Abstract
BackgroundBursicon is a heterodimer neuropeptide responsible for regulating cuticle sclerotization and wing expansion in several insect species. Recent studies indicate that the action of bursicon is mediated by a specific G protein-coupled receptor DLGR2 and the cAMP/PKA signaling pathway. However, little is known regarding the genes that are regulated by bursicon. The identification of bursicon-regulated genes is the focus of this investigation.ResultsWe used DNA microarray analysis to identify bursicon-regulated genes in neck-ligated flies (Drosophila melanogaster) that received recombinant bursicon (r-bursicon). Fifty four genes were found to be regulated by bursicon 1 h post r-bursicon injection, 52 being up-regulated and 2 down-regulated while 33 genes were influenced by r-bursicon 3 h post-injection (24 up-regulated and 9 down-regulated genes). Analysis of these genes by inference from the fly database revealed that these genes encode proteins with diverse functions, including cell signaling, gene transcription, DNA/RNA binding, ion trafficking, proteolysis-peptidolysis, metabolism, cytoskeleton formation, immune response and cell-adhesion. Twenty eight genes randomly selected from the microarray-identified list were verified by real time PCR (qPCR) which supported the microarray data. Temporal response studies of 13 identified and verified genes by qPCR revealed that the temporal expression patterns of these genes are consistent with the microarray data.ConclusionUsing r-bursicon, we identified 87 genes that are regulated by bursicon, 30 of which have no previously known function. Most importantly, all genes randomly selected from the microarray-identified list were verified by real time PCR. Temporal analysis of 13 verified genes revealed that the expression of these genes was indeed induced by bursicon and correlated well with the cuticle sclerotization process. The composite data suggest that these genes play important roles in regulating the cuticle sclerotization and wing expansion processes. The data obtained here will form the basis for future studies aimed at elucidating the exact mechanisms upstream from the secretion of bursicon and its binding to target cells.
Highlights
Bursicon is a heterodimer neuropeptide responsible for regulating cuticle sclerotization and wing expansion in several insect species
Cuticle tanning was detected only in those abdomens injected with the homogenate of the central nervous system (CNS) (0.5 CNS equivalent/fly) (Fig. 1d, positive control) or the purified rbursicon heterodimer expressed in insect High FiveTM cells (Fig. 1e) and mammalian HEK293 cells (Fig. 1f)
We first determined the transcriptional profile of the 18,952 transcripts in the ligated flies injected with r-bursicon α at the two time points noted above i.e. 1 h and 3 h post r-bursicon injection
Summary
Bursicon is a heterodimer neuropeptide responsible for regulating cuticle sclerotization and wing expansion in several insect species. Studies on ecdysial behavior in insects showed that at least six different hormones are released in an orderly manner during the molting cycle to regulate the synthesis and sclerotization (hardening and tanning) of new cuticle [1,2,3,4,5]. The final hormone released in this cascade, the neuropeptide bursicon, was found to trigger sclerotization of the new cuticle four decades ago using the novel neck-ligated blowfly bioassay in which the ligated flies are injected with an extract of the fused thoracic/abdominal ganglion or with hemolymph collected shortly after adult emergence [5,6,7]. Bursicon stimulated wing expansion in newly emerged lepidopteran adults [8,9,10]
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