The beetle's structural protein CPCFC making elytra tough and rigid.

The insect cuticle, which serves as both a protective barrier and an efficient lever system for locomotion, is an extracellular matrix primarily composed of chitin and protein. The cuticle protein CPCFC characterized by a "CFC" motif containing 2 Cys split by the insertion of 5 residues is distributed across most insect species and specifically localized in the hard part of the cuticle. However, their physiological function is not fully understood. Here, we report 2 CPCFC proteins, TcCPCFC1 and TcCPCFC2, derived from the Coleopteran insect Tribolium castaneum. We revealed that TcCPCFC1 and TcCPCFC2 were predominantly expressed during the larval and adult stages of T. castaneum, respectively. The transcription downregulation of TcCPCFC1 significantly decreased the modulus and toughness of the elytral cuticle. We found that TcCPCFC proteins have high binding affinity to chitin. We cloned and produced recombinant TcCPCFC proteins and demonstrated that the addition of TcCPCFC proteins to chitin hydrogel greatly enhanced the hydrogel's modulus and toughness by forming denser chitin fibrous networks. Our findings reveal the functional role of CPCFC proteins in enhancing mechanical properties of insect cuticle, and we validate this process in vitro, and offer a protein candidate for fabrication of advanced chitin-based materials.

Role of cuticular genes in the insect antimicrobial immune response.

Insects are established models for understanding host-pathogen interactions and innate immune mechanisms. The innate immune system in insects is highly efficient in recognizing and opposing pathogens that cause detrimental effects during infection. The cuticular layer which covers the superficial layer of the insect body participates in host defense and wound healing by inducing innate immune responses. Previous studies have started to address the involvement of cuticular genes in conferring resistance to insect pathogens, particularly those that infect by disrupting the insect cuticle. For example, the cuticular gene Transglutaminase (TG) in Drosophila melanogaster plays a structural role in cuticle formation and blood coagulation and also possesses immune properties against pathogenic infection. However, more information is becoming available about the immune function of other cuticular gene families in insects. In this review, we aim to highlight the recent advances in insect cuticular immunity and address the necessity of pursuing further research to fill the existing gaps in this important field of insect immunology. This information will lead to novel strategies for the efficient management of agricultural insect pests and vectors of plant and human disease.

Desert locusts (Schistocerca gregaria) feed with self-sharpening, scissor-like mandibles.

The mandibles of the desert locust Schistocerca gregaria (Forsskål, 1775) are digger-shovel-shaped mouthparts that are part of the locust's exoskeleton formed by the insect cuticle. The cuticle is a polymer-fibre composite, which supports, encases and protects the entire body. Mandibles experience heavy loading and wear due to direct contact with hard and abrasive food, just like teeth, their mineralized analogues in vertebrates. With dual-energy X-ray tomography, we image well-defined regions of zinc (Zn)-enriched cuticle at the mandible cutting edges and quantify the Zn concentrations in these regions. Zn is known to increase stiffness, hardness and wear resistance of the otherwise purely polymeric insect cuticle. In S. gregaria, the position of the Zn-enriched cutting-edge regions relative to one another suggests that the mandibles form a scissor-like cutting tool, which sharpens itself as the mouthparts shear past one another during feeding. Comparing the architecture of these purely polymeric mandibles with the mineralized incisors of rodents, we find fundamental design differences in cutting-tool structure and performance. Locusts' scissors and rodents' carving knives perform different functions, because they act on food that differs significantly in properties and shape: softer, sheet-like material in the case of locusts and harder bulk material in the case of rodents.

Organized assembly of chitosan into mechanically strong bio-composite by introducing a recombinant insect structural protein OfCPH-1.

Chitosan, known for its appealing biological properties in packaging and biomedical applications, faces challenges in achieving a well-organized crystalline structure for mechanical excellence under mild conditions. Herein, we propose a facile and mild bioengineering approach to induce organized assembly of amorphous chitosan into mechanically strong bio-composite via incorporating a genetically engineered insect structural protein, the cuticular protein hypothetical-1 from the Ostrinia furnacalis (OfCPH-1). OfCPH-1 exhibits high binding affinity to chitosan via hydrogen-bonding interactions. Simply mixing a small proportion (0.5 w/w%) of bioengineered OfCPH-1 protein with acidic chitosan precursor induces the amorphous chitosan chains to form fibrous networks with hydrated chitosan crystals, accompanied with a solution-to-gel transition. We deduce that the water shell destruction driven by strong protein-chitosan interactions, triggers the formation of well-organized crystalline chitosan, which therefore offers the chitosan with significantly enhanced swelling resistance, and strength and modulus that outperforms that of most reported chitosan-based materials as well as petroleum-based plastics. Moreover, the composite exhibits a stretch-strengthening behavior similar to the training living muscles on cyclic load. Our work provides a route for harnessing the OfCPH-1-chitosan interaction in order to form a high-performance, sustainably sourced bio-composite.

