Uric acid levels during last larval instar of Manduca sexta, an abrupt transition from excretion to storage in fat body

Levels of uric acid in the whole body of the tobacco hornworm, Manduca sexta increased steadily for the 9 days of the fifth instar. However, concentrations in the haemolymph were lowest during the transition from the feeding stage to the wandering stage (days 3, 4), the time when there was a switch from uric acid excretion by the Malpighian tubule-hindgut system to storage in the fat body. Haemolymph volumes, determined for larvae between 2 and 6 days into the fifth instar by isotope dilution with [¹⁴C]-inulin, were used to calculate rates of incorporation of uric acid into Malpighian tubules and fat body of larvae injected with [¹⁴C]-uric acid. These labelling studies indicated that the Malpighian tubules ceased to remove uric acid from the haemolymph some time between the last 6 hr of day 3 of the fifth instar and the first 18 hr of day 4. At the same period, fat body removed significant quantities of uric acid from the haemolymph. The times of initial decreases and increases in levels of uric acid in haemolymph and fat body, respectively, indicated that storage in the fat body started before cessation of elimination via the Malpighian tubule-hindgut system.     

The influence of ouabain on the elimination of injected and orally applied dyes in Drosophila hydei

The sour dyes azocarmine and indigocarmine are excreted through the Malpighian tubules and the midgut after injection into the body cavity of third instar Drosophila hydei larvae. After injection, the other organs are free of dyes. The epithelium of the midgut does not allow orally applied dyes to pass into the haemolymph. Ouabain diminishes significantly the content of dyes in the cavity of the Malpighian tubules and of the midgut. The maximal concentration of azocarmine decreases in the Malpighian tubules to about 65 per cent and in the midgut to about 70 per cent. Indigocarmine decreases in the Malpighian tubules to about 55 per cent. The content of indigocarmine of the midgut does not change significantly after ouabain injections. As ouabain inhibits active ion transport, the decrease of the concentration of dyes is seen as proof of the coupling of active ion transport processes and of excretion of the dyes. Moreover, this decrease points to an ouabain-sensitive transport mechanism, which is localized in the epithelia of the Malpighian tubules and midgut.     

DNA-RNA bodies in midgut cells of the stick insect, Bacillus rossius.

In the anterior part of the midgut and in the Malpighian tubules of the stick insect Bacillus rossius, about 10% of the epithelial cells develop endonuclear bodies which appear as DNA-RNA masses; in these cells the usual nucleoli are no longer evident. The DNA-RNA bodies are first formed in third instar larvae, become numerous in the fourth instar and persist in adults. In all larval instars and adults a different kind of DNA-body has been noticed in the epithelial cells of the posterior midgut. The DNA-RNA bodies of the anterior midgut and of the Malpighian tubules have been interpreted as the result of somatic gene amplification, whereas the DNA masses of the posterior midgut are likely due to a virus infection.     

Uric acid excretion in larval Manduca sexta

The characteristic coating of frass of last-instar tobacco hornworms, Manduca sexta, reared on artificial diet, proved to be uric acid. Results indicated that uric acid is the major nitrogenous excretory product; during most of the larval feeding stage, 5–7% of the excreta (dry weight basis) was uric acid; only minute quantities of allantoic acid were present. The rate of uric acid excretion was linear for the periods when coated pellets were observed. Abrupt increases in uric acid resulted from delays in pellet expulsion associated with delays in feeding activity. A distinctive coating was not generated by penultimate instar larvae, but abrupt changes in uric acid content did occur, which suggests that the phenomenon of coated frass is directly related to a differential in uric acid concentration. The source of uric acid in the frass was the Malpighian tubule system. The transition period between feeding and the wandering stage was a time of rapid decrease in uric acid excretion; there were only low levels in the last fecal pellets and none in Malpighian ampullae of wandering-stage larvae. Since the first appearance of coated fecal pellets preceded the release of ecdysone by about 24 hr, the involvement of this hormone was not indicated.     

