Arthropod Exoskeletons Research Articles

Effect of carboxymethylated chitosan and cellulose acetate on mechanical properties of PLA blends for biomedical applications

Chitin and chitosan are versatile and valuable biomaterials, with chitosan being abundantly found in the exoskeletons of crustaceans, insects, arthropods, and mollusks. The chemical extraction of chitin involves a series of steps, including deproteinization, demineralization, and decolorization. Chitosan is often combined with various polymeric materials, and polymer blending can enhance the mechanical properties of the resulting bioproduct. In this study, composites of poly(lactic acid) (PLA) with chitosan (Ch) and cellulose acetate (CA) were successfully fabricated using extrusion and injection molding techniques. The chitosan content, ranging from 0 to 7.5 wt. %, was incorporated into the PLA matrix to assess the impact of filler content on the mechanical and morphological properties of the composites. The chitosan powder was first alkalized and then etherified to obtain modified chitosan, specifically o-carboxymethylated chitosan (O-CMCh). The addition of chitosan led to a reduction in tensile strength, flexural strength, modulus of elasticity, and impact strength. However, the hardness of the composite improved with the inclusion of chitosan. The mesoporous structure of chitosan was characterized using nitrogen adsorption and desorption measurements. Morphological analysis revealed that the composite containing 3.75 wt. % chitosan exhibited significantly less fracture surface compared to other composites.

Characterization of chitin and description of its antimicrobial properties obtained from Cydalima perspectalis adults

Chitin is the most abundant biopolymer group after cellulose and forms the exoskeleton of arthropods, the largest animal group. The morphology of chitin differs between and within species. In this study, we determined the physicochemical and biological activity of chitin samples obtained from different body parts of the boxwood moth Cydalima perspectalis for its application as a biotechnological material. The collected chitin samples were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), and elemental analysis. FTIR confirms that the isolation of chitin is successful. The SEM results showed that the surface morphology of the obtained chitins was both fibrous and porous and had a rough surface. As a result of the elemental analysis, the %N values of chitins were calculated as 6.60 on average, and the values were shown to be close to each other. We also investigated the biocompatibility and antimicrobial properties of these chitin samples. We used L929 (mouse fibroblast) cells to perform indirect cytotoxicity experiments and investigated their viability by performing the MTT assay. Our findings showed that the samples had no cytotoxic effect on the L929 cells at 24, 48, and 72 h. The cytotoxic study showed that Escherichia coli, Bacillus subtilis, Staphylococcus aureus bacteria, and Candida albican fungi adhered to chitin surfaces regarding biofilm production. The chitin contents were determined as 21.02% for the head, 5.74% for the body, 32.22% for the wing, 33.53% for the legs, and 2.65% for the pupal shell. Chitin is a material with high potential for use in various fields. Our findings suggested that Cydalima perspectalis can be used as an alternative source of chitin in biomedical applications.

Planctomycetes of the Genus Singulisphaera Possess Chitinolytic Capabilities.

Planctomycetes of the genus Singulisphaera are common inhabitants of soils and peatlands. Although described members of this genus are characterized as possessing hydrolytic capabilities, the ability to degrade chitin has not yet been reported for these bacteria. In this study, a novel Singulisphaera representative, strain Ch08, was isolated from a chitinolytic enrichment culture obtained from a boreal fen in Northern European Russia. The 16S rRNA gene sequence of this isolate displayed 98.2% similarity to that of Singulisphaera acidiphila MOB10T. Substrate utilization tests confirmed that strain Ch08 is capable of growth on amorphous chitin. The complete genome of strain Ch08 determined in this study was 10.85 Mb in size and encoded two predicted chitinases, which were only distantly related to each other and affiliated with the glycoside hydrolase family GH18. One of these chitinases had a close homologue in the genome of S. acidiphila MOB10T. The experimental verification of S. acidiphila MOB10T growth on amorphous chitin was also positive. Transcriptome analysis performed with glucose- and chitin-growth cells of strain Ch08 showed upregulation of the predicted chitinase shared by strain Ch08 and S. acidiphila MOB10T. The gene encoding this protein was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The ability to utilize chitin, a major constituent of fungal cell walls and arthropod exoskeletons, appears to be one of the previously unrecognized ecological functions of Singulisphaera-like planctomycetes.

