Can larval epidermis omit the secretion of a pupal cuticle in a saturniid moth, Samia cynthia ricini?

When the larval tissue is exposed to the hormonal milieu lacking juvenile hormone, adult characters appear directly omitting the pupal stage in some insects but not in others. In Samia cynthia ricini, a species belonging to the latter group, a possible omission of pupal characters was tested by previously untried experiments. Firstly, the possibility that the larval epidermis of only some stages is capable of responding so as to omit to secrete the pupal cuticle was tested. Pieces of larval integument taken from various developmental stages were implanted into developing (pharate) adults. None of these failed to secrete the pupal cuticle. Secondly, pieces of larval integument were first implanted into brainless pupae and left there for a month to eliminate the effect of a trace of juvenile hormone which might have been carried over by the implants. They were then caused to develop, and they again secreted pupal cuticle. It is concluded that the larval epidermis cannot omit secreting pupal cuticle in this species.     

Developmental changes of the antenna and its neurons in the silkworm,Bombyx mori,with special regard to larval-pupal transformation

The larval antenna of Bombyx mori has 13 sensilla and about 52 sensory neurons in its distal portion. The axons form two nerve cords which unite in the cranial hemocoel to supply the brain as the olfactory nerve. The antennal imaginal disc, which is a thick pseudostratified epithelium continuous with the antennal epidermis, thickens markedly during the 5th instar by rapid cell proliferation. At the prepupal stage cell proliferation ceases and the disc everts to form a large pupal antenna. Simultaneously, an extensive cell rearrangement occurs in the antennal epidermis and the disc tissue becomes much thinner because of the abrupt expansion of antennal surface area. The two larval nerve cords thin down markedly by degeneration of axons, but they do not disintegrate totally even after the onset of pupation. The epidermis of the larval antenna forms the distal portion of the pupal antenna, while the imaginal disc forms the more basal portion. Development to the adult antenna occurs almost immediately after the onset of pupation; many adult neurons appear in the simple epidermis facing toward the thick outer side of the newly formed pupal cuticle. By 12 hours after the onset of pupation, these neurons align themselves in many transverse rows which are the first sign of the adult antennal configuration. Addition of these neuronal axons to the once-thinned nerve cords causes resumed thickening of the cords during the first 24 hours and thereafter. Differentiation of adult sensilla begins in the next 24 hours and is almost completed at the third day of pupation, which requires a total of 10 days.     

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.     

Hormonal requirements for the larval-pupal transformation of the epidermis of Manduca sexta in vitro

In the tobacco hornworm, Manduca sexta, metamorphosis occurs in response to two releases of ecdysone that occur 2 days apart. Epidermis was explanted from feeding final-instar larvae before the first release of ecdysone and was cultured in Grace's medium. When exposed to 1 μg/ml of β-ecdysone for 24 hr and then to hormone-free medium for 24 hr, followed by 5 μg/ml of β-ecdysone for 4 days, the epidermis produced tanned pupal cuticle in vitro. During the first 24 hr of exposure to β-ecdysone, the epidermis first changed its cellular commitment to that for pupal cuticle formation (ET₅₀ = 14 hr), then later (by 22 hr) it became committed to tan that cuticle. Then, for most of the pupal cuticle to be tanned, at least a 12-hr period of culture in hormone-free medium was required before the cuticle synthesis was initiated. Consequently, some events prerequisite to sclerotization of pupal cuticle not only occur during the ecdysone-induced change in commitment but also during the ecdysone-free period. When the tissue was preincubated in 3 μg/ml of juvenile hormone (JH I or a mimic epoxygeranylsesamole) for 3 hr and then exposed to both ecdysone and juvenile hormone for 24 hr, it subsequently formed larval cuticle. The optimal conditions for this larval cuticle formation were exposure to 5 μg/ml of β-ecdysone in the presence of 3 μg/ml of epoxygeranylsesamole for 48 hr. When the epidermis was cultured in Grace's medium for 3 days and then exposed to 5 μg/ml of β-ecdysone for 4 days, 70% of the pieces formed pupal cuticle. By contrast, if both ecdysone and JH were added, 77% formed larval cuticle. Therefore, the change from larval to pupal commitment of the epidermal cells requires not only the absence of JH, but also exposure to ecdysone.     

