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.

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.

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.

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.

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.

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.

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.

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.

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.

Model reactions for insect cuticle sclerotization: participation of amino groups in the cross-linking of Manduca sexta cuticle protein MsCP36.

Current theories of sclerotization center on protein cross-linking and dehydration as major factors in the hardening and stability of the insect cuticle. Several studies have reported the identification of catechol-amino acid adducts from sclerotizing cuticle involving histidine, lysine, and tyrosine, though there have been no reports of a catechol linked between two amino acid residues. Previously, we reported an in vitro model system for sclerotization and observed that stable protein oligomers were formed, presumably through cross-links with oxidized catecholamines [Insect Biochem. Mol. Biol. (2006) 36, 353-365]. Using site-directed mutagenesis we created a mutant lacking histidine, rMsCP36(H65A/H178A), to investigate the possible involvement of the two histidine residues of MsCP36 in cross-linking. Surprisingly, this alteration had little or no effect on the formation of protein oligomers as determined by SDS-PAGE analysis. Blocking of the free amino groups in lysyl side chains and the amino-terminus by succinylation diminished, but did not eliminate, cross-linking of either rMsCP36 or rMsCP36(H65A/H178A). We also examined the possibility that cross-linking was due to intermolecular dityrosine linkages. Immunoblot analysis utilizing a monoclonal antibody known to recognize peptidyl dityrosine indicated that dityrosyl cross-links were present. Taken together, these results indicate that lysyl residues are important for the cross-linking of the cuticle protein rMsCP36, but that additional residues other than histidine can also contribute.

Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm, Manduca sexta.

Laccases belong to the group of multicopper oxidases that exhibit wide substrate specificity for polyphenols and aromatic amines. They are found in plants, fungi, bacteria, and insects. In insects the only known role for laccase is in cuticle sclerotization. However, extracting laccase from the insect's cuticle requires proteolysis, resulting in an enzyme that is missing its amino-terminus. To circumvent this problem, we expressed and purified full-length and amino-terminally truncated recombinant forms of laccase-2 from the tobacco hornworm, Manduca sexta. We also purified the endogenous enzyme from the pharate pupal cuticle and used peptide mass fingerprinting analysis to confirm that it is laccase-2. All three enzymes had pH optima between 5 and 5.5 when using N-acetyldopamine (NADA) or N-beta-alanyldopamine-alanyldopamine (NBAD) as substrates. The laccases exhibited typical Michaelis-Menten kinetics when NADA was used as a substrate, with K(m) values of 0.46 mM, 0.43 mM, and 0.63 mM, respectively, for the full-length recombinant, truncated recombinant, and cuticular laccases; the apparent k(cat) values were 100 min(-1), 80 min(-1), and 290 min(-1). The similarity in activity of the two recombinant laccases suggests that laccase-2 is expressed in an active form rather than as a zymogen, as had been previously proposed. This conclusion is consistent with the detection of activity in untanned pupal wing cuticle using the laccase substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Immunoblot analysis of proteins extracted from both tanned and untanned cuticle detected only a single protein of 84 kDa, consistent with the full-length enzyme. With NBAD as substrate, the full-length recombinant and cuticular laccases showed kinetics indicative of substrate inhibition, with K(m) values of 1.9 mM and 0.47 mM, respectively, and apparent k(cat) values of 200 min(-1) and 180 min(-1). These results enhance our understanding of cuticle sclerotization, and may aid in the design of insecticides targeting insect laccases.

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.

Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae.

Laccase (EC 1.10.3.2) is an enzyme with p-diphenol oxidase activity that is a member of a group of proteins collectively known as multicopper, or blue copper, oxidases. Laccase is hypothesized to play an important role in insect cuticle sclerotization by oxidizing catechols in the cuticle to their corresponding quinones, which then catalyze protein cross-linking reactions. To facilitate studies of the structure, function and regulation of insect laccases, we have cloned two cDNAs for laccases from the tobacco hornworm, Manduca sexta (MsLac1 and 2), and one from the malaria mosquito, Anopheles gambiae (AgLac1). The MsLac1 and 2 cDNAs encode proteins of 801 amino acids (aa) and 760 aa, respectively, while the AgLac1 cDNA encodes a protein of 1009 aa. All three cDNAs contain putative secretion signal sequences, and the 10 histidines and one cysteine that form the copper-binding centers, as well as a methionine in the T1 copper center. Novel to the insect laccases, relative to both fungal and plant laccases, is a longer amino-terminal sequence characterized by a unique domain consisting of several conserved cysteine, aromatic, and charged residues. Northern blot analyses identified single transcripts of approximately 3.6, 3.5, and 4.4 kb for MsLac1, MsLac2, and AgLac1, respectively, and also showed that AgLac1 was expressed in all life stages of the mosquito. RT-PCR revealed that the MsLac1 transcript was most abundant in the midgut, Malpighian tubules, and epidermis, whereas the MsLac2 transcript was most abundant in the epidermis. MsLac2 showed strong expression in the pharate pupal and reduced expression in the early pupal epidermis, consistent with the laccases' presumed role in cuticle sclerotization.

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.