Material composition and mechanical properties of the venom-injecting forcipules in centipedes.

BACKGROUND: Centipedes are terrestrial and predatory arthropods that possess an evolutionary transformed pair of appendages used for venom injection-the forcipules. Many arthropods incorporate reinforcing elements into the cuticle of their piercing or biting structures to enhance hardness, elasticity or resistance to wear and structural failure. Given their frequent exposure to high mechanical stress, we hypothesise that the cuticle of the centipede forcipule might be mechanically reinforced. With a combination of imaging, analytical techniques and mechanical testing, we explore the centipede forcipule in detail to shed light on its morphology and performance. Additionally, we compare these data to characteristics of the locomotory leg to infer evolutionary processes. RESULTS: We examined sclerotization patterns using confocal laser-scanning microscopy based on autofluorescence properties of the cuticle (forcipule and leg) and elemental composition by energy-dispersive X-ray spectroscopy in representative species from all five centipede lineages. These experiments revealed gradually increasing sclerotization towards the forcipular tarsungulum and a stronger sclerotization of joints in taxa with condensed podomeres. Depending on the species, calcium, zinc or chlorine are present with a higher concentration towards the distal tarsungulum. Interestingly, these characteristics are more or less mirrored in the locomotory leg's pretarsal claw in Epimorpha. To understand how incorporated elements affect mechanical properties, we tested resistance to structural failure, hardness (H) and Young's modulus (E) in two representative species, one with high zinc and one with high calcium content. Both species, however, exhibit similar properties and no differences in mechanical stress the forcipule can withstand. CONCLUSIONS: Our study reveals similarities in the material composition and properties of the forcipules in centipedes. The forcipules transformed from an elongated leg-like appearance into rigid piercing structures. Our data supports their serial homology to the locomotory leg and that the forcipule's tarsungulum is a fusion of tarsus and pretarsal claw. Calcium or zinc incorporation leads to comparable mechanical properties like in piercing structures of chelicerates and insects, but the elemental incorporation does not increase H and E in centipedes, suggesting that centipedes followed their own pathways in the evolutionary transformation of piercing tools.

The tanning hormone, bursicon, does not act directly on the epidermis to tan the Drosophila exoskeleton.

BACKGROUND: In insects, continuous growth requires the periodic replacement of the exoskeleton. Once the remains of the exoskeleton from the previous stage have been shed during ecdysis, the new one is rapidly sclerotized (hardened) and melanized (pigmented), a process collectively known as tanning. The rapid tanning that occurs after ecdysis is critical for insect survival, as it reduces desiccation, and gives the exoskeleton the rigidity needed to support the internal organs and to provide a solid anchor for the muscles. This rapid postecdysial tanning is triggered by the "tanning hormone", bursicon. Since bursicon is released into the hemolymph, it has naturally been assumed that it would act on the epidermal cells to cause the tanning of the overlying exoskeleton. RESULTS: Here we investigated the site of bursicon action in Drosophila by examining the consequences on tanning of disabling the bursicon receptor (encoded by the rickets gene) in different tissues. To our surprise, we found that rapid tanning does not require rickets function in the epidermis but requires it instead in peptidergic neurons of the ventral nervous system (VNS). Although we were unable to identify the signal that is transmitted from the VNS to the epidermis, we show that neurons that express the Drosophila insulin-like peptide ILP7, but not the ILP7 peptide itself, are involved. In addition, we found that some of the bursicon targets involved in melanization are different from those that cause sclerotization. CONCLUSIONS: Our findings show that bursicon does not act directly on the epidermis to cause the tanning of the overlying exoskeleton but instead requires an intermediary messenger produced by peptidergic neurons within the central nervous system. Thus, this work has uncovered an unexpected layer of control in a process that is critical for insect survival, which will significantly alter the direction of future research aimed at understanding how rapid postecdysial tanning occurs.

Chemical Reactivities of ortho-Quinones Produced in Living Organisms: Fate of Quinonoid Products Formed by Tyrosinase and Phenoloxidase Action on Phenols and Catechols.

