Judith H. Willis

The cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages

Cuticles of Hyalophora cecropia were extracted with guanidine hydrochloride, and their cuticular proteins analyzed on isoelectric focusing gels. Extensive differences were found among the banding patterns from extracts of different cuticular regions within each metamorphic stage. The distribution of isoelectric points from each region correlated with the flexibility of the mature cuticle. Extracts from larval tubercles and head capsule were found to possess many of the same bands as hard pupal and adult cuticles. There were relatively few bands restricted to a single stage, and no band was found in all cuticles of a single stage. Examination of cuticles from pharate animals revealed that the differences found were not a result of differential sclerotization. A Wagner character-state analysis was used to compare the banding patterns. It was concluded that anatomical region is more a determinant of cuticular protein composition than metamorphic stage.

Rates and cycles of oxygen consumption during pupal diapause in Sarcophaga flesh flies.

The rate of oxygen consumption in non-diapausing flesh flies during the pupal and pharate adult stages follows a U-shaped curve with a minimum of 140 to 150 mm3/g. per hr at 25°C. In diapausing pupae the mean QO2 falls to 10 to 20 mm3/g. per hr. Continuous measurements with single diapausing pupae show that the QO2 is not constant; cycles of high oxygen consumption occur with a periodicity of several days. The cycle periodicity differs with species and temperature. The time between QO2 peaks in Sarcophaga crassipalpis is twice as great at 18°C as it is at 25°C. The cycles of high oxygen consumption are close together in early diapause; in mid-diapause the cycles become farther apart, and as the end of diapause is approached the cycles again become closer together. The time of diapause termination appears unrelated to the phase of the QO2 cycles. Injury produces an increase in the metabolic rate, but an acceleration of diapause termination was not observed.

The cuticular proteins of Tenebrio molitor: I. Electrophoretic banding patterns during postembryonic development☆

Abstract Buffer-soluble cuticular proteins of the abdomen of the yellow mealworm, Tenebrio molitor, were analyzed by SDS-polyacrylamide gel electrophoresis. Since the abdominal epidermis of Tenebrio persists throughout the insects life, these cuticular proteins reflect the secretory history of a continuous line of cells during its entire metamorphic developmental program. Twenty-two to thirty-eight bands were detected in extracts of larval cuticle, 11 to 35 in pupal cuticular extracts, and 30 to 41 in extracts from adults. No population polymorphism was apparent, nor was there any sexual dimorphism, in these cuticular proteins. At each metamorphic stage, the cuticular proteins formed a unique banding pattern. Bands unique to the larval and to the adult exocuticular extracts were observed. Extracts from cuticles of freshly ecdysed animals (exocuticle) differed from extracts from animals in which sclerotization and postecdysial (endocuticle) deposition had occurred, both in number of hands and in their molecular weight distributions. Some proteins became less soluble during sclerotization. The majority of the exocuticular bands from all three stages had molecular weights below 25,000; higher-molecular-weight proteins were extracted from postecdysial animals of each stage.

Analysis of the cuticular proteins of Hyalophora cecropia with two dimensional electrophoresis

The soluble cuticular proteins of defined anatomical regions from different metamorphic stages of the giant silkmoth, Hyalophora cecropia, were characterized by two dimensional electrophoresis. As urea concentrations in 2D gels were increased, some of the cuticular proteins from the larval dorsal abdomen decreased in mobility relative to the molecular weight standards. This decrease was also influenced by the pH and ionic strength of the resolving gel. Clustering of proteins into groups, whose members showed similar behavior under different electrophoretic conditions, was indicative of membership in multigene families. By such criteria, common families were found in cuticles with similar mechanical properties from different metamorphic stages, yet there was evidence that different members of a single family were independently regulated.

The Metamorphosis of Arthropodin

Some modern elementary biology texts inform their readers that arthropod cuticles are composed exclusively of the polysaccharide, chitin. But it has been known since 1823 that there is another major nitrogenous component which we now know to be protein. The first to measure the amount of protein in the cuticle was Gottfried Fraenkel. His publication with Rudall in 1940 alerted the scientific community to the fact that about 40% of the dry weight of fly larval cuticle is protein, much of which is soluble in boiling water. They also discovered that less than 10% of the dry weight remained soluble in boiling water after the puparium was tanned, but that protein could still be extracted with 5% KOH, leaving the chitin behind.