Correlations between epidermal cell structure and endogenous hormone titers during the fifth larval instar of the tobacco hornworm, Manduca sexta

Epidermal cell morphology and cuticle production in Manduca sexta are directly influenced by both ecdysterone and juvenile hormone. Up to day 6 of the last larval instar, post-molt endocuticle is continuously deposited even though cells undergo a partial and temporary separation from the overlying cuticle at the time when a small ecdysteroid peak is detected (approximately day 3.5). At about days 6--7 when another, larger ecdysteroid peak is present, apolysis occurs accompanied by the appearance of edcysial droplets. Following apolysis, layers of pupal cuticle are deposited. Increased quantities of rough endoplasmic reticulum characterize the epidermis at times of peak endocuticle deposition (day 3, larval cuticle; day 9, pupal cuticle). Dense pigment inclusions are found in epidermis from the day of ecdysis to the last larval instar until they are eliminated 5 days later. These dense bodies migrate from cell apex to base in the absence of juvenile hormone (or in the presence of a negligible amount of juvenile hormone) and probably contain insecticyanin.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

Purification,properties, and titer of a hemolymph trophic factor in larvae and pupae of Manduca sexta

Purification of a hemolymph protein (hemolymph trophic factor, or HTF) from last instar larvae of Manduca sexta was achieved using Sephadex G15-120 gel filtration and DEAE anion exchange chromatography. Homogeneity was visualized using SDS gel electrophoresis and ampholytic chromatofocusing. HTF was estimated to be a tetrameric protein with a molecular weight of 286 K and a Stokes' radius of 55.3 × 10⁻⁸ cm by agarose bead gel filtration; chromatofocusing suggests an isoionic point > 10. Polyclonal antibodies to HTF were prepared in rabbits and an ELISA was developed. The ELISA was used to titer HTF during the last larval instar and day 1 and 14 of the pupal stage and estimates a maximum of 1.5 mg/ml larval hemolymph on day 6 with a smaller larval peak of 0.75 mg/ml at day 3 and titers of 0.70 and 0.35 mg/ml on the 2 pupal days, respectively. ELISA of aqueous extracts of larval fat body, epidermis, and cuticle demonstrate that HTF comprises nearly a third of the soluble fat body protein and is a lesser component of epidermis and cuticle. The physiological role of HTF has not yet been determined.     

Monoclonal antibodies recognizing larval- and pupal-specific cuticular proteins of Tenebrio molitor (Insecta,Coleoptera)

To study the sequential expression of insect epidermal cells during metamorphosis, a library of monoclonal antibodies (MABs) was prepared against the water-soluble proteins from preecdysial pupal cuticle of Tenebrio molitor. Six selected MABs recognizing only larval and pupal cuticular proteins (CPs) in immunoblot analysis were classified into three types. Type 1 recognized a 21.5 and a 22 kDa polypeptide, type 2, a 26 kDa polypeptide, and type 3, three polypeptides of 18.5, 19.5 and 21.5 kDa. They did not immunoreact with any protein of fat bodies or haemolymph from pharate pupae, suggesting that the antigens originate from the epidermis. The stage-specificity was confirmed by electron microscopic immunogold labelling. Type 1 and 3 MABs recognized antigens characterizing larval and pupal preecdysial sclerotized cuticles, while the antigens recognized by type 2 were localized in the first few lamellae of unsclerotized postecdysial cuticle. When the expression of the adult programme was inhibited by application of a juvenile hormone analogue, the larval-/pupal-specific CPs were detected in the supernumerary pupal cuticle. These results suggest that the genes encoding these proteins are juvenile hormone dependent. These MABs should be useful tools to isolate pupal-specific genes whose regulation sems to be different from that of the adult-specific ones.     