Insect cuticle-inspired design of sustainably sourced composite bioplastics with enhanced strength, toughness and stretch-strengthening behavior.

Insect cuticles that are mainly made of chitin, chitosan and proteins provide insects with rigid, stretchable and robust skins to defend harsh external environment. The insect cuticle therefore provides inspiration for engineering biomaterials with outstanding mechanical properties but also sustainability and biocompatibility. We herein propose a design of high-performance and sustainable bioplastics via introducing CPAP3-A1, a major structural protein in insect cuticles, to specifically bind to chitosan. Simply mixing 10w/w% bioengineered CPAP3-A1 protein with chitosan enables the formation of plastics-like, sustainably sourced chitosan/CPAP3-A1 composites with significantly enhanced strength (∼90 MPa) and toughness (∼20 MJ m -3), outperforming previous chitosan-based composites and most synthetic petroleum-based plastics. Remarkably, these bioplastics exhibit a stretch-strengthening behavior similar to the training living muscles. Mechanistic investigation reveals that the introduction of CPAP3-A1 induce chitosan chains to assemble into a more coarsened fibrous network with increased crystallinity and reinforcement effect, but also enable energy dissipation via reversible chitosan-protein interactions. Further uniaxial stretch facilitates network re-orientation and increases chitosan crystallinity and mechanical anisotropy, thereby resulting in stretch-strengthening behavior. In general, this study provides an insect-cuticle inspired design of high-performance bioplastics that may serve as sustainable and bio-friendly materials for a wide range of engineering and biomedical application potentials.

Adhesive properties of Aphrophoridae spittlebug foam.

Aphrophora alni spittlebug nymphs produce a wet foam from anal excrement fluid, covering and protecting themselves against numerous impacts. Foam fluid contact angles on normal (26°) and silanized glass (37°) suggest that the foam wets various substrates, including plant and arthropod surfaces. The pull-off force depends on the hydration state and is higher the more dry the fluid. Because the foam desiccates as fast as water, predators once captured struggle to free from drying foam, becoming stickier. The present study confirms that adhesion is one of the numerous foam characteristics resulting in multifunctional effects, which promote spittlebugs' survival and render the foam a smart, biocompatible material of biological, biomimetic and biomedical interest. The sustainable 'reuse' of large amounts of excrement for foam production and protection of the thin nymph integument suggests energetic and evolutionary advantages. Probably, that is why foam nests have evolved in different groups of organisms, such as spittlebugs, frogs and fish.

Non-invasive characterization of the elastic protein resilin in insects using Raman spectroscopy.

Resilin is an extremely efficient elastic protein found in the moving parts of insects. Despite many years of resilin research, we are still only just starting to understand its diversity, native structures, and functions. Understanding differences in resilin structure and diversity could lead to the development of bioinspired elastic polymers, with broad applications in materials science. Here, to better understand resilin structure, we offer a novel methodology for identifying resilin-rich regions of the insect cuticle using non-invasive Raman spectroscopy in a model species, the desert locust (Schistocerca gregaria). The Raman spectrum of the resilin-rich semilunar process of the hind leg was compared with that of nearby low-resilin cuticle, and reference spectra and peaks assigned for these two regions. The main peaks of resilin include two bands associated with tyrosine at 955-962 and 1141-1203 cm-1 and a strong peak at 1615 cm-1, attributed to the α-Amide I group associated with dityrosine. We also found the chitin skeletal modes at ~485-567 cm-1 to be significant contributors to spectra variance between the groups. Raman spectra were also compared to results obtained by fluorescence spectroscopy, as a control technique. Principal component analysis of these resulting spectra revealed differences in the light-scattering properties of resilin-rich and resilin-poor cuticular regions, which may relate to differences in native protein structure and relative abundance.

