Self-Assembled Coacervates of Chitosan and an Insect Cuticle Protein Containing a Rebers-Riddiford Motif.

The interactions among biomacromolecules within insect cuticle may offer new motifs for biomimetic material design. CPR27 is an abundant protein in the rigid cuticle of the elytron from Tribolium castaneum. CPR27 contains the Rebers-Riddiford (RR) motif, which is hypothesized to bind chitin. In this study, active magnetic microrheology coupled with microscopy and protein particle analysis techniques were used to correlate alterations in the viscosity of chitosan solutions with changes in solution microstructure. Addition of CPR27 to chitosan solutions led to a 3-fold drop in viscosity. This change was accompanied by the presence of micrometer-sized coacervate particles in solution. Coacervate formation had a strong dependence on chitosan concentration. Analysis showed the existence of a critical CPR27 concentration beyond which a significant increase in particle count was observed. These effects were not observed when a non-RR cuticular protein, CP30, was tested, providing evidence of a structure-function relationship related to the RR motif.

Proteomic and transcriptomic analyses of rigid and membranous cuticles and epidermis from the elytra and hindwings of the red flour beetle, Tribolium castaneum.

The insect cuticle is a composite biomaterial made up primarily of chitin and proteins. The physical properties of the cuticle can vary greatly from hard and rigid to soft and flexible. Understanding how different cuticle types are assembled can aid in the development of novel biomimetic materials for use in medicine and technology. Toward this goal, we have taken a combined proteomics and transcriptomics approach with the red flour beetle, Tribolium castaneum, to examine the protein and gene expression profiles of the elytra and hindwings, appendages that contain rigid and soft cuticles, respectively. Two-dimensional gel electrophoresis analysis revealed distinct differences in the protein profiles between elytra and hindwings, with four highly abundant proteins dominating the elytral cuticle extract. MALDI/TOF mass spectrometry identified 19 proteins homologous to known or hypothesized cuticular proteins (CPs), including a novel low complexity protein enriched in charged residues. Microarray analysis identified 372 genes with a 10-fold or greater difference in transcript levels between elytra and hindwings. CP genes with higher expression in the elytra belonged to the Rebers and Riddiford family (CPR) type 2, or cuticular proteins of low complexity (CPLC) enriched in glycine or proline. In contrast, a majority of the CP genes with higher expression in hindwings were classified as CPR type 1, cuticular proteins analogous to peritrophins (CPAP), or members of the Tweedle family. This research shows that the elyra and hindwings, representatives of rigid and soft cuticles, have different protein and gene expression profiles for structural proteins that may influence the mechanical properties of these cuticles.

Proteomic analysis of pharate pupal molting fluid from the tobacco hornworm, Manduca sexta.

The insect cuticle is a key component of their success, being important for protection, communication, locomotion, and support. Conversely, as an exoskeleton, it also limits the size of the insect and must be periodically molted and a new one synthesized, to permit growth. To achieve this, the insect secretes a solution of chitinases, proteases and other proteins, known collectively as molting fluid, during each molting process to break down and recycle components of the old cuticle. Previous research has focused on the degradative enzymes in molting fluid and offered some characterization of their biochemical properties. However, identification of the specific proteins involved remained to be determined. We have used 2D SDS-PAGE and LC/MS-based proteomic analysis to identify proteins in the molting fluid of the tobacco hornworm, Manduca sexta, undergoing the larval to pupal molt. We categorized these proteins based on their proposed functions including chitin metabolism, proteases, peptidases, and immunity. This analysis complements previous reported work on M. sexta molting fluid and identifies candidate genes for enzymes involved in cuticle remodeling. Proteins classified as having an immune function highlight potential for molting fluid to act as an immune barrier to prevent infections during the cuticle degradation and ecdysis processes. Several proteins known to function in melanin synthesis as an immune response in hemolymph were present in molting fluid. We demonstrated that the bacterium Micrococcus luteus and the entomopathogenic fungus Beauveria bassiana can stimulate activation of phenoloxidase in molting fluid, indicating that the recognition proteins, protease cascade, and prophenoloxidase needed for melanin synthesis are present as a defense against infection during cuticle degradation. This analysis offers insights for proteins that may be important not only for molting in M. sexta but for insects in general.