Tyrosinase catalyzes the oxidation of phenols and catechols (o-diphenols) to o-quinones. The reactivities of o-quinones thus generated are responsible for oxidative browning of plant products, sclerotization of insect cuticle, defense reaction in arthropods, tunichrome biochemistry in tunicates, production of mussel glue, and most importantly melanin biosynthesis in all organisms. These reactions also form a set of major reactions that are of nonenzymatic origin in nature. In this review, we summarized the chemical fates of o-quinones. Many of the reactions of o-quinones proceed extremely fast with a half-life of less than a second. As a result, the corresponding quinone production can only be detected through rapid scanning spectrophotometry. Michael-1,6-addition with thiols, intramolecular cyclization reaction with side chain amino groups, and the redox regeneration to original catechol represent some of the fast reactions exhibited by o-quinones, while, nucleophilic addition of carboxyl group, alcoholic group, and water are mostly slow reactions. A variety of catecholamines also exhibit side chain desaturation through tautomeric quinone methide formation. Therefore, quinone methide tautomers also play a pivotal role in the fate of numerous o-quinones. Armed with such wide and dangerous reactivity, o-quinones are capable of modifying the structure of important cellular components especially proteins and DNA and causing severe cytotoxicity and carcinogenic effects. The reactivities of different o-quinones involved in these processes along with special emphasis on mechanism of melanogenesis are discussed.

Critical Analysis of the Melanogenic Pathway in Insects and Higher Animals.

Animals synthesize melanin pigments for the coloration of their skin and use it for their protection from harmful solar radiation. Insects use melanins even more ingeniously than mammals and employ them for exoskeletal pigmentation, cuticular hardening, wound healing and innate immune responses. In this review, we discuss the biochemistry of melanogenesis process occurring in higher animals and insects. A special attention is given to number of aspects that are not previously brought to light: (1) the molecular mechanism of dopachrome conversion that leads to the production of two different dihydroxyindoles; (2) the role of catecholamine derivatives other than dopa in melanin production in animals; (3) the critical parts played by various biosynthetic enzymes associated with insect melanogenesis; and (4) the presence of a number of important gaps in both melanogenic and sclerotinogenic pathways. Additionally, importance of the melanogenic process in insect physiology especially in the sclerotization of their exoskeleton, wound healing reactions and innate immune responses is highlighted. The comparative biochemistry of melanization with sclerotization is also discussed.

Tanning of the tarsal and mandibular cuticle in adult Anax imperator (Insecta: Odonata) during the emergence sequence.

The arthropod cuticle offers strength, protection, and lightweight. Due to its limit in expandability, arthropods have to moult periodically to grow. While moulting is beneficial in terms of parasite or toxin control, growth and adaptation to environmental conditions, it costs energy and leaves the soft animal's body vulnerable to injuries and desiccation directly after ecdysis. To investigate the temporal change in sclerotization and pigmentation during and after ecdysis, we combined macrophotography, confocal laser scanning microscopy, scanning electron microscopy and histological sectioning. We analysed the tarsal and mandibular cuticle of the blue emperor dragonfly to compare the progress of tanning for structures that are functionally involved during emergence (tarsus/tarsal claws) with structures whose functionality is required much later (mandibles). Our results show that: (i) the tanning of the tarsal and mandibular cuticle increases during emergence; (ii) the tarsal cuticle tans faster than the mandibular cuticle; (iii) the mandibles tan faster on the aboral than on the oral side; and (iv) both the exo- and the endocuticle are tanned. The change in the cuticle composition of the tarsal and mandibular cuticle reflects the demand for higher mechanical stability of these body parts when holding on to the substrate during emergence and during first walking or hunting attempts.

Minimally invasive surgical approach for abdominoscrotal hydrocele treatment: A case report.

Abdominoscrotal hydrocele (ASH) is a rare clinical finding comprising fluid collection between the layers of the tunica vaginalis, extending from the scrotum to the abdominal cavity. At present, there is no unique or recommended management for ASH, and different surgical treatments have been proposed. Despite an open surgical approach being the most common treatment, the use of laparoscopy has also previously been described. The most common intraoperative complication is devascularization of the testis due to damage to the spermatic cord, with consequent orchiectomy. We present a case of ASH treated with minimally invasive surgery, consisting of a right inguinotomy with puncture of the ASH by positioning a mono-J stent avoiding spermatic cord dissection and the risk of testis devascularization. Sclerotization of the hydrocelic sac with iodopovidone through a mono-J stent was performed with healing from ASH and preservation of testicular vascularization. Two months later, magnetic resonance imaging showed the presence of scar tissue replacing the previous ASH cavity.

