Department of Protein Biosynthesis
We present here the analysis of ESTs from the CA of D. punctata compared to a similar effort from Aedes aegypti and Anopheles albimanus. Reciprocal matches between the cockroach and the mosquito ESTs, as well as reference to the Anopheles gambiae and Drosophila genomes in particular allowed us to assign probable function to the majority of genes coding for enzymes of the JH biosynthetic pathway. This has already been validated by the functional identification of three cockroach ESTs as representing methyl farnesoate epoxidase, the last enzyme in JH biosynthesis (). In addition, we identified a number of gene products coding for potential elements of the signaling pathways in the CA. Our study provides a basis for the systematic and comprehensive analysis of molecules that may play an active role in the function and regulation of this endocrine gland.
Protein hormone synthesis is ..
The biosynthesis of insect juvenile hormone (JH) and its neuroendocrine control are attractive targets for chemical control of insect pests and vectors of disease. To facilitate the molecular study of JH biosynthesis, we analyzed ESTs from the glands producing JH, the corpora allata (CA) in the cockroach Diploptera punctata, an insect long used as a physiological model species and compared them with ESTs from the CA of the mosquitoes Aedes aegypti and Anopheles albimanus. The predicted genes were analyzed according to their probable functions with the Gene Ontology classification, and compared to Drosophila and Anopheles gambiae genes. A large number of reciprocal matches in the cDNA libraries of cockroach and mosquito CA were found. These matches defined known and suspected enzymes of the JH biosynthetic pathway, but also several proteins associated with signal transduction that might play a role in the modulation of JH synthesis by neuropeptides. The identification in both cockroach and mosquito CA of homologs of the small ligand binding proteins from insects, Takeout/JH binding protein and retinol-binding protein highlights a hitherto unsuspected complexity of metabolite trafficking, perhaps JH precursor trafficking, in these endocrine glands. Furthermore, many reciprocal matches for genes of unknown function may provide a fertile ground for an in-depth study of allatal-specific cell physiology.
Importantly, peroxidase-processed thyroglobulin within the follicle can act as a reservoir for thyroid hormones in the absence of stimulation for hormone release.
occurring both allosterically and via hormone-receptor ..
In an attempt to reveal the complexity of the insect CA transcriptome, several approaches can be considered. Differential display of mRNA can be useful when the tissues can be cleanly dissected. In the silkworm, mRNA differential display has been used to identify the OMT which is the last step of JH biosynthesis in lepidoptera (). DNA microarray analysis is another potential tool. The transcriptome analysis using whole-genome microarrays gives a complete overview of those genes that are expressed significantly in certain tissues, when these tissues can be experimentally compared to “whole organism” transcriptome signatures. Such an approach has been elegantly described for the Malpighian tubules of Drosophila (). Whole-genome arrays are not yet available for other insects, and their use with Drosophila ring glands which comprise both CA and prothoracic gland cells has not been attempted to our knowledge. In comparison, EST analysis is not dependent on prior knowledge of the whole genome, but relies on identification of the transcripts by homology. A careful analysis of ESTs from various tissues of the silkworm () indicates that about one-third or less of the ESTs found were specific for each tissue-derived library. In contrast, the silkworm prothoracic gland library yielded 49% library-specific ESTs, but a detailed description and analysis is not available.
Thyroid hormone biosynthesis and release - …
Identification of the enzymes involved in the early steps of JH biosynthesis has taken advantage of the conservation of the mevalonate pathway between vertebrates and insects (), but this option was not available for the later, JH-specific steps. Regulatory pathways controlling JH biosynthesis are more difficult to decipher with classical homology inferences. The comparison of cockroach and mosquito ESTs was felt to be a useful tool to discriminate such CA-selective transcripts. Indeed, the CA of mosquitoes are minute (30–40 μm) and closely associated within other cell types; we dissected the CA–CC complex attached to a small piece of aorta (see Fig. 1 of ). It is therefore more difficult to study the physiological control of JH biosynthesis in mosquitoes than in the model species D. punctata. In this species, the CA (CA cells plus neurosecretory and neuromodulatory terminals) are somewhat easier to dissect clean of contaminating tissues, the glands are larger. Most importantly, they produce far more JH than any other insect species studied to date, a thousand fold more than mosquito CA, heroic amounts that can only be compared to the prodigious amounts of JH produced by the CA of adult male Saturniidae, especially Cecropia. It is likely that the enzyme machinery is present in higher amounts, and we could expect a relatively high abundance of the most representative mRNAs. Indeed several ESTs were found for mevalonate kinase, and for the methyl farnesoate epoxidase, CYP15A1. A large number of contigs (70) with predicted Gene Ontology function had a reciprocal match in the cockroach and A. aegypti CA collections of ESTs. Interestingly, an additional 19 contigs of unknown function also had a reciprocal match, suggesting that a significant proportion of the CA transcriptome has so far completely eluded any functional understanding.