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Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1-specific RNA interference (RNAi) disrupted all three types of moult (larval–larval, larval–pupal and pupal–adult) and greatly reduced whole-body chitin content. Exon-specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval-pupal and pupal-adult moults, whereas splice variant 8b was required only for the latter. TcCHS2-specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi-mediated down-regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut. [ABSTRACT FROM AUTHOR]

The insect exoskeleton or cuticle is a complex extracellular matrix consisting of several functional layers. Cuticular proteins (CPs) and the polysaccharide chitin are the major components of the exo- and endocuticular layers or procuticle. During cuticle tanning (sclerotization and pigmentation), CPs are cross-linked by quinones or quinone methides produced by oxidation of catechols catalyzed by the phenoloxidase, laccase. CPs contain a conserved sequence known as the Rebers & Riddiford (R&R) motif, which may function as a chitin-binding domain that helps to coordinate the interaction between chitin fibers and the protein network. More than 100 CP-like genes have been identified in Drosophila melanogaster, with a similar number present in the red flour beetle, Tribolium castaneum. Different CPs have been found in the different developmental stages and in different regions of the exoskeleton, indicating that specific CPs are required to produce a cuticle with the proper morphology and mechanical properties. To study the function(s) of insect CPs, we focused on adult elytra of the T. castaneum. In protein samples extracted from elytra of newly emerged adults, yielding two highly abundant proteins with apparent sizes of 10 and 20 kDa. To characterize these major proteins further, each was digested with trypsin, and the resulting peptides were analyzed by MALDI-TOF mass spectrometry. Comparison of this result with conceptual trypsinization of the computed proteome of Tribolium identified two candidate genes, which we call TcCP10 and TcCP20. We cloned the full-length cDNAs corresponding to these genes. Both proteins belong to the RR-2 family (cuticle DB, ) and possess a putative signal peptide sequence. TcCP20 is an ortholog of the Tenebrio adult cuticle protein, acp22. Both TcCP10 and TcCP20 were mapped to linkage group 3. The expression patterns revealed by real-time PCR showed that the transcript levels of both genes dramatically increase at the pharate adult stage (1 -2 days before molting), and then decline after adult eclosion. RNA interference (RNAi) was used to investigate the functions of TcCP10 and TcCP20. Specific dsRNAs for each gene were injected into last-instar larvae. Following dsRNA injections, expression of TcCP10 and TcCP20 genes was analyzed by real-time PCR and SDS PAGE to evaluate transcript and protein levels. Both genes were substantially and specifically down-regulated at the mRNA and protein levels after RNAi treatment. Injection of ds TcCP10 or ds TcCP20 had no effect on larval-larval, larval-pupal or pupal-adult molts. The elytra of the resulting adults, however, were malformed and abnormal. Elytra of ds TcCP20 adults did not fully elongate and did not extend far enough to cover the abdomen. These elytra were slightly separated and not smooth as compared to those of control insects. Like the ds TcCP20 phenotype, the resulting adults obtained after injection ds TcCP10 also exhibited elytral defects, but they were more severe. These elytra were short, wrinkled, bumpy, warped and fenestrated. CP10-deficient insects could not fold their hind wings properly and eventually died approximately 7 days after eclosion, probably because of dehydration. TcCP10 and TcCP20 were also extracted from the pronotum cuticle of Tribolium. These results demonstrate that two cuticle proteins, TcCP10 and TcCP20, play critical roles in adult cuticle formation of T. castaneum. [ABSTRACT FROM AUTHOR]

