New Polar Constituents in the Pupae of the Silkworm Bombyx mori L. (II): Developmental Changes of the Constituents

A correlation between the quantitative changes in L-methionine analogs, the ratio of D-serine/L-serine during the pupal stage, and metamorphosis was observed. The glycoside appearing at low blood sugar values during the pupal stage was isolated and characterized as D-glucosyl-L-tyrosine. ¹H-NMR indicated the appearance and increase of this glycoside, and Mirrorcle Ray CV4 equipment was used to take X-ray pictures of the pupal bodies. The results indicate that γ-cyclic di-L-glutamate and L-methionine sulfone might be concerned with ammonia assimilation in the pupae, and that D-glucosyl-L-tyrosine served as a switch for the fatty acid (pupal oil) dissimilation hybrid system.     

New polar constituents of the pupae of the silkworm Bombyx mori L. I. Isolation and identification of methionine sulfoxide, methionine sulfone, and gamma-cyclic di-L-glutamate

In addition to serine (L:D = 68:32), methionine sulfoxide (MSO), L-methionine sulfone (L-MSO(2)), and disodium gamma-cyclic di-L-glutamate were identified in a methanol extract of Bombyx mori L. pupae. MSO was isolated in a diastereomeric mixture of L(+)- and D(+)-MSO in a ratio of 99:1. The presence of these compounds in other developmental stages, including eggs, larvae (1st, 4th, 5th, and mature 5th instar), adults, and excrement (feces and urine) was investigated. The L(+)-isomer of MSO was present in extracts of the 1st and 5th instar larvae, adults, and eggs, but was not detected in feces or urine. The D(+)-isomer was found only in pupal stage extracts, and was excreted into the meconium with L(+)-isomer. L-MSO(2) and gamma-cyclic di-L-glutamate were not detected at other insect life stages or in the insect excrement. gamma-Cyclic di-L-glutamate is thought be produced due to blockage of the glutamate synthetic pathway (glutamine synthetase) by L-MSO(2) and Mg(2+). The biochemical role of L-MSO(2) during the pupal life stage remains unknown, but importantly, the stage-specific expression suggests that it is a candidate molecule for the induction of diapause.     

Function of the nuclear receptor FTZ‐F1 during the pupal stage in Drosophila melanogaster

The nuclear receptor βFTZ‐F1 is expressed in most cells in a temporally specific manner, and its expression is induced immediately after decline in ecdysteroid levels. This factor plays important roles during embryogenesis, larval ecdysis, and early metamorphic stages. However, little is known about the expression pattern, regulation and function of this receptor during the pupal stage. We analyzed the expression pattern and regulation of ftz‐f1 during the pupal period, as well as the phenotypes of RNAi knockdown or mutant animals, to elucidate its function during this stage. Western blotting revealed that βFTZ‐F1 is expressed at a high level during the late pupal stage, and this expression is dependent on decreasing ecdysteroid levels. By immunohistological analysis of the late pupal stage, FTZ‐F1 was detected in the nuclei of most cells, but cytoplasmic localization was observed only in the oogonia and follicle cells of the ovary. Both the ftz‐f1 genetic mutant and temporally specific ftz‐f1 knockdown using RNAi during the pupal stage showed defects in eclosion and in the eye, the antennal segment, the wing and the leg, including bristle color and sclerosis. These results suggest that βFTZ‐F1 is expressed in most cells at the late pupal stage, under the control of ecdysteroids and plays important roles during pupal development.     

Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.     

Sublethal effects of Bacillus thuringiensis on the spruce budworm, Choristoneura fumiferana

Exposing larvae of the spruce budworm, Choristoneura fumiferana (Clemens), to sublethal ( 50% lethal dose) levels of Bacillus thuringiensis subsp. kurstaki at various stages of their development significantly increased development time to the pupal stage and reduced pupal size and number of eggs laid per female, but did not affect the proportion of embryonated eggs. The changes in larval development time, pupal weight and fecundity depended on the larval stage that was treated. Exposure of fourth instars delayed larval development and reduced only male pupal weights with no effects on fecundity. Exposure of sixth instars delayed larval development to a lesser extent than exposure of fourth instars but had a pronounced effect on weight of both male and female pupae. The effect on pupal weight was sex dependent, as males tended to be more affected than females. The reduction in male pupal weight did not appear to influence fecundity, because the effect of exposure was explained by the change in female pupal weight. Effects on larval growth and pupal weight were proportional to the dose that was ingested during exposure, and were observed at doses as low as one-tenth of the LD₅₀. Ingestion of an LD₅₀ caused a 29 or 45% delay in development of, respectively, female or male larvae when exposed as fourth instars and a 30% reduction in female pupal weight when larvae were exposed as sixth instars.     

