BACKGROUND
molting is a process common to all arthropods, during which a larger new cuticle is synthesized and the old one is digested and cast off  allowing the animal to grow.
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  of new cuticle  <cit> .
the final hormone released in this cascade, the neuropeptide bursicon, was found to trigger sclerotization of the new cuticle four decades ago using the then 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  <cit> .
bursicon activity in the thoracic-abdominal ganglion was strikingly higher than that of the brain  <cit> .
using this bioassay, bursicon activity was identified in diverse insect orders including diptera, orthoptera, hemiptera, coleoptera and lepidoptera  <cit> .
bursicon also stimulated wing expansion in newly emerged lepidopteran adults  <cit> .
previously, bursicon was thought to be a monomeric neuropeptide with a molecular size ranging from  <dig> to  <dig> kda  <cit> .
more recently, functional bursicon was shown to be a heterodimer consisting of two cystine knot subunits, referred to as bursicon α  and bursicon β  <cit> .
in drosophila melanogaster, bursicon acts via a specific g protein-coupled receptor  dlgr <dig>  encoded by the rickets gene  <cit> .
dlgr <dig>  once activated, is hypothesized to activate the camp/pka signaling pathway  <cit> .
recombinant bursicon  heterodimer was also found to bind to dlgr <dig> with high affinity, to stimulate camp production in vitro and to then initiate cuticle sclerotization in the ligated fruit fly bioassay in vivo  <cit> .
mutation of the bursicon α gene and receptor rickets gene caused defects in cuticle sclerotization and wing expansion  <cit> .
of great interest is the work of davis et al  who showed that tyrosine hydroxylase, the enzyme mediating the conversion of tyrosine to dopa in the metabolic pathway leading to cuticle tanning, is activated by pka via bursicon stimulation of dlgr <dig>  <cit> .
a gene silencing study revealed that injection of the double-stranded bursicon α rna into bombyx mori  pupae significantly reduced the level of bursicon α mrna, resulting in decreased wing expansion in the newly emerged adult moth  <cit> .
however, little is known about the signaling pathway downstream of the bursicon receptor dlgr <dig> and adenylate cyclase, as well as the genes regulated by bursicon.
here, we report the functional analysis and identification of the genes affected by the injection of r-bursicon into the thorax of ligated d. melanogaster just after ecdysis to the adult.
RESULTS
functional analysis of r-bursicon in neck-ligated flies
the r-bursicon protein, expressed in mammalian hek <dig> cells or in insect high five™ cells, was assayed for bursicon activity using the neck-ligated fly assay.
three hours after injection , no sign of cuticle sclerotization was observed in abdomens injected with  <dig>  μl of the supernatant from a cell culture transfected with blank vector  or with r-bursicon α  or with r-bursicon β alone .
cuticle tanning was detected only in those abdomens injected with the homogenate of the cns    or the purified r-bursicon heterodimer expressed in insect high five™ cells  and mammalian hek <dig> cells .
when the neck-ligated flies were injected with r-bursicon and photographed at the indicated time periods , cuticle sclerotization was not visible at  <dig> min, slightly visible at  <dig> h and almost complete by  <dig> h post r-bursicon injection, data consistent with past observations  <cit> .
additionally, these results suggest that the r-bursicon heterodimer expressed in both cell cultures has strong cuticle sclerotization activity in the neck-ligated fly assay and could therefore be used to identify early and late bursicon-regulated genes in the subsequent microarray analyses.
dna microarray identification of early and late bursicon-regulated genes
to identify bursicon-regulated genes, gene expression experiments were performed at  <dig> and  <dig> h after bursicon administration.
we first determined the transcriptional profile of the  <dig>  transcripts  in the ligated flies injected with r-bursicon α  at the two time points noted above i.e.