Pigmentation/melanization is an important physiological event in insect development and is involved in cuticle tanning, wound healing and encapsulation as a defensive response as well as in egg chorion hardening. 'Yellow' protein (dopachrome conversion enzyme, DCE) is involved in the melanin biosynthetic pathway and significantly accelerates pigmentation and melanization reactions. Recent studies have suggested that yellow is a rapidly evolving gene family generating functionally diverse paralogs. However, the exact physiological functions of several yellow genes are still not understood. Tribolium castaneum ( Tc) is an excellent experimental insect to determine the roles of individual yellow genes because it is possible to carry out RNA interference (RNAi) experiments by injecting gene-specific double-stranded RNAs (dsRNAs) into insects at all developmental stages. Injection of dsRNAs for Tcyellow-e ( TcY-e) into last instar larvae had no effect on larval-pupal molting, and the resulting pupae developed normally. The pupal cuticle including the setae, gin traps and urogomphi showed normal tanning. Adult cuticle could also be viewed through the translucent pupal cuticle. Adult cuticle tanning including the head, mandibles and legs was initiated on schedule (pupal day 4-5), indicating that TcY-e is not required for pupal or adult cuticle pigmentation in T. castaneum. The subsequent pupal-adult molt, however, was adversely affected. Although pupal cuticle apolysis and slippage were evident, some of the adults (∼20%) were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults rapidly became completely desiccated. Failure of the pupal-adult molt and desiccation-induced mortality were prevented by maintaining dsRNA TcY-e-treated insects at 100% relative humidity (rh). When the humidity-rescued adults were subsequently moved from 100% rh to 50% rh, they quickly desiccated and died (wild-type insects thrive throughout development at 50% rh). These results suggest that TcY-e has a 'critical waterproofing' function for cuticle rather than cuticle pigmentation. [ABSTRACT FROM AUTHOR]

Insect cuticle tanning (sclerotization and pigmentation) is a complex process that involves the production of quinones and quinone methides from catechols, followed by their oxidative conjugation with cuticular proteins, a process that leads to cuticle hardening and darkening. Using RNA interference (RNAi) methodology, we previously demonstrated that laccase 2 is the gene encoding the enzyme catalyzing cuticle tanning in the red flour beetle, T. castaneum. By searching Beetlebase, we identified several genes that are probably involved in the synthesis of catechols that are potential laccase 2 substrates, such as tyrosine hydroxylase ( TH), dopa decarboxylase ( DDC), dopamine N-acetyltransferase ( NAT) and aspartate α-decarboxylase ( ADC/black). To further clarify the metabolic pathway responsible for cuticle tanning and to determine the influence of different catechols on sclerotization and pigmentation, double stranded RNAs (dsRNAs) for each of those genes were injected into Tribolium and the resulting phenotypes analyzed in terms of morphology, pigmentation, and mRNA levels. [ABSTRACT FROM AUTHOR]

Abstract: Chymotrypsin-like peptidases (CTLPs) of insects are primarily secreted into the gut lumen where they act as digestive enzymes. We studied the gene family encoding CTLPs in the genome of the red flour beetle, Tribolium castaneum. Using an extended search pattern, we identified 14 TcCTLP genes that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. We further analyzed the expression patterns of seven TcCTLP genes at various developmental stages. While some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A), others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP-6C/D/E). TcCTLP genes were expressed predominantly in the midgut, where they presumably function in digestion. However, TcCTLP-6C and TcCTLP-5C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, we performed western blots using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6E suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, we performed systemic RNAi experiments. While injections of five TcCTLP-dsRNAs into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects. [Copyright &y& Elsevier]