The Prospect of Hydrolytic Enzymes from Bacillus Species in the Biological Control of Pests and Diseases in Forest and Fruit Tree Production.

Plant diseases and insect pest damage cause tremendous losses in forestry and fruit tree production. Even though chemical pesticides have been effective in the control of plant diseases and insect pests for several decades, they are increasingly becoming undesirable due to their toxic residues that affect human life, animals, and the environment, as well as the growing challenge of pesticide resistance. In this study, we review the potential of hydrolytic enzymes from Bacillus species such as chitinases, ß-1,3-glucanases, proteases, lipases, amylases, and cellulases in the biological control of phytopathogens and insect pests, which could be a more sustainable alternative to chemical pesticides. This study highlights the application potential of the hydrolytic enzymes from different Bacillus sp. as effective biocontrol alternatives against phytopathogens/insect pests through the degradation of cell wall/insect cuticles, which are mainly composed of structural polysaccharides like chitins, ß-glucans, glycoproteins, and lipids. This study demonstrates the prospects for applying hydrolytic enzymes from Bacillus sp. as effective biopesticides in forest and fruit tree production, their mode of biocidal activity and dual antimicrobial/insecticidal potential, which indicates a great prospect for the simultaneous biocontrol of pests/diseases. Further research should focus on optimizing the production of hydrolytic enzymes, and the antimicrobial/insecticidal synergism of different Bacillus sp. which could facilitate the simultaneous biocontrol of pests and diseases in forest and fruit tree production.

Insect exoskeletons react to hypergravity.

A typical feature of biological materials is their ability to adapt to mechanical load. However, it is not known whether the cuticle exoskeleton, one of the most common biological structures, also shares this trait. Here, we show direct experimental evidence that prolonged exposure to hypergravity conditions affects the morphology and biomechanics of an insect exoskeleton. Locusts were raised for several weeks in a custom-designed centrifuge at various levels of hypergravity. Biomechanical measurements and X-ray microtomography show that up to 3 g load Young's modulus of the tibiae increased by about 67%. Higher gravitational loads however decreased the survival rate, body mass and endocuticle thickness. These results directly show that cuticle exoskeletons can react to hypergravity. This ability has so far only been known for bone endoskeletons and plants. Our findings thus add important context to the discussion on general ultimate factors in the evolution of adaptive biological materials and skeletal systems.

The origins of colour patterns in fossil insects revealed by maturation experiments.

Many fossil insects show monochromatic colour patterns that may provide valuable insights into ancient insect behaviour and ecology. Whether these patterns reflect original pigmentary coloration is, however, unknown, and their formation mechanism has not been investigated. Here, we performed thermal maturation experiments on extant beetles with melanin-based colour patterns. Scanning electron microscopy reveals that melanin-rich cuticle is more resistant to degradation than melanin-poor cuticle: with progressive maturation, melanin-poor cuticle regions experience preferential thinning and loss, yet melanin-rich cuticle remains. Comparative analysis of fossil insects with monotonal colour patterns confirms that the variations in tone correspond to variations in preserved cuticle thickness. These preserved colour patterns can thus be plausibly explained as melanin-based patterning. Recognition of melanin-based colour patterns in fossil insects opens new avenues for interpreting the evolution of insect coloration and behaviour through deep time.

Identification and transcriptional profiling of UV-A-responsive genes in Bemisia tabaci.

Ultraviolet-A (UV-A) radiation directly impacts the growth and spread of Bemisia tabaci. However, the mechanistic pathways of this phenomenon remain unknown. We analyzed B. tabaci transcriptome data after exposure to UV-A radiation for 6 h. The 453 genes were identified whose expression were significantly altered in response to the stress induced by UV-A irradiation. Forty genes were up-regulated, while 413 genes were down-regulated. Enrichment analysis using GO, KEGG, and Genomes databases revealed that the DEGs play key roles in antioxidation and detoxification, protein turnover, metabolic, developmental processes, and immunological response. Among the gene families involved in detoxification, shock, and development, down-regulated DEGs in transcriptional factor gene families were significantly greater than those up-regulated DEGs. Our findings demonstrated that exposure to UV-A stress can suppress immunity and affect the growth and biological parameters of B. tabaci by altering gene regulation. These results suggest a potential utility of UV-A stress in managing B. tabaci under greenhouse conditions.

