Constancy of ascending projections in the metamorphosing brain of the meal-beetle Tenebrio molitor L. (Insecta: Coleoptera)

Summary The fate of ascending projections of thoracic interneurons in the metamorphosing brain of Tenebrio molitor is described. Persistent brain neurons were identified and their fate is described during metamorphosis. The projection sites of ascending elements are invariable throughout metamorphosis both in quantitative and in qualitative terms. Some of these ascending neurons are serotonin-immunoreactive and this set of neurons maintains a constant projection site within the metamorphosing brain. The alterations in the projection sites of these and other ascending neurons in the ventral nerve cord were analysed experimentally. The central projection sites of these persistent ascending neurons are not important for the maintenance of their nerve cord projections throughout metamorphosis. Experimental deletion of ascending neurons which project into the suboesophageal ganglion varies the shape of persistent central neurons.     

Age-related changes in acetylcholinesterase and its molecular forms in various brain areas of rats

A previous study conducted in this laboratory revealed a decrease in total cholinesterase (total ChE) in the cerebral cortex, hippocampus and striatum in aged rats (24 months) of various strains, as compared with young animals (3 months). The purpose of the present experiments was to extend the study to other brain areas (hypothalamus, medulla-pons and cerebellum) and to assess whether this decrease was dependent on the reduction of either specific acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) or both. By using ultracentrifugation on a sucrose gradient, the molecular forms of AChE were evaluated in all the brain areas of young and aged Sprague-Dawley rats. In young rats the regional distribution of total ChE and AChE varied considerably with respect to BuChE. The age-related loss of total ChE was seen in all areas. Although there was a reduction of AChE and, to somewhat lesser extent, of BuChE in the cerebral cortex, hippocampus, striatum, and hypothalamus (but not in the medulla-pons or the cerebellum), the ratio AChE/BuChE was not substantially modified by age. Two molecular forms of AChE, namely G4 (globular tetrameric) and G1 (monomeric), were detected in all the brain areas. Their distribution, expressed as G4/G1 ratio, varied in young rats from about 7.5 for the striatum to about 2.0 for the medulla-pons and cerebellum. The age-related changes consisted in a significant and selective loss of the enzymatic activity of G4 forms in the cerebral cortex, hippocampus, striatum, and hypothalamus, which resulted in a significant decrease of the G4/G1 ratio. No such changes were found in the medullapons or the cerebellum. Since G4 forms have been proposed to be present presynaptically, their age-related loss in those brain areas where acetylcholine plays an important role in neurotransmission may indicate an impairment of presynaptic mechanisms.     

Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

家蚕蛹变态期丝腺组织的退化与细胞凋亡特征

利用形态学观察方法、分子生物学检测方法以及20-羟基蜕皮酮(20-hydroxyecdysone)和放线菌酮(cycloheximide)体外培养方法, 研究了家蚕Bombyx mori 蛹变态期丝腺组织的退化与细胞凋亡特征。显微镜的观察显示家蚕丝腺的逐渐退化发生在吐丝期间。DNA梯度电泳的分析表明程序性细胞死亡(programmed cell death)可能伴随发生在丝腺的退化过程中。在离体培养条件下, 用20-羟基蜕皮酮处理5龄第6天幼虫的丝腺, 导致的细胞凋亡提前于对照, 提示在进入蛹变态期前, 20-羟基蜕皮酮提早激发了介导家蚕丝腺细胞凋亡与水解机制的遗传调控级联系统。上述结果表明, 20-羟基蜕皮酮能够诱导家蚕丝腺组织在蛹变态期发生程序性细胞死亡。     

Constant topological organization of the coleopteran metamorphosing nervous system: analysis of persistent elements in the nervous system of Tenebrio molitor

Evidence is provided that the topological organization of the larval neuropil is preserved during metamorphosis of Tenebrio molitor. Constancies in neuronal organization were due in part to persistence of individual neurons whose morphologies were individually followed. It appears that the phenomenologically static situation of the metamorphosing neuropil is achieved by stabilization and regulation due to cellular interactions. Comparisons are made with features of hemimetabolan postembryogenesis.     

