic form on incubation with phosphatidylinositol-specific phospholipase C and papain, respectively. The effects of papain on solubilization and conversion of trehalase were completely inhibited by leupeptin. These results suggest that, in the silkworm larvae, alkaline phosphatase is anchored in the brush-border membrane via a glycosyl-phosphatidylinositol, while trehalase is associated with the basolateral membrane through a hydrophobic segment of the polypeptide.     

栗精碱对棉铃虫中肠组织可溶型海藻糖酶的抑制效应

【目的】研究一种植物源化合物栗精碱对棉铃虫Helicoverpa armigera(Hübner)5龄幼虫中肠组织可溶型海藻糖酶的活体和离体抑制效果,初步探索其抑制作用。【方法】经过离子交换层析和疏水层析从棉铃虫5龄幼虫中肠组织分离纯化得到可溶型海藻糖酶,通过凝胶过滤层析确定其分子量。分别用不同浓度的抑制剂进行离体和活体抑制研究,得出栗精碱对可溶型海藻糖酶的抑制率和抑制中浓度(IC50),并采用Lineweaver-Burk和Dixon作图法分析抑制类型。【结果】棉铃虫中肠可溶型海藻糖酶比活力为2.022 U/mg,回收率为67.57%,纯化倍数为23.84,分子量约为67 k Da。离体抑制研究表明,3.75,7.5,15和30μmol/L的栗精碱对可溶型海藻糖酶的抑制率分别为42.00%,50.30%,58.32%和67.33%;抑制中浓度(IC50)为7.32μmol/L。通过LineweaverBurk和Dixon作图法,确定栗精碱是棉铃虫中肠组织可溶型海藻糖酶的一种有效的竞争性抑制剂,Ki值为4.9μmol/L。活体抑制研究表明,分别注射浓度为30,15和7.5μmol/L栗精碱10和20 h后,棉铃虫中肠可溶型海藻糖酶活性被显著抑制(P0.05)。相应地,注射后随着时间的延长,血淋巴海藻糖含量连续增加。而注射栗精碱20 h后,血淋巴葡萄糖浓度显著下降,使棉铃虫的能量供给出现障碍。【结论】结果表明,栗精碱是棉铃虫中肠可溶型海藻糖酶的一种有效的抑制剂,可为下一步开发有效的棉铃虫杀虫剂提供理论支持。     

Alkaline proteases in the midgut tissue and digestive fluid of the silkworm,Bombyx mori

In larvae of Bombyx mori proteolytic activity was found in the midgut tissue and digestive fluid. The pH-activity curves of both proteases were very similar and optimal activity was about pH 11.2. The reduction in activity at 50°C, for 10 min was about 80% in digestive fluid protease, and about 40% in tissue protease. HgCl₂ and DFP strongly inhibited protease activity, and the influence was greater in digestive fluid than in the midgut tissue. Most of the tissue proteases was found to be membrane bound enzyme from results of differential centrifugation and column experiments.     

Soluble and particle-bound trehalase in the silk glands during larval-pupal development of the silkworm, Bombyx mori

Trehalase localization in silk glands of the silkworm, Bombyx mori has been studied during larval-pupal development. Subcellular distribution of the silk gland trehalase depends upon larval-pupal development. The activity increases in the soluble fraction with a concomitant decrease in the particle-bound fraction during larval-pupal development. The pH-optimum value of trehalase activity in the particle-bound fraction changes from 6.5 to 6.0, and in the soluble fraction from 5.5 to 4.5 in the course of the silk gland degeneration during metamorphosis.     

Acid trehalase deficiency and extracellular trehalose utilization in Candida utilis

Biochemical characterization of a trehalase, detected in the mid-exponential growth phase of Candida utilis NCIM Y500, has indicated that it was a neutral trehalase and possibly the only trehalase present in this strain. Unlike Saccharomyces cerevisiae and other C. utilis strains, this strain without acid trehalase grew quite well in minimal or complete medium containing trehalose as the sole source of carbon. Both these observations were contradictory to the findings reported for acid trehalase mutants of S. cerevisiae and C. utilis. The trehalase system of the strain is suggested to be similar to that of fungi.     