Serine Protease Networks Mediate Immune Responses in Extra-Embryonic Tissues of Eggs in the Tobacco Hornworm, Manduca sexta.

The melanization and Toll pathways, regulated by a network of serine proteases and noncatalytic serine protease homologs (SPHs), have been investigated mostly in adult and larval insects. However, how these innate immune reactions are regulated in insect eggs remains unclear. Here we present evidence from transcriptome and proteome analyses that extra-embryonic tissues (yolk and serosa) of early-stage Manduca sexta eggs are immune competent, with expression of immune effector genes including prophenoloxidase and antimicrobial peptides. We identified gene products of the melanization and Toll pathways in M. sexta eggs. Through in vitro reconstitution experiments, we demonstrated that constitutive and infection-induced serine protease cascade modules that stimulate immune responses exist in the extra-embryonic tissues of M. sexta eggs. The constitutive module (HP14b-SP144-GP6) may promote rapid early immune signaling by forming a cascade activating the cytokine Spätzle and regulating melanization by activating prophenoloxidase (proPO). The inducible module (HP14a-HP21-HP5) may trigger enhanced activation of Spätzle and proPO at a later phase of infection. Crosstalk between the two modules may occur in transition from the constitutive to the induced response in eggs inoculated with bacteria. Examination of data from two other well-studied insect species, Tribolium castaneum and Drosophila melanogaster, supports a role for a serosa-dependent constitutive protease cascade in protecting early embryos against invading pathogens.

Temporal changes in the physical and mechanical properties of beetle elytra during maturation.

Changes in physical properties of Tenebrio molitor and Tribolium castaneum elytra (hardened forewings) were studied to understand how the development of microstructure and chemical interactions determine cuticle mechanical properties. Analysis of these properties supports a model in which cuticular material is continuously secreted from epidermal cells to produce an extracellular matrix so that the outermost layers mature first. It is hypothesized that enzymatic crosslinking and pigmentation reactions along with dehydration help to stabilize the protein-chitin network within the initial layers of cuticle shortly after eclosion. Mature layers are proposed to bear most of the mechanical loads. The frequency dependence of the storage modulus and the tan δ values decreased during the beginning of maturation, reaching constant values after 48 h post-eclosion. A decrease of tan δ indicates an increase in crosslinking of the material. The water content declined from 75% to 31%, with a significant portion lost from within the open spaces between the dorsal and ventral cuticular layers. Dehydration had a less significant influence than protein crosslinking on the mechanical properties of the elytron during maturation. When Tribolium cuticular protein TcCP30 expression was decreased by RNAi, the tan δ and frequency dependence of E' of the elytron did not change during maturation. This indicates that TcCP30 plays a role in the crosslinking process of the beetle's exoskeleton. This study was inspired by previous work on biomimetic multicomponent materials and helps inform future work on creating robust lightweight materials derived from natural sources. STATEMENT OF SIGNIFICANCE: Examination of changes in the physical properties of the elytra (hardened forewings) of two beetle species advanced understanding of how the molecular interactions influence the mechanical properties of the elytra. Physical characterization, including dynamic mechanical analysis, determined that the outer portion of the elytra matured first, while epidermal cells continued to secrete reactive components until the entire structure reached maturation. RNA interference was used to identify the role of a key protein in the elytra. Suppression of its expression reduced the formation of crosslinked polymeric components in the elytra. Identifying the molecular interactions in the matrix of proteins and polysaccharides in the elytra together with their hierarchical architecture provides important design concepts in the development of biomimetic materials.

Predicted effector molecules in the salivary secretome of the pea aphid (Acyrthosiphon pisum): a dual transcriptomic/proteomic approach.

