Comparison of rates of penetration through insect cuticle of amphiphylic analogs of insect pyrokinin neuropeptides

Rates of penetration through the cuticle of amphiphylic analogs, synthesized by addition of 6-phenylhexanoic acid or 9-fluoreneacetic acid or 1-pyrenebutyric acid to the amino terminus of the pentapeptide Phe-Thr-Pro-Arg-Leu-amide, were assessed by quantitative analysis using reversed phase liquid chromatography. The analogs effectively penetrated the cuticle of both the adult American cockroach and tobacco budworm moth. However, the amounts of analogs that penetrated the cuticle of the cockroach were significantly lower and the rates of penetration were slower than for moth cuticle. Penetration of the analogs through the cuticle was dependent upon the size of the lipidic attachment to the pentapeptide. The 6-phenylhexanoic acid analog penetrated most rapidly followed by the 9-fluoreneacetic acid analog and the 1-pyrenebutyric acid analog penetrated slowest. All of the analogs exhibited an initial rapid period of penetration lasting 2-3 h followed by the establishment of a steady slow release state which lasted between 9-24 h and was dependent upon both the size and surface area of the aromatic lipidic portion of the analog and species of insect to which the analog was applied. The results confirmed the hypothesis that the insect cuticle could be employed as a slow release device for delivery of analogs of insect neuropeptides.     

Hydration and tanning in insect cuticle

The water content of larval and puparial cuticle of Calliphora vomitoria has been measured under differing conditions using nuclear magnetic resonance, differential scanning calorimetry and simple gravimetry. On average, 2.5 molecules of water are associated with each amino acid side chain. This water is not displaced by tanning, even though tanning reduces the overall content of freezable water, suggesting that tanning agents do not interact with polar groups on the protein but increase overall hydrophobicity. This refutes normally accepted concepts of tanning by covalent cross linking. Additionally, covalent cross linking cannot account for the reduction in swellability of the cuticle on tanning.     

Design and mechanical properties of insect cuticle

Since nearly all adult insects fly, the cuticle has to provide a very efficient and lightweight skeleton. Information is available about the mechanical properties of cuticle-Young's modulus of resilin is about 1 MPa, of soft cuticles about 1 kPa to 50 MPa, of sclerotised cuticles 1-20 GPa; Vicker's Hardness of sclerotised cuticle ranges between 25 and 80 kgf mm(-2); density is 1-1.3 kg m(-3)-and one of its components, chitin nanofibres, the Young's modulus of which is more than 150 GPa. Experiments based on fracture mechanics have not been performed although the layered structure probably provides some toughening. The structural performance of wings and legs has been measured, but our understanding of the importance of buckling is lacking: it can stiffen the structure (by elastic postbuckling in wings, for example) or be a failure mode. We know nothing of fatigue properties (yet, for instance, the insect wing must undergo millions of cycles, flexing or buckling on each cycle). The remarkable mechanical performance and efficiency of cuticle can be analysed and compared with those of other materials using material property charts and material indices. Presented in this paper are four: Young's modulus-density (stiffness per unit weight), specific Young's modulus-specific strength (elastic hinges, elastic energy storage per unit weight), toughness-Young's modulus (fracture resistance under various loading conditions), and hardness (wear resistance). In conjunction with a structural analysis of cuticle these charts help to understand the relevance of microstructure (fibre orientation effects in tendons, joints and sense organs, for example) and shape (including surface structure) of this fibrous composite for a given function. With modern techniques for analysis of structure and material, and emphasis on nanocomposites and self-assembly, insect cuticle should be the archetype for composites at all levels of scale.     

A new form of insect cuticle

A hitherto unnoticed, harder form of cuticle, which occurs on the mandibles of the Australian plague locust, Chortoicetes terminifera , is described     

An assembly zone antigen of the insect cuticle

The assembly zone is a morphologically distinct region in the insect integument that lies between the epidermis and its principal secretory product, the lamellate cuticle. Despite its central location in the process of cuticle formation, little is known about its structure or function. Using various antisera we have shown that in Drosophila melanogaster larvae and pupae the assembly zone is antigenically distinct from the overlying lamellate cuticle. This observation suggests that this region does not contain lamellae in the process of assembling but rather is a stable and permeable matrix through which lamellar components travel in the process of cuticle formation. Curiously an antigen present in the assembly zone was also contained in the moulting gel, indicating a heretofore unsuspected chemical relationship between these two materials.     

