Starvation suppresses cell proliferation that rebounds after refeeding in the midgut of the American cockroach, Periplaneta americana

Starvation affects behavior, development, metabolism, reproduction, and longevity in almost all animals including insects. In the American cockroach, Periplaneta americana, we investigated the effect of starvation on organ size and cell proliferation activity of the midgut, over a period of one month, using anti-bromodeoxyuridine (BrdU), and anti-phospho-histone H3 antibodies. Under starvation conditions, the midgut became clear and fragile while its length and diameter were reduced. Both the rate of BrdU-uptake in the nucleus and the mitotic activity shown by anti-phospho-histone H3 antibody decreased under long starvation up to half that of the continuously fed control. Refeeding restored BrdU-uptake and mitosis that overshot the fed control. When casein, starch, or cooking oil was fed as representative nutrient sources to the starved cockroaches, all restored BrdU-uptake, but non-nutrient, talc, did not. This study supports the hypothesis that P. americana has a homeostatic mechanism to regulate the cell population of the midgut epithelium according to changes in the nutritional environment.     

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica)

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and α-amylase were still detected in the midgut of diseased larvae.     

Protease and phospholipase inhibition protect Veneza zonata (Hemiptera Coreidae) against septicemia caused by parasite trypanosomatid 563DT

Veneza zonata (Hemiptera Coreidae) is an insect which causes losses in several crops, and it is also an important vector of lower trypanosomatids. V. zonata specimens were collected on rural properties in Londrina, state of Paraná, Southern Brazil. Inoculation of Leptomonas 563DT into V. zonata hemocoel caused insect death within approximately 24 h, with large bacterial proliferation into their hemocoels. Some bacteria which were found in the digestive tract of those insects, such as Escherichia coli, Providencia rettgeri, and Kluyveria ascorbata, were also found in their hemolymph, which suggests that trypanosomatid crossing into hemocoel caused mechanical lesions in the digestive tract that allowed intestinal bacteria to infect the hemolymph, thereby leading to lethal septicemia. In this study we analysed proteolytic activities from the 563DT Leptomonas strain, which is pathogenic for V. zonata, aiming at evaluating the potential use of this Leptomonas strain for the biocontrol of the insect. The proteolytic action was evaluated on cells and on culture supernatants of trypanosomatids. We also evaluated the gelatinolytic activities, the action over natural and synthetic substrates for aminopeptidases, and the action of protease inhibitors during all trypanosomatid growth stages. A significant reduction in the number of insect deaths was observed when Leptomonas 563DT were incubated with inhibitors of proteases and phospholipases before being inoculated into the insects, which suggests that those enzymes are involved in the pathogenic mechanism.     

Starvation induces apoptosis in the midgut nidi of <Emphasis Type="Italic">Periplaneta americana</Emphasis>: a histochemical and ultrastructural study

The effects of starvation on cell death in the midgut of Periplaneta americana were studied histochemically and ultrastructurally. TUNEL assays showed that cell death began to increase in the columnar cells and nidi, the nests of stem cells and newborn cells from 2 weeks of starvation. A significant increase in cell death occurred in the nidi after 4 weeks of starvation. Cockroaches starved for 4 weeks showed active-caspase-3-like immuno-reactivity both in the columnar cells and nidi, whereas control cockroaches that were fed for 4 weeks showed this reactivity only in the apical cytoplasm of columnar cells. Electron microscopy revealed no chromatin condensation in the nucleus of columnar cells of cockroaches, whether fed or starved for 4 weeks. Starved cockroaches exhibited many small vacuoles in the cytoplasm of some columnar cells and “floating” organelles including nuclei in the lumen. A 4-week starvation induced the appearance of cytoplasmic fragmentation and secondary lysosomes in the nidi. Each fragment contained nuclear derivatives with condensed chromatin, i.e. apoptotic bodies. Mitotic cells were found in some, but not all nidi, even within the same starved sample. Fragmentation was not observed in the nidi of control cockroaches. Thus, starvation increases cell death not only in the columnar cells, but also in the nidi. The cell death in the nidi is presumably apoptosis executed by caspase 3.     