Modulation of host lipid metabolism by virus infection leads to exoskeleton damage in shrimp.

The arthropod exoskeleton provides protection and support and is vital for survival and adaption. The integrity and mechanical properties of the exoskeleton are often impaired after pathogenic infection; however, the detailed mechanism by which infection affects the exoskeleton remains largely unknown. Here, we report that the damage to the shrimp exoskeleton is caused by modulation of host lipid profiles after infection with white spot syndrome virus (WSSV). WSSV infection disrupts the mechanical performance of the exoskeleton by inducing the expression of a chitinase (Chi2) in the sub-cuticle epidermis and decreasing the cuticle chitin content. The induction of Chi2 expression is mediated by a nuclear receptor that can be activated by certain enriched long-chain saturated fatty acids after infection. The damage to the exoskeleton, an aftereffect of the induction of host lipogenesis by WSSV, significantly impairs the motor ability of shrimp. Blocking the WSSV-caused lipogenesis restored the mechanical performance of the cuticle and improved the motor ability of infected shrimp. Therefore, this study reveals a mechanism by which WSSV infection modulates shrimp internal metabolism resulting in phenotypic impairment, and provides new insights into the interactions between the arthropod host and virus.

Formulation and Characterization of Chitosan-Based Mixed-Matrix Scaffold for Tissue Engineering

The use of scaffolds, three-dimensional porous, biodegradable and biocompatible structures, that can be produced from natural polymers, synthetics, ceramics and metals is crucial in the tissue engineering field. Chitosan is a polysaccharide of natural origin, found in the exoskeleton of marine arthropods and in the cell wall of fungi, with enormous popularity in the production of three-dimensional materials for Tissue Engineering, in particular bone repair. This polymer has several advantages in the production of these structures in bone regeneration and repair: biodegradability, biocompatibility, non-toxicity and antimicrobial properties. This study aimed to prepare porous scaffolds, for bone repair of degenerative diseases in the spine with better performance and less secondary effects, based on chitosan and another biopolymer (sodium alginate) with the incorporation of calcium phosphates (hydroxyapatite and β-tricalcium phosphate), for tissue engineering application. The obtained scaffolds were object of a detailed characterization, namely with regard to their porosity through the ethanol method, degradation, positron annihilation spectroscopy (PAS), mechanical properties, scanning electronic microscope (SEM), thermal stability through thermogravimetric analysis (TGA), chemical composition through X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The results obtained showed that the different scaffolds presented pores able to support osteoid matrix growth. The crosslinking of scaffolds was also evaluated and resulted in pores with smaller dimensions and higher regularity in the chitosan-sodium alginate polymer without calcium phosphate scaffold. It was also possible to observe the effect of inorganics on mixed-matrix scaffolds, both morphologically and chemically. These scaffolds showed promising results in terms of mechanical and chemical properties, along with promising porosity for tissue regeneration applications.

Functionally graded structures in the involucre of Job’s tears

Nature is filled with materials that are both strong and light, such as bones, teeth, bamboo, seashells, arthropod exoskeletons, and nut shells. The insights gained from analyzing the changing chemical compositions and structural characteristics, as well as the mechanical properties of these materials, have been applied in developing innovative, durable, and lightweight materials like those used for impact absorption. This research concentrates on the involucres of Job’s tears (Coix lacryma-jobi var. lacryma-jobi), which are rich in silica, hard, and serve to encase the seeds. The chemical composition and structural characteristics of involucres were observed using scanning electron microscopy and energy-dispersive x-ray spectroscopy and optical microscopy with safranin staining. The hardness of the outer and inner surfaces of the involucre was measured using the micro-Vickers hardness test, and the Young’s modulus of the involucre’s cross-section was measured using nanoindentation. Additionally, the breaking behavior of involucres was measured through compression test and three-point bending tests. The results revealed a smooth transition in chemical composition, as well as in the orientation and dimensions of the tissues from the outer to the inner layers of involucres. Furthermore, it was estimated that the spatial gradient of the Young’s modulus is due to the gradient of silica deposition. By distributing the hard, brittle silica in the outer layer and elastoplastic organic components in the middle and inner layers, the involucres effectively respond to compressive and tensile stresses that occur when loads are applied to the outside of the involucre. Furthermore, the involucres are reinforced in both meridional and equatorial directions by robust fibrovascular bundles, fibrous bundles, and the inner layer’s sclerenchyma fibers. From these factors, it was found that involucres exhibit high toughness against loads from outside, making it less prone to cracking.