<dig> h  and  <dig> h  post r-bursicon injection.
the results showed that  <dig>  transcripts were present in the control  <dig> h post r-bursicon injection, which corresponds to  <dig> % of the total number of described drosophila genes while  <dig>  transcripts  were present in the control  <dig> h post r-bursicon injection.
we then compared gene-expression profiles between the control and the r-bursicon heterodimer-injected flies at these two time points.
statistical filtering of the microarray data revealed that the expression of  <dig> transcripts was up- or down-regulated by the r-bursicon heterodimer by at least  <dig> fold  at  <dig> h and  <dig> transcripts at  <dig> h post r-bursicon injection .
more upregulated genes were present at  <dig> h  than at  <dig> h  while more down-regulated genes were revealed at  <dig> h  than at  <dig> h  after r-bursicon injection.
surprisingly, only two genes, cg30080-ra and cg <dig>  involved in gene transcription, were up-regulated at both  <dig> and  <dig> h after r-bursicon injection.
transcription
the genes in bold were detected at both  <dig> and  <dig> h post r-bursicon injection.
the name in parenthesis indicates a functional domain.
n: gene with no known function associated with it.
annotation of  <dig> genes identified  <dig> h post r-bursicon injection using flybase revealed that proteins encoded by these genes belong to diverse functional categories.
among the proteins with known function, the largest group contains those genes involved with dna/rna binding proteins and signaling, each with  <dig> proteins.
the second largest groups contain those genes involved with channel/transporter and metabolic enzymes with  <dig> proteins each.
in addition,  <dig> transcription factors,  <dig> immune response factors, one cytoskeletal component and one cell-adhesion gene product were also identified.
on the other hand,  <dig> out of  <dig> bursicon-regulated genes  encode proteins with as yet unknown functions .
microarray analysis revealed that only  <dig> transcripts were regulated by bursicon at  <dig> h post r-bursicon injection.
among these transcripts, the channel/transporter is the largest group with  <dig> proteins.
the second largest group is the immune response factors with  <dig> proteins.
transcription factors and proteolysis/peptidolysis transcripts, each encoding  <dig> proteins, present the third largest groups followed by  <dig> dna/rna binding proteins,  <dig> cell adhesion proteins, one cytoskelethal component and a signaling protein.
twelve out of  <dig> bursicon regulated genes  encode proteins with unknown functions .
before discussing the possible function of some of these genes in the action of bursicon e.g.
cuticle sclerotization and wing expansion, it was important to verify the results of the microarray analyses and for this we chose real-time pcr analysis.
validation by qpcr
specific primers  were designed to  <dig> randomly selected bursicon-regulated genes   and were utilized for corroborating estimates of gene transcription using qpcr.
the rna samples used were identical to those used for the microarray, i.e.
extracted from the neck-ligated flies either  <dig> or  <dig> h post r-bursicon injection.
the qpcr results revealed that all selected genes were verifiable , i.e., up- or down-regulated by r-bursicon by at least  <dig> fold in the qpcr analysis, a result similar to that obtained for the same genes using microarray analysis .
to gain further insight into how the activity of the bursicon-regulated genes may change between  <dig> and  <dig> h post-injection, qpcr analysis was carried out on  <dig> genes selected from the list of  <dig> verifiable genes.
the transcriptional profiles of these genes change over time.
as expected, all  <dig> genes selected from the flies  <dig> h after r-bursicon injection were up-regulated maximally at  <dig> h, declined sharply thereafter and returned to the basal level by  <dig> h .
these genes include cg10897-ra, cg12348-ra, cg3019-ra, cg9500-ra, cg6386-ra, cg8920-ra, and cg13338-ra.
among  <dig> randomly selected genes from flies  <dig> h after r-bursicon injection, four were up-regulated maximally at  <dig> or  <dig> h, but not at  <dig> h, while  <dig> down-regulated genes reached their lowest levels after  <dig> h or later .
these  <dig> up-regulated genes include cg31193-ra, cg31508-ra, cg <dig>  and cg32491-ra while the  <dig> down-regulated genes were cg32438-ra and cg <dig>  the temporal response patterns of these  <dig> genes obtained by qpcr analysis  were consistent with the microarray data .
discussion
the composite data indicate that the genes described above as being regulated by bursicon are associated with cuticle sclerotization and wing expansion in drosophila.