Aspartate 1-decarboxylase (ADC) and 3,4-dihydroxyphenylalanine decarboxylase (DDC) provide β-alanine and dopamine used in insect cuticle tanning. β-Alanine is conjugated with dopamine to yield N-β-alanyldopamine (NBAD), a substrate for the phenol oxidase laccase that catalyzes the synthesis of cuticle protein cross-linking agents and pigment precursors. We identified ADC and DDC genes in the red flour beetle, Tribolium castaneum (Tc), and investigated their functions. TcADC mRNA was most abundant prior to the pupal-adult molt. Injection of TcADC double-stranded (ds) RNA (ds TcADC) into mature larvae resulted in depletion of NBAD in pharate adults, accumulation of dopamine, and abnormally dark pigmentation of the adult cuticle. Injection of β-alanine, the expected product of ADC, into dsTcADC-treated pupae rescued the pigmentation phenotype, resulting in normal rust-red color. A similar pattern of catechol content consisting of elevated dopamine and depressed NBAD was observed in the genetic black mutants of Tribolium, in which levels of TcADC mRNA were drastically reduced. Furthermore, from the Tribolium black mutant and dsTcADC-injected insects both exhibited similar changes in material properties. Dynamic mechanical analysis of elytral cuticle from beetles with depleted TcADC transcripts revealed diminished cross-linking of cuticular components, further confirming the important role of oxidation products of NBAD as cross-linking agents during cuticle tanning. Injection of dsTcDDC into larvae produced a lethal pupal phenotype, and the resulting grayish pupal cuticle exhibited many small patches of black pigmentation. When dsTcDDC was injected into young pupae, the resulting adults had abnormally dark brown body color, but there was little mortality. Injection of dsTcDDC resulted in more than a 5-fold increase in levels of DOPA, indicating that lack of TcDDC led to accumulation of its substrate, DOPA. [ABSTRACT FROM AUTHOR]

The insect exoskeleton is composed of cuticle primarily formed from structural cuticular proteins (CPs) and the polysaccharide chitin. Two CPs, TcCPR27 and TcCPR18, are major proteins present in the elytron (highly sclerotized and pigmented modified forewing) as well as the pronotum (dorsal sclerite of the prothorax) and ventral abdominal cuticle of the red flour beetle, Tribolium castaneum . Both CPs belong to the CPR family, which includes proteins that have an amino acid sequence motif known as the Rebers & Riddiford (R&R) consensus sequence. Injection of double-stranded RNA (dsRNA) for TcCPR27 and TcCPR18 resulted in insects with shorter, wrinkled, warped and less rigid elytra than those from control insects. To gain a more comprehensive understanding of the roles of CPs in cuticle assembly, we analyzed for the precise localization of TcCPR27 and the ultrastructural architecture of cuticle in TcCPR27- and TcCPR18-deficient elytra. Transmission electron microscopic analysis combined with immunodetection using gold-labeled secondary antibody revealed that TcCPR27 is present in dorsal elytral procuticle both in the horizontal laminae and in vertical pore canals. dsRNA-mediated RNA interference (RNAi) of TcCPR27 resulted in abnormal electron-lucent laminae and pore canals in elytra except for the boundary between these two structures in which electron-dense molecule(s) apparently accumulated. Insects subjected to RNAi for TcCPR18 also had disorganized laminae and pore canals in the procuticle of elytra. Similar ultrastructural defects were also observed in other body wall regions with rigid cuticle such as the thorax and legs of adult T. castaneum . TcCPR27 and TcCPR18 are required for proper formation of the horizontal chitinous laminae and vertical pore canals that are critical for formation and stabilization of rigid adult cuticle. [ABSTRACT FROM AUTHOR]

Abstract: A bioinformatics search of the genome of the red flour beetle, Tribolium castaneum, resulted in the identification of two genes encoding proteins closely related to UDP-N-acetylglucosamine pyrophosphorylases (UAPs), which provide the activated precursor, UDP-N-acetylglucosamine, for the synthesis of chitin, glycoproteins and glycosylphosphoinositide (GPI) anchors of some membrane proteins as well as for the modification of other substrates. This is in contrast to other arthropods whose genomes have been completely sequenced, all of which have only a single copy of this gene. The two T. castaneum UAP genes, TcUAP1 and TcUAP2, share both nucleotide and amino acid sequence identities of about 60%. RT-PCR analysis revealed that the two genes differ in their developmental and tissue-specific patterns of expression. RNA interference (RNAi) indicated roles for TcUAP1 and TcUAP2 at the molt and intermolt stages, respectively: RNAi for TcUAP1 resulted in specific arrest at the larval-larval, larval-pupal or pupal-adult molts, depending on time of injection of double-stranded RNAs, whereas RNAi for TcUAP2 prevented larval growth or resulted in pupal paralysis. Analysis of elytral cuticle indicated loss of structural integrity and chitin staining after RNAi for TcUAP1, but not after RNAi for TcUAP2. Loss of peritrophic matrix (PM)-associated chitin was also observed following RNAi for TcUAP1, but not after RNAi for TcUAP2. Down-regulation of transcripts for either TcUAP gene at the mature adult stage resulted in cessation of oviposition in females, as well as fat body depletion and eventual death in both sexes. These results demonstrate that both TcUAP genes are critical for beetle development and survival, but that only TcUAP1 is clearly associated with synthesis of cuticular or PM chitin. However, both of these genes appear to have additional critical role(s) unrelated to chitin synthesis, presumably in the glycosylation of proteins and/or secondary metabolites. [ABSTRACT FROM AUTHOR]