Arthropods as Vectors of Grapevine Trunk Disease Pathogens: Quantification of Phaeomoniella chlamydospora on Arthropods and Mycobiome Analysis of Earwig Exoskeletons.

Viticulture worldwide is challenged by grapevine trunk diseases (GTDs). Involvement of arthropods in the dissemination process of GTD pathogens, notably esca pathogens, is indicated after detection of associated pathogens on arthropod exoskeletons, and demonstration of transmission under artificial conditions. The present study is the first to quantify spore loads via qPCR of the esca-relevant pathogen Phaeomoniella chlamydospora on arthropods collected in German vineyards, i.e., European earwigs (Forficula auricularia), ants (Formicidae), and two species of jumping spiders (Marpissa muscosa and Synageles venator). Quantification of spore loads showed acquisition on exoskeletons, but most arthropods carried only low amounts. The mycobiome on earwig exoskeletons was described for the first time to reveal involvement of earwigs in the dispersal of GTDs in general. Metabarcoding data support the potential risk of earwigs as vectors for predominantly Pa. chlamydospora and possibly Eutypa lata (causative agent of Eutypa dieback), as respective operational taxonomical unit (OTU) assigned genera had relative abundances of 6.6% and 2.8% in total reads, even though with great variation between samples. Seven further GTD-related genera were present at a very low level. As various factors influence the successful transmission of GTD pathogens, we hypothesize that arthropods might irregularly act as direct vectors. Our results highlight the importance of minimizing and protecting pruning wounds in the field.

Plant root associated chitinases: structures and functions.

Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent K M values of (20-40 µM) for the substrate 4-MU-GlcNAc3. They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5-7 and 30-40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50-60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.

Secondary Reorientation and Hygroscopic Forces in Chitinous Biopolymers and Their Use for Passive and Biochemical Actuation

AbstractPassive actuation is the production of movement or deformation without external power sources or control systems. This phenomenon has been gaining significant attention in the engineering community due to its potential use in energy‐efficient systems, in which part of the functionality is programmed in the material composition to reduce overall cost and complexity. Biological organisms, formed through millennia of evolution in a paradigm of limited resources and energy and at a multiscale equilibrium with their environment, have developed a myriad of outstanding materials and strategies for integrating complex functionalities into their multiscale structural design. In this study, the arthropod exoskeleton's chitinous composition and manufacturing strategies are used to reproduce its complex relations with external forces and water‐driven molecular rearrangement and use them to produce electricity and actuate a mechanical hand. The proposed technology is unique in combining the generation of strong forces and the integration of the principles and materials of biological organisms, leading to significant advances in the design and performance of passive solutions integrated into biological systems for a wide range of applications, such as biorobotics, medical devices, and energy harvesting.

Artificial Arthropod Exoskeletons/Fungi Cell Walls Integrating Metal and Biocatalysts for Heterogeneous Synergistic Catalysis of Asymmetric Cascade Transformations

AbstractA novel and sustainable tandem‐catalysis system for asymmetric synthesis is disclosed, which is fabricated by bio‐inspired self‐assembly of artificial arthropod exoskeletons (AAEs) or artificial fungi cell walls (AFCWs) containing two different types of catalysts (enzyme and metal nanoparticles). The heterogeneous integrated enzyme/metal nanoparticle AAE/AFCW systems, which contain chitosan as the main structural component, co‐catalyze dynamic kinetic resolution of primary amines via a tandem racemization/enantioselective amidation reaction process to give the corresponding amides in high yields and excellent ee. The heterogeneous AAE/AFCW systems display successful heterogeneous synergistic catalysis at the surfaces since they can catalyze multiple reaction cycles without metal leaching. The use of natural‐based and biocompatible structural components makes the AAE/AFCW systems fully biodegradable and renewable, thus fulfilling important green chemistry requirements.

Variation among arthropod taxa in the amino acid content of exoskeleton and digestible tissue.

