Functional Analysis of Insect Molting Fluid Proteins on the Protection and Regulation of Ecdysis

Molting fluid accumulates between the old and new cuticles during periodical ecdysis in Ecdysozoa. Natural defects in insect ecdysis are frequently associated with melanization (an immunity response) occurring primarily in molting fluids, suggesting that molting fluid may impact immunity as well as affect ecdysis. To address this hypothesis, proteomic analysis of molting fluids from Bombyx mori during three different types of ecdysis was performed. Many proteins were newly identified, including immunity-related proteins, in each molting fluid. Molting fluids inhibited the growth of bacteria in vitro. The entomopathogenic fungi Beauveria bassiana, which can escape immune responses in feeding larvae, is quickly recognized by larvae during ecdysis, followed by melanization in molting fluid and old cuticle. Fungal conidia germination was delayed, and no hyphae were detected in the hemocoels of pharate instar insects. Molting fluids protect the delicate pharate instar insects with extremely thin cuticles against microorganisms. To explore the function of molting fluids in ecdysis regulation, based on protein similarity, 32 genes were selected for analysis in ecdysis regulation through RNAi in Tribolium castaneum, a model commonly used to study integument development because RNAi is difficult to achieve in B. mori. We identified 24 molting proteins that affected ecdysis after knockdown, with different physiological functions, including old cuticle protein recycling, molting fluid pressure balance, detoxification, and signal detection and transfer of molting fluids. We report that insects secrete molting fluid for protection and regulation of ecdysis, which indicates a way to develop new pesticides through interrupting insect ecdysis in the future.     

Molting fluid enzymes of the tobacco hornworm,Manduca sexta: Timing of proteolytic and chitinolytic activity in relation to pre-ecdysial development

Developmental profiles for a number of molting fluid (MF) enzyme activities were established and related to the progress of pupal cuticle degradation during the four days that precede the eclosion of adult tobacco hornworms. Cuticle degrading activity, molting fluid protease 1 (MFP-1), and molting fluid protease 2 (MFP-2) all increased in activity at the time that loss of material from the old cuticle occurred. In contrast, chitinase and β-acetylglucosaminidase activities did not parallel weight loss from the old cuticle. These results are consistent with the hypothesis that proteolytic activity is a prerequisite for the action of chitinase on cuticle chitin. © 1993 Wiley-Liss, Inc.     

In vivo biosynthesis of a stage-specific cuticle glycoprotein during early metamorphosis of the medfly Ceratitis capitata

Cuticle proteins of an insect pest, the Medfly Ceratitis capitata, were resolved in polyacrylamide gels and partially characterized. The pupal cuticle was found to be different from cuticles of other insects since more than 80% w/w of the protein is a single mannose-containing polypeptide (PCG-100). The temporally-regulated in vivo biosynthesis and deposition of cuticle proteins was studied by microinjection of [35S]methionine followed by hand dissection of pupal cuticles. The major pupal glycoprotein, PCG-100, is cuticle- and stage-specific and was the earliest to be labeled and deposited. Its synthesis was maximal at around 46 hours after pupariation and then it decreased. The deposited PCG-100 and other minor pupal cuticle proteins become non-extractable at the end of the instar (7 days after pupariation) probably by sclerotization phenomena. These results provide insight into the temporal control of gene expression programs involved in cuticle deposition during medfly metamorphosis.     