The influence of starvation and different diets on the hindgut of isopoda (Mesidotea entomon,Oniscus asellus,Porcellio scaber)

Summary Under conditions of food deprivation, the hindgut epithelium of the experimental animals (Mesidotea entomon, Oniscus asellus, Porcellio scaber) undergoes ultrastructural changes. After the application of different diets it was demonstrated that this part of the alimentary canal contains nutrients, though it is lined by a cuticle. Experimental evidence for the formation of glycogen from glucose offered as the only diet comes from autoradiographic experiments. Amino acids, too, were detected in the hindgut cells soon after refeeding. Lipids, on the other hand, which are first absorbed by the large cells of the midgut glands, were not found in the hindgut epithelium. The existence of lipid inclusions in the hindgut epithelium some weeks after refeeding, however, supports the hypothesis that lipids reach the epithelial cells of the hindgutvia midgut glands and hemolymph.     

Biochemical characterization of digestive α-amylase, α-glucosidase and β-glucosidase in pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

The pistachio green stink bug, Brachynema germari, has 3–5 generations per year and causes severe damages to pistachio crops in Iran. Physiological digestive processes, such as digestive carbohydrases, can be used to design new strategies in IPM programs for controlling this pest. The enzyme α-amylase digests starch during the initial stage of digestion. Complete breakdown of carbohydrates takes place in the midgut where α- and β-glucosidic activities are highest. Alpha-amylase and α- and β-glucosidase activities were found in the midgut and salivary glands of pistachio green stink bug adults. Overall enzyme activities were significantly higher in the midgut than in salivary glands. The highest α-amylase and α- and β-glucosidase activities were in section v3, whereas the lowest activities were in section v4. V_max was higher and K_m was lower in the midgut than in the salivary glands for these enzymes. In the pistachio green stink bug, the optimal pH was pH 5–6.5 and the optimal temperature was 30 °C to 35 °C for these enzymes. Alpha-amylase activity in the midgut and salivary glands decreased as the concentrations of MgCl₂, EDTA and SDS increased. Enzyme activities in both midgut and salivary glands increased in the presence of NaCl, CaCl₂, and KCl. NaCl had a negative effect on alpha-amylase extracted from salivary glands.     

Salivary digestive enzymes of the wheat bug, Eurygaster integriceps (Insecta: Hemiptera: Scutelleridae)

The digestive enzymes from salivary gland complexes (SGC) of Eurygaster integriceps, and their response to starvation and feeding were studied. Moreover, digestive amylases were partially purified and characterized by ammonium sulfate precipitation and gel filtration chromatography. The SGC are composed of two sections, the principal glands and accessory glands. The principal glands are further divided into the anterior lobes and posterior lobes. The SGC main enzyme was α-amylase, which hydrolyzed starch better than glycogen. The other carbohydrases were also present in the SGC complexes. Enzymatic activities toward mannose (α/β-mannosidases) were little in comparison to activities against glucose (α/β-glucosidases) and galactose (α/β-galactosidases), the latter being the greatest. Acid phosphatase showed higher activity than alkaline phosphatase. There was no measurable activity for lipase and aminopeptidase. Proteolytic activity was detected against general and specific protease substrates. Activities of all enzymes were increased in response to feeding in comparison to starved insects, revealing their induction and secretion in response to feeding pulse. The SGC amylases eluted in four major peaks and post-electrophoretic detection of the α-amylases demonstrated the existence of at least five isoamylases in the SGC. The physiological implication of these findings in pre-oral digestion of E. integriceps is discussed.     

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.     

中华真地鳖中肠主要消化酶的活性研究

以中华真地鳖EupolyphagasinensisWalker为研究材料,测定人工饲养和野生地鳖虫在不同生长阶段消化酶的活性以及温度及pH对人工饲养地鳖虫中肠消化酶活性的影响。结果表明,在地鳖虫生长发育过程中,蛋白酶和脂肪酶活性随发育而逐渐增强,淀粉酶活性却随发育而逐渐减弱。在低龄若虫、高龄若虫和成虫阶段,人工饲养地鳖虫蛋白酶活力比野生地鳖虫低,人工饲养地鳖虫淀粉酶和脂肪酶活力比野生地鳖虫高;在30～60℃的范围内,人工饲养地鳖虫蛋白酶、淀粉酶的适宜温度范围为40～50℃,脂肪酶的适宜温度范围为35～45℃;蛋白酶、淀粉酶和脂肪酶的适宜pH范围分别为6.5～7.5,5.6～6.4和7.5～8.5。     

