Shotgun proteomic analysis of wing discs from the domesticated silkworm (Bombyx mori) during metamorphosis

Proteomic profiles from the wing discs of silkworms at the larval, pupal, and adult moth stages were determined using shotgun proteomics and MS sequencing. We identified 241, 218, and 223 proteins from the larval, pupal, and adult moth stages, respectively, of which 139 were shared by all three stages. In addition, there were 55, 37, and 43 specific proteins identified at the larval, pupal, and adult moth stages, respectively. More metabolic enzymes were identified among the specific proteins expressed in the wing disc of larvae compared with pupae and moths. The identification of FKBP45 and the chitinase-like protein EN03 as two proteins solely expressed at the larval stage indicate these two proteins may be involved in the immunological functions of larvae. The myosin heavy chain was identified in the pupal wing disc, suggesting its involvement in the formation of wing muscle. Some proteins, such as proteasome alpha 3 subunits and ribosomal proteins, specifically identified from the moth stage may be involved in the degradation of old cuticle proteins and new cuticle protein synthesis. Gene ontology analysis of proteins specific to each of these three stages enabled their association with cellular component, molecular function, and biological process categories. The analysis of similarities and differences in these identified proteins will greatly further our understanding of wing disc development in silkworm and other insects.     

The cuticle proteins of Drosophila melanogaster: stage specificity

Five stage-specific cuticles are produced during the development of Drosophila. Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.     

The ribosomal proteins of Drosophila melanogaster. I. Characterization in polyacrylamide gel of proteins from larval, adult, and ammonium chloride-treated ribosomes

Summary Ribosomes were isolated from larvae and adult flies, and the purity of the preparation was checked by electron microscopy. The ribosomal proteins were extracted with cold dilute hydrochloric acid, and precipitated with cold acetone. The proteins were characterized by polyacrylamide gel electrophoresis. At pH 3.0 at least 25 bands of different color intensities were resolved, forming a complex pattern.On the basis of electrophoretic mobilities, it was shown that some ribosomal proteins are species-specific, and that larval ribosomes have three protein components more than ribosomes from adult flies.Incubation of the ribosomes with 0.75 M NH₄Cl at a low Mg⁺⁺ concentration lead to detachment of 64% of the ribosomal protein. This detachment of protein molecules was considerably reduced by a five-fold increase of Mg⁺⁺ ions.     

Protein crosslinking by transglutaminase controls cuticle morphogenesis in Drosophila

Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins--two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin-as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.     

利用 CRISPR/Cas9 系统定向编辑黑腹果蝇Osiris24基因

Osiris基因在几丁质沉积过程中表达,可能参与昆虫表皮的发育。本研究利用CRISPR/Cas9 基因编辑系统对Osiris24基因进行编辑,进而观察Osiris24突变体果蝇的性状并且检测Osiris24的表达特征。在Osiris24第1外显子设计2个sgRNA靶位点,插入到pCFD4敲除载体骨架中,同时构建酵母Gal4蛋白序列的供体(donor)载体,将2个载体同时注射到nos-Cas9胚胎中获得G0代转基因果蝇。结果显示,G0代基因编辑阳性率为92.8%,Osiris24纯合突变体在胚胎或1龄幼虫期致死,杂合突变体未观察到可见表型。将阳性G0代雄虫与UAS-GFP雌虫杂交,检测不同龄期和不同组织GFP信号表达情况。结果发现,Osiris24在不同龄期幼虫中均有表达,幼虫期主要在体壁、气管、前肠和后肠高表达,蛹期主要在体壁和翅上表达,推测其在果蝇发育中发挥重要作用,本研究为深入探究Osiris基因功能提供了研究模型。     

