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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]

Abstract: Ion transport peptide (ITP) and ITP-like (ITPL) are highly conserved neuropeptides in insects and crustaceans. We investigated the alternatively spliced variants of ITP/ITPL in Tribolium castaneum to understand their functions. We identified three alternatively spliced transcripts named itp, itpl-1, and itpl-2. Expression patterns of the splice variants investigated by exon-specific in situ hybridization were somewhat different from those previously reported in other insect species. Most importantly, we found for the first time that itpl-1 transcripts are abundantly expressed in the midgut at the late larval stage, showing an expression pattern similar to that of the crustacean hyperglycemic hormone (CHH) in the crab Carcinus maenas. CHH was shown to function by increasing the body volume through fluid absorption, resulting in breakage of the outer shell at the time of molt. Exon-specific RNA interference (RNAi) was designed to distinguish between itp and itpl-1, but we were unable to design a dsRNA uniquely targeting or uniquely excluding itpl-2; therefore, RNAi targeting was limited to either itp/itpl-2 or itpl-1/itpl-2. For dsRNA injections in the larval stages, either RNAi led to gradually increasing mortality in the larval and pupal stages, with 100% cumulative mortality at the time of eclosion or shortly afterward. Developmental deficiencies in the adult tarsal segments were observed after RNAi suppressing either itp/itpl-2 or itpl-1/itpl-2. After dsRNA injections at the pupal stage, the most striking observation was a significant reduction in egg numbers (8% of control) and reduced survival of the offspring (5%) in RNAi targeting itpl-1/itpl-2, while a milder degree of the same phenotype was observed in that targeting itp/itpl-2. [Copyright &y& Elsevier]

Abstract: Insect chitinase (CHT) family proteins are encoded by as many as 16 genes depending upon the species of interest. We have classified these proteins in three species into five different groups based on amino acid sequence similarities (Zhu et al., companion paper). The functions of most of the individual proteins of this family during growth and development are largely unknown. To help determine their enzymatic properties and physiological roles, we expressed representative members belonging to this protein family from Drosophila melanogaster (Dm) and Tribolium castaneum (Tc), and characterized their kinetic and carbohydrate-binding properties. Seven proteins, including DmCHT 4, 5, 9 and DmDS47 from Drosophila, and TcCHT5, TcIDGF2 and TcIDGF4 from Tribolium, belonging to groups I, IV or V of the chitinase-like family were expressed in a baculovirus-insect cell line expression system, purified and characterized. Their enzymatic and chitin-binding properties were compared to those of the well-characterized chitinase, MsCHT535, from Manduca sexta (Ms). All of these proteins, except those belonging to group V that are related to imaginal disc growth factors (IDGFs), exhibited chitinolytic activity against the long polymeric substrate, CM-Chitin-RBV, and/or the short oligomeric substrate, MU-(GlcNAc)3. TcCHT5, DmCHT5 and MsCHT535, which are members of group I chitinases, cleaved both polymeric and oligomeric substrates. Their enzymatic properties, including pH optima, kinetic parameters, and susceptibility to substrate inhibition by chitooligosaccharides, were similar. Two group IV chitinases, DmCHT4 and DmCHT9, also were characterized. DmCHT4 had one optimum pH of 6 towards the polymeric substrate and no detectable chitinolytic activity towards an oligosaccharide substrate. DmCHT9 had high activity from pH 4 to 8 towards the polymeric substrate and exhibited low activity towards the oligosaccharide substrate. The group V proteins, TcIDGF2 and TcIDGF4, contain all of the catalytically critical residues within conserved region II of family 18 chitinases but neither exhibited chitinolytic activity. Another group V protein, DmDS47, which lacks the critical glutamate residue in region II and the C-terminal CBD, also exhibited no chitinolytic activity. However, all three of the group V proteins bound to chitin tightly. A comparison of the amino acid sequences and homology model structures of group V proteins with enzymatically active members of the chitinase family indicated that the presence of additional loops of amino acids within the (βα)8-barrel structure of these proteins interferes with productive substrate binding and/or catalysis. [Copyright &y& Elsevier]

Chitin, a homopolymer of β -1-4-linked N -acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum , dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A ( TcChs-A ) and tryptophan oxygenase ( TcVer ) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A , TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum . [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: The peritrophic matrix (PM) in the midgut of insects consists primarily of chitin and proteins and is thought to support digestion and provide protection from abrasive food particles and enteric pathogens. We examined the physiological roles of 11 putative peritrophic matrix protein (PMP) genes of the red flour beetle, Tribolium castaneum (TcPMPs). TcPMP genes are differentially expressed along the length of the midgut epithelium of feeding larvae. RNAi of individual PMP genes revealed no abnormal developmental phenotypes for 9 of the 11 TcPMPs. However, RNAi for two PMP genes, TcPMP3 and TcPMP5-B, resulted in depletion of the fat body, growth arrest, molting defects and mortality. In situ permeability assays after oral administration of different-sized FITC-dextran beads demonstrated that the exclusion size of the larval peritrophic matrix (PM) decreases progressively from >2 MDa to <4 kDa from the anterior to the most posterior regions of the midgut. In the median midguts of control larvae, 2 MDa dextrans were completely retained within the PM lumen, whereas after RNAi for TcPMP3 and TcPMP5-B, these dextrans penetrated the epithelium of the median midgut, indicating loss of structural integrity and barrier function of the larval PM. In contrast, RNAi for TcPMP5-B, but not RNAi for TcPMP3, resulted in breakdown of impermeability to 4 and 40 kDa dextrans in the PM of the posterior midgut. These results suggest that specific PMPs are involved in the regulation of PM permeability, and that a gradient of barrier function is essential for survival and fat body maintenance. [Copyright &y& Elsevier]