小分子热激蛋白基因HaHSP19.8在棉铃虫抗逆反应中的作用

【目的】小分子热激蛋白(small heat shock protein, sHSP)在昆虫抵御外界环境压力中至关重要。本研究旨在探究小分子热激蛋白sHSP19.8基因在棉铃虫Helicoverpa armigera生长发育、抵御高温胁迫和对Cry1Ac杀虫蛋白抗性机制中的作用,为更深入探析该基因作用机理及棉铃虫的防治奠定基础。【方法】通过PCR结合RACE克隆棉铃虫sHSP19.8基因序列,利用生物信息学软件对该基因序列进行分析;通过qRT-PCR测定Cry1Ac敏感棉铃虫5龄幼虫在40℃高温下处理1 h和2 h及饲喂含30 μg/mL Cry1Ac的人工饲料1 h和2 h后该基因的表达量,并测定抗感Cry1Ac棉铃虫不同发育阶段(1-5龄幼虫、蛹及成虫)和5龄幼虫不同组织(前肠、中肠、后肠、马氏管及表皮)中该基因的表达模式。【结果】获得了棉铃虫sHSP19.8基因的全长cDNA序列,命名为HaHSP19.8(GenBank登录号: XP_021195228.1),长608 bp,开放阅读框长528 bp,编码175个氨基酸残基,具有小分子热激蛋白的典型α-晶体结构域(α-crystallin domain, ACD)。该基因受40℃高温和30 μg/mL Cry1Ac杀虫蛋白诱导时在Cry1Ac敏感棉铃虫5龄幼虫中均过量表达;在Cry1Ac敏感棉铃虫整个发育阶段和5龄幼虫各组织中均表达,其中在成虫和5龄幼虫以及5龄幼虫表皮、马氏管和中肠内表达量较高;但是该基因在Cry1Ac抗性品系各个发育阶段和5龄幼虫各组织中表达量相比敏感品系都显著较低。【结论】结果说明HaHSP19.8参与棉铃虫生长发育和生理生化的过程,帮助昆虫抵御外界环境压力,并可能参与到棉铃虫对Cry1Ac的抗性机制中。     

F污染桑叶对家蚕繁殖力的影响

对不同抗Ｆ性蚕品种的幼虫短期添食梯度浓度ＮａＦ研究Ｆ对桑蚕生殖的影响,结果表明,只要个体能正常羽化产卵,Ｆ对怀卵数、产卵数、产卵率和孵化率都无明显影响,低浓度的Ｆ对强抗Ｆ性品种蚕的生殖似有促进作用;但５龄期添食高浓度的Ｆ可引起怀卵数、产卵数和孵化率下降;Ｆ主要港集在马氏管、中肠等组织中,蚕卵中Ｆ的含量极低     

Ultrastructural changes in the gut and the Malpighian tubules of Epilachna varivestis after application of the new antibiotic Nikkomycin involve an osmoregulatory defect

The effect of the antibiotic Nikkomycin was investigated on the Malpighian tubules and the gut of fourth-instar larvae of the Mexican bean beetle, Epilachna varivestis. Within the Malpighian tubules, three different stages in cell alterations can be recognized. A stage of increased activity (Stage A), and two stages of dedifferentiation (Stages B and C) which are distinguishible by characteristic mitochondrial morphology. In Stage C individuals, when Malpighian tubule function stops entirely, alterations in the midgut take place, that are signs of increased activity. Measurements of hemolymph osmotic pressure showed that there is a considerable increase to a higher level which is maintained. Compared with the ultrastructural data, the regulation of osmotic pressure on a higher level may, in part, be the result of compensation for the failure of Malpighian tubule function by the midgut.     