among the total  <dig> transcripts identified at  <dig> and  <dig> h post r-bursicon injection,  <dig> showed significant similarities to known proteins or functional domains in the drosophila data base while  <dig> did not.
in drosophila, bursicon binds with high affinity and specificity to the dlgr <dig> that is encoded by rickets  <cit> .
dlgr <dig>  once activated leads to the activation of the camp/pka signaling pathway  <cit> .
however, the signaling pathway downstream of pka remains conjectural.
among the  <dig> bursicon-regulated genes we identified,  <dig> genes were found to control cell signaling .
this group includes a ras-related protein , a g-protein activator-odorant receptor 59c  etc .
ras proteins are considered to be very important molecular switches for a wide variety of signaling pathways that control such processes as cytoskeletal integrity, proliferation, cell adhesion, apoptosis, and cell migration  <cit> .
ras-related protein is often activated via phosphorylation by pka.
in yeast the ras signaling pathway controls cell growth via pka, resulting in the regulation of the elongation step of the rna polymerase ii transcription process  <cit> .
perhaps in drosophila the ras-related protein cg2849-rb identified in the microarray analysis  might be activated directly by pka i.e.
act as a downstream component of pka in the bursicon signaling pathway.
bursicon is a neuropeptide that not only induces cuticle hardening and tanning but also regulates wing expansion and subsequent epidermal cell death  <cit> .
ras proteins have the ability to activate transforming signals as well as signals that regulate apoptosis  <cit> .
in the rat <dig> fibroblast cell line for example, the activated h-rasr <dig> is a potent inducer of apoptosis in response to serum starvation  <cit>  and that may also be the case in the insect wing.
in addition, microarray analysis also detected a fmrfamide , which in the clam is thought to play an important role in the regulation of heart rate, muscle contraction and blood pressure  <cit> .
such controls are necessary for wing expansion and fmrfamide may play an analogous role in the insect.
our microarray analyses also detected three antibacterial peptides, attacin-like  and cecropin .
although no direct association between bursicon and antibacterial peptides is obvious, the newly ecdysed insect is soft  and more susceptible to injury.
perhaps a more vigorous antibacterial defense is necessary at this time when there may be perforations in the soft cuticle due to predators or simple contact injuries.
three humoral factor turandot genes  are also induced by bursicon .
the turandot gene family is considered to be immune-related in insects, and is induced under a wide range of adverse conditions such as infection, heat, oxidizing agents, wounding and aging  <cit> .
however, under normal conditions, some turandot genes, such as turandot c, turandot f, etc, are expressed during specific developmental periods, indicating that these genes are associated with normal physiological morphogenetic processes  <cit> .
our results showed that three turandot genes  can be induced in the ligated flies injected with r-bursicon, suggesting that these genes are not only involved in specific response reactions to abnormal conditions, but may also be involved in the cuticle sclerotization process.
using crude thoracic/abdominal ganglion homogenates, mills and whitehead demonstrated that bursicon enhanced the uptake of tyrosine by hemocytes  <cit> .
this result is consistent with the result of post  <cit> .
they speculated that one of bursicon's functions was to increase the membrane permeability of hemocytes to tyrosine which would then be converted to n-acetyldopamine.
although we do not have direct evidence to support this claim, the microarray data did reveal that  <dig> channels/transporters were regulated by bursicon.
these channel proteins/transporters may be involved in the uptake of tyrosine by hemocytes, the earliest precursor for tanning agent biosynthesis.
cuticle sclerotization is a complex process, during which many genes detected in our studies are induced.
the induction of these genes requires corresponding transcription-associated factors and dna/rna binding proteins.
the present results identified several transcription-associated genes and dna/rna binding proteins .
for example, cg <dig> encodes eukaryotic sm and sm-like  proteins involving pre-mrna splicing, telomere replication, and mrna degradation  <cit> .
most interesting, a trans-splicing drosophila gene mod   was found to be up-regulated by bursicon in our microarray screening.