Endocrine regulation of reproduction and diapause in the boll weevil,Anthonomus grandis Boheman

A review of the literature for the hormonal control of reproduction and diapause in Coleoptera is presented. The role of juvenile hormone and juvenile hormone esterase (JHE) in the control of the different life history strategies of the boll weevil are examined. Elevated levels of hemolymph JHE were found to be positively correlated with survival throughout the winter in South Texas population of weevils. Winter weevils were examined for hemolymph vitellogenin (Vg) and their subsequent survival was monitored. The majority of weevils surviving beyond ten weeks had no hemolymph Vg. We conclude that elevated hemolymph JHE and the absence of Vg are good predictors of survivors in the South Texas population. The hormonal basis of diapause termination was examined using hormone treatment and implant therapy. We were unsuccessful in our attempt to induce post-diapause reproductive development with all of our treatments. Only weevils given access to a food source were capable of reproductive development. Our recent experimental findings in the control of Vg synthesis and uptake in the boll weevil are reviewed. Arch. Insect Biochem. Physiol. 35:455–477, 1997. © 1997 Wiley-Liss, Inc.     

Developmental and environmental regulation of antifreeze proteins in the mealworm beetle Tenebrio molitor.

The yellow mealworm beetle, Tenebrio molitor, contains a family of small Cys-rich and Thr-rich thermal hysteresis proteins that depress the hemolymph freezing point below the melting point by as much as 5. 5 degrees C (DeltaT = thermal hysteresis). Thermal hysteresis protein expression was evaluated throughout development and after exposure to altered environmental conditions. Under favorable growth conditions, small larvae (11-13 mg) had only low levels of thermal hysteresis proteins or thermal hysteresis protein message, but these levels increased 10-fold and 18-fold, respectively, by the final larval instar (> 190 mg), resulting in thermal hysteresis > 3 degrees C. Exposure of small larvae (11-13 mg) to 4 weeks of cold (4 degrees C) caused an approximately 20-fold increase in thermal hysteresis protein concentration, well in excess of the less than threefold developmental increase seen after 4 weeks at 22 degrees C. Exposure of large larvae (100-120 mg) to cold caused 12-fold and sixfold increases in thermal hysteresis protein message and protein levels, respectively, approximately double the maximum levels they would have attained in the final larval instar at 22 degrees C. Thus, thermal hysteresis increased to similar levels (> 4 degrees C) in the cold, irrespective of the size of the larvae (the overwintering stage). At pupation, thermal hysteresis protein message levels decreased > 20-fold and remained low thereafter, but thermal hysteresis activity decreased much more slowly. Exposure to cold did not reverse this decline. Desiccation or starvation of larvae had comparable effects to cold exposure, but surprisingly, short daylength photoperiod or total darkness had no effect on either thermal hysteresis or message levels. As all environmental conditions that caused increased thermal hysteresis also inhibited growth, we postulate that developmental arrest is a primary factor in the regulation of T. molitor thermal hysteresis proteins.     

Preferential inhibition of acetylcholinesterase molecular forms in rat brain

The effect of eight different acetylcholinesterase inhibitors (AChEIs) on the activity of acetylcholinesterase (AChE) molecular forms was investigated. Aqueous-soluble and detergent-soluble AChE molecular forms were separated from rat brain homogenate by sucrose density sedimentation. The bulk of soluble AChE corresponds to globular tetrameric (G₄), and monomeric (G₁) forms. Heptylphysostigmine (HEP) and diisopropylfluorophosphate were more selective for the G₁ than for the G₄ form in aqueous-soluble extract. Neostigmine showed slightly more selectivity for the G₁ form both in aqueous- and detergent-soluble extracts. Other drugs such as physostigmine, echothiophate, BW284C51, tetrahydroaminoacridine, and metrifonate inhibited both aqueous- and detergent-soluble AChE molecular forms with similar potency. Inhibition of aqueous-soluble AChE by HEP was highly competitive with Triton X-100 in a gradient, indicating that HEP may bind to a detergent-sensitive non-catalytic site of AChE. These results suggest a differential sensitivity among AChE molecular forms to inhibition by drugs through an allosteric mechanism. The application of these properties in developing AChEIs for treatment of Alzheimer disease is considered.Special issue dedicated to Dr. Morris H. Aprison.     