Sclerotin and lipid in the waterproofing of the insect cuticle

In all the cuticles studied waterproofing is effected by extracuticular material, a mixture of sclerotin precursors and lipids, exuded from the tubular filaments of the pore canals. In Rhodnius larval abdomen it is a layer of thickness similar to the outer epicuticle, believed to be composed of 'sclerotin' and wax, in Schistocerca larval sternal cuticle and in Carausius sternal cuticle it is similar. In Tenebrio adult sternal cuticle of the abdomen, in both the extracuticular exudation and the contents of the distal endings of the tubular filaments, the wax component is obscured by hard 'sclerotin'. In Manduca larva a very thin layer of 'sclerotin' and wax is covered by an irregular wax layer, average 0.75 micron, twice the thickness of the inner epicuticle. In Periplaneta and Blattella the abdominal cuticle is covered by a soft waxy layer, often about 1 micron thick, which is mixed with argentaffin material. Below this is a very thin waterproof layer of wax and 'sclerotin' continuous with the contents of the tubular filaments, which is readily removed by adsorptive dusts. In Apis adult abdominal terga free wax plus sclerotin precursors form a thin layer which is known to be removed by adsorptive dusts. In Calliphora larva there is a very thin layer of the usual mixed wax and sclerotin and below this a thick (0.5 micron) layer, lipid staining and strongly osmiophil, likewise extracuticular and exuded from the epicuticular channels. This material (which is often called 'outer epicuticle') has the same staining and resistance properties as the true outer epicuticle on which it rests. In the abdomen of Calliphora adult the waterproofing wax-sclerotin mixture forms a thin layer over the entire cuticle including the surface of the microtrichia. There is also a thin detachable layer of free wax on the surface.     

Chlorinated tyrosine derivatives in insect cuticle

A method for quantitative measurement of 3-monochlorotyrosine and 3,5-dichlorotyrosine in insect cuticles is described, and it is used for determination of their distribution in various cuticular regions in nymphs and adults of the desert locust, Schistocerca gregaria. The two chlorinated tyrosine derivatives were present in all analyzed regions in mature adult locusts, the highest concentrations were found in the sclerotized cuticle of femur and tibia, but significant amounts were also present in the unsclerotized arthrodial membranes. Small amounts of the two amino acids were obtained from pharate, not-yet sclerotized cuticle of adult femur and tibia, the amounts increased rapidly during the first 24 h after ecdysis and more slowly during the next two weeks. Control analyses using stable isotope dilution mass spectrometry have confirmed that the chlorinated tyrosines are not artifacts formed during sample hydrolysis. Mono- and dichlorotyrosine are also present in cuticular samples from other insect species, such as the beetle, Tenebrio molitor, the moth Hyalophora cecropia, the cockroach Blaberus craniifer, and the bug Rhodnius prolixus, but not in the sclerotized puparial cuticle of the blowfly, Calliphora vicina, or in sclerotized ootheca from the cockroach, Periplaneta americana. Cuticular sclerotization and formation of chlorotyrosines occur simultaneously in locust legs; sclerotized cuticles tend to have a higher content of chlorotyrosines than unsclerotized cuticles, but it is concluded that the chlorotyrosines are not just a by-product from the sclerotization process.     