Degradation of cuticle during larval-pupal and pupal-adult development of the housefly, Musca domestica

Abstract. The patterns of changes in cuticle weight, its chitin content and chitinase activity have been studied during postembryonic development of the housefly, Musca domestica L. During pupariation the larval cuticle loses weight. During the early part of this weight-loss the decline in chitin content parallels the overall change in cuticle weight. A simultaneous elevation in chitinase activity suggests that at this time the larval cuticle is being enzymatically degraded. Later weight loss may be due to sclerotization. No significant changes in cuticle weight or its chitin content occur in pharate cuticle until one day before eclosion. However, a peak of chitinase activity found at mid-late pupal stage suggests the timing of pupal cuticle breakdown.     

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.     

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.     

Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titre during the last larval instar of the tobacco hornworm, Manduca sexta

During the fifth larval instar of Manduca sexta the commitment of the epidermis to the synthesis of pupal cuticle is presumably affected by a small increase in ecdysteroid titre when juvenile hormone levels are minimal. Two sequential rounds of DNA synthesis without an intervening mitosis occur at about this time, resulting in polyploidy of the epidermis. There is a definite temporal correlation between the first peak of ecdysone and the second round of DNA synthesis and indirect evidence has been presented which suggests that this small increase in ecdysteroid titre actually initiates the second period of DNA synthesis. Further, it appears that large doses of ecdysteroids do not elicit the same response as smaller doses at a specific developmental stage, indicating that the different physiological effects of ecdysteroids (reprogramming and apolysis) may be dependent upon the relative concentration of the hormone. Following mitosis which takes place on approximately day 6 of the last instar, the epidermis undergoes apolysis and secretes pupal cuticle, expressing the commitment made 4.5 days earlier. These results support the ‘quantal mitosis’ theory of cytodifferentiation since the covert differentiative event occurs during a period of DNA synthesis and since mitosis precedes the expression of that event.     

蓖麻蚕个体发育中蜕皮甾类滴度的变化

用放射免疫分析法(RIA)测定了蓖麻蚕(Philosamia cynthia rieini)从卵期到成虫个体发育整个过程的蜕皮甾类(MH)水平.卵期在6天时有一个MH峰.一龄到四龄各龄均有一个MH峰,出现在停食前一天,导致幼虫蜕皮.五龄期有两个MF峰.第一个小峰出现在第三天,使进食的幼虫向预蛹转化;第二个高峰在上簇两天后,导致蛹表皮的形成.与其它鳞翅目昆虫一样,蛹期只有一个MH峰,发生在蛹期的前半段.成虫期血淋巴内MH含量很低.     

Hormonal regulation and patterning of the broad-complex in the epidermis and wing discs of the tobacco hornworm, Manduca sexta

Expression of Manduca Broad-Complex (BR-C) mRNA in the larval epidermis is under the dual control of ecdysone and juvenile hormone (JH). Immunocytochemistry with antibodies that recognize the core, Z2, and Z4 domains of Manduca BR-C proteins showed that BR-C appearance not only temporally correlates with pupal commitment of the epidermis on day 3 of the fifth (final) larval instar, but also occurs in a strict spatial pattern within the abdominal segment similar to that seen for the loss of sensitivity to JH. Levels of Z2 and Z4 BR-C proteins shift with Z2 predominating at pupal commitment and Z4 dominant during early pupal cuticle synthesis. Both induction of BR-C mRNA in the epidermis by 20-hydroxyecdysone (20E) and its suppression by JH were shown to be independent of new protein synthesis. For suppression JH must be present during the initial exposure to 20E. When JH was given 6 h after 20E, suppression was only seen in those regions that had not yet expressed BR-C. In the wing discs BR-C was first detected earlier 1.5 days after ecdysis, coincident with the pupal commitment of the wing. Our findings suggest that BR-C expression is one of the first molecular events underlying pupal commitment of both epidermis and wing discs.     

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.     