Expression and purification of snustorr snarlik protein from Plutella xylostella.

Snustorr snarlik (Snsl) is a type of extracellular protein essential for insect cuticle formation and insect survival, but is absent in mammals, making it a potential selective target for pest control. Here, we successfully expressed and purified the Snsl protein of Plutella xylostella in Escherichia coli. Two truncated forms of Snsl protein, Snsl 16-119 and Snsl 16-159, were expressed as a maltose-binding protein (MBP) fusion protein and purified to a purity above 90% after a five-step purification protocol. Snsl 16-119, forming stable monomer in solution, was crystallized, and the crystal was diffracted to a resolution of ∼10 Å. Snsl 16-159, forming an equilibrium between monomer and octamer in solution, was shown to form rod-shaped particles on negative staining electron-microscopy images. Our results lay a foundation for the determination of the structure of Snsl, which would improve our understanding of the molecular mechanism of cuticle formation and related pesticide resistance and provide a template for structure-based insecticide design.

Comparative Material and Mechanical Properties among Cicada Mouthparts: Cuticle Enhanced with Inorganic Elements Facilitates Piercing through Woody Stems for Feeding.

Adult cicadas pierce woody stems with their mouthparts to feed on xylem, suggesting the presence of cuticular adaptations that could increase hardness and elastic modulus. We tested the following hypotheses: (a) the mouthpart cuticle includes inorganic elements, which augment the mechanical properties; (b) these elements are abundant in specific mouthpart structures and regions responsible for piercing wood; (c) there are correlations among elements, which could provide insights into patterns of element colocalization. We used scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) to investigate mouthpart morphology and quantify the elemental composition of the cuticle among four cicada species, including periodical cicadas (Magicicada sp.). Nanoindentation was used to quantify hardness and elastic modulus of the mandibles. We found 12 inorganic elements, including colocalized manganese and zinc in the distal regions of the mandible, the structure most responsible for piercing through wood; nanoindentation determined that these regions were also significantly harder and had higher elastic modulus than other regions. Manganese and zinc abundance relates to increased hardness and stiffness as in the cuticle of other invertebrates; however, this is one of the first reports of cuticular metals among insects with piercing-sucking mouthparts (>100,000 described species). The present investigation provides insight into the feeding mechanism of cicadas, an important but understudied component of their life traits.

Fatty acid binding protein is required for chitin biosynthesis in the wing of Drosophila melanogaster.

Chitin, the major structural polysaccharide in arthropods such as insects and mites, is a linear polymer of N-acetylglucosamine units. The growth and development of insects are intimately coupled with chitin biosynthesis. The membrane-bound ß-glycosyltransferase chitin synthase is known to catalyze the key polymerization step of N-acetylglucosamine. However, the additional proteins that might assist chitin synthase during chitin biosynthesis are not well understood. Recently, fatty acid binding protein (Fabp) has been suggested as a candidate that interacts with the chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster. Here, using split-ubiquitin membrane yeast two-hybrid and pull-down assays, we have demonstrated that the Fabp-B splice variant physically interacts with Kkv in vitro. The global knockdown of Fabp in D. melanogaster using RNA interference (RNAi) induced lethality at the larval stage. Moreover, in tissue-specific RNAi experiments, silenced Fabp expression in the epidermis and tracheal system caused a lethal larval phenotype. Fabp knockdown in the wings resulted in an abnormal wing development and uneven cuticular surface. In addition to reducing the chitin content in the first longitudinal vein of wings, Fabp silencing also caused the loss of procuticle laminate structures. This study revealed that Fabp plays an important role in chitin synthesis and contributes to a comprehensive understanding of the complex insect chitin biosynthesis.

Comparative Analysis of Epicuticular Lipids in Locusta migratoria and Calliptamus italicus: A Possible Role in Susceptibility to Entomopathogenic Fungi.

