Locust Cuticle Research Articles

Effect of sample treatment on the elastic modulus of locust cuticle obtained by nanoindentation.

Cuticle is one of the most abundant, but least studied, biological composites. As a result, it has contributed very little to the field of biomimetics. An important step to overcome this problem is to study cuticle biomechanics by means of accurate mechanical measurements. However, due to many reasons, mechanical testing on fresh cuticle specimens is not always possible. Hence, researchers often use stored specimens to measure properties of arthropod cuticle. Our knowledge about the influence of different treatment methods on cuticle properties is currently very limited. In this study, we investigated the effect of freezing, desiccation, and rehydration on the elastic modulus of the hind tibial cuticle of locusts obtained by nanoindentation. We found that all the mentioned treatments significantly influence cuticle properties. This is in contrast to previous reports suggesting that freezing did not significantly influence the elastic modulus of native cuticle specimens tested in bending. In the light of our data, we suggest that changes of the elastic modulus of cuticle are not solely due to changes of the water content. Our results provide a platform for more accurate measurements of cuticle properties.

Biomechanical strategies to reach a compromise between stiffness and flexibility in hind femora of desert locusts

Insect cuticle can reach a wide range of material properties, which is thought to be the result of adaptations to applied mechanical stresses. Biomechanical mechanisms behind these property variations remain largely unknown. To fill this gap, here we performed a comprehensive study by simultaneous investigation of the microstructure, sclerotization and the elasticity modulus of the specialized cuticle of the femora of desert locusts. We hypothesized that, considering their different roles in jumping, the femora of fore-, mid- and hind legs should be equipped with cuticles that have different mechanical properties. Surprisingly, our results showed that the hind femur, which typically bears higher stresses, has a lower elasticity modulus than the fore and mid femora in the longitudinal direction. This is likely due to the lower sclerotization and different microstructure of the hind femur cuticle. This allows for some deformability in the femur wall and is likely to reduce the risk of mechanical failure. In contrast to both other femora, the hind femur is also equipped with a set of sclerotized ridges that are likely to provide it with the required stiffness to withstand the mechanical loads during walking and jumping. This paper is one of only a few comprehensive studies on insect cuticle, which advances the current understanding of the relationship between the structure, material property and function in this complex biological composite. Statement of significanceInsect cuticle is a biological composite with strong anisotropy and wide ranges of material properties. Using an example of the femoral cuticle of desert locusts, we measured the elasticity modulus, microstructure and sclerotization level of the cuticle. Our results show that, although the hind femur withstands most of the stress during locomotion, it has a lower elasticity modulus than the fore and mid femora. This is likely to be a functional adaption to jumping, in order to allow small deformations of the femur wall and reduce the risk of material failure. Our results deepen the current understanding of the structure-material-function relationship in the complex insect cuticle.

Silencing of transcription factor E93 inhibits adult morphogenesis and disrupts cuticle, wing and ovary development in Locusta migratoria

Ecdysone-induced protein 93F (E93) plays important roles during the metamorphosis process in insects. In this study, a cDNA of the LmE93 gene was identified from the transcriptome of Locusta migratoria, which consists of the 3378-nucleotide open-reading frame (ORF) and encodes 1125 amino acids with helix-turn-helix (HTH) motifs. Reverse transcription quantitative polymerase chain reaction analysis revealed that LmE93 was highest expressed in ovary. The LmE93 expression level was markedly low from the 3rd to 4th instar nymphs, and greatly increased in 1-day-old 5th instar nymphs with a peak on middle nymphal days, then declined in the late nymphal days. Moreover, injected dsLmE93 into 4th and 5th instar nymphs greatly reduced LmE93 transcripts, respectively, and prevented the process of metamorphosis, causing supernumerary nymphal stages. Hematoxylin-eosin staining of the integument showed that the apolysis occurred in advance in 4th instar nymphs, and old cuticle degradation was decreased in dsLmE93-injected locusts of 5th instar nymphs. Smaller and no fully developed wings with reduced columns between the anterior and posterior regions were found in N6 and N7 supernumerary nymphs. In addition, the development of the ovary in dsLmE93-injected locusts was severely blocked, the yolk was almost not formed and there was no development of ovarioles. The results indicated that LmE93 play key roles in the metamorphosis, cuticle, wing and ovarian development of locusts.