Ecdysone-binding proteins in haemolymph and tissues of Drosophila hydei larvae

An α-ecdysone-binding protein fraction, approx. mol. wt. 120,000, has been demonstrated in haemolymph of Drosophila hydei late third instar larvae. The protein has been partly characterized by Sephadex G-25 filtration, hydroxylapatite chromatography, density gradient centrifugation, and ezyme digestion experiments. The protein-steroid complex appears to be heat stable. Binding of labelled ecdysone to the protein fraction is significantly reduced in competition experiments using unlabelled ecdysones.An ecdysone-binding protein fraction has been detected in hand-isolated total alimentary tract tissues (predominantly midgut, Malpighian tubules, and salivary glands) and in mass-isolated midgut and Malpighian tubules. The sedimentation properties of this protein-hormone complex are similar to those of the complex found in haemolymph.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

烟草甲谷胱甘肽S-转移酶基因LsGSTe1的表达及其与甲酸乙酯耐受性的关系

【目的】探究烟草甲Lasioderma serricorne谷胱甘肽S-转移酶(glutathione S-transferase, GST)基因LsGSTe1的分子特性和生物学功能。【方法】在烟草甲转录组数据的基础上,利用RT-PCR技术扩增LsGSTe1基因,并进行生物信息学分析;采用qPCR技术检测LsGSTe1在烟草甲不同发育阶段(低龄幼虫、高龄幼虫、蛹、低龄成虫、高龄成虫)及高龄幼虫不同组织(表皮、中肠、脂肪体、马氏管)中的表达水平,以及在甲酸乙酯熏蒸胁迫后的5龄幼虫中的表达变化。进一步采用RNAi技术沉默烟草甲5龄幼虫LsGSTe1基因,通过生物测定分析烟草甲对熏蒸剂甲酸乙酯的敏感性变化。【结果】获得LsGSTe1基因的全长cDNA序列(GenBank登录号:MN480468),开放阅读框长684 bp,编码227个氨基酸,N端和C端均存在催化保守位点。系统发育分析表明该基因属于GSTs的Epsilon家族。qPCR结果表明,LsGSTe1在烟草甲不同发育阶段均有表达,且在高龄幼虫期的表达量较高;表达部位主要在幼虫脂肪体,其次为中肠和表皮,而在马氏管中的表达量最低。LC     

甜菜夜蛾围食膜肠粘蛋白基因SeM-8的克隆、序列分析及在不同组织中的表达检测

昆虫肠粘蛋白（invertebrate intestinal mucin, IIM）是围食膜（peritrophic membrane, PM）的重要蛋白组分之一,在保护昆虫免受微生物侵染方面起着非常重要的作用。本研究以甜菜夜蛾Spodoptera exigua 5龄幼虫中肠总RNA为模板,根据已知的甜菜夜蛾肠粘蛋白SeIIM-8基因的cDNA序列（GenBank登录号：ABW06596）设计引物,利用RT-PCR技术扩增得到甜菜夜蛾肠粘蛋白基因SeM-8,序列分析发现其开放阅读框（open reading frame, ORF）长2 703 bp,编码900个氨基酸残基,预测分子量为95.7 kDa。序列分析表明,其氨基酸序列（GenBank登录号：GU255994）与棉铃虫Helicoverpa armigera（Hübner）,粘虫Mamestra configurata和小菜蛾Plutella xylostella肠粘蛋白的一致性分别为48%,49% 和45%。Western blot分析结果表明,SeM-8粘蛋白在甜菜夜蛾即将孵化的卵和5龄幼虫的围食膜、中肠和粪便中都有存在,在5龄幼虫马氏管、脂肪体、唾腺、血淋巴等组织部位没有发现。本研究为进一步研究IIM结构和功能,寻找生物防治新靶标奠定了理论基础。     

家蚕中的5-氧脯氨酸酶及保幼激素类似物对它的影响

本工作首次在家蚕Bombyx mori的马氏管、中肠、丝腺及脂肪体等组织中测到了γ-谷氨酰循环中一个关键酶——5-L-氧脯氨酸酶的活力.该酶以马氏管中活力最高,在蚕的中肠、血淋巴中均存在有游离的5-氧脯氨酸.观察了保幼激素类似物(JHA)处理后,家蚕中肠、丝腺和脂肪体中5-L-氧脯氨酸酶活力的变化,同时观察了血淋巴中5-氧脯氨酸含量的变化.对该酶及γ-谷氨酰循环在蚕体中氨基酸转运上的可能作用进行了讨论.     