Imaginal disc ribosomal proteins of D. melanogaster

Summary The ribosomal proteins from undifferentiated imaginal discs of Drosophila melanogaster were analyzed by two-dimensional gel electrophoresis and compared with the ribosomal protein pattern of adult flies. It is shown that the ribosomal proteins from these discs are qualitatively identical with those of adult flies except that two acidic proteins are missing in the discs. This heterogeneity is discussed in terms of the functional roles these two proteins may carry in connection with disc differentiation.Paper No. 6 of the present series. Paper No. 5 is by Lambertsson, A. G. Molec. gen. Genet. 139, 145–156 (1975)     

Haemolymph protein patterns in developing tobacco hornworm larvae

Quantitative changes in haemolymph proteins from each physiological phase of the last three larval instars of the tobacco hornworm, Manduca sexta, were studied by means of disk electrophoresis. Twelve anodical migrating protein bands were found, six of which occurred only sporadically. Total protein concentration increased from pharate third instar to late fifth instar larvae, then decreased slightly in the pharate pupal stages. Some individual bands showed cyclic patterns within each instar similar to the overall cyclic pattern of total protein, whereas other bands showed different patterns or no pattern.     

Body size and cell size in Drosophila: the developmental response to temperature

In Drosophila, like most ectotherms, development at low temperature reduces growth rate but increases final adult size. Cultures were shifted from 25 degrees C to low (16.5 degrees C) or to high (29 degrees C) temperature at regular intervals through larval and pupal stages, and the flies of both sexes showed an increase or decrease, respectively, in the size of thorax, wing and abdominal tergite. Size changes in the wing blade resulted from changes in the size of the epidermal cells (with only a small increase in cell number in males reared at low temperature). The temperature-shifts became less effective as they were made at successively later developmental stages, demonstrating a cumulative effect of temperature on adult size. The thorax and wing develop from the same imaginal disc, with most cell division occurring in larval stages, but they differ in timing of temperature sensitivity, which extends only to pupariation or into the late pupal stage, respectively. Growth of the adult abdomen occurs largely after pupariation but its size is temperature-sensitive through both larval and pupal stages. We discuss growth control in Drosophila and the likely effects of temperature on food assimilation, growth efficiency and allocation of nutrients to the production of different tissues.     

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.     

RNA metabolism during metamorphosis of Drosophila hydei

Determinations of total protein and RNA at various times after the beginning of the pharate pupal stage of Drosophila hydei, revealed an increase in both substances during the first 25 hr and a sudden decrease thereafter until 52 hr. From this time on the total amount of both protein and RNA increases slightly until emergence of the flies at 160 hr after the beginning of the pharate pupal stage. A similar pattern of changes was recorded for the total radioactivity as well as the specific activity of RNA labelled with ³H-uridine after the injection of the isotope immediately before the beginning of the pharate pupal stage.The migration profile of RNA labelled with ³H-uridine during larval development, revealed that shortly after the onset of the pharate pupal stage an essentially normal larval pattern consisting of major fractions of 28, 18, 8 to 9, and 4 to 7 S RNA. At 52 hr only the low molecular weight RNA was present. The ‘normal’ pattern was restored at the time of emergence of the flies, indicating re-utilization of degradation products of previously labelled RNA.     

Effects of natal habitat odour, reinforced by adult experience, on choice of oviposition site in the mosquito Aedes aegypti

The effects of natal experience on the oviposition behaviour of adult female mosquitoes were investigated in the laboratory using Aedes aegypti (L.) (Diptera: Culicidae). 'Treatment' mosquitoes were exposed to a dilute repellent (inducing stimulus) in their breeding water (aquatic stages) and/or in the air (adults) during various combinations of life stages [larval only (L regime); larval and pupal (LP regime); larval, pupal and emergent adult (LPE regime); larval, pupal, emergent adult and adult (LPEA regime); pupal, emergent adult and adult (PEA regime); adult only (A regime)]. 'Control' mosquitoes were raised in an identical manner, but were not exposed to the inducing stimulus. The oviposition behaviour of treatment and control females was assessed in an oviposition assay that presented a choice of water with or without the inducing stimulus. Of the 435 mosquitoes tested in the experiment, 176 were non-distributors (i.e. laid all of their eggs in only one of the choices). Treatment females (distributors plus non-distributors) reared in the presence of the inducing stimulus throughout their lives (LPEA regime) showed a significant preference for the oviposition option containing the inducing stimulus (24/36 females) compared with corresponding controls (5/39 females). Distributors reared under the LPEA and PEA regimes also showed this preference (6/6 treatment vs. 2/29 control females, and 13/18 treatment vs. 7/23 control females, respectively). Females that had been exposed to the inducing stimulus as either immatures or adults only showed no preference for, and some showed an aversion to, the treatment oviposition option. This is interpreted as evidence for a natal habitat preference induction (NHPI) in this species, albeit one that requires extensive reinforcement in the adult stage. This adult experience-reinforced NHPI (AER-NHPI) is discussed in terms of its adaptive significance for container breeders, the possible timing mechanism and sensory basis of induction and potential practical consequences.     