Functional analysis of two chitinase genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by RNA interference

Insect chitinases are a multigene family that is encoded by a rather large and diverse group of genes. The main function of chitinases is to digest the chitin contained in tissues such as the cuticles and gut lining during molting. In this study, we examined the role of a chitinase (SeChi) and a bacterial type chitinase (SeChi-h) during the pupation and eclosion stages of Spodoptera exigua. First, efficient silencing of the SeChi and SeChi-h genes through specific double-stranded RNA (dsRNA) injection led to a significant reduction in the mRNA levels of SeChi and SeChi-h. Additionally, different phenotypic defects were observed at the pupal and adult stages after injection of the SeChi and SeChi-h dsRNAs. After injecting SeChi dsRNA in the pupal stage, the cuticle of the head split open and the pupal cuticle was visible under the old larval cuticle. However, after injecting the SeChi-h dsRNA, animals died without exhibiting any special phenotypes. At the adult death stage, animals injected with dsSeChi could not shed their pupal shell completely, and their old cuticles remained attached to their head or chest. However, the main lethal phenotype was that insects did not emerge after dsSeChi-h injection. Additionally, the average survival rates of S. exigua were 52.02% and 40.38% at the pupal and adult stages, respectively, after injection with SeChi dsRNA. For the insects injected with SeChi-h dsRNA, the survival rates were 72.38% and 48.52%, respectively. These results suggest that SeChi and SeChi-h may have different biologic functions during the pupal-adult molting.     

Biochemical evidence of DNA transport from the silk gland to the fat body of the silkworm, Bombyx mori

β-DNA, a component of DNA found in the pupal fat body of the silkworm, Bombyx mori, has the same GC content but a smaller molecular weight than typical silkworm DNA (α-DNA). Its origin and time of synthesis were studied by MAK column chromatography of phenol extracts after labelling with radioactive precursors.The DNA components of the fat body changed greatly during the early pupal stage, the β-DNA showing a striking increase relative to α-DNA. Thymidine-6-³H and phosphoric acid-³²P injected into the animals 1 day before analysis caused labelling of α-DNA, but not of β-DNA of the fat body, indicating that β-DNA was not synthesized during the stage of its appearance in the fat body.On the other hand, injection of thymidine-6-³H into 2-day-old fifth instar larvae, when DNA of the silk gland was being actively synthesized, gave high incorporation of the isotope into β-DNA of the pupal fat body. The sudden appearance of highly labelled β-DNA in the fat body during the early pupal stage as well as the occurrence of β-DNA in both the silk gland and fat body suggested that DNA might move from the silk gland to the fat body.It is possible that the fat body stores DNA as a nutrient from the degenerating silk gland.     

家蚕滞育激素－性信息素合成激活肽基因的表达

家蚕滞育激素－性信息素合成激活肽基因的表达徐卫华（中国农业科学院蚕业研究所,江苏镇江,２１２０００）山下兴亚（名古屋大学农学院,日本名古屋,４６４－０１）关键词滞育激素－性信息素合成激活肽基因;发育阶段;表达;家蚕昆虫是地球上最繁盛的物种,占地球上生...     

L-Glutamate retrieved with the moulting fluid is processed by a glutamine synthetase in the pupal midgut of Calpodes ethlius

From apolysis until pupal ecdysis, the pharate pupa of the Brazilian Skipper (Calpodes ethlius) lies wrapped in a prepupal shell composed of the larval cuticle and an ecdysial space (ES) filled with enzyme-rich moulting fluid (MF). In the 4h before ecdysis the pharate pupa drinks the moulting fluid through its mouth and anus, and transfers the cuticular degradation products to its midgut (MG). At the same time, extra fluid passes across the body wall of the pharate pupa and flushes out the ES. The MF is recovered at an overall rate of 70μl/h and reabsorbed across the pharate pupal midgut at about 26μl/h. L-Glutamate was found to be the dominant amino acid in the moulting fluid. Total MF glutamate peaked at 850nmol about 8h before pupal ecdysis (P-8), but by ecdysis it had dropped to nearly zero as the MF became diluted with new fluid and was consumed. The drop in glutamate in the ES coincided with a rise in the glutamine content of the fluid in the midgut lumen. The highest rate of glutamine synthesis occurred in midguts isolated from pharate pupae actively drinking MF (P相似文献   