the mod gene of d. melanogaster has been reported to encode a chromatin protein and has been independently identified through mutations isolated for their effects on position effect variegation , the properties of insulator sequences, correct path finding of growing nerve cells, meiotic pairing of chromosomes, and apoptosis  <cit> .
besides the above mentioned genes, there are  <dig> other transcription-related genes  detected in our microarray analysis.
these genes possibly play pivotal roles in regulating the expression of genes encoding enzymes required for the cuticle sclerotization process.
sclerotization is a metabolic process, during which enzymes mediate a significant number of reactions.
these enzymes include diphenoloxidases, laccases, peroxidase, tyrosine hydroxylase , dopa decarboxylase  etc  <cit> .
it is a little surprising that our microarray analysis did not detect transcriptional changes of these enzymes.
however, it should be pointed out that that not all genes are regulated at the transcriptional level.
for example, th mediates the conversion of tyrosine to dihydroxyphenylalanine  and is a key enzyme during the tanning process.
however, th mrna level is at its highest  <dig> h before eclosion and remains unchanged until  <dig> hours after eclosion  <cit> .
it seems that the activity of th during the sclerotization process is not regulated at the transcriptional level, th being transiently activated during cuticle sclerotization by a post-translational mechanism, i.e.
phosphorylation by pka  <cit> .
post-translational mechanisms may be in force here.
another example is ddc, which catalyzes the conversion of dopa to dopamine, a compound of central importance for sclerotization.
the ddc mrna level peaks at  <dig> hours before eclosion and decreases thereafter.
it is almost unchanged or decreases minutely from  <dig> h to  <dig> h after eclosion.
ddc is transcribed and translated before eclosion to ensure that enzyme activity is present when substrate becomes available  <cit> .
therefore, it is not surprising that microarray analysis does not detect the changes in some genes encoding enzymes whose activation is required for the normal processes of tanning and sclerotization.
most interestly, our microrray analysis identified  <dig> metabolic enzymes whose transcripts were up-regulated significantly at  <dig> h, but not at  <dig> h after r-bursicon administration, suggesting that these enzymes may be actively involved in the early sclerotization process, perhaps in the synthesis of agents required for tanning and hardening.
sclerotization is a process of cross-linking protein to protein chains, chitin to chitin chains, and protein to chitin chains  <cit> .
our microarray analysis also detected a cuticular protein gene 50ca  with a chitin-binding domain and a gene, cg10140-ra that appears to be involved in chitin metabolism.
although their exact role in cuticle formation is not known, the possibility exists that they function to bind chitin and/or play important roles in chitin metabolism.
tanning requires energy in the form of atp  and the cg3649-ra gene detected in the microarray analysis has a sugar transporter domain and may help supply metabolic energy for melanization.
it is therefore evident that further research is necessary to identify with certainty those genes involved with specific steps in the tanning and sclerotization processes.
our data are tantalizing but only suggestive at this point.
early studies indicate that the brain triggers the release of bursicon from the fused thoracic/abdominal ganglion to the hemolymph by nervous stimuli leading to cuticle sclerotization  <cit> .
more recent investigations revealed that both bursicon protein and transcripts are present in the thoracic/abdominal ganglion, but not in the brain, of d. melanogaster  <cit>  and musca domestica  <cit> , thus confirming the early observation  <cit> .
cuticle sclerotization is a brief but complex process occurring after each molt and the genes involved in this process are not expected to be present in large number.
the  <dig> genes identified in the microarray analysis reflect this estimation.
although the functions and interrelationship of these genes in the cuticle sclerotization process is conjectural at present, the microarray data provide a foundation for further investigation of the functions of these identified genes, and such studies may allow a better understanding of the mechanisms of the cuticle sclerotization process in insects and other arthropods.
sclerotization of the exoskeleton is a primary reason for the success of insects on this planet and the series of reactions constituting sclerotization and its control affects more animal species than any other series of phylum-specific reactions.
it is most worthy of future research and we believe that the present study lays the basis for such investigations at another level.