Abstract: Cuticle tanning in insects involves simultaneous cuticular pigmentation and hardening or sclerotization. The dynamic mechanical properties of the highly modified and cuticle-rich forewings (elytra) from Tribolium castaneum (red flour beetle) wild-type and body color mutant strains were investigated to relate body coloration and elytral mechanical properties. There was no statistically significant variation in the storage modulus E′ among the elytra from jet, cola, sooty and black mutants or between the mutants and the wild-type GA-1 strain: E′ averaged 5.1±0.6GPa regardless of body color. E′ is a power law function of oscillation frequency for all types. The power law exponent, n, averaged 0.032±0.001 for elytra from all genotypes except black; this small value indicated that the elytra are cross-linked. Black elytra, however, displayed a significantly larger n of 0.047±0.004 and an increased loss tangent (tan δ), suggesting that metabolic differences in the black mutant strain result in elytra that are less cross-linked and more pigmented than the other types. These results are consistent with the hypothesis that black elytra have a β-alanine-deficient and dopamine-abundant metabolism, leading to greater melanin (black pigment) production, probably at the expense of cross-linking of cuticular proteins mediated by N-β-alanyldopamine quinone. [ABSTRACT FROM AUTHOR]

Abstract: Querying the genome of the red flour beetle, Tribolium castaneum, with the Drosophila melanogaster Yellow-y (DmY-y) protein sequence identified 14 Yellow homologs. One of these is an ortholog of DmY-y, which is required for cuticle pigmentation (melanization), and another is an ortholog of DmY-f/f2, which functions as a dopachrome conversion enzyme (DCE). Phylogenetic analysis identified putative T. castaneum orthologs for eight of the D. melanogaster yellow genes, including DmY-b, -c, -e, -f, -g, -g2, -h and -y. However, one clade of five beetle genes, TcY-1-5, has no orthologs in D. melanogaster. Expression profiles of all T. castaneum yellow genes were determined by RT-PCR of pharate pupal to young adult stages. TcY-b and TcY-c were expressed throughout all developmental stages analyzed, whereas each of the remaining yellow genes had a unique expression pattern, suggestive of distinct physiological functions. TcY-b, -c and -e were all identified by mass spectrometry of elytral proteins from young adults. Eight of the 14 genes showed differential expression between elytra and hindwings during the last three days of the pupal stage when the adult cuticle is synthesized. Double-stranded RNA (dsRNA)-mediated transcript knockdown revealed that TcY-y is required for melanin production in the hindwings, particularly in the region of the pterostigma, while TcY-f appears to be required for adult cuticle sclerotization but not pigmentation. [Copyright &y& Elsevier]