Coevolution of the glucose dehydrogenase gene and the ejaculatory duct in the genus Drosophila

The glucose dehydrogenase gene (Gld) in Drosophila melanogaster exhibits a unique spatial and temporal pattern of expression. GLD expression switches from a non-sex-limited state at the pupal stage to a male-limited state at the adult stage. At the adult stage, the enzyme is restricted to the ejaculatory duct. Within the genus Drosophila, the ejaculatory duct has undergone a simple morphological divergence. In order to determine whether correlated changes in GLD expression had occurred, GLD activity during the pupal and adult stages was determined for several Drosophila species. It was found that virtually all of the species exhibit pupal GLD activity, whereas only those species with an expanded ejaculatory duct express male-limited GLD. The results of interspecific genital imaginal disc transplantation experiments indicate that the expanded morphology and GLD expression do not require any species- or sex-specific diffusible factors. An apparent regulatory polymorphism exists within the D. takahashii species with respect to male-limited GLD expression.      

Genetic and environmental sources of egg size variation in the butterfly Bicyclus anynana

By dividing families of the tropical butterfly, Bicyclus anynana, among different larval (including early pupal) and adult (including late pupal) temperatures, we investigate the genetic and environmental effects on egg size. Both sources of variation affected egg size to similar extents. As previously found in other arthropods, egg size tended to increase at lower temperatures. Our data suggest that the plastic response in egg size can be induced during the pupal stage. Females reared as larvae at the same high temperature tended to lay larger eggs when transferred to a lower temperature, either as prepupae or pupae, compared to those remaining at the high temperature. Additionally, females reared as larvae at different temperatures, but maintained at the same temperature from the early pupal stage onwards, laid larger eggs after larval growth at a low temperature. Heritability estimates for egg size were about 0.4 (parent-offspring regression) and 0.2 (variance component estimates using the full-sib families). Although there seemed to be some variation in the plastic response to temperature among families, genotype-environment interactions were nonsignificant.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

Broad specifies pupal development and mediates the 'status quo' action of juvenile hormone on the pupal-adult transformation in Drosophila and Manduca

The understanding of the molecular basis of the endocrine control of insect metamorphosis has been hampered by the profound differences in responses of the Lepidoptera and the Diptera to juvenile hormone (JH). In both Manduca and Drosophila, the broad (br) gene is expressed in the epidermis during the formation of the pupa, but not during adult differentiation. Misexpression of BR-Z1 during either a larval or an adult molt of Drosophila suppressed stage-specific cuticle genes and activated pupal cuticle genes, showing that br is a major specifier of the pupal stage. Treatment with a JH mimic at the onset of the adult molt causes br re-expression and the formation of a second pupal cuticle in Manduca, but only in the abdomen of DROSOPHILA: Expression of the BR isoforms during adult development of Drosophila suppressed bristle and hair formation when induced early or redirected cuticle production toward the pupal program when induced late. Expression of BR-Z1 at both of these times mimicked the effect of JH application but, unlike JH, it caused production of a new pupal cuticle on the head and thorax as well as on the abdomen. Consequently, the 'status quo' action of JH on the pupal-adult transformation is mediated by the JH-induced re-expression of BR.     

三种微小RNA在意大利蜜蜂工蜂蛹期发育过程中的表达谱及潜在功能

[目的]意大利蜜蜂Apis mellifera ligustica是生产性能优良的西方蜜蜂Apis mellifera亚种.本研究旨在利用分子生物学手段揭示3种微小RNA(microRNA,miRNA) ame-miR-13b,ame-miR-100和ame-miR-bantam调控蛹期变态发育的分子机理.[方法]通过...     

