BACKGROUND
environmental changes are an obvious source of stress for an organism.
insects inhabiting variable environments employ a number of adaptations to survive adverse conditions.
developmental arrest, called diapause in insects, is one evolutionary adaptation utilized to endure unfavorable conditions  <cit> .
as a strategy for surviving unfavorable environmental conditions, diapause can occur in various developmental stages including egg, larva, pupa or adult, resulting in a programmed arrest of development coupled with other physiological changes  <cit> .
diapause is a dynamic process consisting of several successive phases: pre-diapause, diapause, and post-diapause, and each phase may comprise some sub-phases, e.g., the diapause phase is divided into diapause initiation, maintenance, and termination  <cit> .
the hormonal regulation of diapause has been well defined, but the molecular mechanism of diapause is unclear.
using pulse labeling combined with 2-dimensional electrophoresis and elimination hybridization, changes in protein synthesis  <cit>  and gene expression  <cit>  were firstly identified in the diapausing pupal brain of sarcophaga crassipalpis, suggesting that diapause is a unique developmental pathway rather than a simple shutdown of gene expression  <cit> .
suppression subtractive hybridization  has been used to evaluate diapause-specific gene expression in culex pipiens  <cit>  and s. crassipalpis  <cit> .
recently, a systemic investigation of transcript profiling of nondiapause and diapause pupae has been conducted using microarray technique in s. crassipalpis  <cit> .
in addition, proteomic method has been used to identify differentially expressed proteins in the brains of s. crassipalpis  <cit>  and helicoverpa armigera  <cit> .
many genes and proteins related to diapause have been identified, but differentially expressed genes during diapause initiation are rarely reported  <cit> .
it is yet unknown why individual insects can switch from direct development to arrested development.
the cotton bollworm h. armigera , an agriculturally important pest, enters pupal diapause for survival in winter.
after pupation, the diapause-destined pupae will enter diapause within 7-- <dig> days, because day  <dig> of pupae can not develop towards adults, even though these pupae are incubated in a suit conditions.
the physiological characteristics of diapause are observed on day  <dig> pupae, such as low ecdysone titer and unmoved eyespots, so differentially expressed genes as diapause instructions may be issued at an earlier pupal stage.
thus, we focused on gene expression in day  <dig> and  <dig> of pupae.
as the programmable center of diapause, the brain is the most important organ to release instructions for diapause initiation  <cit> .
to understand the molecular mechanism of diapause initiation, we searched for differentially expressed genes during pupal diapause initiation in h. armigera by using ssh.
meanwhile, the differentially expressed genes in nondiapause individuals were also investigated to search those genes expressed at low level in diapause-destined individuals.
RESULTS
general statistics from two ssh libraries
two subtracted cdna libraries enriched in diapause- or development-correlative genes were constructed by using ssh.
one was the f  library expected to be enriched in diapause up-regulated cdnas.
the f library was obtained using the mrnas from the diapause-destined pupal brains of days 1-- <dig> after pupation as the "tester", and mrnas from the nondiapause pupal brains of days 1-- <dig> as the "driver".
the other is the r  library to enrich cdnas up-regulated in nondiapause-destined  pupal brain by reversing the "tester" and "driver" mrnas.
a total of over  <dig> clones were randomly picked and subjected to colony pcr, and sequencing was carried out for  <dig> and  <dig> cdna clones selected at random from the f and r libraries, respectively.
after removing poor quality sequences, we finally obtained  <dig> unique sequences in the f library and  <dig> unique sequences in the r library.
all the sequences  were compared against available databases to find similarities with known sequences.
we carried out dynamic translation , and only matched sequences with an e-value lower than 10- <dig> were considered to be homologous sequences.
sequences with an e-value higher than 10- <dig> were labeled undescribed.
homologous sequences accounted for  <dig> % of the sequences in the f library and  <dig> % in the r library.
the homologous sequences are shown in additional file  <dig>  among the homologous sequences,  <dig> in the f library  and  <dig> in the r library  lacked annotation.
additionally, the number of no mapping sequences was four in the f library and three in the r library, respectively.
all of these data are summarized in figure  <dig>
gene ontology analysis
most genes isolated from the two libraries have not been identified, so that we here define these genes as putatively up-regulated genes from the f library and putatively down-regulated genes from the r library as shown in table  <dig>
hardp-a355
hardp-c556
hardp-c941
hardp-b1408
hardp-a22
hardp-a112
hardp-a345
hardp-c509
transcripts were classified according to the function of homologous genes in other species.
the putatively up-regulated genes at diapause initiation are shown in italics, and the putatively down-regulated genes are shown in bold
gene ontology analysis was carried out using the blast2go program  <cit> .
sequences were classified into the three ontology categories: biological process , molecular function and cellular component .
in the category "biological process", the most frequent process was cellular process , followed by metabolic process  and biological regulation .
in the category "molecular function", binding was the most frequent activity , followed by catalytic activity .
significant differences were observed in structural molecular activity  and transcription regulator activity categories .