The cuticular proteins of Tenebrio molitor: II. Patterns of synthesis during postembryonic development☆

The program of synthesis for the soluble cuticular proteins of Tenebrio molitor was determined by following the incorporation of labeled leucine after a 4-hr pulse in vivo. Soluble proteins were extracted from labeled cuticles and separated on SDS-polyacrylamide slab gels; individual gel slices were counted. The synthetic patterns of larvae and pupae were similar to one another but distinct from the adult pattern. At each stage, the preecdysial pattern was unlike that of postecdysial animals. Distinct periods of synthesis were detected for different proteins. One protein was synthesized and deposited throughout cuticle formation in all three metamorphic stages. One group was synthesized only after ecdysis, while synthesis and secretion of other proteins were restricted to the preecdysial period. Some cuticular proteins never acquired detectable label.

Partial amino acid sequences of cuticular proteins from Hyalophora cecropia

Abstract The amino-terminal amino acid sequences for seven cuticular proteins from Hyalophora cecropia are reported. Proteins were purified by blotting two dimensional acrylamide gels onto acid-etched glass fiber filters, and the proteins were sequenced without further elution. The sequences of the serine-rich proteins from rigid cuticles revealed a new family of cuticular proteins, with features reminiscent of the amino-termini of certain vertebrate neurofilament proteins, members of the intermediate filament protein family which includes keratins. The proteins from flexible cuticles showed sequence similarity to proteins previously sequenced for Drosophila, Manduca and Sarcophaga . Proteins with identical electrophoretic mobility from two different metamorphic stages or from two anatomical regions within a single stage had identical amino-terminal sequences.

Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages

Abstract Post-translational modifications are a conspicuous feature of the proteins of vertebrate extracellular matrices such as cartilage. Yet this feature remains virtually unexplored with insect cuticle, a situation this paper begins to remedy. Cuticular proteins were extracted from cuticles of Hyalophora cecropia and separated on isoelectrofocusing and 2D gels. Periodic acid-Schiff reagent stained several proteins from flexible cuticles and a few proteins from rigid cuticles, indicating that some proteins were glycosylated. Elucidation of the specific nature of this glycosylation came from probing electrophoretically separated cuticular proteins blotted onto nitrocellulose with biotinylated lectins. Most major cuticular proteins did not react; minor cuticular proteins and molecules which do not stain with Coomassie blue were found to bind lectins specific for mannose and N -acetylgalactosamine. Limited binding was also detected with lectins specific for N -acetylglucosamine, galactose and fucose. No sialic acid was detected using either lectins or neuraminidase digestion. The amount of glycosylation was greatest in proteins extracted from flexible cuticles. Although several proteins stained with Alcian blue indicating presence of sulfation, 35 S which had been incorporated at low levels in cuticular proteins corresponded to [ 35 S]methionine. No indication of the presence of mammalian-type glycosaminoglycans in insect cuticles was obtained after treatment with chondroitinase or nitrous acid. The functional significance of the modifications detected remains unknown. No evidence for phosphorylated proteins or lipoproteins was found.

Control of cuticle formation by wing imaginal discs in vitro.

Abstract Wing discs from late final-instar Ephestia larvae form only pupal cuticle when immediately implanted into pupae which subsequently undergo metamorphosis. However, either pupal or adult structures are made in vitro depending on (1) the ecdysterone dose and/or (2) disc cell proliferation. Continuous culture in ecdysterone (0.5–5.0 μg/ml) results in the appearance of transparent cuticle. On the basis of several criteria, this untanned cuticle is postulated to be scaleless adult cuticle. Discs pulsed with 0.5 μg/ml ecdysterone for 48–120 hr, or with 5.0 μg/ml for 24 hr, formed tanned cuticle. Lower doses of ecdysterone (i.e., 0.5 μg/ml for 24 hr or continuous exposure to 0.05 μg/ml) trigger adult scale formation. Enhancement of [ 3 H]thymidine incorporation by these latter doses suggests the occurrence of disc cell divisions and polyploidization. The choice between pupal and adult pathways by wing discs of this age can be controlled exclusively by ecdysterone; juvenile hormone need not be involved in vitro .

Molecular polymorphism of ciliary proteins from different species of the ciliate Tetrahymena.

Ciliary proteins from five different Tetrahymena species were analyzed by means of SDS-polyacrylamide gel electrophoresis. Of at least 32 different polypeptides, only 2 were found to have identical molecular weights in all species. In any comparison of 2 species, a maximum of 60% and at least 20% of the proteins had indistinguishable molecular weights, depending on the combination examined. Extensive codominance of ciliary proteins was found in a F1 hybrid. No interspecific cross-reactions occurred in double-diffusion tests involving the cilia from different species and antisera produced against them in rabbits.