Retroactive maintains cuticle integrity by promoting the trafficking of Knickkopf into the procuticle of Tribolium castaneum.

Molting, or the replacement of the old exoskeleton with a new cuticle, is a complex developmental process that all insects must undergo to allow unhindered growth and development. Prior to each molt, the developing new cuticle must resist the actions of potent chitinolytic enzymes that degrade the overlying old cuticle. We recently disproved the classical dogma that a physical barrier prevents chitinases from accessing the new cuticle and showed that the chitin-binding protein Knickkopf (Knk) protects the new cuticle from degradation. Here we demonstrate that, in Tribolium castaneum, the protein Retroactive (TcRtv) is an essential mediator of this protective effect of Knk. TcRtv localizes within epidermal cells and specifically confers protection to the new cuticle against chitinases by facilitating the trafficking of TcKnk into the procuticle. Down-regulation of TcRtv resulted in entrapment of TcKnk within the epidermal cells and caused molting defects and lethality in all stages of insect growth, consistent with the loss of TcKnk function. Given the ubiquity of Rtv and Knk orthologs in arthropods, we propose that this mechanism of new cuticle protection is conserved throughout the phylum.

Gene families of cuticular proteins analogous to peritrophins (CPAPs) in Tribolium castaneum have diverse functions.

The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal 'stiff-jointed' gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species.

Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function.

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.

Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum.

Ecdysis behavior in arthropods is driven by complex interactions among multiple neuropeptide signaling systems. To understand the roles of neuropeptides and their receptors in the red flour beetle, Tribolium castaneum, we performed systemic RNA interference (RNAi) experiments utilizing post-embryonic injections of double-stranded (ds) RNAs corresponding to ten gene products representing four different peptide signaling pathways: eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon. Behavioral deficiencies and developmental arrests occurred as follows: RNAi of (1) eh or eth disrupted preecdysis behavior and prevented subsequent ecdysis behavior; (2) ccap interrupted ecdysis behavior; and (3) bursicon subunits resulted in wrinkled elytra due to incomplete wing expansion, but there was no effect on cuticle tanning or viability. RNAi of genes encoding receptors for those peptides produced phenocopies comparable to those of their respective cognate neuropeptides, except in those cases where more than one receptor was identified. The phenotypes resulting from neuropeptide RNAi in Tribolium differ substantially from phenotypes of the respective Drosophila mutants. Results from this study suggest that the functions of neuropeptidergic systems that drive innate ecdysis behavior have undergone significant changes during the evolution of arthropods.

Vasopressin-like peptide and its receptor function in an indirect diuretic signaling pathway in the red flour beetle.

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 (EC(50)=1.5 nM) 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.

Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects.

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.

Chitin synthase genes in Manduca sexta: characterization of a gut-specific transcript and differential tissue expression of alternately spliced mRNAs during development.

Chitin, the linear homopolymer of beta-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.