in the last category, "cellular component" the most frequent activity was macromolecular complex , and membrane-enclosed lumen  appeared to be different between the two ssh libraries.
identification of differentially expressed transcripts
to test the reliability of the two ssh libraries, we randomly selected  <dig> genes from each library to investigate their expression levels in diapause- and nondiapause-destined brains at the early pupal stage by rt-pcr.
in the f library,  <dig> transcripts were expressed highly in diapause-destined individuals, with hardp-c <dig> being the exception .
in the r library,  <dig> transcripts were expressed highly in nondiapause pupae, with harnp- <dig> and harnp- <dig> being the exceptions .
furthermore, the levels of four transcripts from the f library were confirmed by northern blot analysis.
as shown in figure 3c, their expression was higher in diapause-destined pupae.
these results show that the two ssh libraries are reliable.
expression patterns at diapause initiation
to obtain some clues about the functions of the genes from the ssh library, the expression patterns of four selected genes in the brain were investigated during early pupal development by rt-pcr and western blot analysis.
the four genes encoded ubiquitin-like protein smt <dig> , mn superoxide dismutase , sericotropin  and translated controlled tumor protein , which were assessed by northern blot analysis above .
all four mrnas were expressed higher during early pupal development in diapause-destined individuals, especially sumo from day  <dig> to day  <dig>  mnsod from day  <dig> to day  <dig>  sericotropin from day  <dig> to day  <dig>  and tctp from day  <dig> to day  <dig> .
the four protein levels reflected their mrna levels .
apparently, high expression of these genes at the early pupal stage is likely to be associated with pupal diapause initiation.
metabolism and energy
nine genes, including four high- and five low-expression genes, were assigned to the metabolism and energy category .
two enzymes, aldolase  and enolase , which were up-regulated during diapause initiation, participate in glycolysis.
in contrast, an enzyme fructose- <dig> -bisphosphatase  , which is important in gluconeogenesis, was down-regulated at diapause initiation.
aconitase  and malate synthase , which are important components of the tricarboxylic acid  cycle, are down-regulated at diapause initiation.
additionally, a set of transcripts  encoding proteins involved in atp generation were up-regulated at diapause initiation.
atp synthase f <dig> subunit  <dig>  is a key component of atp synthase.
cytochrome c oxidase subunit  <dig>  and cytochrome c oxidase subunit 7c  are two components of the respiratory chain in mitochondria.
two genes related to lipid metabolism were found in the r library: harnp- <dig> and harnp- <dig> were down-regulated at diapause initiation.
apolipoprotein d  is closely associated with the enzyme lecithin:cholesterol acyltransferase and is involved in lipoprotein metabolism.
lipase  participates in the lipid degradation process.
stress resistance
eight genes were assigned to the stress resistance category , all up-regulated at diapause initiation.
hsp <dig>  acts as a molecular chaperone to protect cellular proteins from denaturation and contributes to the cold tolerance of insects  <cit> .
another group of transcripts that was up-regulated at diapause initiation was related to antioxidation: ferritin , ferritin light chain , mnsod , glutathione s-transferase  and bombyrin .
the last two transcripts activated in response to stress are related to dna repair: rad <dig>  and integrator complex subunit  <dig> .
rad <dig> functions in uv-damaged dna repair post-replication, and integrator complex subunit  <dig> is a component of the sensor of ssdna  complex, which is required for efficient homologous recombination-dependent repair of double-strand breaks  <cit> .
signaling pathway
several signaling pathways are involved in the regulation of developmental arrest, such as the guanylyl cyclase pathway, tgfβ-like pathway, insulin-like pathway, and steroid hormone pathway  <cit> .
in this study, the transcription of the akt gene  was up-regulated.
akt is an important protein in the insulin-like pathway .
in contrast, calmodulin protein kinase ii   and arginine kinase  are down-regulated during diapause initiation.
calmodulin-dependent signaling is required for development, and camk ii is a key member of this signaling pathway.
argk is a phosphotransferase that catalyzes the reaction between l-arginine and atp to produce l-phospho-arginine and adp, and it functions in the regulation of atp level, as creatine kinase in vertebrates  <cit> .
cell cycle
six transcripts down-regulated at diapause initiation were cell cycle regulators .
cyclin-dependent kinase   <dig>  is a member of the cdk family, which are important regulators of cell cycle progression.
cdk <dig> is also a coactivator involved in regulated gene transcription of nearly all rna polymerase ii--dependent genes.
the 80-kda mcm3-associated protein  interacts with mcm <dig>  which is a factor that allows the dna to undergo a single round of replication per cell cycle and is required for dna replication and cell proliferation  <cit> .
gtp-binding nuclear protein ran  is involved in chromatin condensation and cell cycle control.
mcm <dig> , as a dna replication licensing factor, participates in cell cycle regulation.
septin  <dig>  is required for the progression through mitosis  <cit> .
transcription factor dp- <dig>  can stimulate e2f-dependent transcription and promote the transcription of a number of genes whose products are involved in cell cycle regulation or in dna replication  <cit> .