Control of pupal commitment in the imaginal disks of Precis coenia (Lepidoptera: Nymphalidae)

When final (5th) instar larvae of Precis coenia were treated with the juvenile hormone analog (JHA) methoprene, they underwent a supernumerary larval molt, except for certain regions of their imaginal disks, which deposited a normal pupal cuticle. Evidently those regions had already become irreversibly committed to pupal development at the time JHA was applied. By applying JHA at successively later times in the instar, the progression of pupal commitment could be studied. Pupal commitment in the proboscis, antenna, eye, leg and wing imaginal disks occurred in disk-specific patterns. In each imaginal disk there were distinct initiation sites where pupal commitment began during the first few hours of the final larval instar, and from which commitment spread across the remainder of the disk over a 2- to 3-day period. The initiation sites were not always located in homologous regions of the various disks. As a rule, pupal commitment also spread from imaginal disk tissue to surrounding epidermal tissue. The regions of pupal commitment in all disks except those of the wings, coincided with the regions of growth of the disk. Only portions of the disk that had undergone cell division and growth underwent pupal commitment. Shortening the growth period did not prevent pupal commitment in the wing imaginal disk, indicating that, in this disk at least, a normal number of cell divisions was not crucial in reprogramming of disk cells for pupal cuticle synthesis. The apparent growth spurt of imaginal disks that occurs during the last part of the final larval instar is merely the final stage of normal and constant exponential growth. Juvenile hormone (JH) and ecdysteroids appeared to play little role in the regulation of normal imaginal disk growth. Instead, growth of the disks may be under intrinsic control. Interestingly, even though endogenous fluctuation in JH titers do not affect imaginal disk growth, exogenous JHA proved able to inhibit both pupal commitment, cell movement, and growth of the disks during the last larval instar. This function of JH could be important under certain adverse conditions, such as when metamorphosis is delayed in favor of a supernumerary larval molt.     

Hormonal control of pupal coloration in the painted lady butterfly Vanessa cardui

Pupae of the painted lady butterfly Vanessa cardui exhibit pupal color polyphenism consisting of white, dark and intermediate types. We investigated environmental factors affecting pupal coloration and the physiological mechanisms underlying the control of pupal color polyphenism in this species. Over 80% of larvae reared at 16 °C developed into pupae of dark types, whereas over 82% of larvae at 32 °C developed into pupae of white types irrespective of long/short-day photoperiod conditions. When mature larvae reared at 32 °C were ligatured between thoracic and abdominal parts at three different pharate pupal stages, all of the head-thoracic parts developed into white pupae regardless of pupal stage, but all abdominal parts ligatured at the early pharate pupal stage only developed into dark pupae. These results indicate that temperature during larval stages is an important element affecting pupal coloration as an environmental cue in V. cardui, and that a factor(s) inducing white pupae is released from head-thoracic parts under conditions of high temperature. Additionally, when ligatured abdomens destined to develop into dark pupae were treated with crude extracts prepared from the central nervous system, all of the ligatured abdomens developed into white pupae at a level dependent on dose and pupal stage. These results suggest that the factor inducing white pupae is a key molecule controlling pupal color polyphenism in V. cardui.     

Effects of photoperiod and temperature on pupal diapause induction of grape berry moth Lobesia botrana

Abstract. The grape berry moth, Lobesia botrana Denn. & Sciff. (Lepidoptera: Tortricidae), one of the most injurious pest of grape berries in Greece, is a multivoltine species that overwinters as diapausing pupae. The effects of several diel and non-diel photoperiods and of temperature, experienced by eggs and larvae, on pupal diapause induction were investigated. The diapause response curve was of Type I (long day type) and the determining factor was the duration of scotophase (> 11 h), regardless of the duration of photophase. However, at very short (< 4 h) photoperiods, the incidence of diapause was also high. Diapause was positively and significantly correlated with the egg-larval developmental time, pupal mortality and the duration of the pupal stage. Eggs and larvae reared under LD 12 : 12 h photoperiod and various temperatures (from 12 to 30 °C) produced diapausing pupae (almost 100%), but the duration of the pupal stage (intensity of diapause) increased with increasing temperature. Under continuous darkness, however, the percentage diapause decreased with increasing temperature. Single and double 1-h light pulses were applied systematically at various times during the scotophase of six diapause-inducing diel photoperiods. Two photosensitive points in time (called A and B) were revealed, during which illumination resulted in a significant decrease of diapause induction. The decrease was much greater during the first sensitive period (early in scotophase) rather than in the second (late in the scotophase).