The relationship between aphids and their host plants is thought to be functionally analogous to plant-pathogen interactions. Although virulence effector proteins that mediate plant defenses are well-characterized for pathogens such as bacteria, oomycetes, and nematodes, equivalent molecules in aphids and other phloem-feeders are poorly understood. A dual transcriptomic-proteomic approach was adopted to generate a catalog of candidate effector proteins from the salivary glands of the pea aphid, Acyrthosiphon pisum. Of the 1557 transcript supported and 925 mass spectrometry identified proteins, over 300 proteins were identified with secretion signals, including proteins that had previously been identified directly from the secreted saliva. Almost half of the identified proteins have no homologue outside aphids and are of unknown function. Many of the genes encoding the putative effector proteins appear to be evolving at a faster rate than homologues in other insects, and there is strong evidence that genes with multiple copies in the genome are under positive selection. Many of the candidate aphid effector proteins were previously characterized in typical phytopathogenic organisms (e.g., nematodes and fungi) and our results highlight remarkable similarities in the saliva from plant-feeding nematodes and aphids that may indicate the evolution of common solutions to the plant-parasitic lifestyle.

A protein from the salivary glands of the pea aphid, Acyrthosiphon pisum, is essential in feeding on a host plant.

In feeding, aphids inject saliva into plant tissues, gaining access to phloem sap and eliciting (and sometimes overcoming) plant responses. We are examining the involvement, in this aphid-plant interaction, of individual aphid proteins and enzymes, as identified in a salivary gland cDNA library. Here, we focus on a salivary protein we have arbitrarily designated Protein C002. We have shown, by using RNAi-based transcript knockdown, that this protein is important in the survival of the pea aphid (Acyrthosiphon pisum) on fava bean, a host plant. Here, we further characterize the protein, its transcript, and its gene, and we study the feeding process of knockdown aphids. The encoded protein fails to match any protein outside of the family Aphididae. By using in situ hybridization and immunohistochemistry, the transcript and the protein were localized to a subset of secretory cells in principal salivary glands. Protein C002, whose sequence contains an N-terminal secretion signal, is injected into the host plant during aphid feeding. By using the electrical penetration graph method on c002-knockdown aphids, we find that the knockdown affects several aspects of foraging and feeding, with the result that the c002-knockdown aphids spend very little time in contact with phloem sap in sieve elements. Thus, we infer that Protein C002 is crucial in the feeding of the pea aphid on fava bean.

Phylogenetic and sequence analyses of insect transferrins suggest that only transferrin 1 has a role in iron homeostasis.

Iron is essential to life, but surprisingly little is known about how iron is managed in nonvertebrate animals. In mammals, the well-characterized transferrins bind iron and are involved in iron transport or immunity, whereas other members of the transferrin family do not have a role in iron homeostasis. In insects, the functions of transferrins are still poorly understood. The goals of this project were to identify the transferrin genes in a diverse set of insect species, resolve the evolutionary relationships among these genes, and predict which of the transferrins are likely to have a role in iron homeostasis. Our phylogenetic analysis of transferrins from 16 orders of insects and two orders of noninsect hexapods demonstrated that there are four orthologous groups of insect transferrins. Our analysis suggests that transferrin 2 arose prior to the origin of insects, and transferrins 1, 3, and 4 arose early in insect evolution. Primary sequence analysis of each of the insect transferrins was used to predict signal peptides, carboxyl-terminal transmembrane regions, GPI-anchors, and iron binding. Based on this analysis, we suggest that transferrins 2, 3, and 4 are unlikely to play a major role in iron homeostasis. In contrast, the transferrin 1 orthologs are predicted to be secreted, soluble, iron-binding proteins. We conclude that transferrin 1 orthologs are the most likely to play an important role in iron homeostasis. Interestingly, it appears that the louse, aphid, and thrips lineages have lost the transferrin 1 gene and, thus, have evolved to manage iron without transferrins.

Development of a new method for collecting hemolymph and measuring phenoloxidase activity in Tribolium castaneum.