transcription and translation
six genes related to transcription and translation were also found in the two ssh libraries .
two genes, cg <dig> , which is predicted to have transcriptional repressor activity, and sumo , which always represses the activity of transcription factors, were up-regulated at diapause initiation.
in contrast, four genes were down-regulated in diapause-type pupae: pleomorphic adenoma gene  <dig>  is a transcription factor whose activation results in up-regulation of target genes, such as insulin-like growth factor   <cit> .
elongation factor  <dig> delta  facilitates the events of translational elongation, resulting in promotion of protein biosynthesis.
oocyte zinc finger protein xlcof <dig>  functions in transcriptional regulation.
reptin  acts as a transcriptional activator, and also as an essential cofactor for the normal function of myc, so it is required for cellular proliferation and growth  <cit> .
discussion
the insect brain is the center of developmental control and serves as the repository of the diapause program  <cit> .
in pupal diapause species, photoperiodic signal is perceived by larval brain during diapause induction.
then gene expression changes affected by photoperiod are first present in diapause preparation phase which follows diapause induction to regulate specific metabolism for diapause  <cit> .
it is well known that after pupation, a shut-down of prothoracicotropic hormone  in the brain and ecdysteroids in the prothoracic gland cause diapause initiation  <cit> .
meola and adkisson demonstrated that the shut-down of ptth is found in day  <dig> of pupal brain of helicoverpa zea, a closely related species to h. armigera  <cit> .
thus, these differentially expressed genes isolated from the two libraries in day 1- <dig> pupal brain of h. armigera for diapause initiation are in response to hormones, but not photoperiodic signal.
in h. armigera, the photosensitive stage for diapsuse induction is from 5th instar to early stage of 6th instar.
this is little different compared to h. armigera population from okayama , whose photosensitive stage for diapause induction is the early fifth instar  <cit> .
after pupation, h. armigera diapause-type pupae are transferred into l14:10d photoperiod, all pupae will enter diapause, and all pupae will develop without diapause even if nondiapause-type pupae are transferred into l10:14d photoperiod .
apparently, photoperiod regime does not affect pupal diapause or development.
the most remarkable characteristic of insect diapause is strong metabolic suppression.
for example, in diapausing pupae of the flesh fly, sarcophaga argyrostoma, the metabolic rate is approximately 90% lower than in nondiapause counterparts  <cit> .
therefore, diapause was thought to represent a shutdown in gene expression.
however, joplin et al.
<cit>  and flannagan et al.
<cit>  demonstrated that diapause should be a unique developmental pathway rather than a simple shutdown of gene expression.
recently, the proteomic analysis of the brain at diapause initiation has been reported, suggesting that the expression of many diapause-specific genes in the brain accompanies certain down-regulated genes  <cit> .
thus, identification of diapause-associated genes at diapause initiation is the first step to understand the complex process of diapause.
in the present paper, we isolated  <dig> diapause-specific mrnas from h. armigera brain using ssh, and the subset of these genes with sequences similar to known genes in genbank were classified according to their functions.
furthermore, we evaluated their mrna expression at diapause initiation by rt-pcr and northern blot analysis, and investigated the expression patterns of four important genes by rt-pcr and western blot analysis, showing that these genes may be associated with diapause initiation.
from the ssh f library, we found a high percentage of undescribed sequences .
some sequences may correspond to 3' or 5' untranslated regions , so it is impossible to find their homologues in protein databases.
however, most of these undescribed sequences can be classified as novel genes related to h. armigera pupal diapause initiation, because only a few genes related to developmental arrest have been identified.
the large percentage of unknown  genes in the f library shows that diapause is a complex physiological process involving a number of unknown genes in the regulation of developmental arrest.
we also constructed an r library to identify specific genes expressed in nondiapause individuals.
the up-regulated gene expression in nondiapause pupae identified from the r library usually corresponded to down-regulated expression in diapause-type pupae , so these genes from the r library will help us to identify the genes associated with insect diapause if these differentially expressed genes in diapause-destined pupae are further characterized.
a total of  <dig> sequences from the two libraries that were homologous to known genes were obtained.
according to gene ontology analysis, most genes belonged to cellular process and metabolic process in the category of "biological process"; this implies that the insect brain at diapause initiation focuses on alteration of cellular and metabolic state.
signaling and transcriptional regulator activity also showed significant differences between the two libraries.
up-regulation of signaling genes and down-regulation of transcriptional regulators at diapause initiation indicate that signaling pathways are changed, global transcription levels are down-regulated, and diapause does require a unique gene expression regulatory mechanism.
the quality and reliability of the two ssh libraries were validated by investigating gene expression difference between diapause- and nondiapause-destined pupae.
the two libraries were quite reliable, so the ssh method was useful to search for genes related to pupal diapause initiation.
subsequently, the expression patterns of four genes were detected by rt-pcr and western blot analysis.
all four genes were expressed higher at both the mrna and protein levels during early pupal development in diapause-destined individuals than their nondiapause-destined counterparts.