Tyrosine metabolism for insect cuticle tanning

Insects have become one of the most successful animal groups in diversity and numbers through the development of a multifunctional exoskeleton and skin, which must be shed periodically in order for them to grow and develop into adults. The evolutionary choice of certain structural materials for the assembly and stabilization of a cuticle with remarkable mechanical and chemical properties has allowed insects to invade terrestrial environments and to evolve flight mechanics for dispersion relatively early in geological history. Diphenolic compounds derived from tyrosine play a central role in sclerotization or tanning of the new cuticle. The phenolic amino acid is stored during larval feeding, and it is mobilized for the production of both structural proteins and diphenolic tanning precursors that are transported into the cuticle. The latter compounds permeate the cuticle and serve as precursors for quinonoid derivatives that both sclerotize and pigment the exoskeleton. This report focuses on how tyrosine and derived diphenolic structures are stored as inactive molecules in preecdysial stages, and how they are released and metabolized to tanning chemicals that stabilize the new cuticle.     

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.     

Cell culture techniques for studying insect cuticle

Evidence that biosynthetic pathways critical to the formation of insect cuticle are retained in continuous insect cell lines opens new possibilities for research on the cuticle system. Recent findings indicate that chitin, molting hormone, and catecholamines are all produced by a vesicle cell line derived from embryos of the cockroach Blattella germanica. The chitin that is formed by this cell line is particulate and does not show the characteristic featherlike crystalline structure found in mature cuticle. The molting hormone is produced as ecdysone and is released into the culture medium. The addition of 20-hydroxyecdysone to the cultures increases the production of chitin fourfold. These responses are similar to those found in insect organ cultures.     

Structural lipids in the insect cuticle and the function of the oenocytes

Stabilized lipid (cuticulin), combined with protein, serves to stiffen the cuticle before sclerotization occurs. It is always present in large amounts in exocuticle that will later be tanned to form sclerotin. It is plentiful also in the untanned mesocuticle, including the tracheal taenidia, as well as pore canals, egg shell and spermatophore sheath. Stabilized lipid present in small amounts between the laminae of the endocuticle may perhaps be concerned in reversible stiffening and plasticisation. The oenocytes appear to be the source of the precursors for cuticulin formation in both larva and reproducing adult.     

Degradation of insect cuticle by Paecilomyces farinosus proteases

The entomopathogenic fungus Paecilomyces farinosus showed proteolytic activity in both solid and semi-liquid culture with gelatin as sole N and C source. Semi-liquid cultures were used to characterise proteases. Zymography of crude culture filtrates showed several bands of gelatin degradation in electrophoresis gels. Gel filtration chromatography of these filtrates revealed two peaks of proteolytic activity. Ion-exchange absorption eliminated gelatin from culture filtrates while retaining activity and was used to semipurify P. farinosus proteases. Semipurified culture filtrates had basic pH (8.5 approx.) optimum for proteolytic activity. Treatment of these filtrates with effectors revealed that P. farinosus proteases are serine proteases containing sulphydryl groups. Isoelectrofocusing combined with zymography revealed the presence of several active basic isoforms. Larvae of the lepidopteran Galleria mellonella showed cuticle damage and protein release 1h after incubation with semipurified extracts of P. farinosus. These results indicate that proteolytic enzymes could be involved in insect host penetration by P. farinosus.     

Water balance across the cuticle of a soil insect.

1. The water regime in soil commonly approaches equilibrium of water potential with the insects living there. 2. Even under these conditions, non-equilibrium processes have a significant effect on water movement through the cuticle of soil insects. 3. Measurements of water potential on either side of the cuticle of Costelytra zealandica larvae showed that equilibrium is not reached while the insect is alive. There is an active outward flow of water by thermoosmosis associated with the flow of heat from the insect.     

The nature of driving forces for passive water transport through arthropod cuticle

1. 1.|We demonstrate using thermodynamic arguments that water loss through arthropod epicuticle is well described by a linear law relating water flux to transmembrane vapour pressure drop.

2. 2.|The relationship applies equally to systems where the liquid or vapour exist on either side of a membrane.

3. 3.|An earlier claim by some workers that water diffusion through arthropod epicuticle is proportional to chemical potential drop across the membrane is found to be theoretically unjustified.

4. 4.|Recent measurements with Periplaneta cuticle support the prediction that flux at a given temperature is proportional to the difference in vapour pressure.