OBJECTIVE: Hemolymph plays many important roles in the physiology of an insect throughout its lifetime; however, for small-bodied insects, studies are lacking because of the difficulties encountered while collecting hemolymph. The objective of our study was to develop a method to collect hemolymph plasma from various stages of Tribolium castaneum and to evaluate phenoloxidase activity in the plasma samples. We first designed a procedure for easily and quickly collecting clear hemolymph plasma from T. castaneum. RESULTS: By using this method, we collected approximately 5 µl plasma from 30 individuals at the larval, pupal or adult stages. And then, we studied the expression of phenoloxidase by performing western blot analysis of the plasma samples and found that phenoloxidase is present in hemolymph in each developmental stage. We also measured phenoloxidase activity in control plasma and plasma treated with Gram-positive bacteria, Micrococcus luteus. Phenoloxidase activity was greater in some of the M. luteus-treated plasma samples compared with control samples. Thus, we developed a method to collect hemolymph plasma that is suitable for studies of phenoloxidase activity.

Characterization of the multicopper oxidase gene family in Anopheles gambiae.

The multicopper oxidase (MCO) family of enzymes includes laccases, which oxidize a broad range of substrates including diphenols, and several oxidases with specific substrates such as iron, copper or ascorbic acid. We have identified five putative MCO genes in the genome of Anopheles gambiae and have cloned cDNAs encompassing the full coding region for each gene. MCO1 mRNA was detected in all developmental stages and in all of the larval and adult tissues tested. We observed an increase in MCO1 transcript abundance in the midguts and Malphighian tubules of adult females following a blood meal and in adult abdominal carcasses in response to an immune challenge. Two alternatively spliced isoforms of MCO2 mRNA were identified. The A isoform of MCO2 was previously detected in larval and pupal cuticle where it probably catalyzes sclerotization reactions (He, N., Botelho, J.M.C., McNall, R.J., Belozerov, V., Dunn, W.A., Mize, T., Orlando, R., Willis, J.H., 2007. Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry. Insect Biochem. Mol. Biol. 37, 135-146). The B isoform was transcriptionally upregulated in ovaries in response to a blood meal. MCO3 mRNA was detected in the adult midgut, Malpighian tubules, and male reproductive tissues; like MCO1, it was upregulated in response to an immune challenge or a blood meal. MCO4 and MCO5 were observed primarily in eggs and in the abdominal carcass of larvae. A phylogenetic analysis of insect MCO genes identified putative orthologs of MCO1 and MCO2 in all of the insect genomes tested, whereas MCO3, MCO4 and MCO5 were found only in the two mosquito species analyzed. MCO2 orthologs have especially high sequence similarity, suggesting that they are under strong purifying selection; the A isoforms are more conserved than the B isoforms. The mosquito specific group shares a common ancestor with MCO2. This initial study of mosquito MCOs suggests that MCO2 may be required for egg development or eggshell tanning in addition to cuticle tanning, while MCO1 and MCO3 may be involved in metal metabolism or immunity.

The Manduca sexta serpinome: Analysis of serpin genes and proteins in the tobacco hornworm.

Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and some viruses. They adopt a variety of physiological functions, including regulation of immune system, modulation of apoptosis, hormone transport and acting as storage proteins. Most members of the serpin family are inhibitors of serine proteinases. In this study, we searched the genome of Manduca sexta and identified 32 serpin genes. We analyzed the structure of these genes and the sequences of their encoded proteins. Three M. sexta genes (serpin-1, serpin-15, and serpin-28) have mutually exclusive alternatively spliced exons encoding the carboxyl-terminal reactive center loop of the protein, which is the site of interaction with target proteases. We discovered that MsSerpin-1 has 14 splicing isoforms, including two undiscovered in previous studies. Twenty-eight of the 32 M. sexta serpins include a putative secretion signal peptide and are predicted to be extracellular proteins. Phylogenetic analysis of serpins in M. sexta and Bombyx mori indicates that 17 are orthologous pairs, perhaps carrying out essential physiological functions. Analysis of the reactive center loop and hinge regions of the protein sequences indicates that 16 of the serpin genes encode proteins that may lack proteinase inhibitor activity. Our annotation and analysis of these serpin genes and their transcript profiles should lead to future advances in experimental study of their functions in insect biochemistry.

Model reactions for insect cuticle sclerotization: cross-linking of recombinant cuticular proteins upon their laccase-catalyzed oxidative conjugation with catechols.