Digestive amylase and pectinase activity in the larvae of alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil Hypera postica is a serious economic pest in most alfalfa grown in many countries worldwide. Digestive α-amylase and pectinase activities of larvae were investigated using general substrates. Midgut extracts from larvae showed an optimum activity for α-amylase against starch at acidic pH (pH 5.0). α-Amylase from larval midgut was more stable at mildly acidic pH (pH 5–6) than highly acidic and alkaline pH. The enzyme showed its maximum activity at 35°C. α-Amylase activity was significantly decreased in the presence of Ca²⁺, Mg²⁺ and sodium dodecylsulfate. On the contrary, K⁺ and Na⁺ did not significantly affect the enzyme activity. Zymogram analysis revealed the presence of one band of α-amylase activity in in-gel assays. Pectinase activity was assayed using agarose plate and colorimetric assays. Optimal pH for pectinase activity in the larval midgut was determined to be pH 5.0. Pectinase enzyme is more stable at pH 4.0–7.0 than highly acidic and alkaline pH. However, the enzyme was more stable at slightly acidic pH (pH 6.0) when incubation time increased. Maximum activity for the enzyme incubated at different temperatures was observed to be 40°C. Optimum pH activity for α-amylase and pectinase is not completely consistent with the pH prevailing in the larval midgut. This is the first report of the presence of pectinase activity in H. postica.     

粘虫中肠α-淀粉酶活性的敏感性研究

研究了不同酶反应缓冲体系、pH值、氯离子浓度以及噁唑哒嗪对5龄2日粘虫 Pseudaletia separata Walker 中肠α－淀粉酶活性的影响。结果表明,乙酸-乙酸钠缓冲体系（pH 5.8）和磷酸氢二钠-磷酸二氢钠缓冲体系（pH 8.0）有利于增强α-淀粉酶活性,比活力最高分别达到4.49和4.97。在乙酸-乙酸钠缓冲体系（pH 5.8）中,5、10、20、40和80 mmol/L氯离子浓度引起α-淀粉酶活性呈现先减弱后增强的变化规律,而在磷酸氢二钠-磷酸二氢钠缓冲体系（pH 8.0）中仅呈现减弱的趋势。1.4 mmol/L噁唑哒嗪对α-淀粉酶活性的抑制率可达70%,但抑制程度随着反应体系中蛋白含量的增加而逐渐降低。     

Fatty acid incorporation by Rhodnius prolixus midgut

[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.     

Tricarboxylic Acid Cycle Enzymes in Tetrahymena pyriformis after Infection of the Cockroach Periplaneta americana1

SYNOPSIS. Activities of enzymes of the tricarboxylic acid cycle in extracts of Tetrahymena pyriformis S, axenically recovered after living in the hemocoel of female cockroaches Periplaneta americana for 48 hr, were compared with activities in ciliates not exposed to the cockroach. Malic dehydrogenase activity was depressed after recovery from the cockroach; isocitric and succinic dehydrogenases and α-ketoglutaric oxidase activities were unchanged. Citrate synthetase activity was increased, and pyruvi oxidase activity decreased, after ciliates had been in the cockroach. These alterations persisted for several hundred generations after recovery from the insect.     

Sunn pest,Eurygaster integriceps Putton (Hemiptera: Scutelleridae), digestive α-amylase, α-glucosidase and β-glucosidase

Morphology, pH and carbohydrate hydrolyzing enzyme activities of the Sunn pest gut were investigated in this study. The Sunn pest midgut is separated into the first ventriculus (V1), the second ventriculus (V2), the third ventriculus (V3) and the fourth ventriculus (V4). The first three regions of the midgut were acidic (pH 5.0–5.2), while the fourth region of the midgut and rectum was moderately acidic (pH 6.2–6.4 and pH 6.5–6.8, respectively). Activity of α-amylase was highest at pH 6 to 7, which correlates with the pH of the midgut. The optimum pH for α-glucosidase and β-glucosidase is 4 to 6 and 5 to 6, respectively. Different gut regions had different carbohydrate hydrolyzing enzyme activities. Carbohydrate hydrolyzing enzyme activities in V2 and V4 were the same, but activities in V1 were slightly higher than in V2 and V4 and lower than in V3. Levels of α- and β-glucosidase activities were similar in various midgut sections. However, the V3 had the highest activity followed by V4, V2, V1, respectively.     