CONCLUSIONS
bursicon is a neuropeptide that regulates cuticle sclerotization and wing expansion in insects, critical processes for insect survival.
in these processes, many genes are up- and down-regulated.
in this report, we demonstrated the presence of a set of genes that are sensitive to r-bursicon stimulation and the data were verified by qpcr analysis.
this gene set supplies unique and intriguing candidates for a more complete understanding of bursicon action by the use of specific gene deletion and over-expression experiments that may lead to a functional dissection of some of these genes in the cuticle sclerotization process.

 molting is a process common to all arthropods, during which a larger new cuticle is synthesized and the old one is digested and cast off allowing the animal to grow. 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 of new cuticle. the final hormone released in this cascade, the neuropeptide bursicon, was found to trigger sclerotization of the new cuticle four decades ago using the then 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. bursicon activity in the thoracic-abdominal ganglion was strikingly higher than that of the brain. using this bioassay, bursicon activity was identified in diverse insect orders including diptera, orthoptera, hemiptera, coleoptera and lepidoptera. bursicon also stimulated wing expansion in newly emerged lepidopteran adults. previously, bursicon was thought to be a monomeric neuropeptide with a molecular size ranging from to kda. more recently, functional bursicon was shown to be a heterodimer consisting of two cystine knot subunits, referred to as bursicon α and bursicon β. in drosophila melanogaster, bursicon acts via a specific g protein-coupled receptor dlgr encoded by the rickets gene. dlgr once activated, is hypothesized to activate the camp/pka signaling pathway. recombinant bursicon heterodimer was also found to bind to dlgr with high affinity, to stimulate camp production in vitro and to then initiate cuticle sclerotization in the ligated fruit fly bioassay in vivo. mutation of the bursicon α gene and receptor rickets gene caused defects in cuticle sclerotization and wing expansion. of great interest is the work of davis et al who showed that tyrosine hydroxylase, the enzyme mediating the conversion of tyrosine to dopa in the metabolic pathway leading to cuticle tanning, is activated by pka via bursicon stimulation of dlgr. a gene silencing study revealed that injection of the double-stranded bursicon α rna into bombyx mori pupae significantly reduced the level of bursicon α mrna, resulting in decreased wing expansion in the newly emerged adult moth. however, little is known about the signaling pathway downstream of the bursicon receptor dlgr and adenylate cyclase, as well as the genes regulated by bursicon. here, we report the functional analysis and identification of the genes affected by the injection of r-bursicon into the thorax of ligated d. melanogaster just after ecdysis to the adult.  functional analysis of r-bursicon in neck-ligated flies the r-bursicon protein, expressed in mammalian hek cells or in insect high five™ cells, was assayed for bursicon activity using the neck-ligated fly assay. three hours after injection, no sign of cuticle sclerotization was observed in abdomens injected with μl of the supernatant from a cell culture transfected with blank vector or with r-bursicon α or with r-bursicon β alone. cuticle tanning was detected only in those abdomens injected with the homogenate of the cns or the purified r-bursicon heterodimer expressed in insect high five™ cells and mammalian hek cells. when the neck-ligated flies were injected with r-bursicon and photographed at the indicated time periods, cuticle sclerotization was not visible at min, slightly visible at h and almost complete by h post r-bursicon injection, data consistent with past observations. additionally, these results suggest that the r-bursicon heterodimer expressed in both cell cultures has strong cuticle sclerotization activity in the neck-ligated fly assay and could therefore be used to identify early and late bursicon-regulated genes in the subsequent microarray analyses. dna microarray identification of early and late bursicon-regulated genes to identify bursicon-regulated genes, gene expression experiments were performed at and h after bursicon administration. we first determined the transcriptional profile of the transcripts in the ligated flies injected with r-bursicon α at the two time points noted above i.e. h and h post r-bursicon injection. the results showed that transcripts were present in the control h post r-bursicon injection, which corresponds to % of the total number of described drosophila genes while transcripts were present in the control h post r-bursicon injection. we then compared gene-expression profiles between the control and the r-bursicon heterodimer-injected flies at these two time points. statistical filtering of the microarray data revealed that the expression of transcripts was up- or down-regulated by the r-bursicon heterodimer