The quinone-tanning hypothesis for insect cuticle sclerotization proposes that N-acylcatecholamines are oxidized by a phenoloxidase to quinones and quinone methides, which serve as electrophilic cross-linking agents to form covalent cross-links between cuticular proteins. We investigated model reactions for protein cross-linking that occurs during insect cuticle sclerotization using recombinant pupal cuticular proteins from the tobacco hornworm, Manduca sexta, fungal or recombinant hornworm laccase-type phenoloxidase, and the cross-linking agent precursor N-acylcatecholamines, N-beta-alanydopamine (NBAD) or N-acetyldopamine (NADA). Recombinant M. sexta pupal cuticular proteins MsCP36, MsCP20, and MsCP27 were expressed and purified to near homogeneity. Polyclonal antisera to these recombinant proteins recognized the native proteins in crude pharate brown-colored pupal cuticle homogenates. Furthermore, antisera to MsCP36, which contains a type-1 Rebers and Riddiford (RR-1) consensus sequence, also recognized an immunoreactive protein in homogenates of larval head capsule exuviae, indicating the presence of an RR-1 cuticular protein in a very hard, sclerotized and nonpigmented cuticle. All three of the proteins formed small and large oligomers stable to boiling SDS treatment under reducing conditions after reaction with laccase and the N-acylcatecholamines. The optimal reaction conditions for MsCP36 polymerization were 0.3mM MsCP36, 7.4mM NBAD and 1.0U/mul fungal laccase. Approximately 5-10% of the monomer reacted to yield insoluble oligomers and polymers during the reaction, and the monomer also became increasingly insoluble in SDS solution after reaction with the oxidized NBAD. When NADA was used instead of NBAD, less oligomer formation occurred, and most of the protein remained soluble. Radiolabeled NADA became covalently bound to the MsCP36 monomer and oligomers during cross-linking. Recombinant Manduca laccase (MsLac2) also catalyzed the polymerization of MsCP36. These results support the hypothesis that during sclerotization, insect cuticular proteins are oxidatively conjugated with catechols, a posttranslational process termed catecholation, and then become cross-linked, forming oligomers and subsequently polymers.

Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum.

Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress-response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails.

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta.

Manduca sexta, known as the tobacco hornworm or Carolina sphinx moth, is a lepidopteran insect that is used extensively as a model system for research in insect biochemistry, physiology, neurobiology, development, and immunity. One important benefit of this species as an experimental model is its extremely large size, reaching more than 10 g in the larval stage. M. sexta larvae feed on solanaceous plants and thus must tolerate a substantial challenge from plant allelochemicals, including nicotine. We report the sequence and annotation of the M. sexta genome, and a survey of gene expression in various tissues and developmental stages. The Msex_1.0 genome assembly resulted in a total genome size of 419.4 Mbp. Repetitive sequences accounted for 25.8% of the assembled genome. The official gene set is comprised of 15,451 protein-coding genes, of which 2498 were manually curated. Extensive RNA-seq data from many tissues and developmental stages were used to improve gene models and for insights into gene expression patterns. Genome wide synteny analysis indicated a high level of macrosynteny in the Lepidoptera. Annotation and analyses were carried out for gene families involved in a wide spectrum of biological processes, including apoptosis, vacuole sorting, growth and development, structures of exoskeleton, egg shells, and muscle, vision, chemosensation, ion channels, signal transduction, neuropeptide signaling, neurotransmitter synthesis and transport, nicotine tolerance, lipid metabolism, and immunity. This genome sequence, annotation, and analysis provide an important new resource from a well-studied model insect species and will facilitate further biochemical and mechanistic experimental studies of many biological systems in insects.

Annotation and expression analysis of cuticular proteins from the tobacco hornworm, Manduca sexta.