Abstract: This study is focused on the characterization and expression of genes in the red flour beetle, Tribolium castaneum, encoding proteins that possess one or more six-cysteine-containing chitin-binding domains related to the peritrophin A domain (ChtBD2). An exhaustive bioinformatics search of the genome of T. castaneum queried with ChtBD2 sequences yielded 13 previously characterized chitin metabolic enzymes and 29 additional proteins with signal peptides as well as one to 14 ChtBD2s. Using phylogenetic analyses, these additional 29 proteins were classified into three large families. The first family includes 11 proteins closely related to the peritrophins, each containing one to 14 ChtBD2s. These are midgut-specific and are expressed only during feeding stages. We propose the name “Peritrophic Matrix Proteins” (PMP) for this family. The second family contains eight proteins encoded by seven genes (one gene codes for 2 splice variants), which are closely related to gasp/obstructor-like proteins that contain 3 ChtBD2s each. The third family has ten proteins that are of diverse sizes and sequences with only one ChtBD2 each. The genes of the second and third families are expressed in non-midgut tissues throughout all stages of development. We propose the names “Cuticular Proteins Analogous to Peritophins 3” (CPAP3) for the second family that has three ChtBD2s and “Cuticular Proteins Analogous to Peritophins 1 (CPAP1) for the third family that has 1 ChtBD2. Even though proteins of both CPAP1 and CPAP3 families have the “peritrophin A” domain, they are expressed only in cuticle-forming tissues. We determined the exon–intron organization of the genes, encoding these 29 proteins as well as the domain organization of the encoded proteins with ChtBD2s. All 29 proteins have predicted cleavable signal peptides and ChtBD2s, suggesting that they interact with chitin in extracellular locations. Comparison of ChtBD2s-containing proteins in different insect species belonging to different orders suggests that ChtBD2s are ancient protein domains whose affinity for chitin in extracellular matrices has been exploited many times for a range of biological functions. The differences in the expression profiles of PMPs and CPAPs indicate that even though they share the peritrophin A motif for chitin binding, these three families of proteins have quite distinct biological functions. [Copyright &y& Elsevier]

Abstract: The expression profiles of nine genes encoding chitin deacetylase (CDA)-like proteins were studied during development and in various tissues of the red flour beetle, Tribolium castaneum, by RT-PCR. TcCDA1, TcCDA2 and TcCDA5 were expressed throughout all stages of development, while TcCDA6–9 were expressed predominantly during larval feeding stages. In situ hybridization experiments revealed that both TcCDA1 and TcCDA2 were expressed in epidermal cells. Polyclonal antibody to TcCDA1 detected an immunoreactive protein in larval tracheae. TcCDA6 through TcCDA9, which belong to a distinct subgroup of gut-specific CDAs, were transcribed in the cells lining the midgut, including epithelial cells. TcCDA3 was expressed in the thoracic muscles, whereas TcCDA4 was expressed in early imaginal appendages. To study the function(s) of individual TcCDA genes, double-stranded RNAs (dsRNA) specific for each gene were injected into insects at different developmental stages and the phenotypes were monitored. No visible phenotypic changes were observed after injection of dsRNAs for TcCDA3 to 9, whereas injection of dsRNAs for TcCDA1 or TcCDA2 affected all types of molts, including larval–larval, larval–pupal and pupal–adult. Insects treated with these dsRNAs could not shed the old cuticle and were trapped in their exuviae. Interestingly, unique and very dissimilar adult phenotypes were observed after injection of dsRNAs that specifically down-regulated either of the two alternatively spliced transcripts of TcCDA2, namely TcCDA2a or TcCDA2b. These results reveal functional specialization among T. castaneum CDA genes and splice variants. [Copyright &y& Elsevier]