Group I CDAs are responsible for a selective CHC-independent cuticular barrier in Locusta migratoria

Chitin deacetylases including CDA1 and CDA2, containing a chitin deacetylase domain and an LDL domain, have been reported to be essential for cuticle structure differentiation in different insect species. However, it is yet unexplored whether CDA1 and CDA2 activity is needed for the function of the cuticle as a barrier against pathogen and xenobiotics penetration. In this study, we studied the efficiency of fungal infection in the migratory locust Locusta migratoria in dependence of LmCDA1 and LmCDA2 function. Second instar nymphs injected with dsRNA against LmCDA1 and LmCDA2 transcripts were less resistant against the infection by the fungus Metarhizium anisopliae than control nymphs. At the same time, permeability to organophosphorus pesticides was increased in these nymphs. Interestingly, the CHC amounts at the cuticle surface were unaffected upon LmCDA1 and LmCDA2 reduction. These results suggest that the barrier function of the locust cuticle not only depends on surface CHCs, but also on an intact procuticle.

Natural arrangement of micro-strips reduces shear strain in the locust cuticle during power amplification

As one of the key components in power amplification of locust, semi-lunar process (SLP) cuticle has five different portions where its portion II stores a large amount of strain energy when locusts kick. The SLP portion II is composed of regular chitin micro-strips that non-uniformly arranged in its transverse plane. However, it is still unknown whether the natural arrangement of micro-strips affects the power amplification of the SLP cuticle. To address this, SLP portion II samples of adult locusts were mechanically tested along three orthogonal directions, corresponding to the length, width, and thickness axes of the micro-strips, respectively. Four different arrangements of micro-strips in the portion II were compared using finite element method. It was found that the micro-strip set of the portion II is orthotropic elastic and its elastic modulus is maximal in the length axis of the micro-strip. The inclusion of the micro-strips offers more strain energy than the isotropic portion II (without micro-strips). Compared to the horizontally- and vertically-arrangements of the micro-strips, their natural arrangement does not affect the strain energy but reduces the in-plane shear strain in the portion II. This research helps us deeply understand the power amplification mechanism of locusts and may guide optimization of catapult device design in bioinspired robots.

Epidermal Cell Surface Structure and Chitin-Protein Co-assembly Determine Fiber Architecture in the Locust Cuticle.

The geometrical similarity of helicoidal fiber arrangement in many biological fibrous extracellular matrices, such as bone, plant cell wall, or arthropod cuticle, to that of cholesteric liquid mesophases has led to the hypothesis that they may form passively through a mesophase precursor rather than by direct cellular control. In search of direct evidence to support or refute this hypothesis, here, we studied the process of cuticle formation in the tibia of the migratory locust, Locusta migratoria, where daily growth layers arise by the deposition of fiber arrangements alternating between unidirectional and helicoidal structures. Using focused ion beam/scanning electron microscopy (FIB/SEM) volume imaging and scanning X-ray scattering, we show that the epidermal cells determine an initial fiber orientation, from which the final architecture emerges by the self-organized co-assembly of chitin and proteins. Fiber orientation in the locust cuticle is therefore determined by both active and passive processes.

Cuticle sclerotization determines the difference between the elastic moduli of locust tibiae

A striking characteristic of insect cuticle is the wide range of its material property values, with respect to stiffness, strength and toughness. The elastic modulus of cuticle, for instance, ranges over seven orders of magnitude in different structures and different species. Previous studies suggested that this characteristic is influenced by the microstructure and sclerotization of cuticle. However, the relative role of the two factors in determining the material properties of cuticle is unknown. Here we used a combination of scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and nanoindentation, to investigate the effect of microstructure and sclerotization on the elastic modulus of tibiae of desert locusts. Our results showed that tibial cuticle is an anisotropic material with the highest elastic modulus along the tibial axis. This is likely because majority of the fibers in the cuticle are oriented along this axis. We also found that the hind tibia has a significantly higher elastic modulus, compared with the fore and mid tibiae. This is likely due to the higher sclerotization level of the hind tibia cuticle, and seems to be an adaptation to the locust locomotion by jumping, in which axial loads in the hind tibiae may reach several times the insect body weight. Our results suggest that while sclerotization determines the difference between the elastic moduli of the tibiae, anisotropic properties of each tibia is controlled by the specific fiber orientation. Our study provides one of only a few comprehensive investigations on insect cuticle, and helps to better understand the structure-material-function relationship in this complex biological composite. Statement of significanceInsect cuticle is a biological composite with strong anisotropy and wide ranges of material properties. Using an example of the tibial cuticle of desert locusts, we examined the role of two influential factors on the elastic modulus of cuticle: microstructure and sclerotization. Our results suggested the strong influence of sclerotization on the variation of the elastic modulus among fore, mid and hind tibiae, and that of the microstructure on the anisotropy of each tibia. Our results deepens the current understanding of the structure-material-function relationship in complex insect cuticle.