Morphological comparison of pupal wing cuticle patterns in butterflies

Butterfly wing color-patterns are determined in the prospective wing tissues during the late larval and early pupal stages. To study the cellular differentiation process of wings, morphological knowledge on pupal wings is prerequisite. Here we systematically examined morphological patterns of the pupal wing cuticular surface in a wide variety of nymphalid butterflies in relation to adult color-patterns. Several kinds of pupal wing patterns corresponding to particular adult color-pattern elements were widely observed in many species. Especially noteworthy were the pupal "focal" spots corresponding to the adult border ocelli system, which were detected in many species of Nymphalinae, Apaturinae, Argynninae, Satyrinae, and Danainae. Striped patterns on the pupal wing cuticle seen in some species of Limenitinae, Ariadnae, and Marpesiinae directly corresponded to those of the adult wings. In Vanessa cardui, eyespot-like pattern elements were tentatively produced during development in the wing tissue underneath the pupal spots and subsequently erased, suggesting a mechanism for producing novel color-patterns in the course of development and evolution. The pupal focal spots reasonably correlated with the adult eyespots in size in Precis orithya and Ypthima argus. We physically damaged the pupal focal spots and their corresponding cells underneath in these species, which abolished or inhibited the formation of the adult eyespots. Taken together, our results clarified that pupal cuticle patterns were often indicative of the adult color-patterns and apparently reflect molecular activity of organizing centers for the adult color-pattern formation at least in nymphalid butterflies.     

Butterfly Wings Are Three-Dimensional: Pupal Cuticle Focal Spots and Their Associated Structures in Junonia Butterflies

Butterfly wing color patterns often contain eyespots, which are developmentally determined at the late larval and early pupal stages by organizing activities of focal cells that can later form eyespot foci. In the pupal stage, the focal position of a future eyespot is often marked by a focal spot, one of the pupal cuticle spots, on the pupal surface. Here, we examined the possible relationships of the pupal focal spots with the underneath pupal wing tissues and with the adult wing eyespots using Junonia butterflies. Large pupal focal spots were found in two species with large adult eyespots, J. orithya and J. almana, whereas only small pupal focal spots were found in a species with small adult eyespots, J. hedonia. The size of five pupal focal spots on a single wing was correlated with the size of the corresponding adult eyespots in J. orithya. A pupal focal spot was a three-dimensional bulge of cuticle surface, and the underside of the major pupal focal spot exhibited a hollowed cuticle in a pupal case. Cross sections of a pupal wing revealed that the cuticle layer shows a curvature at a focal spot, and a positional correlation was observed between the cuticle layer thickness and its corresponding cell layer thickness. Adult major eyespots of J. orithya and J. almana exhibited surface elevations and depressions that approximately correspond to the coloration within an eyespot. Our results suggest that a pupal focal spot is produced by the organizing activity of focal cells underneath the focal spot. Probably because the focal cell layer immediately underneath a focal spot is thicker than that of its surrounding areas, eyespots of adult butterfly wings are three-dimensionally constructed. The color-height relationship in adult eyespots might have an implication in the developmental signaling for determining the eyespot color patterns.     