Abstract: The synthesis of chitin, the β-1,4-linked polymer of N-acetylglucosamine, is catalyzed by chitin synthase (CHS). Chitin is essential for the structural integrity of the exoskeletal cuticle and midgut peritrophic membrane (PM) of insects. To study the functions of the two chitin synthase genes, TcCHS-A and TcCHS-B, during embryonic and adult development in the red flour beetle, Tribolium castaneum, RNA interference (RNAi) experiments were carried out. When dsRNA for TcCHS-A was injected into male or female pharate adults, all insects died 5–7 d after the adult molt, and the females failed to oviposit prior to death. When dsTcCHS-A was injected into young adults 1–2 d post-eclosion, a similar lethal phenotype was obtained after 5 d and no oviposition occurred. When dsTcCHS-A injections were delayed until after adult maturation (7–10 d post-eclosion), the treated females did oviposit and the resulting embryos appeared to develop normally. However, the chitin content of the eggs was dramatically reduced, the embryos became twisted and enlarged, and the eggs did not hatch. Adults treated with dsRNA for TcCHS-B exhibited little or no chitin in their PM and died about 2 wk after injection. None of the TcCHS-B-treated females oviposited, which was probably a secondary effect caused by starvation. These results extend our previous findings that CHS genes are required for all types of molt. The present study also demonstrates that these genes have additional roles in embryonic and adult development. [Copyright &y& Elsevier]

Abstract: Enzymes belonging to the β-N-acetylhexosaminidase family cleave chitin oligosaccharides produced by the action of chitinases on chitin into the constituent N-acetylglucosamine monomer. Four genes encoding putative chitooligosaccharidolytic β-N-acetylhexosaminidases (hereafter referred to as N-acetylglucosaminidases (NAGs)) in the red flour beetle, Tribolium castaneum, namely TcNAG1, TcFDL, TcNAG2, and TcNAG3, and three other related hexosaminidases were identified by searching the recently completed genome [Tribolium Genome Sequencing Consortium, 2007. The first genome sequence of a beetle, Tribolium castaneum, a model for insect development and pest biology. Nature, submitted for publication]. Full-length cDNAs for all four NAGs were cloned and sequenced, and the exon–intron organization of the corresponding genes was determined. Analyses of their developmental expression patterns indicated that, although all four of the NAGs are transcribed during most developmental stages, each gene had a distinct spatial and temporal expression pattern. TcNAG1 transcripts are the most abundant, particularly at the late pupal stage, while TcNAG3 transcripts are the least abundant, even at their peak levels in the late larval stages. The function of each NAG during different developmental stages was assessed by observations of lethal phenotypes after gene-specific double-stranded RNA (dsRNA)-mediated transcript depletion as verified by real-time PCR. TcNAG1 dsRNA was most effective in interrupting all three types of molts: larval–larval, larval–pupal, and pupal–adult. Treated insects died after failing to completely shed their old cuticles. Knockdown of transcripts for the other three NAG genes resulted in phenotypes similar to those of TcNAG1 dsRNA-treated insects, but the effects were somewhat variable and less severe. Sequence comparisons with other enzymatically characterized insect homologs suggested that TcFDL, unlike the other NAGs, may have a role in N-glycan processing in addition to its apparent role in cuticular chitin turnover. These results support the hypothesis that TcNAGs participate in chitin turnover and/or N-glycan processing during insect development and that each NAG fulfills an essential and distinct function. [Copyright &y& Elsevier]

Abstract: A bioinformatics-based investigation of three insect species with completed genome sequences has revealed that insect chitinase-like proteins (glycosylhydrolase family 18) are encoded by a rather large and diverse group of genes. We identified 16, 16 and 13 putative chitinase-like genes in the genomic databases of the red flour beetle, Tribolium castaneum, the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, respectively. Chitinase-like proteins encoded by this gene family were classified into five groups based on phylogenetic analyses. Group I chitinases are secreted proteins that are the most abundant such enzymes in molting fluid and/or integument, and represent the prototype enzyme of the family, with a single copy each of the catalytic domain and chitin-binding domain (ChBD) connected by an S/T-rich linker polypeptide. Group II chitinases are unusually larger-sized secreted proteins that contain multiple catalytic domains and ChBDs. Group III chitinases contain two catalytic domains and are predicted to be membrane-anchored proteins. Group IV chitinases are the most divergent. They usually lack a ChBD and/or an S/T-rich linker domain, and are known or predicted to be secreted proteins found in gut or fat body. Group V proteins include the putative chitinase-like imaginal disc growth factors (IDGFs). In each of the three insect genomes, multiple genes encode group IV and group V chitinase-like proteins. In contrast, groups I–III are each represented by only a singe gene in each species. [Copyright &y& Elsevier]