The BmChi-h gene, a bacterial-type chitinase gene of Bombyx mori, encodes a functional exochitinase that plays a role in the chitin degradation during the molting process

The silkworm, Bombyx mori, has been recently demonstrated to contain a bacterial-type chitinase gene (BmChi-h) in addition to a well-characterized endochitinase gene (BmChitinase). The deduced amino acid sequence of BmChi-h showed extensive structural similarities with chitinases from bacteria such as Serratia marcescens chiA and baculoviruses (v-CHIA). Bacterial-type chitinase genes have not been found from any eukaryotes and viruses except for lepidopteran insects and lepidopteran baculoviruses. Thus, it was suggested that BmChi-h may be derived from a bacterial or baculovirus chitinase gene via horizontal gene transfer. In this report, we investigated the biological function of BmChi-h. Our enzymological study indicated that a chitinase encoded by BmChi-h has exo-type substrate preference, which is the same as S. marcescens chiA and v-CHIA, and different from BmChitinase, which has endo-type substrate preference. An immunohistochemical study revealed that BmChi-h localizes in the chitin-containing tissues during the molting stages, indicating that it plays a role in chitin degradation during molting. These results suggest that BmChi-h (exochitinase) and BmChitinase (endochitinase) may catalyze a native chitin by a concerted mechanism. Cloning and comparison of BmChi-h orthologues revealed that bacterial-type chitinase genes are highly conserved among lepidopteran insects, suggesting that the utilization of a bacterial-type chitinase during the molting process may be a general feature of lepidopteran 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.     

Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum

Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.     

Localization of molting chitinase in insect cuticle

Chitinase activity in molting larvae of Manduca sexta is localized in old cuticle; it is not quantitatively extracted during homogenization, has good activity at the pH of molting fluid, and preferentially utilizes endogenous cuticle chitin as substrate. It is concluded that cuticle chitinase is the physiologically active molting enzyme in Manduca.     

The class A chitin synthase gene of Spodoptera exigua: molecular cloning and expression patterns

Chitin synthase (CHS) is an important enzymatic component required for chitin formation in the cuticles and cuticular linings of other tissues. In the present study, a new CHS gene was characterized from the beet army worm Spodoptera exigua (Hübner) (Se). Homologous alignment and phylogenetic analysis of S. exigua CHS (SeCHS) with other related proteins suggest that SeCHS belongs to the class A CHS family (SeCHSA). Northern blot analysis revealed that SeCHSA is transcribed preferentially in the cuticle and tracheae. Further investigation indicated that SeCHSA mRNA is highly expressed in the early and late stages of each larval instar, and consistently expressed in high level during the pupal stage. Using antibody specific for CHS, SeCHS was further localized in the underlying epidermal cells of the integument and tracheal cells, but not in the fat body or Malpighian tubules. These data suggest that SeCHS plays an important role in cuticle formation and development of S. exigua.     

The mechanism of sensory transduction in a mechanoreceptor. Functional stages in campaniform sensilla during the molting cycle

This paper describes the ultrastructural modifications that cockroach campaniform sensilla undergo at three major stages in the molting cycle and finds that the sensilla are physiological functional at all developmental stages leading to ecdysis. Late stage animals on the verge of ecdysis have two completely separate cuticles. The campaniform sensillum sends a 220-mum extension of the sensory process through a hole in its cap in the new (inner) cuticle across a fluid-filled molting space to its functional insertion in the cap in the old (outer) cuticle. Mechanical stimulation of the old cap excites the sensillum. The ultrastructural geometry of late stage sensilla, coupled with the observation they are physiolgically functional, supports the hypotheses (a) that sensory transduction occurs at the tip of the sensory process, and (b) that cap identation causes the cap cuticle to pinch the tip of the sensory process, thereby stimulating the sensillum.     

昆虫几丁质酶基因的分子特性概述

昆虫几丁质酶是分解昆虫体壁和中肠围食膜几丁质的重要酶类。已从烟草天蛾、家蚕等多种昆虫中分离到几丁质酶的cDNA和DNA序列。昆虫几丁质酶基因有着相似的分子特性,这些特性可为构建杀虫工程菌及转几丁质酶基因植物奠定基础。作者结合自己在该领域的工作,着重就昆虫几丁质酶基因结构特点,基因的拷贝数,基因在体内的时空表达以及异源表达及活性测定等多个方面的研究方法和研究进展进行了较为全面地介绍。