Helicoidal Organization of Chitin in the Cuticle of the Migratory Locust Requires the Function of the Chitin Deacetylase2 Enzyme (LmCDA2)

In the three-dimensional extracellular matrix of the insect cuticle, horizontally aligned microfibrils composed of the polysaccharide chitin and associated proteins are stacked either parallel to each other or helicoidally. The underlying molecular mechanisms that implement differential chitin organization are largely unknown. To learn more about cuticle organization, we sought to study the role of chitin deacetylases (CDA) in this process. In the body cuticle of nymphs of the migratory locust Locusta migratoria, helicoidal chitin organization is changed to an organization with unidirectional microfibril orientation when LmCDA2 expression is knocked down by RNA interference. In addition, the LmCDA2-deficient cuticle is less compact suggesting that LmCDA2 is needed for chitin packaging. Animals with reduced LmCDA2 activity die at molting, underlining that correct chitin organization is essential for survival. Interestingly, we find that LmCDA2 localizes only to the initially produced chitin microfibrils that constitute the apical site of the chitin stack. Based on our data, we hypothesize that LmCDA2-mediated chitin deacetylation at the beginning of chitin production is a decisive reaction that triggers helicoidal arrangement of subsequently assembled chitin-protein microfibrils.

LmCYP4G102: An oenocyte-specific cytochrome P450 gene required for cuticular waterproofing in the migratory locust, Locusta migratoria.

Cytochrome P450 superfamily proteins play important roles in detoxification of xenobiotics and during physiological and developmental processes. To contribute to our understanding of this large gene family in insects, we have investigated the function of the cytochrome P450 gene LmCYP4G102 in the migratory locust Locusta migratoria. Suppression of LmCYP4G102 expression by RNA interference (RNAi) does not interfere with moulting but causes rapid loss of body weight - probably due to massive loss of water, and death soon after moulting. Accordingly, maintaining these animals at 90% relative humidity prevented lethality. Consistently, RNAi against LmCYP4G102 provoked a decrease in the content of cuticular alkanes, which as an important fraction of cuticular hydrocarbons have been shown to confer desiccation resistance. In addition, the cuticle of LmCYP4G102-knockdown locusts was fragile and easier deformable than in control animals. Presumably, this phenotype is due to decreased amounts of cuticular water that is reported to modulate cuticle mechanics. Interestingly, LmCYP4G102 was not expressed in the epidermis that produces the cuticle but in the sub-epdiermal hepatocyte-like oenocytes. Together, our results suggest that the oenocyte-specific LmCYP4G102 plays a critical role in the synthesis of cuticular hydrocarbons, which are important for cuticle waterproofing and mechanical stability in L. migratoria

Specific Activation of the G Protein-coupled Receptor BNGR-A21 by the Neuropeptide Corazonin from the Silkworm, Bombyx mori, Dually Couples to the Gq and Gs Signaling Cascades

Corazonin, an undecapeptide neurohormone sharing a highly conserved amino acid sequence across Insecta, plays different physiological roles in the regulation of heart contraction rates, silk spinning rates, the induction of dark color and morphometric phase changes, and ecdysis. Corazonin receptors have been identified in Drosophila melanogaster, Manduca sexta, and Musca domestica. However, detailed information on the signaling and major physiological functions of corazonin and its receptor is largely unknown. In the current study, using both the mammalian cell line HEK293 and insect cell lines BmN and Sf21, we paired the Bombyx corazonin neuropeptide as a specific endogenous ligand for the Bombyx neuropeptide G protein-coupled receptor A21 (BNGR-A21), and we therefore designated this receptor as BmCrzR. Further characterization indicated that synthetic BmCrz demonstrated a high affinity for and activated BmCrzR, resulting in intracellular cAMP accumulation, Ca(2+) mobilization, and ERK1/2 phosphorylation via the Gq- and Gs-coupled signaling pathways. The direct interaction of BmCrzR with BmCrz was confirmed by a rhodamine-labeled BmCrz peptide. Moreover, experiments with double-stranded RNA and synthetic peptide injection suggested a possible role of BmCrz/BmCrzR in the regulation of larval growth and spinning rate. Our present results provide the first in-depth information on BmCrzR-mediated signaling for further elucidation of the BmCrz/BmCrzR system in the regulation of fundamental physiological processes.