An insect sclerotization-inspired antifouling armor on biomedical devices combats thrombosis and embedding.

Thrombus formation and tissue embedding significantly impair the clinical efficacy and retrievability of temporary interventional medical devices. Herein, we report an insect sclerotization-inspired antifouling armor for tailoring temporary interventional devices with durable resistance to protein adsorption and the following protein-mediated complications. By mimicking the phenol-polyamine chemistry assisted by phenol oxidases during sclerotization, we develop a facile one-step method to crosslink bovine serum albumin (BSA) with oxidized hydrocaffeic acid (HCA), resulting in a stable and universal BSA@HCA armor. Furthermore, the surface of the BSA@HCA armor, enriched with carboxyl groups, supports the secondary grafting of polyethylene glycol (PEG), further enhancing both its antifouling performance and durability. The synergy of robustly immobilized BSA and covalently grafted PEG provide potent resistance to the adhesion of proteins, platelets, and vascular cells in vitro. In ex vivo blood circulation experiment, the armored surface reduces thrombus formation by 95 %. Moreover, the antifouling armor retained over 60 % of its fouling resistance after 28 days of immersion in PBS. Overall, our armor engineering strategy presents a promising solution for enhancing the antifouling properties and clinical performance of temporary interventional medical devices.

Hemocyanin with phenoloxidase activity in the chitin matrix of the crayfish gastrolith.

Gastroliths are transient extracellular calcium deposits formed by the crayfish Cherax quadricarinatus von Martens on both sides of the stomach wall during pre-molt. Gastroliths are made of a rigid chitinous organic matrix, constructed as sclerotized chitin-protein microfibrils within which calcium carbonate is deposited. Although gastroliths share many characteristics with the exoskeleton, they are simpler in structure and relatively homogeneous in composition, making them an excellent cuticle-like model for the study of cuticular proteins. In searching for molt-related proteins involved in gastrolith formation, two integrated approaches were employed, namely the isolation and mass spectrometric analysis of proteins from the gastrolith matrix, and 454-sequencing of mRNAs from both the gastrolith-forming and sub-cuticular epithelia. SDS-PAGE separation of gastrolith proteins revealed a set of bands at apparent molecular masses of 75-85 kDa; mass spectrometry data matched peptide sequences from the deduced amino acid sequences of seven hemocyanin transcripts. This assignment was then examined by immunoblot analysis using anti-hemocyanin antibodies, also used to determine the spatial distribution of the proteins in situ. Apart from contributing to oxygen transport, crustacean hemocyanins were previously suggested to be involved in several aspects of the molt cycle, including hardening of the new post-molt exoskeleton via phenoloxidation. The phenoloxidase activity of gastrolith hemocyanins was demonstrated. It was also noted that hemocyanin transcript expression during pre-molt was specific to the hepatopancreas. Our results thus reflect a set of functionally versatile proteins, expressed in a remote metabolic tissue and dispersed via the hemolymph to perform different roles in various organs and structures.

Disruption of tetrahydrobiopterin (BH4) biosynthesis pathway affects cuticle pigmentation in Henosepilachna vigintioctopunctata.