The insect cuticle is a unique material that covers the exterior of the animal as well as lining the foregut, hindgut, and tracheae. It offers protection from predators and desiccation, defines body shape, and serves as an attachment site for internal organs and muscle. It has demonstrated remarkable variations in hardness, flexibility and elasticity, all the while being light weight, which allows for ease of movement and flight. It is composed primarily of chitin, proteins, catecholamines, and lipids. Proteomic analyses of cuticle from different life stages and species of insects has allowed for a more detailed examination of the protein content and how it relates to cuticle mechanical properties. It is now recognized that several groups of cuticular proteins exist and that they can be classified according to conserved amino acid sequence motifs. We have annotated the genome of the tobacco hornworm, Manduca sexta, for genes that encode putative cuticular proteins that belong to seven different groups: proteins with a Rebers and Riddiford motif (CPR), proteins analogous to peritrophins (CPAP), proteins with a tweedle motif (CPT), proteins with a 44 amino acid motif (CPF), proteins that are CPF-like (CPFL), proteins with an 18 amino acid motif (18 aa), and proteins with two to three copies of a C-X5-C motif (CPCFC). In total we annotated 248 genes, of which 207 belong to the CPR family, the most for any insect genome annotated to date. Additionally, we discovered new members of the CPAP family and determined that orthologous genes are present in other insects. We established orthology between the M. sexta and Bombyx mori genes and identified duplication events that occurred after separation of the two species. Finally, we utilized 52 RNAseq libraries to ascertain gene expression profiles that revealed commonalities and differences between different tissues and developmental stages.

Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt.

In the insect cuticle, structural proteins (CPs) and the polysaccharide chitin are the major components. It has been hypothesized that CPs are cross-linked to other CPs and possibly to chitin by quinones or quinone methides produced by the laccase2-mediated oxidation of N-acylcatechols. In this study we investigated functions of TcCP30, the third most abundant CP in protein extracts of elytra (wing covers) from Tribolium castaneum adults. The mature TcCP30 protein has a low complexity and highly polar amino acid sequence. TcCP30 is localized with chitin in horizontal laminae and vertically oriented columnar structures in rigid cuticles, but not in soft and membranous cuticles. Immunoblot analysis revealed that TcCP30 undergoes laccase2-mediated cross-linking during cuticle maturation in vivo, a process confirmed in vitro using recombinant rTcCP30. We identified TcCPR27 and TcCPR18, the two most abundant proteins in the elytra, as putative cross-linking partners of TcCP30. RNAi for the TcCP30 gene had no effect on larval and pupal growth and development. However, during adult eclosion, ~70% of the adults were unable to shed their exuvium and died. These results support the hypothesis that TcCP30 plays an integral role as a cross-linked structural protein in the formation of lightweight rigid cuticle of the beetle.

Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity.

Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism.

Analysis of chitin-binding proteins from Manduca sexta provides new insights into evolution of peritrophin A-type chitin-binding domains in insects.

In insects, chitin is a major structural component of the cuticle and the peritrophic membrane (PM). In nature, chitin is always associated with proteins among which chitin-binding proteins (CBPs) are the most important for forming, maintaining and regulating the functions of these extracellular structures. In this study, a genome-wide search for genes encoding proteins with ChtBD2-type (peritrophin A-type) chitin-binding domains (CBDs) was conducted. A total of 53 genes encoding 56 CBPs were identified, including 15 CPAP1s (cuticular proteins analogous to peritrophins with 1 CBD), 11 CPAP3s (CPAPs with 3 CBDs) and 17 PMPs (PM proteins) with a variable number of CBDs, which are structural components of cuticle or of the PM. CBDs were also identified in enzymes of chitin metabolism including 6 chitinases and 7 chitin deacetylases encoded by 6 and 5 genes, respectively. RNA-seq analysis confirmed that PMP and CPAP genes have differential spatial expression patterns. The expression of PMP genes is midgut-specific, while CPAP genes are widely expressed in different cuticle forming tissues. Phylogenetic analysis of CBDs of proteins in insects belonging to different orders revealed that CPAP1s from different species constitute a separate family with 16 different groups, including 6 new groups identified in this study. The CPAP3s are clustered into a separate family of 7 groups present in all insect orders. Altogether, they reveal that duplication events of CBDs in CPAP1s and CPAP3s occurred prior to the evolutionary radiation of insect species. In contrast to the CPAPs, all CBDs from individual PMPs are generally clustered and distinct from other PMPs in the same species in phylogenetic analyses, indicating that the duplication of CBDs in each of these PMPs occurred after divergence of insect species. Phylogenetic analysis of these three CBP families showed that the CBDs in CPAP1s form a clearly separate family, while those found in PMPs and CPAP3s were clustered together in the phylogenetic tree. For chitinases and chitin deacetylases, most of phylogenetic analysis performed with the CBD sequences resulted in similar clustering to the one obtained by using catalytic domain sequences alone, suggesting that CBDs were incorporated into these enzymes and evolved in tandem with the catalytic domains before the diversification of different insect orders. Based on these results, the evolution of CBDs in insect CBPs is discussed to provide a new insight into the CBD sequence structure and diversity, and their evolution and expression in insects.