by at least fold at h and transcripts at h post r-bursicon injection. more upregulated genes were present at h than at h while more down-regulated genes were revealed at h than at h after r-bursicon injection. surprisingly, only two genes, cg30080-ra and cg involved in gene transcription, were up-regulated at both and h after r-bursicon injection. transcription the genes in bold were detected at both and h post r-bursicon injection. the name in parenthesis indicates a functional domain. n: gene with no known function associated with it. annotation of genes identified h post r-bursicon injection using flybase revealed that proteins encoded by these genes belong to diverse functional categories. among the proteins with known function, the largest group contains those genes involved with dna/rna binding proteins and signaling, each with proteins. the second largest groups contain those genes involved with channel/transporter and metabolic enzymes with proteins each. in addition, transcription factors, immune response factors, one cytoskeletal component and one cell-adhesion gene product were also identified. on the other hand, out of bursicon-regulated genes encode proteins with as yet unknown functions. microarray analysis revealed that only transcripts were regulated by bursicon at h post r-bursicon injection. among these transcripts, the channel/transporter is the largest group with proteins. the second largest group is the immune response factors with proteins. transcription factors and proteolysis/peptidolysis transcripts, each encoding proteins, present the third largest groups followed by dna/rna binding proteins, cell adhesion proteins, one cytoskelethal component and a signaling protein. twelve out of bursicon regulated genes encode proteins with unknown functions. before discussing the possible function of some of these genes in the action of bursicon e.g. cuticle sclerotization and wing expansion, it was important to verify the results of the microarray analyses and for this we chose real-time pcr analysis. validation by qpcr specific primers were designed to randomly selected bursicon-regulated genes and were utilized for corroborating estimates of gene transcription using qpcr. the rna samples used were identical to those used for the microarray, i.e. extracted from the neck-ligated flies either or h post r-bursicon injection. the qpcr results revealed that all selected genes were verifiable, i.e., up- or down-regulated by r-bursicon by at least fold in the qpcr analysis, a result similar to that obtained for the same genes using microarray analysis. to gain further insight into how the activity of the bursicon-regulated genes may change between and h post-injection, qpcr analysis was carried out on genes selected from the list of verifiable genes. the transcriptional profiles of these genes change over time. as expected, all genes selected from the flies h after r-bursicon injection were up-regulated maximally at h, declined sharply thereafter and returned to the basal level by h. these genes include cg10897-ra, cg12348-ra, cg3019-ra, cg9500-ra, cg6386-ra, cg8920-ra, and cg13338-ra. among randomly selected genes from flies h after r-bursicon injection, four were up-regulated maximally at or h, but not at h, while down-regulated genes reached their lowest levels after h or later. these up-regulated genes include cg31193-ra, cg31508-ra, cg and cg32491-ra while the down-regulated genes were cg32438-ra and cg the temporal response patterns of these genes obtained by qpcr analysis were consistent with the microarray data. discussion the composite data indicate that the genes described above as being regulated by bursicon are associated with cuticle sclerotization and wing expansion in drosophila. among the total transcripts identified at and h post r-bursicon injection, showed significant similarities to known proteins or functional domains in the drosophila data base while did not. in drosophila, bursicon binds with high affinity and specificity to the dlgr that is encoded by rickets. dlgr once activated leads to the activation of the camp/pka signaling pathway. however, the signaling pathway downstream of pka remains conjectural. among the bursicon-regulated genes we identified, genes were found to control cell signaling. this group includes a ras-related protein, a g-protein activator-odorant receptor 59c etc. ras proteins are considered to be very important molecular switches for a wide variety of signaling pathways that control such processes as cytoskeletal integrity, proliferation, cell adhesion, apoptosis, and cell migration. ras-related protein is often activated via phosphorylation by pka. in yeast the ras signaling pathway controls cell growth via pka, resulting in the regulation of the elongation step of the rna polymerase ii transcription process. perhaps in drosophila the ras-related protein cg2849-rb identified in the microarray analysis might be activated directly by pka i.e. act as a downstream component of pka in the bursicon signaling pathway. bursicon is a neuropeptide that not only induces