Tetrahydrobiopterin (BH4) is produced from guanosine triphosphate (GTP) under catalyzation of GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase (SR), among others. In Drosophila melanogaster, BH4 and other pteridines are required for cuticle tanning and eye pigmentation. In this study, two Hvgtpch (Hvgtpch-a and Hvgtpch-b), an Hvptps and an Hvsr transcripts were identified in a serious defoliator Henosepilachna vigintioctopunctata. Hvgtpch-a and Hvgtpch-b were highly expressed just before and/or right after the molt, in contrast to Hvptps and Hvsr. RNA interference (RNAi) by injection of a dsgtpch targeting the common fragment of Hvgtpch-a and Hvgtpch-b into the third instar larvae caused albino fourth-instar larvae and pupae. Around 80% of the Hvgtpch RNAi larvae failed to pupate. The remaining 20% of Hvgtpch RNAi pupated beetles did not completely remove the larval/pupal exuviae after emerged as adults and eventually died. Depletion of Hvgtpch at the fourth instar stage resulted in under-pigmented pupae and adults, with significantly low pupation and emergence rates. The Hvgtpch RNAi adults rarely moved and fed on plant leaves; they died within a week after emergence. Silence of Hvptps or Hvsr at the third- and fourth-instar stages led to similar but less serious phenotypes, with lowest influence in the Hvsr RNAi ladybirds. Moreover, RNAi of Hvgtpch, Hvptps or Hvsr did not affect coloration of the larval ocelli and pupal/adult compound eyes. Therefore, our results demonstrated that pteridines are involved in melanin formation but not in eye pigmentation in H. vigintioctopunctata. Moreover, our findings will enable the development of a dsgtpch-based pesticide to control H. vigintioctopunctata larvae.

Insect phenoloxidase and its diverse roles: melanogenesis and beyond.

Insect life on earth is greatly diversified despite being exposed to several infectious agents due to their diverse habitats and ecological niche. One of the major factors responsible for their successful establishment is having a powerful innate immune system. The most common and effective method used by insects in recognizing pathogen and non-self-substances is the melanization process among others. The key enzyme involved in melanin biosynthesis is the copper containing humoral defense enzyme, phenoloxidase (PO). This review focused on understanding about PO and that had been in research for nearly a century. The review elaborates about evolutionary significance of PO in arthropods, its relationship with mammalian tyrosinases, various substrates, activators and inhibitors involved in the activation of phenoloxidase cascade, as it requires an integrated system of activation that vary among insect species. The enzyme also plays a vital role in insect immunity by involving in several other immune functions like sclerotization, wound healing, opsonization, encapsulation and nodule formation. Further, gene knock down or knock out of PO genes and inhibition of PO-melanization cascade by several mechanisms can also be considered as promising future alternative to control serious pests by making them highly susceptible to any targeted attack.

Catecholamine Derivatives as Novel Crosslinkers for the Synthesis of Versatile Biopolymers.

Catecholamine metabolites are not only involved in primary metabolism, but also in secondary metabolism, serving a diverse array of physiologically and biochemically important functions. Melanin, which originates from dopa and dopamine, found in the hair, eye, and skin of all animals, is an important biopolymeric pigment. It provides protection against damaging solar radiation to animals. N-Acetyldopamine and N-ß-alanyldopamine play a crucial role in the hardening of the exoskeletons of all insects. In addition, insects and other arthropods utilize the melanogenic process as a key component of their defense systems. Many marine organisms utilize dopyl peptides and proteins as bonding materials to adhere to various substrata. Moreover, the complex dopa derivatives that are precursors to the formation of the exoskeletons of numerous marine organisms also exhibit antibiotic properties. The biochemistry and mechanistic transformations of different catecholamine derivatives to produce various biomaterials with antioxidant, antibiotic, crosslinking, and gluing capabilities are highlighted. These reactivities are exhibited through the transient and highly reactive quinones, quinone methides, and quinone methide imine amide intermediates, as well as chelation to metal ions. A careful consideration of the reactivities summarized in this review will inspire numerous strategies for synthesizing novel biomaterials for future medical and industrial use.

Ultrastructural analysis of beetle larva cuticles during infection with the entomopathogenic fungus, Beauveria bassiana.