apparently, these genes from the ssh library may reflect differential expression between diapause- and nondiapause-destined pupae for promoting diapause initiation.
based on the functions of the putatively up- and down-regulated genes , we have proposed a possible mechanism for diapause initiation.
changes in metabolism and energy
the brain of early diapause-destined pupae releases instructions to switch from development to diapause, so changes in metabolism and energy must be involved in the process.
at diapause initiation, insects need to store energy and synthesize some specific compounds for cold hardiness, such as cryoprotectants.
from the two ssh libraries, some transcripts function in metabolism and energy.
aldolase catalyzes the fourth step in glycolysis, which cleaves fructose  <dig>  6-bisphosphate and generates dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.
dihydroxyacetone phosphate is synthesized into glycerol, and glyceraldehyde 3-phosphate enters the glycolytic pathway to generate energy.
enolase is an enzyme that catalyzes the ninth step of the glycolytic pathway, resulting in the formation of phosphoenolpyruvate  and pyruvate.
fbp catalyzes the conversion of fructose- <dig> -bisphosphate to fructose-6-phosphate, a key step between glycolysis and gluconeogenesis.
to the best of our knowledge, gluconeogenesis and glycolysis are coordinated so that one way is relatively inactive while the other is highly active.
as shown in figure 5a, the down-regulation of fbp and up-regulation of aldolase and enolase suggest that gluconeogenesis diminished at diapause initiation, and glycerol biosynthesis is accelerated by glycolysis.
glycerol protects insects from cold stress.
meanwhile, the possible up-regulation of aldolase and enolase are responsible for generating pyruvate, which is also elevated in s. crassipalpis during pupal diapause  <cit> , and pyruvate enters the glycolytic pathway to generate energy.
aconitase and malate synthase, which participate in the tca cycle, are down-regulated.
this result implies that the down-regulated aconitase and malate synthase may directly repress the tca cycle.
in diapause pupae of the flesh fly, s. crassipalpis, the tca cycle is suppressed, and the metabolic intermediates from the tca cycle are also reduced  <cit> .
therefore, inhibition of the tca cycle and enhancement of glycolysis indicate that anaerobic metabolism is predominant at diapause initiation.
in fact, respiration in diapause individuals is significantly lower than in nondiapause individuals  <cit> , which is consistent with the decreased metabolic rate in diapause-destined individuals, and inhibition of the tca cycle in the brain helps diapause individuals save energy.
enhancement of anaerobic metabolism has also been reported in recent studies of larval diapauses in the pitcher plant mosquito, wyeomyia smitbii  <cit> , and embryonic diapauses in the cricket, allonemobius socius  <cit> .
additionally, three transcripts  for atp generation were up-regulated at diapause initiation.
atp synthase f <dig> subunit  <dig>  plays a role in the production of atp from adp.
cytochrome c oxidase is a component of the respiratory chain in mitochondria.
cytochrome c oxidase subunit  <dig>  transfers electrons from cytochrome c to the bimetallic center of the catalytic subunit  <dig>  cytochrome c oxidase subunit 7c  is one of the nuclear-coded polypeptide chains of cytochrome c oxidase, the terminal oxidase in mitochondrial electron transport.
such a change of cytochrome c oxidase subunits during diapause has been reported in c. pipiens  <cit> .
these observations suggest that energy demand still high during pupal diapause initiation.
as shown in table 1a, the transcripts associated with lipid metabolism  are down-regulated in diapause-destined pupal brain.
down-regulation of lipase has also been reported in early stage of diapause c. pipiens, but it is up-regulated in late diapause, suggesting that fatty acid-oxidation is suppressed in early diapause  <cit> .
the down-regulation of apolipoprotein d and lipase implies that diapause individuals first utilize sugar as energy and store lipid for use during long diapause periods.
stress resistance
during the long overwintering phase, diapause pupae must encounter various stress challenges.
the expressions of some specific genes are evoked in response to environmental stress  <cit> , and stress resistance is important for the survival of diapause individuals.
hsp <dig> functions as a molecular chaperone to protect cellular proteins from denaturation in many species, including diptera, lepidoptera, coleoptera and hymenoptera  <cit> .
in addition, a small hsp, hsp <dig>  identified by proteomic analysis, is more abundant in the brain of h. armigera pupae at diapause initiation  <cit> .
thus, the up-regulated hsp <dig> at diapause initiation plays a role in cold-hardiness for overwintering .
moreover, up-regulation of hsp has also been reported under short day-length conditions, and hsp up-regulation could represent a molecular exaptation to diapause  <cit> .
ferritin is the primary iron storage protein, and it functions in scavenging oxygen radicals  <cit> .
ferritin and ferritin light chain are up-regulated at diapause initiation, as reported in nasonia  <cit>  and in s. crassipalpis  <cit> .
mnsod is also up-regulated at diapause initiation.
in caenorhabditis elegans, mnsod participates in the regulation of both longevity and dauer formation  as a physiological redox signaling modulators  <cit> .
