Mary Goldberg

Studies on the hardening and darkening of insect cuticles

Abstract The protein components of hardened and unhardened insect cuticles have been analysed for their amino acid composition. These analyses show that the puparia of muscoid flies and the ootheca of the cockroach, which have been extensively studied as examples of sclerotization, differ markedly from insects in which hardening of the cuticle occurs after an ecdysis. β-Alanine and cyst(e)ine take part in the hardening of cuticles. Melanins occur in darkly coloured cuticles and the melanins may be either of the indole type or a mixture of indole and catechol types. During the formation of the puparium in Lucilia cuprina changes take place in the composition of the larval cuticle which are similar to those which occur during insect ecdysis. Approximately 20 per cent of both the protein and chitin of the larval cuticle were either absorbed during puparium formation and pupation or were converted to moulting membranes, or both. About the same amount of tanning material was added to the cuticle.

The o-diphenoloxidases of fly larvae

Abstract A water-soluble o -diphenoloxidase occurs in the cuticles of the larvae of six species of flies belonging to three genera. The enzymic activity of the cuticular extract increases rapidly as the larvae approach the time for puparium formation but falls to zero just when puparium formation begins. As indicated by the activities on different substrates and by the K m and V values the enzymes from the different species of flies are very similar. The preferred substrates are dopamine and 4-methylcatechol. The larval haemolymph of Lucilia cuprina contains a pro- o -diphenoloxidase which, when activated by the cuticular extract, oxidizes a wide range of phenols but is most active on dopamine and 4-methyl catechol. The activator in the cuticular extract is stable when heated to 80°C for 5 min. When activated by sodium dodecylsulphate the proenzyme only oxidizes dopamine and 4-methylcatechol, it is without monophenoloxidase activity. In contrast to the cuticular enzyme the proenzyme is present in the haemolymph of larvae just as they are forming their puparia. The role of o -diphenoloxidases in fly larvae is discussed.

Comparative study of some expanding arthropod cuticles: The relation between composition, structure and function

Abstract The abdominal cuticles of the adult female ticks Argas (Persicargas) robertsi and Boophilus microplus, sp der Badumna insignis, tsetse fly Glossina morsitans morsitans, sheep ked Melophagus ovinus and locust Locusta migratoria migratorioides and of the fifth-instar larva of the bug Rhodnius prolixus stretch greatly, some of them quite rapidly, when they are feeding, laying eggs or carrying a developing larva or egg mass. During this expansion the epicuticle, which is convoluted, unfolds and the underlying endocuticle stretches. There is an increase in the volume of each of the cuticles on expansion. The fine structures of those cuticles which go through cycles of expansion and contraction do not become disrupted. Cuticles with acidic proteins have much higher chitin contents than those with basic proteins. Plasticization, i.e. breaking of intermolecular non-covalent bonds, precedes rapid expansion of cuticles but is unnecessary for slow expansion. The compositions of the cuticles and the properties of the proteins are discussed in relation to the expansions which take place in the cuticles.

Molecular crosslinks in cuticles

Abstract The number of acid-stable covalent crosslinks in sclerotized cuticles is small but the potential for non-covalent crosslinks is high. Calliphora vicina larval cuticular protein binds up to at least 125% of its weight of tannin. In fly puparia tanning material, in addition to forming covalent crosslinks, is non-covalently bound. 1,2-Diaminoethane extracted more protein from cuticles than other reagents and this result, with further observations, requires that theories for sclerotization of locust cuticle be reappraised. Non-sclerotized cuticles contain a glycoprotein composed of approximately 3 parts chitin and 2 parts protein by weight. In Oryctes rhinoceros the amino acid compositions of pharate adult cuticle and adult exocuticle differ, those of sclerotized pronotum and ventral abdominal cuticle are similar and that of pronotal endocuticle is typical of sclerotized cuticles and not of soft cuticles.