Isolation of two cDNA clones coding for larval hemolymph proteins of Galleria mellonella

Galleria mellonella a group of four larval hemolymph proteins (LHP) (74, 76, 81 and 82 kDa), which had been earlier shown to be storage proteins, exhibit a stage-specific synthetic pattern. The 82 kDa LHP is synthesized only in day-3 to day-5 last instar larvae, while the other three LHPs are synthesized both in the penultimate (six) and the last instar larvae. None of these LHPs are synthesized in day-0 last instar. With a view to isolate one or more cDNA clones corresponding to these LHPs a cDNA library was prepared in pBR322 starting with poly(A)⁺ RNA from day-5 last instar larval fat body. By differential screening of 714 clones with poly(A)⁺ RNA 39 day-5 larval stage-specific clones were isolated. Two of these clones, designated as 26–38 and 17–36, had 1200–1300 base pair cDNA inserts. Their cDNA inserts did cross hybridize to each other, exhibited different restriction endonuclease digestion patterns and hybridized in northern blots to transcrips of different sizes, thereby suggesting that they represent two separate genes. In addition, the genomic fragments that hybridized in southern blots to the two cDNAs differed in their size. On translation, mRNAs hybrid selected by 26–38 and 17–36 cDNAs produced 76 and 79 kDa polypeptides respectively. Both these genes are expressed in the fat body but not in the midgut, silk glands, Malpighian tubules or carcass. While 26–38 was expressed both in the sixth and seventh (last) instars, 17–36 was expressed only in the last instar. On the basis of tissue and developmental stage specificity of their expression and the sizes of their hybrid selected translation products, these clones are tentatively identified as two LHP-specific cDNA clones. The genes coding for these LHPs appear to be single copy genes.     

Routine alfalfa (Medicago sativa) cutting program and its influence on pea aphid (Acyrthosiphon pisum Harris) populations

Summary Larvae of Cerura vinula L. were raised at 18, 25 and 30°C in order to test the effect of temperature on the speed of the entire larval development. The time required decreases hyperbolically, with increasing temperature. The optimum temperature seems to be 25°C since all larvae raised at this temperature developed completely. In contrast, some of the larvae at 18 and at 30°C were unable to molt from instar 2 to instar 3, causing high loss of larvae. Further development and metamorphosis were normal. At 30°C, some of the larvae died before the pupal molt; the remaining larvae showed morphological deficiencies upon pupal molt.Mit dankenswerter Unterstützung der Deutschen Forschungsgemeinschaft.     

Genes controlling posterior gut development in theDrosophila embryo

Our present detailed understanding of the genetic mechanisms controlling segmentation has been made possible, in large part, by comprehensive screens of cuticular morphology that identified genes involved in epidermal patterning. To systematically identify genes involved in internal morphogenesis, specifically development of the gut, we have screened mutant embryos produced by a collection of 53 embryonic lethal mutations affecting embryonic pattern formation or differentiation, and a collection of 161 deficiencies covering, in aggregate, approximately 70% of the genome. Staining with the anti-crumbs antibody was used to characterize the Malpighian tubules and hindgut, as well as other internal organs. The geneshuckebein, tailless andwingless, and two previously undescribed loci at 24C/D and 68D/E, are required to establish the primordia for the posterior midgut and hindgut/Malpighian tubules. A locus in region 30A/C is required for extension of the midgut epithelium to surround the yolk, and region 36E/37F is required for outbudding of the Malpighian tubule primordia. Several deficiencies were identified that uncover loci with specific effects on the morphogenesis (elongation, lumen formation) of the hindgut and Malpighian tubules and on the formation of constrictions in the midgut.     