gst and bombyrin also have antioxidant function and are up-regulated in the brain, as reported in proteomic analysis of h. armigera  <cit> .
oxidative stress can damage tissues and cellular components during diapause.
therefore, the up-regulation of transcripts of antioxidant proteins will protect diapause individuals from oxidative stress .
rad <dig> is a nucleotide excision repair  gene that, functions in dna repair and protein degradation  <cit> .
integrator complex subunit  <dig>  which is also called soss-a, is involved in sensing ssdna and maintaining genome stability  <cit> .
up-regulation of genes related to dna repair  in diapause has not been reported previously.
however, it is possible that dna lesions occur under extreme environmental conditions during diapause, and the integrity of dna is crucial for re-starting the development into an adult when diapause is terminated.
therefore, these up-regulated genes at diapause initiation mainly respond to stress resistance for insect survival in rigorous environmental conditions .
signaling pathways
genes involved in signaling pathway were also found in the ssh library .
akt is an essential component of the insulin signaling pathway for glucose uptake to synthesize sugar and also as an activator of the target of rapamycin  pathway to increase protein synthesis  <cit> .
insect organs and tissues need to accumulate a large store of sugar as energy and other substances, such as antifreeze agents glycerol and sorbitol, for use during a long diapause phase.
therefore, enhanced akt transcription reflects increased sugar metabolism in diapause-destined pupal brain, and akt participates in the regulation of energy reserves and in response to environmental stress at the onset of diapause .
calmodulin signaling, which is involved in the regulation of neuronal development and plasticity  <cit> , is down-regulated at diapause initiation in h. armigera  <cit> .
in this study, camk ii, which modulates synaptic plasticity, learning, and memory  <cit> , was down-regulated.
argk was also down-regulated at diapause initiation, and high expression of argk, which is a developmental signal, was closely correlated with pupal development .
thus, down-regulation of camk ii and argk may cause developmental arrest at diapause initiation .
cell cycle
during diapause, the cell cycle is arrested in the embryo of b. mori  <cit>  and in the brains of s. crassipalpis  <cit>  and chymomyza costata  <cit> .
cyclin-dependent kinase  <dig> is a kinase partner of cyclin c, interacts with the large subunit of rna polymerase ii, and then participates in the regulation of the g1/s transition of mitosis  <cit> .
more than 97% of the brain cells become arrested in the g0/g <dig> phase in the diapause pupae of s. crassipalpis  <cit> .
proteomic analysis of sitodiplosis mosellana has found a strong up-regulation of inhibitor of nuclear factor kappa-b kinase interacting protein isoform  <dig>  during diapause, which contributes to inhibiting cell division during diapause  <cit> .
therefore, cyclin-dependent kinase  <dig> and five other transcripts down-regulated in the brain at diapause initiation may cause cell cycle arrest, inducing the insect to enter diapause .
transcription and translation
transcription and translation are two major energetic costs in cellular development.
to reduce energy consumption, many genes are silenced during diapause  <cit> .
in this study, several genes involved in the regulation of transcription and translation were identified .
the down-regulation of transcription factor plag <dig> may result in the modulation of downstream target genes  <cit> .
the down-regulation of elongation factor  <dig> delta indicates that translation is also suppressed at diapause initiation.
in addition, some transcripts of proteins involved in transcription were up-regulated at diapause initiation: hardp-c <dig> is homologous to drosophila cg <dig>  which contains the conserved mynd and set domains found in human smyd homologues.
drosophila smyd represses transcription  <cit> .
smt <dig>  is a reversible post-translational protein modifier that usually represses the activity of transcriptional activators  <cit> .
thus, we conclude that the down-regulation of plag <dig> and elongation factor  <dig> delta and the up-regulation of transcriptional repressors and sumo lead to the global down-regulation of transcription and translation at diapause initiation .
CONCLUSIONS
in this study, differentially expressed genes at the early pupal stage of diapause- and nondiapause-destined individuals were isolated and identified that may be involved in regulation of diapause initiation.
diapause initiation is an intriguing developmental process with a complex molecular mechanism.
based on the above results, we suggest a possible molecular mechanism for diapause initiation.
transition of metabolism and energy utilization.
in addition to a decrease of metabolic activity, metabolic pathways are also changed in diapause-destined pupae at diapause initiation.
anaerobic metabolism predominates, and sugars and polylols accumulate in the brain.
enhancement of stress resistance.
the antifreeze agents glycerol and sorbitol as well as hsp, gst, and others are heavily synthesized to protect the insect from rigorous environmental conditions.
regulation of cellular development.
the cell cycle is arrested, resulting in repression of pupal development toward adulthood.
repression of transcription and translation.
the up-regulation of transcriptional repressors, down-regulation of translational activators, and increased protein sumoylation result in decreases of both gene transcription and protein translation at diapause initiation.
this idea awaits detailed experimental investigation in the future.
materials and methods
animals
h. armigera larvae were reared on an artificial diet at 20°c with a l14:d <dig>  and a l10:d <dig>  photoperiod.