Cuticular lipids protect insects from desiccation and may determine resistance to fungal pathogens. Nonetheless, the trade-off between these lipid functions is still poorly understood. The migratory locust Locusta migratoria and the Italian locust Calliptamus italicus have dissimilar hygrothermal preferences: L. migratoria inhabits areas near water bodies with a reed bed, and C. italicus exploits a wide range of habitats and prefers steppes and semideserts with the predominance of sagebrushes. This paper presents significant differences between these species' nymphs in epicuticular lipid composition (according to gas chromatography with mass spectrometry) and in susceptibility to Metarhizium robertsii and Beauveria bassiana. The main differences in lipid composition are shifts to longer chain and branched hydrocarbons (di- and trimethylalkanes) in C. italicus compared to L. migratoria. C. italicus also has a slightly higher n-alkane content. Fatty acids showed low concentrations in the extracts, and L. migratoria has a wider range of fatty acids than C. italicus does. Susceptibility to M. robertsii and the number of conidia adhering to the cuticle proved to be significantly higher in C. italicus, although conidia germination percentages on epicuticular extracts did not differ between the species. We propose that the hydrocarbon composition of C. italicus may be an adaptation to a wide range of habitats including arid ones but may make the C. italicus cuticle more hospitable for fungi.

A Blueprint of Microstructures and Stage-Specific Transcriptome Dynamics of Cuticle Formation in Bombyx mori.

Insect cuticle is critical for the environmental adaptability and insecticide resistance of insects. However, there is no clear understanding of the structure and protein components of the cuticle during each developmental stage of holometabolous insects, and knowledge about the protein components within each layer is vague. We conducted serial sectioning, cuticular structure analysis, and transcriptome sequencing of the larval, pupal, and adult cuticles of Bombyx mori. The deposition processes of epicuticle, exocuticle, and endocuticle during larval, pupal, and adult cuticle formation were similar. Transcriptome analysis showed that these cuticle formations share 74% of the expressed cuticular protein (CP) genes and 20 other structural protein genes, such as larval serum protein and prisilkin. There are seven, six, and eleven stage-specific expressed CP genes in larval, pupal, and adult cuticles, respectively. The types and levels of CP genes may be the key determinants of the properties of each cuticular layer. For example, the CPs of the RR-2 protein family with high contents of histidine (His) are more essential for the exocuticle. Functional analysis suggested that BmorCPAP1-H is involved in cuticle formation. This study not only offers an in-depth understanding of cuticle morphology and protein components but also facilitates the elucidation of molecular mechanisms underlying cuticle formation in future studies.

Antifungal roles of adult-specific cuticular protein genes of the red flour beetle, Tribolium castaneum.

The insect cuticle is a composite structure that can further be divided into a few sub-structural layers. Its large moiety comprises a lattice of chitin fibrils and structural proteins, both of which are stabilized by covalent bonding among them. The cuticle covers the whole surface of insect body, and thus has long been suggested for the involvement in defense against entomopathogens, especially entomopathogenic fungi that infect percutaneously. We have been addressing this issue in the past few years and have so far demonstrated experimentally that chitin synthase 1, laccase2 as well as benzoquinone synthesis-related genes of Tribolium castaneum have indispensable roles in the antifungal host defense. In the present study we focused on another major component of the insect cuticular integument, structural cuticular proteins. We chose three genes coding for adult-specific cuticular proteins, namely CPR4, CPR18 and CPR27, and examined their roles in forming immunologically sound adult cuticular integuments. Analyses of developmental expression revealed that the three genes showed high level expression in the pupal stage. These results are consistent with their proposed roles in constructing cuticle of adult beetles. The RNA interference-mediated gene knockdown was employed to silence these genes, and the administration of double strand RNAs in pupae resulted in the adults with malformed elytra. The single knockdown of the three genes attenuated somewhat the defense of the resulting adult beetles against Beauveria bassiana and Metarhizium anisopliae, but statistical analyses indicated no significant differences from controls. In contrast, the double or triple knockdown mutant beetles displayed a drastic disruption of the host defense against the two entomopathogenic fungal species irrespective of the combination of targeted cuticular protein genes, demonstrating the important roles of the three cuticular protein genes in conferring robust antifungal properties on the adult cuticle. Scanning electron microscopic observation revealed that the germination of conidia attached on the adult body surface was still suppressed after the gene knockdown as in the case of wild-type beetles, suggesting that the weakened antifungal phenotypes resulted from the combined knockdown of the adult-specific cuticular protein genes could not be accounted for by the disfunction of secretion/retention of fungistatic benzoquinone derivatives.

Cuticular Hydrocarbon Trails Released by Host Larvae Lose their Kairomonal Activity for Parasitoids by Solidification.