BACKGROUND: Beauveria bassiana is one of the commercially available entomopathogenic fungi (EPF), and a number of isolates with high virulence and broad host spectrum have been used to control agricultural and forest pests. Although the functional importance of genes in EPFs' pathogenesis have been extensively studied, the precise ultrastructural mechanism of the fungal infection, particularly penetration of the host insect cuticles, is not well understood. RESULTS: In this study, we investigated the morphology and ultrastructure of the larval cuticle of the red flour beetle, Tribolium castaneum, after treatment with B. bassiana ERL1170 expressing an enhanced green fluorescent protein (Bb-eGFP). The Bb-eGFP showed high virulence against the larvae, with approximately 90% mortality at 48 h after treatment (HAT) and 100% at 72 HAT under our infection conditions. In these larvae, the regions of the body wall with flexible cuticles, such as the ventral and ventrolateral thorax and abdomen, became darkly melanized, but there was little to no melanization in the rigid dorsal cuticular structures. Confocal microscopy and transmission electron microscopy (TEM) indicated that germinated conidia on the surface of the larval cuticle were evident at 6 HAT, which formed penetration pegs and began to penetrate the several cuticle layers/laminae by 12 HAT. The penetration pegs then developed invading hyphae, some of which passed through the cuticle and reached the epidermal cells by 24 HAT. The larval cuticle was aggressively and extensively disrupted by 48 HAT, and a number of outgrowing hyphae were observed at 72 HAT. CONCLUSIONS: Our results indicate that Bb-eGFP is capable of infection and penetrating T. castaneum larvae shortly after inoculation (~24 HAT) at the body regions with apparently flexible and membranous cuticles, such as the ventral intersegmental regions and the ventrolateral pleura. This study provides details on the histopathogenesis of the host cuticle by infection and penetration of EPFs, which can facilitate the management of insect pests. © 2022 Society of Chemical Industry.

Material Properties and Morphology of Prestomal Teeth in Relation to the Feeding Habits of Diptera (Brachycera).

Prestomal teeth are cuticular projections on the mouthparts of some fly species that rasp surfaces when feeding. Although prestomal teeth morphology has been reported for several fly species, their material properties have not been investigated. Here we report the morphology, elemental composition, extent of sclerotization, hardness, and elastic modulus of prestomal teeth and relate these findings to feeding habits. Scanning electron microscopy revealed that species categorized as flower visitors have a large labellum with numerous pseudotracheae and lack prestomal teeth, generalist species have these same features but with prestomal teeth, and specialist species that feed on blood or other insects have a smaller labellum with few or no pseudotracheae and relatively large prestomal teeth. Confocal microscopy revealed that prestomal teeth are heavily sclerotized and the labellum contains resilin, an elastomeric protein. Hardness and elastic modulus were explored with nanoindentation and showed that the insectivorous Scathophaga stercoraria had the hardest prestomal teeth and the highest modulus. Energy dispersive x-ray spectroscopy revealed that prestomal teeth had low concentrations of inorganic elements, suggesting that hardness might be partially supplemented by inorganic elements. Our findings indicate that prestomal teeth morphology and material properties relate more to feeding habits than to phylogeny.

Expression profiles of tyrosine metabolic pathway genes and functional analysis of DOPA decarboxylase in puparium tanning of Bactrocera dorsalis (Hendel).

BACKGROUND: Tanning is an important physiological process with critical roles in cuticle pigmentation and sclerotization. Previous studies have shown that insect cuticle tanning is closely associated with the tyrosine metabolism pathway, which consists of a series of enzymes. RESULTS: In this study, 24 tyrosine metabolism pathway genes were identified in the oriental fruit fly Bactrocera dorsalis (Hendel) genome. Gene expression profiles throughout 15 developmental stages of B. dorsalis were established based on our previous RNA sequencing data, and we found that 13 enzyme genes could be involved in the process of pupariation. Accordingly, a tyrosine-mediated tanning pathway during the pupariation of B. dorsalis was predicted and a critical enzyme, 3,4-dihydroxyphenylalanine (DOPA) decarboxylase (DDC), was used to explore its possible roles in formation of the puparium. First, a real-time quantitative polymerase chain reaction confirmed that BdDDC had an epidermis-specific expression pattern, and was highly expressed during larval metamorphosis in B. dorsalis. Subsequent disruption of BdDDC by feeding 5-day-old larvae with DDC inhibitor (l-α-methyl-DOPA) could lead to: (i) a significant decrease in BdDDC enzyme activity and dopamine concentration; (ii) defects in puparium pigmentation; (iii) impairment of the morphology and less thickness of the puparium; and (iv) lower pupal weight and obstacles to eclosion. CONCLUSION: This study provided a potential tyrosine metabolic pathway that was responsible for insect tanning during pupariation, and the BdDDC enzyme has been shown to have crucial roles in larval-pupal tanning of B. dorsalis. © 2021 Society of Chemical Industry.