cuticle hardening and tanning but also regulates wing expansion and subsequent epidermal cell death. ras proteins have the ability to activate transforming signals as well as signals that regulate apoptosis. in the rat fibroblast cell line for example, the activated h-rasr is a potent inducer of apoptosis in response to serum starvation and that may also be the case in the insect wing. in addition, microarray analysis also detected a fmrfamide, which in the clam is thought to play an important role in the regulation of heart rate, muscle contraction and blood pressure. such controls are necessary for wing expansion and fmrfamide may play an analogous role in the insect. our microarray analyses also detected three antibacterial peptides, attacin-like and cecropin. although no direct association between bursicon and antibacterial peptides is obvious, the newly ecdysed insect is soft and more susceptible to injury. perhaps a more vigorous antibacterial defense is necessary at this time when there may be perforations in the soft cuticle due to predators or simple contact injuries. three humoral factor turandot genes are also induced by bursicon. the turandot gene family is considered to be immune-related in insects, and is induced under a wide range of adverse conditions such as infection, heat, oxidizing agents, wounding and aging. however, under normal conditions, some turandot genes, such as turandot c, turandot f, etc, are expressed during specific developmental periods, indicating that these genes are associated with normal physiological morphogenetic processes. our results showed that three turandot genes can be induced in the ligated flies injected with r-bursicon, suggesting that these genes are not only involved in specific response reactions to abnormal conditions, but may also be involved in the cuticle sclerotization process. using crude thoracic/abdominal ganglion homogenates, mills and whitehead demonstrated that bursicon enhanced the uptake of tyrosine by hemocytes. this result is consistent with the result of post. they speculated that one of bursicon's functions was to increase the membrane permeability of hemocytes to tyrosine which would then be converted to n-acetyldopamine. although we do not have direct evidence to support this claim, the microarray data did reveal that channels/transporters were regulated by bursicon. these channel proteins/transporters may be involved in the uptake of tyrosine by hemocytes, the earliest precursor for tanning agent biosynthesis. cuticle sclerotization is a complex process, during which many genes detected in our studies are induced. the induction of these genes requires corresponding transcription-associated factors and dna/rna binding proteins. the present results identified several transcription-associated genes and dna/rna binding proteins. for example, cg encodes eukaryotic sm and sm-like proteins involving pre-mrna splicing, telomere replication, and mrna degradation. most interesting, a trans-splicing drosophila gene mod was found to be up-regulated by bursicon in our microarray screening. the mod gene of d. melanogaster has been reported to encode a chromatin protein and has been independently identified through mutations isolated for their effects on position effect variegation, the properties of insulator sequences, correct path finding of growing nerve cells, meiotic pairing of chromosomes, and apoptosis. besides the above mentioned genes, there are other transcription-related genes detected in our microarray analysis. these genes possibly play pivotal roles in regulating the expression of genes encoding enzymes required for the cuticle sclerotization process. sclerotization is a metabolic process, during which enzymes mediate a significant number of reactions. these enzymes include diphenoloxidases, laccases, peroxidase, tyrosine hydroxylase, dopa decarboxylase etc. it is a little surprising that our microarray analysis did not detect transcriptional changes of these enzymes. however, it should be pointed out that that not all genes are regulated at the transcriptional level. for example, th mediates the conversion of tyrosine to dihydroxyphenylalanine and is a key enzyme during the tanning process. however, th mrna level is at its highest h before eclosion and remains unchanged until hours after eclosion. it seems that the activity of th during the sclerotization process is not regulated at the transcriptional level, th being transiently activated during cuticle sclerotization by a post-translational mechanism, i.e. phosphorylation by pka. post-translational mechanisms may be in force here. another example is ddc, which catalyzes the conversion of dopa to dopamine, a compound of central importance for sclerotization. the ddc mrna level peaks at hours before eclosion and decreases thereafter. it is almost unchanged or decreases minutely from h to h after eclosion. ddc is transcribed and translated before eclosion to ensure that enzyme activity is present when substrate becomes available. therefore, it is not surprising that microarray analysis does not detect