Some old concepts and new findings on hormonal control of insect morphogenesis

By means of the artificially induced heterochronic developmental deviations represented by local prothetelies and metathetelies it has been possible to investigate the individual developmental fates of ontogenetically different tissues, such as larval, pupal, and adult epidermal cells, in one and the same body and under the identical concentration of juvenile hormone (JH) in the haemolymph.In contrast to the widely accepted hormonal theories which claim that the kind of morphogenesis is determined by large, intermediate, and low titres of JH, the heterochronic character of the tissues never developed into a uniform population of homomorphic epidermal cells. Instead, in the presence of effective amounts of JH the heterochronic pattern has been fully preserved and carried on into the next developmental instar. Moreover, in the absence of the effective JH amounts the ontogenetically different tissues, such as larval and pupal epidermal cells, simultaneously undergo their respective morphogenetic developments, i.e. larval-pupal and pupal-adult morphogenesis in the same hormonal milieu. It is concluded that the selective factor in determination of the kind of morphogenetical changes is not an altered JH titre but the extant, previously attained degree of ontogenetic structural differentiation. It has been demonstrated that JH can temporarily and reversibly inhibit the morphogenetic progress at quite different ontogenetic levels but it cannot cause a ‘reversal of metamorphosis’ at any of these levels.Under specific experimental conditions the larval epidermal cells can undergo pupal and adult morphogenesis without secreting the pupal cuticle. However, the pupal morphogenetic interstage, whether with the cuticle or without the pupal cuticle, constitutes an obligatory developmental step. Further, it appears that an absence of JH may represent an important condition but not a real cause of insect metamorphosis, as presumed in some other hormonal concepts. Thus, chromosomal duplications or cellular divisions in the absence of JH have not committed the cells to morphogenesis unless provided by an additional stimulus of endogenous prothoracic gland hormone or exogenous ecdysterone. An important factor in understanding the hormonal control of insect morphogenesis is the critical timing of the respective morphogenetic steps. This corresponds closely with the duration of the pharate phases in insect development. Possible hormonal mechanisms concerned in the regulation of morphogenesis in endopterygote insects have been outlined.     

Isolation and developmental expression of two nuclear receptors, MHR4 and betaFTZ-F1, in the tobacco hornworm, Manduca sexta

The cDNAs for two members of the nuclear receptor superfamily were isolated from the tobacco hornworm, Manduca sexta. The deduced amino acid sequence of MHR4 shows 93-95% identity in the DNA-binding domain and the first portion of the hinge (D) region with the germ cell nuclear factor (GCNF)-related factors (GRFs) of the silkworm, Bombyx mori, and the mealworm, Tenebrio molitor, and with a genomic sequence from the fruit fly, Drosophila melanogaster. Northern blot hybridization showed that a 7.5 kb MHR4 mRNA appeared in Manduca abdominal epidermis just as the ecdysteroid titer began to decline during the larval molt, disappeared about 12 h later, then transiently reappeared shortly before larval ecdysis. During the pupal and adult molts, a similar pattern of expression was seen (the very end of the adult molt was not studied). At peak times of expression in the epidermis, MHR4 mRNA was also present in fat body and the central nervous system (CNS). The deduced amino acid sequence of Manduca FTZ-F1 is 100% and 96% identical to that of B. mori and Drosophila betaFTZ-F1, respectively, in the DNA-binding domain and the adjacent hinge region including the FTZ-F1 box. Northern blot analysis showed that the >9.5 kb betaFTZ-F1 mRNA appeared in Manduca epidermis during the decline of the ecdysteroid titer in the larval, pupal and adult molts as the first peak of MHR4 mRNA declined, then it disappeared in the larval and pupal molts before the second peak of MHR4 appeared. betaFTZ-F1 mRNA was also found in fat body and the CNS at the time of peak expression in the epidermis during the larval and pupal molts. Both MHR4 and betaFTZ-F1 mRNAs were found in the testis during the onset of spermatogenesis in the prepupal period.     

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.