after pupation, the two types of pupae were moved to the same conditions .
under these conditions, all nondiapause pupae developed toward adults, and more than 95% of diapause type pupae entered diapause.
the developmental stages were synchronized at each molt by collecting new larvae or pupae.
all tissues were dissected in insect saline containing  <dig> % nacl, and stored at -80°c until use.
suppression subtractive hybridization
we constructed two subtracted cdna libraries  to detect high gene expression in diapause- and nondiapause-destined individuals at the early pupal stage  using the pcr-select™ cdna subtraction kit .
in the f library, diapause type pupae were used as the "tester", and nondiapause pupa as the "driver".
in the r library, the "tester" and "driver" were reversed.
after pupation, diapause- and nondiapause-destined pupae were incubated with the same condition 20°c and a short daylength  for 2- <dig> days before dissection.
total rna from day 1- <dig> brains of diapause- and nondiapause-destined pupae was isolated using a guanidinium thiocyanate-chloroform method  <cit> .
the mrna was obtained according to the manufacturer's protocols of quickprep micro mrna purification kit .
double-stranded cdnas were synthesized from  <dig>  μg of polya+ mrna and digested with rsai to obtain shorter blunt-ended cdna.
the "tester" cdna was subdivided into two populations, which were ligated to adaptor  <dig> and adaptor 2r, respectively.
two hybridizations were performed with the tester and driver cdnas.
in the first hybridization, the amount of driver cdna was  <dig> times more than the tester cdna.
as a result, cdnas that were not up-regulated were hybridized by driver cdnas, only the up-regulated tester cdnas were left as single strand.
in the second hybridization, the amount of driver cdna was  <dig> times more than the tester cdna.
as a result, the hybridized cdnas were eliminated, leaving only the unhybridized cdnas.
the entire population of unhybridized molecules was then subjected to pcr to amplify target cdna fragments .
only the molecules of the tester sample, which were ligated to the two different adaptors, could be amplified exponentially.
a second pcr amplification was performed using nested primers to get a low-background, high-level enrichment of the differentially expressed sequences.
the pcr products were analyzed by 2% agarose gel electrophoresis.
products from the secondary pcrs were inserted into pmd18-t by t/a cloning .
the recombinant plasmid dnas were transformed into xl- <dig> blue competent cells.
the dna from recombinant clones was isolated and sequenced .
bioinformatics analysis
all contigs and singlets were annotated according to the go classification and the hierarchical structure using the blast2go suite  <cit> .
the blast2go program, which assigns the go terms based on the blast definitions, was applied with an e-value < 10- <dig>  if a transcript was annotated with more than one go category, it was split equally among them.
rna extraction and rt-pcr
total rna was extracted from the brain using the acid-guanidine method  <cit> .
first-strand cdna was synthesized using  <dig> μg of total rna at 37°c for  <dig> h, with an m-mlv reverse transcription system .
the primers used to identify of differentially expressed transcripts by rt-pcr are presented in additional file  <dig>  the pcr reactions were subjected to 22-- <dig> cycles consisting of 94°c for  <dig> s; 55°c for  <dig> s; 72°c for  <dig> min.
actin was used as an internal standard.
northern blot hybridization
total rna  from the brain of day 1-- <dig> diapause- and nondiapause-destined pupae was separated on a  <dig> % agarose gel containing  <dig>  mol/l formaldehyde, and transferred to a nylon membrane .
probes for hybridization were labeled with -dctp using the random primer labeling kit .
after prehybridization for  <dig> h in 5× sspe  containing 50% formamide, 5× denhardt's solution,  <dig> % sds, and  <dig> μg/ml salmon sperm dna, the radiolabeled probe was added and hybridization was conducted overnight at 42°c  <cit> .
after hybridization, the membrane was washed in  <dig> × sspe at 42°c three times and exposed to x-ray film  overnight at -70°c.
polyclonal antibody generation and western blot analysis
the orfs of four genes  were amplified by pcr, using primers that contained restriction sites.
the pcr product was digested by the appropriate restricted enzymes, then purified and subcloned into the pet28a vector.
the recombinant pet plasmid was transfected into bl <dig> cells and induced by iptg.
the e. coli pellet was solubilized in  <dig> m urea in  <dig> mm tris-hcl buffer, ph  <dig> , followed by ni-nta column purification.
purified recombinant proteins were used to generate polyclonal antibodies in rabbit.
proteins for western blotting were extracted from the brain and sg or the brain--sg complexes of pupae, quantified by the bradford method  <cit> , and stored at -80°c.
protein  was separated by 15% sds-page and transferred onto an immobilon-p transfer membrane .
the immuno-reactivity was tested with antiserum, and then incubated with goat anti-rabbit igg , and protein was detected using the novex chemiluminescent substrates .
authors' contributions
whx designed the research project, bb performed experiments.
bb and whx wrote the paper, and approved the final manuscript.
supplementary material
additional file 1
table s <dig> checklist of known genes in the two libraries.
checklist of the sequences of known genes in the f library.