The physiological role of the peritrophic membrane and trehalase: Digestive enzymes in the midgut and excreta of starved larvae of Rhynchosciara

Amylase, cellulase, trehalase, aminopeptidase and trypsin were determined using the midgut and trehalose using the haemolymph of starved and of subsequently fed larvae of Rhynchosciara americana. Midgut trehalase activity decreases steadily during starvation and increases again on feeding, whereas haemolymph trehalose titres remain constant, suggesting that trehalase is a true digestive enzyme. The decrease in amylase, cellulase and trypsin activity in the midgut during starvation is of the same order as that recovered from the excreta. Since this finding is exactly what one would expect if enzyme production stops in response to starvation, this supports the hypothesis that synthesis that synthesis of these enzymes is controlled. The excretion rate of amylase, cellulase and trypsin is very low in comparison to their activity inside the peritrophic membrane and the travel time of the food bolus through the gut. It is proposed that the peritrophic membrane separates two extracellular sites for digestion as an adaptation to conserve secreted enzymes. This could be accomplished by the existence of an endo-ectoperitrophic circulation of the enzymes involved in the initial attack on the food and by restricting to the ectoperitrophic fluid the enzymes which participate only in intermediary digestion of food.     

Antiviral proteins in midgut of the silkworm, Bombyx mori, fed on different food sources

Midgut proteins from the silkworms, Bombyx mori, fed on an artificial diet containing dried powder of the spiral blue-green alga, Spirulina platensis, had a very low fluorescence intensity. This indicated that chlorophyll in this alga is not utilizable for synthesizing the red fluorescent protein (RFP) which has been considered as an effective antiviral protein in silkworm midgut. Many electrophoretic bands of the midgut proteins from the mulberry-fed silkworms emitted red fluorescence under UV light. It is thus concluded that the RFP as a whole is more than one midgut protein. Enzymatic analyses showed that silkworms fed on mulberry leaves and artificial diets had similar protease activities, but they had different phospholipase C activities. Therefore, it is suggested that the antiviral activity of purified RFP in the silkworm gut juice is attributable to phospholipase C.

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Résumé Les protéines de l'intestin moyen de Bombyx mori, nourri avec un aliment artificiel contenant de la poudre séchée de l'algue bleue spiralée, Spirulina pratensis, avaient une très faible fluorescence. Cesi indique que la chlorophylle a de cette algue n'est pas utilisable pour la synthèse de la protéine fluorescente rouge (RFP), considérée comme une protéine antivirale efficace de l'intestin moyen. Après séparation par électrophorèse sur gel de polyacrylamine DISC plusieurs bandes de la protéine de l'intestin moyen de B. mori élevé sur mûrier émettaient une fluorescence rouge en lumière U.V. On peut conclure que RFP est un complexe plutôt qu'une protéine particulière de l'intestin moyen. Les analyses enzymatiques ont montré que la phospholipase C et non les protéases ont une activité antivirale dans la RFP purifiée provenant de vers à soie nourris à partir de mûrier ou sur régime artificiel.

     

Changes in lipid droplet localization during embryogenesis of the silkworm, Bombyx mori

Lipid droplets are considered one of the most important energy sources in lepidopteran eggs during late embryogenesis, but the process of their incorporation into the embryo is as yet unknown. The present study focused on the process of transition of lipid droplets from the extraembryonic yolk to the embryo of the silkworm Bombyx mori, using morphological and biochemical approaches. The morphological study revealed that the incorporation of lipid droplets from the extraembryonic yolk into the embryo occurs at three points and in three different ways during the development of the embryo. Some lipid droplets were translocated directly from the extraembryonic yolk to the embryo before the blastokinesis stage. However, the majority of lipid droplets together with the other components of the extraembryonic yolk were incorporated in the embryo via both morphogenetic inclusion during dorsal closure and ingestion of the extraembyonic yolk by the developing caterpillar prior to hatching. Similar results were obtained from the biochemical study. Thus, we propose that there are three steps in the incorporation of lipid droplets from the extraembryonic yolk into the embryo. In addition, morphological and biochemical data concerning the total amount of lipid droplets in the egg suggested that lipid droplets were mainly consumed during late embryogenesis, seeming to synchronize with tracheal development.     

The biochemical effects of potassium chloride on the silkworm, （ Bombyx mori L.）

The supplementation with 50, 100 and 150μg/mL potassium chloride to the fifth instar larvae of the silkworm Bombyx mori on fat body glycogen, protein, total lipids and haemolymph protein and trehalose were analyzed. The fat body glycogen and protein and haemolymph protein were increased significantly in all the treated groups; whereas fat body total lipids increased only in 100 and 150μg/mL and haemolymph trehalose increased only in 150μg/mL potassium chloride-treated groups when compared with those of the corresponding parameters of the carrier controls.