Molecular identification, expression and function analysis of peroxidasin in Chilo suppressalis.

Peroxidasin plays a unique role in the formation and stability of extracellular matrix (ECM) in the animal kingdom; however, it was only characterized in Diptera, not in other insect orders. In this study peroxidasin (CsPxd) was first identified and characterized from Chilo suppressalis, a lepidopteran pest. CsPxd complementary DNA with a 4080 bp open reading frame encodes a peptide of 1359 amino acids; the derived amino acid sequence of CsPxd harbors the typical structural characteristics of peroxidasin family in heme-peroxidase superfamily, including the signal peptide at N-terminal, leucine-rich repeat domain, Ig-loop motifs and peroxidase domain, signifying the extracellular location of protein and the involvement in ECM formation. Eukaryotic expression reveals CsPxd protein displays peroxidase activity on H2 O2 , justifying the membership of peroxidase. Phyletic analysis shows the monophyletic evolution pattern of peroxidasin in insect phyle, and moreover only one peroxidasin is present in each species of insects, suggesting its evolutionary conservation on function. Peroxidasin messenger RNA is mainly expressed in egg and the final instar larvae stage. Injection of peroxidasin double-stranded RNA into the final instar larvae impacts the cuticle sclerotization during the metamorphosis from larvae to pupa, and eventually lead to lethality of larvae and pupa. These results suggest the presence of collagen crosslink in chorion and cuticle of insects, and indicate peroxidasin plays a role in the development of chorion and cuticle; furthermore peroxidasin might be the one of potential target genes for pest control using RNA interference.

Tyrosine hydroxylase is crucial for pupal pigmentation in Zeugodacus tau (Walker) (Diptera: Tephritidae).

Tyrosine hydroxylase (TH) is the initial enzyme responsible for cuticle sclerotization and pigmentation in many insect species, but to date, no direct functional studies have focused on TH in Zeugodacus tau. Here, the 3336-bp full-length cDNA of TH was isolated from Z. tau, a notorious horticultural pest infesting fruits and vegetables. qRT-polymerase chain reaction revealed that ZtTH transcripts were highly abundant at the time of pupal tanning and during adult emergence and were expressed in the midgut, integument and head of molting larvae. The pupation and eclosion rates gradually decreased when the 1st-instar larvae were fed diets containing higher concentrations of the TH inhibitor 3-iodo-tyrosine (3-IT). Moreover, pupal weights were significantly decreased, and abnormal uncolored phenotypes were observed after 20 mg/g 3-IT was incorporated into the diet. In addition, the suppression of TH function (mediated by RNA interference) led to a decrease in TH mRNAs and eclosion rates, accompanied by less-pigmented phenotypes. There was a severe impairment of larval-pupal cuticle tanning, leading to pupae with less yellowish pigment or that were completely white and transparent, when we injected 2 µL of 24.4 mM or 73.27 mM 3-IT into 3rd-instar larvae or prepupae. These results suggest that TH is an important enzyme for the normal growth and pupal pigmentation of Z. tau and that TH is a potential gene target for use in the control of Z. tau.

Extracellular activity of NBAD-synthase is responsible for colouration of brown spots in Ceratitis capitata wings.

After the emergence of the Ceratitis capitata imago, the pale and folded wings are expanded and sclerotized to acquire the definitive form and to stabilize the cuticle. The wings of this fly show a specific pattern of brownish and black spots. Black spots are pigmented by melanin, whereas there was scarce information about the development of the brownish spots. N-beta-alanydopamine (NBAD) is the main tanning precursor in C. capitata body cuticle, and we hypothesized that it may be responsible for the colouration of the brownish spots. We determined the topology and timing of NBAD synthesis and deposition to attain the species-specific colouration pattern. We demonstrated that during the first hours the colour of the brownish spots was principally determined by the tanning of the hairs. Haemolymph circulation through the veins is required to tan the wings. We confirmed that soon after wing spreading, most of the wing epidermal cells disappeared. Thus, the tanning of the brown spots was accomplished when the wing lamina was devoid of cells. NBAD synthase (NBAD-S; Ebony protein in D. melanogaster) activity in wings was detected in pharate adults and lasted several days after the emergence, even after the end of the tanning process. This observation is in contrast to epidermal NBAD-S activity in the body, where it was nearly undetectable 48 h post emergence. Our results indicate that NBAD-S was exported and deposited into the extracellular matrix of the brown spot areas before cell death and that tanning occurs through gradual export of NBAD precursors (dopamine and b-alanine) from veins.

