Cuticular Proteins in Insects and Crustaceans

Comparisons between crustacean and insect cuticles are hamperedby the paucity of cuticular protein sequences for the former.Sufficient complete sequences are available for insect cuticularproteins to allow recognition of conserved motifs and relationshipsamong proteins that reflect the type of cuticle from which theyhave been extracted. All five sequences from an arachnid andtwo of 14 from crustaceans have a motif found in the largestgroup of insect cuticular proteins. Numerous insights have beengained from studying insect cuticular proteins and their genes.These insights have been summarized in hopes of encouraginginterest in building on the foundations laid by Dorothy Skinnerwith the exoskeleton of Gecarcinus.     

Ascaris suum: partial isolation and characterization of hypodermis from the adult female

A method was developed to remove the muscle from body wall strips of adult female Ascaris suum resulting in a hypodermis cuticle preparation. Optimum treatment for obtaining the hypodermis cuticle was a 15 min incubation with trypsin (2.0 mg/ml) at room temperature, followed by mechanical removal of the muscle. The hypodermis cuticle prepared in this manner incorporated radiolabeled amino acids into cuticular and hypodermal proteins; incorporation was inhibited by protein synthesis inhibitors. Characterization of the hypodermal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the hypodermis apparently contains proteins that differ from those of the cuticle and that the hypodermis of adult A. suum appears to lack cuticle protein precursors. This result will now allow detailed biochemical and physiological investigations of the hypodermis, a tissue which is critical for cuticle synthesis.     

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.     

Mass spectrometrical analysis of cuticular proteins from the wing of Hebemoia glaucippe (Linnaeus, 1758) (Lepidoptera: Pieridae)

Although several insect cuticular genes and proteins are annotated and an arthropod cuticular database is available, mass spectrometrical data on cuticular proteins and their post-translational modifications are limited. Wings from Hebemoia glaucippe were analyzed by scanning electron microscopy or homogenized, proteins were extracted and run on 2DE. In-gel digestion was carried out by using trypsin, chymotrypsin and Asp-N and subsequently the resulting peptides and post-translational modifications were identified by ion trap tandem mass spectrometry (nano-LC-ESI-MS/MS; HCT). A complex wing skeleton and the cuticle of H. glaucippe were demonstrated. Cuticle protein 18.6, isoform A, pupal cuticle protein, cuticular protein CPR59A and two putative proteins, putative cuticular protein B2DBJ and putative cuticle protein CPG31 with two expression forms were identified. Two phosphorylation sites on the same peptide, T213 and S214, were identified on putative cuticle protein CPG31, quinone formation was observed at Y76 on cuticular protein CPR59A probably indicating the presence of post-translational modifications. The results may be relevant for the interpretation of mechanoelastic and physical properties of these proteins. Along with the extraordinary architecture the proteinaceous matrix is probably representing or allowing the unusual aerodynamic function of the butterfly wing. Moreover, the results may be important for mechanisms of insecticide and drought resistance.     

借助斑马鱼EST数据库从鲤鱼微卫星序列中寻找蛋白编码基因(英文）

应用 in silico的方法,利用Blastn 和Blastx 搜索引擎,将鲤鱼微卫星序列与GenBank数据库进行同源序列比对。利用Blastn,将侧翼序列长度>50 bp的875个鲤鱼微卫星序列与斑马鱼的EST数据库首先进行比对,结果找到了121个同源序列。随后采用Blastx搜索蛋白质数据库,有94个微卫星位点存在同源蛋白。除了33个假定和3个未知蛋白外,剩余的58个微卫星位点被成功地进行了功能注释,而且其中的7个位点已经定位在了鲤鱼连锁图谱上。另外,通过PCR-SSCP的方法,将两个与鲤鱼微卫星侧翼序列相匹配的斑马鱼EST序列开发成鲤鱼的STS标记,并将其中的一个标记HLJZe33定位到鲤鱼连锁图谱上。以上研究结果表明,通过比较基因组研究,模式生物斑马鱼的很多遗传和基因组资源都可以被利用到鲤鱼的基因组研究中。     

Studies on the Ultrastructure and Histochemistry of Plant Cuticles: The Cuticular Membrane of Agave americana L. in situ

The outer epidermal wall of Agave americana leaves was examinedin order to gain more information about the location and chemicalconstitution of the structural components. In middle aged leavesthe wall comprised six layers which were designated epicuticularwax, cuticle proper, exterior and interior cuticular layer,exterior and interior cellin wall. A lamellated structure, consistingof a series of electron translucent lamellae of uniform thicknessalternating with opaque ones of variable thickness, was observedin the thin cuticle proper on the outside of the cuticular membrane,even without heavy metal treatment. The cuticular layers underneathformed the bulk of the cuticular membrane and they also hadtwo components, an amorphous matrix permeated by a reticulumof fibrillae. Cutin, detected with osmium and with iodine/iodine-sulphuricacid–silver proteinate, was a major component of the opaquelamellae of the cuticle proper and the matrix of the cuticularlayer. Carbohydrates were absent from the cuticle proper butwere detected specifically in the fibrillae of the cuticularlayer and in the cellin wall. Pectic material seemed to be presenton both sides of the junction between cuticular membrane andcellin wall, but no discrete zone corresponding to light microscopicalobservations was detected in the electron microscope. Althoughthe lucent lamellae of the cuticle proper were tentatively ascribedto wax there was no structural or ultrahistochemical evidencefor the wax component of the cuticular layer. The various ultrahistochemicalreactions are discussed in relation to the known chemical compositionof the membrane. Agave americana L., epidermis wall, cuticular membrane, cuticle proper, cuticular layer, ultrahistochemistry, wax     

Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.