the changes in some genes encoding enzymes whose activation is required for the normal processes of tanning and sclerotization. most interestly, our microrray analysis identified metabolic enzymes whose transcripts were up-regulated significantly at h, but not at h after r-bursicon administration, suggesting that these enzymes may be actively involved in the early sclerotization process, perhaps in the synthesis of agents required for tanning and hardening. sclerotization is a process of cross-linking protein to protein chains, chitin to chitin chains, and protein to chitin chains. our microarray analysis also detected a cuticular protein gene 50ca with a chitin-binding domain and a gene, cg10140-ra that appears to be involved in chitin metabolism. although their exact role in cuticle formation is not known, the possibility exists that they function to bind chitin and/or play important roles in chitin metabolism. tanning requires energy in the form of atp and the cg3649-ra gene detected in the microarray analysis has a sugar transporter domain and may help supply metabolic energy for melanization. it is therefore evident that further research is necessary to identify with certainty those genes involved with specific steps in the tanning and sclerotization processes. our data are tantalizing but only suggestive at this point. early studies indicate that the brain triggers the release of bursicon from the fused thoracic/abdominal ganglion to the hemolymph by nervous stimuli leading to cuticle sclerotization. more recent investigations revealed that both bursicon protein and transcripts are present in the thoracic/abdominal ganglion, but not in the brain, of d. melanogaster and musca domestica, thus confirming the early observation. cuticle sclerotization is a brief but complex process occurring after each molt and the genes involved in this process are not expected to be present in large number. the genes identified in the microarray analysis reflect this estimation. although the functions and interrelationship of these genes in the cuticle sclerotization process is conjectural at present, the microarray data provide a foundation for further investigation of the functions of these identified genes, and such studies may allow a better understanding of the mechanisms of the cuticle sclerotization process in insects and other arthropods. sclerotization of the exoskeleton is a primary reason for the success of insects on this planet and the series of reactions constituting sclerotization and its control affects more animal species than any other series of phylum-specific reactions. it is most worthy of future research and we believe that the present study lays the basis for such investigations at another level.  bursicon is a neuropeptide that regulates cuticle sclerotization and wing expansion in insects, critical processes for insect survival. in these processes, many genes are up- and down-regulated. in this report, we demonstrated the presence of a set of genes that are sensitive to r-bursicon stimulation and the data were verified by qpcr analysis. this gene set supplies unique and intriguing candidates for a more complete understanding of bursicon action by the use of specific gene deletion and over-expression experiments that may lead to a functional dissection of some of these genes in the cuticle sclerotization process. 
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 which supported the microarray data. temporal response studies of identified and verified genes by qpcr revealed that the temporal expression patterns of these genes are consistent with the microarray data. temporal analysis of verified genes revealed that the expression of these genes was indeed induced by bursicon and correlated well with the cuticle sclerotization process. bursicon is a heterodimer neuropeptide responsible for regulating cuticle sclerotization and wing expansion in several insect species. the composite data suggest that these genes play important roles in regulating the cuticle sclerotization and wing expansion processes. recent studies indicate that the action of bursicon is mediated by a specific g protein-coupled receptor dlgr and the camp/pka signaling pathway.
 bursicon 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 dlgr 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.  we used dna microarray analysis to identify bursicon-regulated genes in neck-ligated flies that received recombinant bursicon. fifty four genes were found to be regulated by bursicon h post r-bursicon injection, being up-regulated and down-regulated while genes were influenced by r-bursicon h post-injection. 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 which supported the microarray data. temporal response studies of identified and verified genes by qpcr revealed that the temporal expression patterns of these genes are consistent with the microarray data.  using r-bursicon, we identified genes that are regulated by bursicon, 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 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. 