Arthropod consumption provides amino acids to invertebrates and vertebrates alike, but not all amino acids in arthropods may be digestible as some are bound in the exoskeleton. Consumers may not be able to digest exoskeleton in significant amounts or avoid it entirely (e.g., extraoral digestion). Hence, measures that do not separate digestible amino acids from those in exoskeleton may not accurately represent the amino acids available to consumers. Additionally, arthropods are taxonomically diverse, and it remains unclear if taxonomic differences also reflect differences in amino acid availability. Thus, we tested: (1) if there were consistent differences in the content and balance of amino acids between the digestible tissue and exoskeleton of arthropods and (2) if arthropod Orders differ in amino acid content and balance. We measured the amino acid content (mg/100 mg dry mass) and balance (mg/100 mg protein) of whole bodies and exoskeleton of a variety of arthropods using acid hydrolysis. Overall, there was higher amino acid content in digestible tissue. There were also significant differences in the amino acid balance of proteins in digestible tissue and exoskeleton. Amino acid content and balance also varied among Orders; digestible tissues of Hemiptera contained more of some essential amino acids than other Orders. These results demonstrate that arthropod taxa vary in amino acid content, which could have implications for prey choice by insectivores. In addition, exoskeleton and digestible tissue content differ in arthropods, which means that whole body amino acid content of an arthropod is not necessarily a predictor of amino acid intake of a predator that feeds on that arthropod.

Study on mechanical behaviors and failure mechanism of polyurethane matrix composites inspired by mussel chemistry and arthropod exoskeleton structures under dynamic impact

Polyurethane elastomer (PUE), an elastic polymer material of lightweight and unexceptionable mechanical properties, is widely used into the military protective structural materials to resist dynamic impact and absorb energy. However, the mechanical and thermal properties of PUE need to be enhanced due to failure damage caused by the thermal stress softening under high-speed impact. Inspired by the mussel chemistry and the exoskeleton structures of arthropods, catechol and polyethyleneimine were used to functionalize micro carbon fibers (MCFs) to enhance the mechanical and thermal properties of PUE composites. Compared with Pure PUE, the static compression modulus of PUE composite with a filler content of 2.0 wt.% was increased by 99.25%. The dynamic maximum strength and energy absorption were improved by 74.08% and 102.92%, respectively. Besides, the modified MCFs could significantly enhance the viscoelasticity and thermal conductivity characteristics of composites. The enhancement of impact resistance and energy absorption capability of the composites are mainly due to the synergistic effect of tough mussel adhesion proteins and enhancing MCFs. Thereinto, MCFs could provide incredible strength, and plenty of crosslinking networks based on phenol-amine chemistry were served as supporting structures to resist dynamic impact by effective energy dissipation. Therefore, this work can provide a way to prepare PUE composites with excellent thermal and mechanical performance, and it is beneficial to understand the deformation and failure mechanisms of PUE and other soft polymer materials under dynamic impact.

Modulating of Bouligand Structure and Chirality Constructed Bionically Based on the Self-Assembly of Chitin Whiskers.

Chitin can self-assemble into a liquid crystal phase with supramolecular chirality and Bouligand structure, which is widely found in the exoskeletons of arthropods. However, bionically replicating this structure via the self-assembly of chitin whiskers (CHWs) is still a challenge. Here, the effects of several internal and external parameters on the self-assembly of CHWs were revealed based on liquid crystal phase, chirality, Bouligand structure, and rheological properties. The formation of chiral liquid crystal phase and Bouligand structure largely depends on the concentration of CHWs and, meanwhile, is affected by the aspect ratio and zeta potential of CHWs and the self-assembly time. Impressively, introducing electrolytes and changing pH significantly affect the thickness of the electrical double layer, thereby also affecting the self-assembly of CHWs. This study offers a comprehensive understanding of CHWs' self-assembly process, which is beneficial for the bionic design of new nature-inspired functional materials with chiral characteristic and Bouligand structure.

Recent Advances in Chitosan-Based Applications-A Review.