Large scale expressed sequence tag (EST) analysis of Metarhizium acridum infecting Locusta migratoria reveals multiple strategies for fungal adaptation to the host cuticle

To infect its insect host, the entomopathogenic fungus Metarhizium acridum has to breach the physical barrier of the host cuticle to gain entry into insect tissue. To identify virulence-associated genes to improve the biocontrol efficacy of M. acridum, it is necessary to understand the genes expressed by M. acridum during its infection of the insect host cuticle. In this study, we performed a large scale gene expression analysis of M. acridum during locust cuticle infection. We report the identification of 4,739 unique expressed sequence tags (ESTs), of which 80 % have not been previously identified. Analysis of the ESTs provided evidence that M. acridum may employ several different adaptation strategies to infect locust cuticle, such as metabolism reprogramming, protein secretion, endocytosis, cell-cycle-regulated morphogenesis and effector-dependent suppression of host immune responses. By searching against the pathogen-host interaction database, several genes associated with different adaptation processes were found to potentially encode important virulence genes. Gene expression pattern analysis by semi-quantitative RT-PCR revealed that M. acridum differentially regulates subsets of genes in response to different infection niches between the host cuticle and hemolymph. The present study provides novel candidate genes to investigate the biology of insect infection by M. acridum.

Quantitative determination of catecholic degradation products from insect sclerotized cuticles

Acid hydrolysates of cuticle from various insect species were quantitatively analyzed for five catecholic amino acid adducts. Four of the adducts are ketocatechols; in three of them the amino acid moiety, either lysine, glycine or β-alanine, is connected via its amino group to the α-carbon atom of 3,4-dihydroxyacetophenone, in the fourth a tyrosine residue is connected to the same position via its phenolic group. The fifth adduct contains histidine linked via its imidazole-ring to the β-position of the dopamine sidechain. The three ketocatecholic adducts containing α-amino acids were obtained in significant yields from adult cuticles of the locust Schistocerca gregaria, the cockroaches Blaberus craniifer and Periplaneta americana, and the beetles Pachynoda sinuata and Tenebrio molitor, but only in trace amounts from larval and pupal cuticles of T. molitor, pupal cuticles of the moths Manduca sexta and Hyalophora cecropia, and puparia of the blowfly Calliphora vicina. The β-alanine-containing ketocatechol was not obtained from cuticle of locusts and T. molitor larvae and pupae, but it was present in the hydrolysates of the other cuticles. The β-histidine–dopamine adduct was obtained from all the cuticles, the highest yield was obtained from adult P. sinuata and the lowest yield was from adult S. gregaria. The β-histidine–dopamine adduct is derived from the product formed by reaction of p-quinone methides of N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD) with histidine residues in the cuticular proteins. The ketocatecholic adducts are assumed to be degradation products of crosslinks formed when oxidized dehydro-NADA reacts with the cuticular proteins. The insect species investigated appear to use both pathways for sclerotization, but to widely differing extents; the dehydro-NADA pathway dominates in cuticles which are exposed to strong deforming forces, such as those of adult locusts and cockroaches, and the p-quinone methide pathway dominates in cuticle of lepidopteran pupae and blowfly puparia, which are not exposed to strong mechanical forces but have to be effectively protected against microbial and fungal attacks.

Regulation of cuticle-degrading subtilisin proteases from the entomopathogenic fungi, Lecanicillium spp: implications for host specificity

The ability to produce cuticle-degrading proteases to facilitate host penetration does not distinguish per se entomopathogenic fungi from saprophytes. However, adapted pathogens may produce host-protein specific enzymes in response to cues. This possibility prompted an investigation of the regulation of isoforms of the subtilisin Pr1-like proteases from five aphid-pathogenic isolates of Lecanicillium spp. Significant differences were found in substrate specificity and regulation of Pr1-like proteases between isoforms of the same isolate and between different isolates. For example, the pI 8.6 isoform from KV71 was considerably more active against aphid than locust cuticle and was induced specifically by N-acetylglucosamine (NAG). Isoform pI 9.1 from the same isolate was only produced on insect cuticle while most other isoforms were more prominent on chitin containing substrates but not induced by NAG. The ability to regulate isoforms independently may allow production at critical points in host penetration. Appearance of proteases (not subtilisins) with pI 4.2 and 4.4 only on aphid cuticle was a possible link with host specificity of KV71. The absence of C or N metabolite repression in subtilisins from KV42 is unusual for pathogen proteases and may help to account for differences in virulence strategy between aphid-pathogenic isolates of Lecanicillium longisporum (unpublished data).

