Characterization and expression of the beta-N-acetylhexosaminidase gene family of Tribolium castaneum.

Enzymes belonging to the beta-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 beta-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.

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.

Characterization of two chitin synthase genes of the red flour beetle, Tribolium castaneum, and alternate exon usage in one of the genes during development.

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.

Genome sequence of Aedes aegypti, a major arbovirus vector.

We present a draft sequence of the genome of Aedes aegypti, the primary vector for yellow fever and dengue fever, which at approximately 1376 million base pairs is about 5 times the size of the genome of the malaria vector Anopheles gambiae. Nearly 50% of the Ae. aegypti genome consists of transposable elements. These contribute to a factor of approximately 4 to 6 increase in average gene length and in sizes of intergenic regions relative to An. gambiae and Drosophila melanogaster. Nonetheless, chromosomal synteny is generally maintained among all three insects, although conservation of orthologous gene order is higher (by a factor of approximately 2) between the mosquito species than between either of them and the fruit fly. An increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to An. gambiae suggests that members of these protein families underpin some of the biological differences between the two mosquito species.