Comparative chemistry of arthropod cuticular proteins

Abstract 1. 1. Differences in the amino acid compositions of the proteins in soft arthropod cuticles are explained by polymorphisms due to mutational changes in the nucleotide sequences which synthesize the amino acids. The protein components of these cuticles are essentially the same. 2. 2. In sclerotized cuticles the alanine, glycine or leucine content of the protein is increased and that of the hydrophilic amino acids decreased. 3. 3. Extensible cuticles have protein components similar to one another. 4. 4. The findings are discussed in relation to the evolution of the Arthropoda.

A method for determinations of microgram amounts of chitin in arthropod cuticles

Abstract A method is described for determining microgram amounts of chitin in arthropod cuticles. The method, adapted from one described for assaying plant material for fungal content, is based on estimating soluble chitosan derivatives and is not affected by the presence of nonchitinous cuticular components. There is good agreement between the results given by the micromethod and those given by a semimicro method based on material insoluble in hot, dilute alkali. As a qualitative test for chitin the limit for the method is

Some conformational studies of larval cuticular protein from Calliphora vicina

Protein extracted from lipid-free late instar larval cuticles of Calliphora vicina consists of a mixture of similar proteins which, when in solution (phosphate buffer pH 6 or 0.05 M NaCl), exist largely in a disordered conformation as shown by infrared (IR) and optical rotary dispersion (ORD) data. ORD showed the presence of about 13% α-helical and 20% β-structures. IR absorption showed the proteins, when in the solid state, to be largely in the β-conformation. Associations take place between some of the proteins, as shown by electrophoresis and light scattering, but tyrosyl-, tryptophanyl- and phenylalanyl-residues are not involved in such associations.

A study of a melanic mutant of the blowfly Lucilia cuprina

Abstract The melanic mutant of the blowfly Lucilia cuprina (Wied.) is homozygous for three recessive characters, yellow eyes, rusty body, and a black puparium whereas the normal wild strain has red eyes, black body, and a chestnut-brown puparium. In the mutant the three pigment characters appear to be independent. The puparium of the mutant has a layer of melanin just inside the epicuticular layer. A study of the cuticular o -diphenoloxidases, the dopa-decarboxylases and the utilization of radioactive phenolic compounds shows the same process of sclerotization to occur in both strains at the time of puparium formation. The larval haemolymph of the mutant contains a prophenoloxidase which, when activated by an extract of the cuticle, has activity on tyrosine and dopa. This activity increases greatly as the larva approaches the time for puparium (and melanin) formation and is presumably responsible for the formation of melanin. In the mutant, sclerotization and melanization are independent processes. The same prophenoloxidase appears to be in the normal wild strain larval haemolymph but the necessary activator is not in the cuticle; the activator in the cuticle causes the pro-enzyme to become highly active only on substrates such as dopamine and 4-methylcatechol. Cystine is present in hydrolysates of puparia of both strains and, at least in the normal wild strain, accounts for all the sulphur present.

The non-covalent binding of two insect cuticular proteins by a chitin

Abstract The amounts of the larval cuticular proteins bound non-covalently by Scylla serrata chitin depend on both the pH and the ionic strength of the solution. Binding is greatest at the isoelectric point of the protein and at low salt concentrations. The binding of some proteins is partly reversible and complete removal of bound protein requires solvents able to break hydrogen bonds. The cuticular proteins are heterogeneous and some minor components of low molecular weight are not bound by the chitin. Those components which are bound have similar affinities for the chitin. Although crab chitin can bind 2.4 times its weight of Calliphora cuticular protein the binding of proteins in insect cuticle is less as only the outer surfaces of the chitin microfibrils are accessible to protein and the non-covalent binding is further limited by covalently bound protein. It is likely that chitin in the cuticle is saturated with protein. Chemical modifications of the proteins lead to the conclusion that the non-covalent binding between the chitin and the proteins is non-specific.

Composition of the oothecae of three Orthoptera

Abstract Oothecae of Periplaneta americana contained 6·5 per cent calcium and this was the only major cation present. Only 35 per cent of the calcium was present as calcium oxalate and in view of the absence of other anions it is suggested that the remainder may be present as a calcium-protein complex. Oothecae of Blattella germanica and Orthodera ministralis, although containing much less calcium, gave similar results. Chitin is absent from the oothecae of these three Orthoptera.