Aspects of cuticular sclerotization in the locust, Scistocerca gregaria, and the beetle, Tenebrio molitor

The number of reactive amino groups in cuticular proteins decreases during the early period of insect cuticular sclerotization, presumably due to reaction with oxidation products of N-acetyldopamine (NADA) and N- β-alanyldopamine (NBAD). We have quantitated the decrease in cuticular N-terminal amino groups and lysine ε-amino groups during the first 24 h of sclerotization in adult locusts, Schistocerca gregaria, and in larval and adult beetles, Tenebrio molitor, as well as the increase in β-alanine amino groups in Tenebrio cuticle. The results indicate that nearly all glycine N-terminal groups and a significant part of the ε-amino groups from lysine residues are involved in the sclerotization process in both locusts and Tenebrio. A pronounced increase in the amount of free β-alanine amino groups was observed in cuticle from adult Tenebrio and to a lesser extent also in Tenebrio larval cuticle, but from locust cuticle no β-alanine was obtained. Hydrolysis of sclerotized cuticles from locusts and Tenebrio by dilute hydrochloric acid released a large number of compounds containing amino acids linked to catecholic moieties. Products have been identified which contain histidine residues linked via their imidazole group to the β-position of various catechols, such as dopamine, 3,4-dihydroxyphenyl-ethanol (DOPET), and 3,4-dihydroxyphenyl-acetaldehyde (DOPALD), and a ketocatecholic compound has also been identified composed of lysine linked via its ε-amino group to the α-carbon atom of 3,4-dihydroxyacetophenone. Some of the hydrolysis products have previously been obtained from sclerotized pupal cuticle of Manduca sexta [Xu, R., Huang, X., Hopkins, T.L., Kramer, K.J., 1997. Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle. Insect Biochemistry and Molecular Biology 27, 101–108; Kerwin, J.L., Turecek, F., Xu, R., Kramer, K.J., Hopkins, T.L., Gatlin, C.L., Yates, J.R., 1999. Mass spectrometric analysis of catechol-histidine adducts from insect cuticle. Analytical Biochemistry 268, 229–237; Kramer, K.J., Kanost, M.R., Hopkins, T.L., Jiang, H., Zhu, Y.C., Xu, R., Kerwin, J.L., Turecek, F., 2001. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385–392], but the lysine-dihydroxyacetophenone compound and the histidine–DOPALD adduct have not been reported before. It is suggested that the compounds are derived from NADA and NBAD residues which were incorporated into the cuticle during sclerotization, and that the lysine-dihydroxyacetophenone as well as the DOPET and DOPALD containing adducts are degradation products derived from cross-links between the cuticular proteins, whereas the dopamine-containing adducts are derived from a non-crosslinking reaction product.

Developmental and Transcriptional Responses to Host and Nonhost Cuticles by the Specific Locust Pathogen Metarhizium anisopliae var. acridum

Transcript patterns elicited in response to hosts can reveal how fungi recognize suitable hosts and the mechanisms involved in pathogenicity. These patterns could be fashioned by recognition of host-specific topographical features or by chemical components displayed or released by the host. We investigated this in the specific locust pathogen Metarhizium anisopliae var. acridum. Only host (Schistocerca gregaria) cuticle stimulated the full developmental program of germination and differentiation of infection structures (appressoria). Cuticle from beetles (Leptinotarsa decimlineata) repressed germination while cuticle from hemipteran bugs (Magicicada septendecim) allowed germination but only very low levels of differentiation, indicating that the ability to cause disease can be blocked at different stages. Using organic solvents to extract insects we identified a polar fraction from locusts that allowed appressorial formation against a flat plastic (hydrophobic) surface. Microarrays comprising 1,730 expressed sequence tags were used to determine if this extract elicits different transcriptional programs than whole locust cuticle or nonhost extracts. Of 483 differentially regulated genes, 97% were upregulated. These included genes involved in metabolism, utilization of host cuticle components, cell survival and detoxification, and signal transduction. Surprisingly, given the complex nature of insect epicuticle components and the specific response of M. anisopliae var. acridum to locusts, very similar transcript profiles were observed on locust and beetle extracts. However, the beetle extract cluster was enriched in genes for detoxification and redox processes, while the locust extract upregulated more genes for cell division and accumulation of cell mass. In addition, several signal transduction genes previously implicated in pathogenicity in plant pathogens were only upregulated in response to locust extract, implying similarities in the regulatory circuitry of these pathogens with very different hosts.