checklist of the sequences those were homologous to known genes in the r library.
all transcripts were compared to the sequences in genbank using blastx.
sequence description, minimum e-value and similarity are shown in the table.
click here for file
additional file 2
table s <dig> comparison of the two libraries according to gene ontology criteria .
click here for file
additional file 3
figure s <dig> classification of ssh ests according to gene ontology criteria .
classification of ssh ests according to gene ontology criteria .
gene ontology analysis was carried out on the transcripts isolated from the two ssh libraries by blast2go program.
the molecular function combined graph was made based on ontology level  <dig>   classification of ssh ests according to gene ontology criteria .
the cellular component combined graph was made based on ontology level  <dig> by blast2go.
click here for file
additional file 4
table s <dig> primer sequences used in pcr.
click here for file
acknowledgements
this study was supported by a grant-in-aid for the natural scientific foundation  from the national natural science foundation of china, and the major state basic research developmental program  from the ministry of science and technology of china.

 environmental changes are an obvious source of stress for an organism. insects inhabiting variable environments employ a number of adaptations to survive adverse conditions. developmental arrest, called diapause in insects, is one evolutionary adaptation utilized to endure unfavorable conditions. as a strategy for surviving unfavorable environmental conditions, diapause can occur in various developmental stages including egg, larva, pupa or adult, resulting in a programmed arrest of development coupled with other physiological changes. diapause is a dynamic process consisting of several successive phases: pre-diapause, diapause, and post-diapause, and each phase may comprise some sub-phases, e.g., the diapause phase is divided into diapause initiation, maintenance, and termination. the hormonal regulation of diapause has been well defined, but the molecular mechanism of diapause is unclear. using pulse labeling combined with 2-dimensional electrophoresis and elimination hybridization, changes in protein synthesis and gene expression were firstly identified in the diapausing pupal brain of sarcophaga crassipalpis, suggesting that diapause is a unique developmental pathway rather than a simple shutdown of gene expression. suppression subtractive hybridization has been used to evaluate diapause-specific gene expression in culex pipiens and s. crassipalpis. recently, a systemic investigation of transcript profiling of nondiapause and diapause pupae has been conducted using microarray technique in s. crassipalpis. in addition, proteomic method has been used to identify differentially expressed proteins in the brains of s. crassipalpis and helicoverpa armigera. many genes and proteins related to diapause have been identified, but differentially expressed genes during diapause initiation are rarely reported. it is yet unknown why individual insects can switch from direct development to arrested development. the cotton bollworm h. armigera, an agriculturally important pest, enters pupal diapause for survival in winter. after pupation, the diapause-destined pupae will enter diapause within 7-- days, because day of pupae can not develop towards adults, even though these pupae are incubated in a suit conditions. the physiological characteristics of diapause are observed on day pupae, such as low ecdysone titer and unmoved eyespots, so differentially expressed genes as diapause instructions may be issued at an earlier pupal stage. thus, we focused on gene expression in day and of pupae. as the programmable center of diapause, the brain is the most important organ to release instructions for diapause initiation. to understand the molecular mechanism of diapause initiation, we searched for differentially expressed genes during pupal diapause initiation in h. armigera by using ssh. meanwhile, the differentially expressed genes in nondiapause individuals were also investigated to search those genes expressed at low level in diapause-destined individuals.  general statistics from two ssh libraries two subtracted cdna libraries enriched in diapause- or development-correlative genes were constructed by using ssh. one was the f library expected to be enriched in diapause up-regulated cdnas. the f library was obtained using the mrnas from the diapause-destined pupal brains of days 1-- after pupation as the "tester", and mrnas from the nondiapause pupal brains of days 1-- as the "driver". the other is the r library to enrich cdnas up-regulated in nondiapause-destined pupal brain by reversing the "tester" and "driver" mrnas. a total of over clones were randomly picked and subjected to colony pcr, and sequencing was carried out for and cdna clones selected at random from the f and r libraries, respectively. after removing poor quality sequences, we finally obtained unique sequences in the f library and unique sequences in the r library. all the sequences were compared against available databases to find similarities with known sequences. we carried out dynamic translation, and only matched sequences with an e-value lower than 10- were considered to be homologous sequences. sequences with an e-value higher than 10- were labeled undescribed. homologous sequences accounted for % of the sequences in the f library and % in the r library. the homologous sequences are shown in additional file among the homologous sequences, in the f library and in the r library lacked annotation. additionally, the number of no mapping sequences was four in the f library and three in the r library, respectively. all of these data are summarized in figure gene ontology analysis most genes isolated from the two libraries have not been identified, so that we here define these genes as putatively up-regulated genes from the f library and putatively down-regulated genes from the r library as shown in table hardp-a355 hardp-c556 hardp-c941 hardp-b1408 hardp-a22 hardp-a112 hardp-a345 hardp-c509 transcripts were classified according to the function of homologous genes in other species. the putatively up-regulated genes at diapause initiation are shown in italics, and the putatively