Absorption of dietary triacylglycerol by lipolysis and lipid resynthesis in the mesenteron of larvalAeshna cyanea (insecta,odonata)

Summary Voluntary uptake of triolein, margarine, and lipid-rich natural food (Tubifex) by fasting dragonfly larvae (Aeshna cyanea) led to heavy accumulations of lipid absorption droplets in the enterocytes within 2 days, while subsequent lipid clearance of the midgut epithelium took several weeks depending on the ingested lipid load. No endocytotic lipid uptake was observed after application of a molecular-dispersed fat dye. The smallest lipid droplets first appeared in the subapical groundplasm of the enterocytes and showed a reversible increase in size on their way towards the base. Lipid droplets were also observed at appropriate intervals after oral administration of oleic acid, after feeding margarine in the cold, and after injection of triolein into the isolated midgut.Comparative biochemical analysis after triolein feeding evidenced release of lipase and hydrolytic liberation of FA from TG in the midgut lumen, as well as time-dependent accumulations of TG in the midgut epithelium and of DG in the hemolymph.Oral injection of [¹⁴C] oleic acid was followed by its rapid absorption into the midgut epithelium, where it was utilized for the synthesis of MG and esterification to DG and TG. Discharge of radioactive lipid into the hemolymph occurred in the form of FA and DG, while the rectal fat body showed approximately equal labeling of the FA, DG, and TG fractions.Abbreviations AG acylglycerol - DG diacylglycerol - ER endoplasmic reticulum - FA fatty acid - MG monoacylglycerol - TG triacylglycerol Dedicated to Prof. Dr. Dr. R.Lehmensick, Bonn, in honor of his 85^th birthday.     

Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.     

HaNPV在宿主内的复制及其对宿主蛋白质代谢的影响

对棉铃虫Helicoverpaarmigera的中肠、血淋巴及脂肪体进行SDS-PAGE蛋白质分析表明：感染早期,HaNPV对棉铃虫的蛋白质合成有刺激作用,晚期则表现出抑制作用。感染96 h和120 h脂肪体有较高水平的多角体蛋白合成。电镜观察表明：①HaNPV感染棉铃虫除病毒在中肠复制后经出芽进入血腔感染脂肪体等组织外,还可能存在病毒粒子直接经中肠进入血腔而感染脂肪体这样的途径;②中肠与血腔之间存在屏障结构;③中肠不仅是一次感染时病毒粒子复制的场所,它完全可能被病毒粒子二次感染。在中肠细胞中没有发现多角体的形成。     

逆境条件下小麦种子活力与种子萌发相关酶活性及其基因表达的关系

分析不同基因型小麦品种逆境萌发过程中种子萌发相关酶活性及基因表达差异,明确在逆境条件下,种子活力与种子萌发相关酶活性及基因表达量的关系.通过标准发芽试验和逆境(冷浸、人工老化、干旱胁迫)发芽试验,测定4个小麦品种种子活力、萌发过程中可溶性总糖和可溶性蛋白含量、α-淀粉酶活性、半胱氨酸蛋白酶活性及相关基因表达量.结果表明:干旱、人工老化和冷浸胁迫3种逆境对种子活力都有一定影响.不同萌发条件下,可溶性总糖含量呈现先小幅度升高后小幅度降低再迅速升高的趋势;而可溶性蛋白含量随着萌发时间的延长呈现逐渐下降的趋势.α-淀粉酶活性整体呈现逐渐升高的趋势,但在冷浸胁迫处理后,豫农949和轮选061的α-淀粉酶活性在萌发60 h后出现下降.半胱氨酸蛋白酶活性整体呈先降低后升高的趋势,但在干旱胁迫条件下,豫农949、豫麦49-198和轮选061的半胱氨酸蛋白酶活性呈现先升高后降低再升高的趋势.不同逆境萌发条件下,α-AMY(α-淀粉酶基因)表达量整体呈先上升后下降的趋势.冷浸胁迫处理后,轮选061的α-AMY表达量高于对照,在其他逆境萌发条件下,4个品种的α-AMY表达量均低于对照;人工老化处理后,长4738的CP(半胱氨酸蛋白酶基因)表达量与对照差异不显著,在其他逆境萌发条件下,4个品种的CP表达量均高于对照.种子萌发期间,不同萌发条件下α-淀粉酶和半胱氨酸蛋白酶活性与其基因表达并没有直接关系,α-淀粉酶活性与可溶性总糖含量达到显著正相关,半胱氨酸蛋白酶活性与可溶性蛋白含量的相关性不显著.在标准发芽条件下,α-淀粉酶活性与活力指数呈显著正相关,而在逆境萌发过程中,其相关性不显著.冷浸胁迫处理后,半胱氨酸蛋白酶活性与活力指数呈显著正相关,但在标准发芽、干旱胁迫、人工老化处理后,其相关性不显著.