Sequence determination of three cuticular proteins and isoforms from the migratory locust, Locusta migratoria, using a combination of Edman degradation and mass spectrometric techniques

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.

Purification and sequence determination of a yellow protein from sexually mature males of the desert locust, Schistocerca gregaria

A yellow protein from abdominal cuticle of the desert locust, Schistocerca gregaria, has been purified and its amino acid sequence determined. The yellow color comes from bound carotene, the protein is only deposited in the epidermis and cuticle of male locusts during their sexual maturation, and the deposition is dependent upon a sufficiently high titer of juvenile hormone. The sequence of the protein is atypical for a cuticular protein, but it has some similarity to a putative juvenile hormone binding protein from Manduca sexta. It is suggested that the protein is involved in the transport of carotenes from internal tissues to epidermis and cuticle of the locust.

Matrix proteins from insect pliable cuticles: are they flexible and easily deformed?

Proteins from pliable cuticle of locusts, Schistocerca gregaria, and silk moth larvae, Hyalophora cecropia, were studied in solution by means of a fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid (ANS), which is much more fluorescent in non-polar media than in polar media. An intense ANS-fluorescence was observed in the presence of the cuticular proteins at pH-values close to their acidic isoelectric points, and the fluorescence decreased markedly when pH was increased to neutrality or when small amounts of denaturants were added. Aggregation and eventual precipitation of both H. cecropia and locust proteins were obtained by addition of neutral salts, and the aggregation was accompanied by an increased ANS-fluorescence intensity. A decreased ANS-fluorescence was observed at salt concentrations too low to cause visible aggregation of the H. cecropia proteins, probably due to weakened electrostatic interactions between chain segments, but such a decrease was not observed for the locust proteins. The changes in intensity of ANS-fluorescence induced by addition of small amounts of denaturants or salts to solutions of the proteins indicate that more hydrophobic residues are exposed to the solvent, when either hydrophobic interactions or electrostatic attractions between chain segments are weakened. The result is a less compact protein structure, where fewer and smaller hydrophobic clusters are available for protecting ANS-molecules from the quenching effects of water. The effects of denaturants on ANS-fluorescence in the presence of the cuticular proteins are different from those observed for globular proteins, such as hen egg albumen, and the differences can be explained by the suggestion that the cuticular proteins do not have a precisely folded and densely packed hydrophobic core comparable to that present in native globular proteins, and that accordingly they do not undergo a process of denaturation corresponding to that of globular proteins. The behaviour of the cuticular proteins resembles that decribed for unordered, randomly coiled, thermally agitated polymer chains, whose hydrodynamic volumes depend upon the composition of the medium. It is proposed that the major part of the peptide chains of the cuticular proteins are in an unordered, random structure both when the proteins are in solution and when present in the intact cuticle; probably only the chain regions involved in binding the proteins to chitin will have a well-defined spatial organisation.

The synergistic effects of Metarhizium anisopliae (Metschikoff) with the acyl urea insecticides teflubenzuron and diflubenzuron for Schistocerca gregaria (Orthoptera: Acrididae)

The acyl urea insecticide teflubenzuron (=Nomolt -) caused loss of appetite, ecdysial failure and eventual death in 4th instar Schistocerca gregaria. The entomopathogenic fungus Metarhizium anisopliae also caused mortality due to mycosis among nymphs of the locust. Enhanced mortality occurred following dual application of the growth regulator and the fungus, which at one combination was synergistic. In another experiment, the fungus was exposed to cuticle treated with acetone (the carrier for the insecticide). This reduced the effectiveness of the fungus. An ultrastructural study showed the effect of the joint application of another acyl urea diflubenzuron (= Dimlin-) and Metarhizium anisopliae on locust cuticle. Disruption of the lamellae pattern was observed in some areas of cuticle. This is consistent with localised disruption of chitin synthesized during treatment with Dimlin. This compound, that has a short half-life in the locust, was applied to the food at 5 day interval over a 15 day period. Clearing zones in the cuticle around penetrant hyphae suggested action of cuticle degrading fungal enzymes. These zones were more extensive around gyphae in Dimlin-affected cuticle suggesting possible enhanced hydrolysis of cuticle protein by fungal proteases in the absence of chitin. SINET: Ethiopian Journal of Science Vol. 24, No. 1 (June 2001), pp. 113-125 Key words/phrases: Diflubenzuron, Metarhizium anisopliae, Schistocerca gregaria, teflubenzuron

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.