家蚕蛹期雌雄生殖腺蛋白质双向电泳比较分析

分析家蚕Bombyx mori雌雄生殖腺细胞蛋白质,有利于发现性别分化相关的功能蛋白质,探讨生殖腺发育相关基因的表达调控机理。本研究利用蛋白质双向凝胶电泳和图像分析技术,分析家蚕蛹期第2天的雌雄生殖腺细胞蛋白质。结果表明: 在雄蚕生殖腺蛋白质电泳图谱中共检测到435个蛋白斑点,其中特异性蛋白斑点73个,占总蛋白斑点数的16.8％;雌蚕生殖腺的电泳图谱中有417个蛋白斑点,其中特异性蛋白斑点55个,占总蛋白斑点数的13.2％。雌雄能匹配的蛋白斑点有362对,匹配率达85.0％。     

Patterns of proteins synthesized during development of Caenorhabditis elegans.

Two-dimensional gel electrophoresis has been used to analyze proteins synthesized during postembryonic development of the nematode Caenorhabditis elegans. This organism is favorable for these studies because it has a limited number of cells, it is genetically well-defined, and its development is currently under investigation in several laboratories. ³⁵S-Labeled E. coli was used for continuous and pulse labeling of C. elegans during its four juvenile larval stages and as a gravid adult. After continuous labeling or pulse labeling for 1 hr, 600–800 individual spots can be resolved on a 2D gel using fluorography and 2 weeks of exposure. Proteins that represent 0.0017% of the total sample can be detected. Exposure for 12 weeks reveals only 100 additional spots even though the films are not saturated. It therefore appears that the frequency distribution of proteins decreases significantly beyond these 800 most abundant proteins that can be fractionated on an O'Farrell gel. When the patterns of pulse-labeled proteins of the five developmental stages were compared, 113 proteins could be seen to undergo modulation at one or more of the developmental stages. A maximum number of changes was seen in the transition from the L4 to the adult stages when 11% of the total spots either appeared, disappeared, or changed in intensity. As controls, different preparations of the same developmental stage were compared and revealed considerable fluctuation, 2.6–4.8%. These fluctuations are presumed to be due to variations in growth conditions during culture of the organism. Continuous label experiments reveal a distinct set of proteins that undergo turnover and/or modification during development. Some of these proteins are absent in only one stage, indicating that stable proteins are also modulated. But nearly all of the proteins seen in a continuous label are also seen in a pulse label indicating that most of the major proteins are always present and always synthesized.     

A comprehensive study of genic variation in natural populations of Drosophila melanogaster. VII. Varying rates of genic divergence as revealed by two-dimensional electrophoresis.

Four sibling species from the melanogaster subgroup (Drosophila melanogaster, D. simulans, D. sechellia, and D. mauritiana) were studied for genetic divergence, by high-resolution two-dimensional protein electrophoresis (2DE) coupled with ultrasensitive silver staining. A total of eight tissues from larval and adult developmental stages representing both gonadal (germ-line) and nongonadal (somatic) tissues were analyzed for protein divergence between species. Close to 400 polypeptides (protein spots) were scored from each tissue and species, and protein divergence was measured on the basis of qualitative differences (presence/absence) of protein spots in pairwise species comparisons. The observed levels of genic divergence varied among tissues and among species. When larval hemolymph proteins (which are known to be highly polymorphic) were excluded, there was no evidence to suggest that either the larval or adult-stage proteins, as a whole, are more diverged than the other; variation between different tissues rather than between developmental stages appears to be the most significant factor affecting genetic divergence between species. The reproductive tissue (testis and accessory gland) showed more divergence than did the nonreproductive tissue; D. melanogaster testis (from both larvae and adult males) showed the highest level of divergence. In view of the previous observation that D. simulans, D. mauritiana, and D. sechellia show similar but significantly less reproductive isolation from each other than from D. melanogaster, the present results suggest a correlation between the levels of reproductive-tract-protein divergence and the degree of reproductive isolation in these species.