Abstract: Chitin, the linear homopolymer of β-1,4-linked N-acetylglucosamine, is produced by the enzyme chitin synthase (CHS). In general, this insoluble polysaccharide is found in two major extracellular structures in insects, the cuticle that overlays the epidermis and the peritrophic membrane (PM) that lines the midgut. Based on amino acid sequence similarities, insect CHSs are divided into two classes, A and B, and to date no more than two CHS genes have been identified in any single insect species. In species where both CHSs have been identified, one class A CHS and one class B CHS are always present. This finding suggests that these two genes may encode enzymes that synthesize chitin in different epithelial tissues. In our laboratory, we previously characterized transcripts for a class A CHS gene (MsCHS1) from the tobacco hornworm, Manduca sexta. We observed the expression of this gene in the larval epidermis, suggesting that the encoded enzyme functions to synthesize cuticular chitin. In this paper, we characterize a second chitin synthase gene (MsCHS2) belonging to class B and its cDNA from Manduca and show that it is expressed only in the midgut. This cDNA contains an open reading frame of 4575 nucleotides, which encodes a conceptual protein that is 1524 amino acids in length and is predicted to contain 16 transmembrane spans. Northern blot analysis of RNA isolated from anterior, medial, and posterior sections of the midgut from feeding larvae indicate that MsCHS2 is primarily expressed in the anterior midgut, with transcript levels tapering off in the medial and posterior midgut. Analysis of the MsCHS2 gene sequence indicates the absence of an alternate exon in contrast to the MsCHS1 gene, which yields two transcripts, MsCHS1a and MsCHS1b. RT-PCR analysis of the differential expression of these alternately spliced transcripts reveals that both splice variants are present in the epidermis. However, the ratio of the two alternately spliced transcripts varies during development, with MsCHS1a being generally more predominant. Southern blot analysis using a probe specific for CHS indicated that Manduca has only two CHS genes, akin to other insect species. Results from an analysis of expression of both genes in different tissues and developmental times indicate that the MsCHS1 enzyme is used for the synthesis of chitin in the cuticle and tracheae, whereas MsCHS2 is utilized exclusively for the synthesis of PM-associated chitin in the midgut. [Copyright &y& Elsevier]

Two chitin synthase (CHS) genes of the red flour beetle, Tribolium castaneum, were sequenced and their transcription patterns during development examined. By screening a BAC library of genomic DNA from T. castaneum (Tc) with a DNA probe encoding the catalytic domain of a putative Tribolium CHS, several clones that contained CHS genes were identified. Two distinct PCR products were amplified from these BAC clones and confirmed to be highly similar to CHS genes from other insects, nematodes and fungi. The DNA sequences of these genes, TcCHS1 and TcCHS2, were determined by amplification of overlapping PCR fragments from two of the BAC DNAs and mapped to different linkage groups. Each ORF was identified and full-length cDNAs were also amplified, cloned and sequenced. TcCHS1 and TcCHS2 encode transmembrane proteins of 1558 and 1464 amino acids, respectively. The TcCHS1 gene was found to use alternate exons, each encoding 59 amino acids, a feature not found in the TcCHS2 gene. During development, Tribolium expressed TcCHS1 predominantly in the embryonic and pupal stages, whereas TcCHS2 was prevalent in the late larval and adult stages. The alternate exon 8a of TcCHS1 was utilized over a much broader range of development than exon 8b. We propose that the two isoforms of the TcCHS1 enzyme are used predominantly for the formation of chitin in embryonic and pupal cuticles, whereas TcCHS2 is utilized primarily for the synthesis of peritrophic membrane-associated chitin in the midgut. [Copyright &y& Elsevier]