Dopamine-Mediated Sclerotization of Regenerated Chitin in Ionic Liquid.

Chitin is a promising structural material for biomedical applications, due to its many advantageous properties and abundance in nature. However, its usage and development in the biomedical field have been stagnant, because of chitin's poor mechanical properties in wet conditions and the difficulties in transforming it into an applicable form. To overcome these challenges, we created a novel biomimetic chitin composite. This regenerated chitin, prepared with ionic liquid, showed improved mechanical properties in wet conditions by mimicking insect cuticle and squid beak sclerotization, i.e., catechol-meditated cross-linking. By ionic liquid-based heat treatment, dopamine oxidation produced melanin-like compounds and dopamine-meditated cross-links without any solvent evaporation and oxidant utilization. The dopamine-meditated sclerotization increased the ultimate tensile strength (UTS) of the regenerated chitin by 2.52-fold, measured after six weeks of phosphate-buffered saline (PBS) submersion. In addition, the linear swelling ratio (LSR) of the chitin film was reduced by about 22%. This strategy raises a possibility of using regenerated chitin as an artificial hard tissue in wet conditions.

Incorporation of calliphorin into the cuticle of the developing blowfly, Calliphora vicina.

The stage-specific appearance of calliphorin in cuticles of Calliphora vicina was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The fate of the protein, injected into last instar larvae, was pursued by autoradiography of histological sections. Fractionation of sclerotized pupal cuticle in buffer-soluble, urea-soluble and NaOH-soluble fractions shows that calliphorin forms covalent and non-covalent links with other cuticle components. Calliphorin traverses the epidermal cells and enters the cuticle in an undegraded state and appears to be an important constituent of the sclerotizing system.

Detection of haemocyte proteins in the integument of the developing Mediterranean fruit flyCeratitis capitata : Evidence that certain haemocyte proteins serve as tyrosine carriers.

Studies of the synthesis of integumental proteins during the feeding and non-feeding stages ofCeratitis capitata demonstrated stage specificity. The synthetic profile changed dramatically, showing a maximum of protein synthesis just before the larval wandering stage, followed by an abrupt decline. The comparison between synthetic and accumulation profiles indicated that some polypeptides must be internalized into the integument from the haemolymph. The major haemolymph proteins or arylphorins have already been documented to be incorporated into the integument. In the present work, we demonstrated the interalization of some haemocyte proteins into the integument. For that purpose, polyclonal antibodies were raised against total haemocyte proteins. Immunoblot analysis of haemocyte salt extractable proteins revealed that the protein bands at 36, 54, 58, 84, 110 and 130 kDa were immunoreactive with the total haemocyte antibodies. Cell-free protein synthesis, organ culture experiments and immunoblot analysis indicated that the 36-, 54- and 58-kDa polypeptides were synthesized only in the haemocytes and were probably internalized into the integument from the serum. The 36-kDa polypeptide was also demonstrated to be internalized into the fat body of white puparia. The immunofluorescence experiments suggested that the internalization of haemocyte proteins first occurs into the epidermal cells and then into the cuticle. The presence of haemocyte proteins in the integument was also demonstrated by immunofluorescence experiments in twoC. capitata mutants. These mutations affect the darkening and stiffening of the cuticle. The demonstration of 36-, 54- and 58-kDa haemocyte polypeptides in the integument reveals a hitherto unknown function of this cell type. Moreover, the demonstration of tyrosine binding to the 54- and 58-kDa polypeptides points to their potential involvement in the sclerotization process in the cuticle.