Inhibitory effects of actinomycin D and cycloheximide on neuronal death in adult Manduca sexta

A decline in circulating 20-hydroxyecdysone permits the emergence of the adult Manduca sexta moth; this endocrine signal also triggers the death of approximately half of the neurons in the unfused abdominal ganglia of the moth central nervous system. This programmed death of neurons was markedly reduced by treatment with either actinomysin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor). Similar results were found after addition of these agents to ventral nerve cord cultures. The effectiveness of these treatments in delaying or blocking neuronal death depended upon their time of administration relative to the normal time of post-emergence death in the particular motoneuron under study: late-dying neurons, for example, could still be saved by these treatments even after early-dying neurons had already initiated degeneration. In both intact moths and cultured ventral nerve cords, the ability of actinomycin D to prevent neuronal death waned at the same time at which replacement of the steroid hormone could no longer block neuronal death. This suggests that the steroid commitment point represents the time at which the genes that mediate cell death are transcribed. Cycloheximide remained effective in delaying or blocking neuronal death until shortly before the onset of degeneration, suggesting that ongoing protein synthesis is essential for the initiation of the degeneration response. 1994 John Wiley & Sons, Inc.     

Balanced intake of protein and carbohydrate maximizes lifetime reproductive success in the mealworm beetle,Tenebrio molitor (Coleoptera: Tenebrionidae)

Recent developments in insect gerontological and nutritional research have suggested that the dietary protein:carbohydrate (P:C) balance is a critical determinant of lifespan and reproduction in many insects. However, most studies investigating this important role of dietary P:C balance have been conducted using dipteran and orthopteran species. In this study, we used the mealworm beetles, Tenebrio molitor L. (Coleoptera: Tenebrionidae), to test the effects of dietary P:C balance on lifespan and reproduction. Regardless of their reproductive status, both male and female beetles had the shortest lifespan at the protein-biased ratio of P:C 5:1. Mean lifespan was the longest at P:C 1:1 for males and at both P:C 1:1 and 1:5 for females. Mating significantly curtailed the lifespan of both males and females, indicating the survival cost of mating. Age-specific egg laying was significantly higher at P:C 1:1 than at the two imbalanced P:C ratios (1:5 or 5:1) at any given age throughout their lives, resulting in the highest lifetime reproductive success at P:C 1:1. When given a choice, beetles actively regulated their intake of protein and carbohydrate to a slightly carbohydrate-biased ratio (P:C 1:1.54–1:1.64 for males and P:C 1:1.3–1:1.36 for females). The self-selected P:C ratio was significantly higher for females than males, reflecting a higher protein requirement for egg production. Collectively, our results add to a growing body of evidence suggesting the key role played by dietary macronutrient balance in shaping lifespan and reproduction in insects.     

双斑恩蚜小蜂寄生对烟粉虱体内保幼激素和20-羟基蜕皮酮含量的影响

用反相高效液相色谱法（RP-HPLC）测定了烟粉虱Bemisia tabaci体内保幼激素和20-羟基蜕皮酮的滴度。结果显示,被双斑恩蚜小蜂Encarsia bimaculata寄生的烟粉虱高龄若虫体内的保幼激素滴度显著高于（P<0.05）对照即未被寄生的高龄若虫,20-羟基蜕皮酮（20-E）滴度显著低于（P<0.05）对照,与未被寄生的低龄若虫滴度差异不显著,说明双斑恩蚜小蜂寄生后可以将烟粉虱高龄若虫的激素含量保持在低龄若虫的水平,从而调节烟粉虱高龄若虫的生长发育。烟粉虱伪蛹和成虫与对照体内保幼激素含量,烟粉虱伪蛹与对照之间20-E含量差异均不显著（P>0.05）。     