Abstract: Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin®) are in wide use to control various insect pests. Although this class of compounds is known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored product pest Tribolium castaneum (red flour beetle) utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects and reduced chitin amounts in the larval cuticle, the peritrophic matrix and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unexpectedly unaffected, but many genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification pathways were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle turned out to be a good model organism for investigating the global effects of bioactive materials such as insect growth regulators and other insecticides. The results of this study recapitulate all of the different DFB-induced symptoms in a single model insect, which have been previously found in several different insect species, and further illustrate that DFB treatment causes a wide range of effects at the molecular level. [Copyright &y& Elsevier]

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: 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: The use of native promoters to drive transgene expression has facilitated overexpression studies in Drosophila and other insects. We identified 12 Tubulin family members from the genome sequence of the red flour beetle, Tribolium castaneum, and used the promoter from one of these to drive constitutive expression of a transgene. The activity of the T. castaneum α-Tubulin1 (TcαTub1) putative promoter was pre-tested in conjunction with an eye-color gene, T. castaneum vermilion (Tcv), by transient expression in Tcv-deficient embryos. Such embryos showed complete rescue of larval eyespot pigmentation. We also examined the TcαTub1 expression pattern in germline transformants using the enhanced green fluorescent protein (EGFP) reporter. Beetles transformed with this piggyBac-based reporter ubiquitously expressed EGFP at all stages. [Copyright &y& Elsevier]

Abstract: The insect arginine vasopressin-like (AVPL) peptide is of special interest because of its potential function in the regulation of diuresis. Genome sequences of the red flour beetle Tribolium castaneum yielded the genes encoding AVPL and AVPL receptor, whereas the homologous sequences are absent in the genomes of the fruitfly, malaria mosquito, silkworm, and honeybee, although a recent genome sequence of the jewel wasp revealed an AVPL sequence. The Tribolium receptor for the AVPL, the first such receptor identified in any insect, was expressed in a reporter system, and showed a strong response (EC50=1.5nM) to AVPL F1, the monomeric form having an intramolecular disulfide bond. In addition to identifying the AVPL receptor, we have demonstrated that it has in vivo diuretic activity, but that it has no direct effect on Malpighian tubules. However, when the central nervous system plus corpora cardiaca and corpora allata are incubated along with the peptide and Malpighian tubules, the latter are stimulated by the AVPL peptide, suggesting it acts indirectly. Summing up all the results from this study, we conclude that AVPL functions as a monomer in Tribolium, indirectly stimulating the Malpighian tubules through the central nervous system including the endocrine organs corpora cardiaca and corpora allata. RNA interference in the late larval stages successfully suppressed mRNA levels of avpl and avpl receptor, but with no mortality or abnormal phenotype, implying that the AVPL signaling pathway may have been near-dispensable in the early lineage of holometabolous insects. [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 investigation of four insect species with annotated genome sequences identified a family of genes encoding chitin deacetylase (CDA)-like proteins, with five to nine members depending on the species. CDAs (EC 3.5.1.41) are chitin-modifying enzymes that deacetylate the β-1,4-linked N-acetylglucosamine homopolymer. Partial deacetylation forms a heteropolysaccharide that also contains some glucosamine residues, while complete deacetylation produces the homopolymer chitosan, consisting exclusively of glucosamine. The genomes of the red flour beetle, Tribolium castaneum, the fruit fly, Drosophila melanogaster, the malaria mosquito, Anopheles gambiae, and the honey bee, Apis mellifera contain 9, 6, 5 and 5 genes, respectively, that encode proteins with a chitin deacetylase motif. The presence of alternative exons in two of the genes, TcCDA2 and TcCDA5, increases the protein diversity further. Insect CDA-like proteins were classified into five orthologous groups based on phylogenetic analysis and the presence of additional motifs. Group I enzymes include CDA1 and isoforms of CDA2, each containing in addition to a polysaccharide deacetylase-like catalytic domain, a chitin-binding peritrophin-A domain (ChBD) and a low-density lipoprotein receptor class A domain (LDLa). Group II is composed of CDA3 orthologs from each insect species with the same domain organization as group I CDAs, but differing substantially in sequence. Group III includes CDA4s, which have the ChBD domain but do not have the LDLa domain. Group IV comprises CDA5s, which are the largest CDAs because of a very long intervening region separating the ChBD and catalytic domains. Among the four insect species, Tribolium is unique in having four CDA genes in group V, whereas the other insect genomes have either one or none. Most of the CDA-like proteins have a putative signal peptide consistent with their role in modifying extracellular chitin in both cuticle and peritrophic membrane during morphogenesis and molting. [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: The whole genome sequence of Tribolium castaneum, a worldwide coleopteran pest of stored products, has recently been determined. In order to facilitate accurate annotation and detailed functional analysis of this genome, we have compiled and analyzed all available expressed sequence tag (EST) data. The raw data consist of 61,228 ESTs, including 10,704 obtained from NCBI and an additional 50,524 derived from 32,544 clones generated in our laboratories. These sequences were amassed from cDNA libraries representing six different tissues or stages, namely: whole embryos, whole larvae, larval hindguts and Malpighian tubules, larval fat bodies and carcasses, adult ovaries, and adult heads. Assembly of the 61,228 sequences collapsed into 12,269 clusters (groups of overlapping ESTs representing single genes), of which 10,134 mapped onto 6463 (39%) of the 16,422 GLEAN gene models (i.e. official Tribolium gene list). Approximately 1600 clusters (13% of the total) lack corresponding GLEAN models, despite high matches to the genome, suggesting that a considerable number of transcribed sequences were missed by the gene prediction programs or were removed by GLEAN. We conservatively estimate that the current EST set represents more than 7500 transcription units. [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]