CREB isoform represses yolk protein gene expression in the mosquito fat body.

In mosquitoes, the steroid 20-hydroxyecdysone (20E) is the main regulator of yolk protein precursor (YPP) gene expression. However, peptide hormones have also been implicated. To investigate involvement of the cAMP-mediated signal-transduction cascade in regulation of mosquito vitellogenic events, we cloned an Aedes aegypti cAMP response element binding protein (AaCREB). The AaCREB contained the domains characteristic to members of the cAMP response element binding protein (CREB) family of transcription factors: a kinase inducible domain region and a bZIP domain responsible for DNA binding and protein dimerization. In the mosquito fat body (site of YPP gene expression), the AaCREB gene was constitutively expressed and produced a transcript of 3.5-4 kb. In vitro fat body organ culture experiments demonstrated that elicitors of the cAMP signal-transduction pathway attenuated 20E-stimulated YPP gene expression. Cell transfection analysis indicated that AaCREB served as a potent repressor of transcription (designated AaCREBr). The role of AaCREBr as a transcriptional repressor supported the electrophoretic mobility shift assay (EMSA) with nuclear extracts from vitellogenic fat bodies. This analysis detected CREB-specific band-shift complexes in nuclear extracts at 24 and 36 h post-blood meal (PBM), when YPP gene expression reaches its peak then terminates. Examination of the regulatory regions of two major YPP genes, vitellogenin (Vg) and vitellogenic carboxypeptidase (VCP), revealed the presence of putative CREB response elements (CREs). These elements competed with the CRE consensus sequence for binding of in vitro-expressed AaCREBr. We propose that AaCREBr functions as a repressor of YPP gene expression at the time of vitellogenesis termination in the fat body.

Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta.

Glycosyltransferases are enzymes that synthesize oligosaccharides, polysaccharides and glycoconjugates. One type of glycosyltransferase is chitin synthase, a very important enzyme in biology, which is utilized by insects, fungi, and other invertebrates to produce chitin, a polysaccharide of beta-1,4-linked N-acetylglucosamine. Chitin is an important component of the insect's exoskeletal cuticle and gut lining. To identify and characterize a chitin synthase gene of the tobacco hornworm, Manduca sexta, degenerate primers were designed from two highly conserved regions in fungal and nematode chitin synthase protein sequences and then used to amplify a similar region from Manduca cDNA. A full-length cDNA of 5152 nucleotides was assembled for the putative Manduca chitin synthase gene, MsCHS1, and sequencing of genomic DNA verified the contiguity of the sequence. The MsCHS1 cDNA has an ORF of 4692 nucleotides that encodes a transmembrane protein of 1564 amino acid residues with a mass of approximately 179 kDa (GenBank no. AY062175). It is most similar, over its entire length of protein sequence, to putative chitin synthases from other insects and nematodes, with 68% identity to enzymes from both the blow fly, Lucilia cuprina, and the fruit fly, Drosophila melanogaster. The similarity with fungal chitin synthases is restricted to the putative catalytic domain, and the MsCHS1 protein has, at equivalent positions, several amino acids that are essential for activity as revealed by mutagenesis of the fungal enzymes. A 5.3-kb transcript of MsCHS1 was identified by northern blot hybridization of RNA from larval epidermis, suggesting that the enzyme functions to make chitin deposited in the cuticle. Further examination by RT-PCR showed that MsCHS1 expression is regulated in the epidermis, with the amount of transcript increasing during phases of cuticle deposition.