Periodic muscular activity and its possible functions in pupae of Tenebrio molitor

Abstract. During pupal development, Tenebrio molitor L. show regular periods of rhythmic muscular contractions and associated body movements. These periods of activity last 2.5-5.8 min and are more frequent in newly ecdysed pupae ( c. 3h^-1). They become less frequent ( c. 1.5 h^-1) when the basal metabolism reaches its lowest level. In the pharate adult stage the clear pattern of muscular activity disappears.
Muscular activity is temperature-dependent and is commonly absent below 20C. Muscular activity did not disturb the cyclic output of CO₂, which is characteristic of metamorphosis. The heart shows characteristic periods (1–3 min) of activity during pupal development. The frequency of these heart pulsation periods depends on metabolic rate. Heart pumping was correlated mostly with muscular contractions. Therefore we suggest that the main physiological function of muscular activity is to support circulation.     

Biochemical and biophysical characterization of collagen in Tenebrio molitor L. (Coleoptera, Tenebrionidae)

The collagen from the mesenteric sheath of the tenebrionid insect Tenebrio molitor was extracted by limited pepsin digestion and purified. This collagen was characterized using CM-cellulose chromatography, sodium-dodecylsulfate disc-gel electrophoresis and aminoacid analysis. This molecule was found to be assembled from three identical alpha chains and could be represented by the formula (alpha) 3. The amino acid composition is characteristic of collagen (one-third glycine, high iminoacid content), with high content of hydroxylysine and low content of alanine. Cyanogen bromide digests of these chains indicated that they are not related to any of the known invertebrate or vertebrate chains of interstitial collagens. The molecular weight (M = 280000D) and length (290 nm) were typical, and the banding patterns of the segment-long-spacing crystallites (SLS) and of the reconstitued fibrils were very similar to type I collagen. The denaturation temperature (Td) was 30.7 degrees C and correlated with the total pyrrolidine content as observed in other collagens (von Hippel & Wong's relation). It was concluded that the collagen from this insect showed the classical biochemical and biophysical features of other invertebrate interstitial "primitive" collagens.     

Developmental changes in the protein kinase activity of chick brain

The protein kinase activity in the postmitochondrial and postribosomal fractions from chick brain at various stages of development was examined. It has been found, that the overall level of protein kinases activity, assayable under the experimental conditions, increases during embryogenesis, sharply decreases at the hatch, and again increases thereafter. The subcellular distribution of protein kinases alters during ontogenetic development. The embryonal protein kinases of both subcellular fractions differ in the protein substrate specificity, cAMP- and salts-sensitivity from those of the adult ones. Thanks to use of ribosomal proteins as an exogenous substrates it was possible to visualize the developmental changes in the protein kinase pattern.     

黄粉甲幼虫抗菌物质的诱导及其抗菌活性

采用饥饿法、紫外线照射法和针刺法处理黄粉甲Tenebriomolitor 6龄幼虫后均能诱导其 产生抗菌物质,收集的血淋巴上清液对真菌有抑制作用,对细菌无抑制作用;经热处理后的血 淋巴上清液则对细菌有抑制作用,而对真菌无抑制作用。SDS-PAGE检测结果发现,与未诱导的 对照相比经诱导的黄粉甲幼虫血淋巴中,原有的一类大分子蛋白质如分子量分别为97kD、44 kD和37 kD左右的蛋白质缺失;而ESI-MS分析结果显示诱导后比诱导前黄粉甲幼虫血淋巴中有 小分子物质产生,推测可能是此类缺失蛋白质分解为小分子量的抗菌肽,从而表现出抗菌活性 。