Ultrastructure of the larval Malpighian tubules in <Emphasis Type="Italic">Terrobittacus implicatus</Emphasis> (Mecoptera: Bittacidae)

The larvae of Bittacidae, a cosmopolitan family in Mecoptera, have an interesting habit of spraying the body surface with soil through the anus after hatching, and each molts. The fine structure of Malpighian tubules, however, remains largely unknown in the larvae of Bittacidae to date. Here, we studied the ultrastructure of the larval Malpighian tubules in the hangingfly Terrobittacus implicatus (Huang & Hua) using scanning and transmission electron microscopy. The larvae of T. implicatus have six elongate Malpighian tubules at the junction of the midgut and hindgut. The tubule comprises a basal lamina, a single-layered epithelium, and a central lumen. The basal plasma membranes of the epithelial cells are conspicuously infolded and generate a labyrinth. The epithelium consists of two types of cells: large principal cells and scattered stellate cells. Mitochondria and cisterns of rough endoplasmic reticulum are numerous in the principal cells but are sparsely distributed in the stellate cells, indicating that the principal cells are active in transport. On the other hand, spherites are only abundant in the principal cells and are likely associated with the soil-spraying habit of the larvae.     

Heat shock phenomena in Chironomus tentans

Incubation of 4th instar larvae of Chironomus tentans at elevated temperatures leads in salivary and Malpighian chromosomes to the appearance of 4–5 new puffs. Previously present puffs, particularly Balbiani rings in salivary chromosomes, become drastically reduced. The reactions of region IV-5C and Balbiani ring 1 and 2 in salivary glands are quantitatively analyzed. Statistically significant heat shock effects are observed already after 5 min and reach a maximum between 30 and 60 min. The effective temperature range is small (between 33 to 40 ° C) with an optimum at 37 ° C. Above 40 ° C, i.e., at overheat shock temperatures, heat shock reactions are suppressed. Larvae heat or overheat shocked for 1–7 h or 15–30 min, respectively, survive when returned to normal culturing temperatures. The recovery from heat shock of the puffing pattern occurs in two phases: a fast one (10–20 min) and a slow one (up to 5 h) sometimes separated by a period of backlash. Quenching of overheat shocked larvae does not result in a delayed heat shock reaction.     

蓖麻蚕芳基酰酰胺酶的研究

芳基酰酰胺酶(E.C.3,5,1,13)为水解N-酰基芳香胺的酶,本工作对该酶在蓖麻蚕个体发育中的活力变化及其性质进行了观察和研究,结果如下:ⅰ)该酶存在于蚕个体发育中的各个阶段,广泛分布在蚕体内各组织。在中肠、马氏管组织的活力在熟蚕时最大,结茧后明显下降。在五龄中前期,蛹中后期,脂肪体和真皮的酶活力均出现高峰,以蛹中后期活力最高。该酶在中枢神经系统中有相当高的活力,在后部丝腺、蚕卵都显示活力,但血淋巴中没有可检出的活力。ⅱ)蓖麻蚕各组织的芳基酰酰胺酶,绝大部分活力存在于细胞浆。对N-乙酰对硝基苯胺容易水解,但对γ-谷氨酰对硝基苯胺和γ-谷氨酰α-萘胺均不能水解。钙、钴阳离子能促进酶活力;瞟呤霉素有明显抑制酶活力的作用,但L-γ-谷氨酸(0-羧基)苯肼无抑制作用。从该酶在蚕体广泛分布,并随变态而活力有明显变化以及对底物芳香胺N-乙酰基团显示特异性等特点来看,它可能具有多种功能,并与芳香胺的代谢有关。