Successful host search by parasitic wasps is often mediated by host-associated chemical cues. The ectoparasitoid Holepyris sylvanidis is known to follow chemical trails released by host larvae of the confused flour beetle, Tribolium confusum, for short-range host location. Although the hexane-extractable trails consist of stable, long-chain cuticular hydrocarbons (CHCs) with low volatility, the kairomonal activity of a trail is lost two days after release. Here, we studied whether this loss of kairomonal activity is due to changes in the chemical trail composition induced by microbial activity. We chemically analyzed trails consisting of hexane extracts of T. confusum larvae after different time intervals past deposition under sterile and non-sterile conditions. GC-MS analyses revealed that the qualitative and quantitative pattern of the long-chain CHCs of larval trails did not significantly change over time, neither under non-sterile nor sterile conditions. Hence, our results show that the loss of kairomonal activity of host trails is not due to microbially induced changes of the CHC pattern of a trail. Interestingly, the kairomonal activity of trails consisting of host larval CHC extracts was recoverable after two days by applying hexane to them. After hexane evaporation, the parasitoids followed the reactivated host trails as they followed freshly laid ones. Cryo-scanning electron microscopy showed that the trails gradually formed filament-shaped microstructures within two days. This self-assemblage of CHCs was reversible by hexane application. Our study suggests that the long-chain CHCs of a host trail slowly undergo solidification by a self-assembling process, which reduces the accessibility of CHCs to the parasitoid's receptors as such that the trail is no longer eliciting trail-following behavior.

Epicuticular hydrocarbons of the redbanded stink bug Piezodorus guildinii (Heteroptera: Pentatomidae): sexual dimorphism and alterations in insects collected in insecticide-treated soybean crops.

BACKGROUND: The redbanded stink bug Piezodorus guildinii (Heteroptera: Pentatomidae) is one of the most important species affecting soybean crops in southern South America. Capillary gas chromatography coupled to mass spectrometry was used to characterize the epicuticular hydrocarbon profiles of field-collected insects, and to identify differences in their composition between fifth-instar nymphs and adults, males and females, and between bugs collected in insecticide-treated and insecticide-free soybean crops. RESULTS: Straight chain saturated n-C27 and n-C29, and monomethyl and dimethyl chains of C31 and C33 were the most abundant compounds. A group of volatile hydrocarbons with n-C13 and n-C15 as the predominant compounds were also detected. The hydrocarbon pattern was different between nymphs and adults, either males or females. Heneicosene was almost exclusively detected in adult males and was the most important component to differentiate between both sexes, followed by tricosadiene. The total hydrocarbon amount was significantly higher in nymphs, males and females collected in insecticide-treated fields compared with insects obtained from untreated fields. CONCLUSION: Differences were found in the epicuticular hydrocarbon pattern among nymphs and adults, as well as sexual dimorphism in adult stink bugs. Interestingly, an alteration was also found in the hydrocarbon profile of insects collected in insecticide-treated soybean crops and its relevance is discussed within a pest management context.

Principal component analysis of the biological characteristics of entomopathogenic fungi in nutrient-limited and cuticle-based media.

Media formulated with insect cuticle (0.5% and 1%; Sphequit Sph®), with a reduction in nutrients (» Sabouraud dextrose agar + yeast [SDAY]) and commercial media (potato dextrose agar, Sabouraud dextrose agar) were evaluated for the cultivation of Beauveria bassiana, Cordyceps javanica (Isaria javanica [Bally] Samson & Hywel-Jones), and Metarhizium robertsii. By using principal component analysis, it was determined that the » SDAY and Sph formulations have greater advantages than commercial media for the development of fungi. The » SDAY and Sph (0.5% and 1%) improved hydrophobicity, radial growth rate, germination, conidia yield, and virulence in B. bassiana; in M. robertsii, they favored conidia yield, germination, and virulence, and in C. javanica, the » SDAY and Sph 0.5% media enhanced conidia yield, germination, radial growth rate, and virulence. We suggest that these formulations are an alternative to commercial culture media as they are cheaper and appropriate to improve the growth characteristics and virulence of the three strains evaluated. Some applications of culture media are suggested, and the importance of multivariate analysis as an exploratory tool to carry out the choice of culture media in a suitable way for the development of mycoinsecticides is also discussed.