Chitosan derived from chitin gas gathered much interest as a biopolymer due to its known and possible broad applications. Chitin is a nitrogen-enriched polymer abundantly present in the exoskeletons of arthropods, cell walls of fungi, green algae, and microorganisms, radulae and beaks of molluscs and cephalopods, etc. Chitosan is a promising candidate for a wide variety of applications due to its macromolecular structure and its unique biological and physiological properties, including solubility, biocompatibility, biodegradability, and reactivity. Chitosan and its derivatives have been known to be applicable in medicine, pharmaceuticals, food, cosmetics, agriculture, the textile and paper industries, the energy industry, and industrial sustainability. More specifically, their use in drug delivery, dentistry, ophthalmology, wound dressing, cell encapsulation, bioimaging, tissue engineering, food packaging, gelling and coating, food additives and preservatives, active biopolymeric nanofilms, nutraceuticals, skin and hair care, preventing abiotic stress in flora, increasing water availability in plants, controlled release fertilizers, dye-sensitised solar cells, wastewater and sludge treatment, and metal extraction. The merits and demerits associated with the use of chitosan derivatives in the above applications are elucidated, and finally, the key challenges and future perspectives are discussed in detail.

Chitosan Based Materials in Cosmetic Applications: A Review

This review provides a report on the properties and recent advances in the application of chitosan and chitosan-based materials in cosmetics. Chitosan is a polysaccharide that can be obtained from chitin via the deacetylation process. Chitin most commonly is extracted from cell walls in fungi and the exoskeletons of arthropods, such as crustaceans and insects. Chitosan has attracted significant academic interest, as well as the attention of the cosmetic industry, due to its interesting properties, which include being a natural humectant and moisturizer for the skin and a rheology modifier. This review paper covers the structure of chitosan, the sources of chitosan used in the cosmetic industry, and the role played by this polysaccharide in cosmetics. Future aspects regarding applications of chitosan-based materials in cosmetics are also mentioned.

Chitosan in the protection of agricultural crops against phytopathogens agents

Mexico is considered one of the main vegetable producing countries, however, its production is affected by several phytopathogens, whose control of these pathogens is based on the use of agrochemicals, which cause consequent environmental and health damages. In 2018, Mexico ranked 5th in the world in the consumption of fungicides and bactericides in agriculture. For this reason, new alternatives to control phytopathogens are important, with natural compounds with antifungal properties such as chitosan, a biopolymer of N-acetyl-D-glucosamine and D-glucosamine units linked by β-(1-4) bonds derived from the deacetylation of chitin with alkalysis (NaOH) or enzymatic hydrolysis. Chitosan is extracted from exoskeletons of arthropods. Research shows that it inhibits the growth of phytopathogens, due to its polycationic nature depending on molecular weight and deacetylation percentage. With other antifungal properties, they are inducer of phytoalexins, promotion of plant growth and biostimulant of beneficial microorganisms, due to its useful and economic potential agricultural application in technological innovation.

Structural Analysis and Construction of a Thermostable Antifungal Chitinase.

Chitin is a biopolymer of N-acetyl-d-glucosamine with β-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the β-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme's thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. IMPORTANCE We solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (Tm) 6.9°C higher than wild type (WT) and a half-life at 60°C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including 5 proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme. These mutations do not affect its high antifungal activity and chitinase activity, and the principle for the construction of the thermostable chitinase was well explained by its crystal structure. Our results provide a useful strategy to enhance the thermostability of this enzyme family and to use the thermostable mutant as a seed for antifungal agents for practical use.

PROSPECTS OF INDUSTRIAL PRODUCTION OF CHITIN-GLUCAN COMPLEXES FROM FUNGAL CULTURES

The paper is devoted to the problem of producing chitin-glucan complexes (CGC) as an alternative to chitin and chitosan. These biopolymers are present in arthropod exoskeletons and other skeletal elements, as well as in fungi and algae cell walls. Chitin-glucan complexes are widely used as sorbents, fillers, and agents for enzyme immobilization in a range of fields including medicine, biotechnology, food industry (particularly in brewing to remove sediment), cosmetology, agriculture, and manufacturing. Due to their fungicidal and antibacterial properties, these biopolymers are used as plant protection products and as biofertilizers. They also have a lot of potential for regenerative medicine and tissue therapy because of their biocompatibility and non-toxicity. The paper considers the main methods for isolation of chitin-glucan complexes and features of commercial production of chitin-[A2] gluconate complexes from fungal cultures, with a focus on the mild alkaline process and the use of an enzymatic treatment to improve production and obtain a product with a stable degree of deacetylation. Also, the paper discusses potential producers and optimal cultivation conditions for obtaining the maximum amount of biomass, which is directly proportional to the amount of biomass produced and is dependent on strain, cultivation, and isolation process conditions. The cost of these polymers largely depends on the cost and availability of raw materials, so chitosan and chitin derived from fungal cultures are more environmentally friendly and relatively inexpensive, owing to their ability to be cultivated in industrial waste, lack of seasonality, and availability of producers.

Silencing of Chitin-Binding Protein with PYPV-Rich Domain Impairs Cuticle and Wing Development in the Asian Citrus Psyllid, Diaphorina citri.

Simple SummaryMolting is extremely important for insect growth and development, which is accompanied the degradation of old cuticle and synthesis of new cuticle. Chitin and proteins, as major components of insect cuticle, maintain the rigidity of the exoskeleton. The functions of chitin-binding proteins have not, to date, been characterized in Diaphorina citri. In the current study, we identified a cuticle protein (DcCP64) according to chitin column purification and LC-MS/MS analysis. Silencing of DcCP64 induced an abnormal phenotype and increased the permeability of the abdomen and wings. Additionally, the mortality and malformation rate significantly increased, and the molting rate decreased after inhibition of DcCP64. Transcriptome sequencing analysis revealed that up-regulated DEGs were mainly related to oxidative phosphorylation, whereas down-regulated DEGs were mainly involved in MAPK and FoxO signaling pathways. Our results provide a basis for further functional research on DcCP64 in D. citri.Chitin is a major component of the arthropod exoskeleton, always working together with chitin-binding proteins to maintain the functions of extracellular structures. In the present study, we identified a cuticle protein 64 from Diaphorina citri using a chitin-binding assay. Bioinformatics analysis revealed that DcCP64 contained eight conserved PYPV motifs but lacked a Rebers–Riddiford (R–R) consensus and other chitin-binding domains. RT-qPCR analysis suggested that DcCP64 had the highest expression level in the wing and fifth-instar nymph stage. Knockdown of DcCP64 by RNA interference (RNAi) resulted in a malformed-wing phenotype, higher mortality and decreased molting rate. Furthermore, transcriptomics analysis revealed that 1244 differentially expressed genes (DEGs) were up-regulated and 580 DEGs were down-regulated, compared with dsDcCP64 groups and dsGFP groups. KEGG enrichment analysis revealed that up-regulated DEGs were mainly related to oxidative phosphorylation, whereas down-regulated DEGs were mainly involved in the MAPK and FoxO signaling pathways. Moreover, inhibition of DcCP64 significantly affected the cuticle surface, and increased the permeability of the abdomen and wings. Further chitin- and cellulose-binding assay confirmed the chitin-binding properties of recombinant DcCP64 in vitro. These results indicate that DcCP64 might play an important role in the cuticle and wing development of D. citri.

Chitin and cuticle proteins form the cuticular layer in the spinning duct of silkworm

Chitin is found in the exoskeleton and peritrophic matrix of arthropods, but recent studies have also identified chitin in the spinning duct of silk-spinning arthropods. Here, we report the presence and function of chitin and cuticle proteins ASSCP1 and ASSCP2 in the spinning duct of silkworm. We show that chitin and these proteins are co-located in the cuticular layer of the spinning duct. Ultrastructural analysis indicates that the cuticular layer has a multilayer structure by layered stacking of the chitin laminae. After knocking down ASSCP1 and ASSCP2, the fine structure of this layer was disrupted, which had negative impacts on the mechanical properties of silk. This work clarifies the function of chitin in the spinning duct of silkworm. Chitin and cuticle proteins are the main components of the cuticular layer, providing the shearing stress during silk fibrillogenesis and regulating the final mechanical properties of silk. Statement of significanceRecent studies have identified chitin in the spinning duct of silk-spinning arthropods. However, the role of chitin in this specific organ remains unclear. This study reports that chitin and cuticle proteins form the cuticular layer, a unique structure of the spinning duct of silkworm. This layer with a precise laminate structure gives the spinning duct flexible properties, provides shearing forces for silk fibrillogenesis, and contributes to silk final mechanical properties. Our work clarifies the component, ultrastructure, and biological significance of the silkworm cuticular layer, describes the specific process of silk fiber formation, and proposes new molecular targets (chitin and cuticle proteins) for the improvement of animal silks.