down-regulated genes are shown in bold gene ontology analysis was carried out using the blast2go program. sequences were classified into the three ontology categories: biological process, molecular function and cellular component. in the category "biological process", the most frequent process was cellular process, followed by metabolic process and biological regulation. in the category "molecular function", binding was the most frequent activity, followed by catalytic activity. significant differences were observed in structural molecular activity and transcription regulator activity categories. in the last category, "cellular component" the most frequent activity was macromolecular complex, and membrane-enclosed lumen appeared to be different between the two ssh libraries. identification of differentially expressed transcripts to test the reliability of the two ssh libraries, we randomly selected genes from each library to investigate their expression levels in diapause- and nondiapause-destined brains at the early pupal stage by rt-pcr. in the f library, transcripts were expressed highly in diapause-destined individuals, with hardp-c being the exception. in the r library, transcripts were expressed highly in nondiapause pupae, with harnp- and harnp- being the exceptions. furthermore, the levels of four transcripts from the f library were confirmed by northern blot analysis. as shown in figure 3c, their expression was higher in diapause-destined pupae. these results show that the two ssh libraries are reliable. expression patterns at diapause initiation to obtain some clues about the functions of the genes from the ssh library, the expression patterns of four selected genes in the brain were investigated during early pupal development by rt-pcr and western blot analysis. the four genes encoded ubiquitin-like protein smt, mn superoxide dismutase, sericotropin and translated controlled tumor protein, which were assessed by northern blot analysis above. all four mrnas were expressed higher during early pupal development in diapause-destined individuals, especially sumo from day to day mnsod from day to day sericotropin from day to day and tctp from day to day. the four protein levels reflected their mrna levels. apparently, high expression of these genes at the early pupal stage is likely to be associated with pupal diapause initiation. metabolism and energy nine genes, including four high- and five low-expression genes, were assigned to the metabolism and energy category. two enzymes, aldolase and enolase, which were up-regulated during diapause initiation, participate in glycolysis. in contrast, an enzyme fructose- -bisphosphatase, which is important in gluconeogenesis, was down-regulated at diapause initiation. aconitase and malate synthase, which are important components of the tricarboxylic acid cycle, are down-regulated at diapause initiation. additionally, a set of transcripts encoding proteins involved in atp generation were up-regulated at diapause initiation. atp synthase f subunit is a key component of atp synthase. cytochrome c oxidase subunit and cytochrome c oxidase subunit 7c are two components of the respiratory chain in mitochondria. two genes related to lipid metabolism were found in the r library: harnp- and harnp- were down-regulated at diapause initiation. apolipoprotein d is closely associated with the enzyme lecithin:cholesterol acyltransferase and is involved in lipoprotein metabolism. lipase participates in the lipid degradation process. stress resistance eight genes were assigned to the stress resistance category, all up-regulated at diapause initiation. hsp acts as a molecular chaperone to protect cellular proteins from denaturation and contributes to the cold tolerance of insects. another group of transcripts that was up-regulated at diapause initiation was related to antioxidation: ferritin, ferritin light chain, mnsod, glutathione s-transferase and bombyrin. the last two transcripts activated in response to stress are related to dna repair: rad and integrator complex subunit. rad functions in uv-damaged dna repair post-replication, and integrator complex subunit is a component of the sensor of ssdna complex, which is required for efficient homologous recombination-dependent repair of double-strand breaks. signaling pathway several signaling pathways are involved in the regulation of developmental arrest, such as the guanylyl cyclase pathway, tgfβ-like pathway, insulin-like pathway, and steroid hormone pathway. in this study, the transcription of the akt gene was up-regulated. akt is an important protein in the insulin-like pathway. in contrast, calmodulin protein kinase ii and arginine kinase are down-regulated during diapause initiation. calmodulin-dependent signaling is required for development, and camk ii is a key member of this signaling pathway. argk is a phosphotransferase that catalyzes the reaction between l-arginine and atp to produce l-phospho-arginine and adp, and it functions in the regulation of atp level, as creatine kinase in vertebrates. cell cycle six transcripts down-regulated at diapause initiation were cell cycle regulators. cyclin-dependent kinase is a member of the cdk family, which are important regulators of cell cycle progression. cdk is also a coactivator involved in regulated gene transcription of nearly all rna polymerase ii--dependent genes. the 80-kda mcm3-associated protein interacts with mcm which is a factor that allows the dna to undergo a single round of replication per cell cycle and is required for dna replication and cell proliferation. gtp-binding nuclear protein ran is involved in chromatin condensation and cell cycle control. mcm, as a dna replication licensing factor, participates in cell cycle regulation. septin is required for the progression through mitosis. transcription factor dp- can stimulate e2f-dependent transcription and promote the transcription of a number of genes whose products are involved in cell cycle regulation or in dna replication. transcription and translation six genes related to transcription and translation were also found in the two ssh libraries. two genes, cg, which is predicted to have transcriptional repressor activity, and sumo, which always represses the activity of transcription factors, were up-regulated at diapause initiation. in contrast, four genes were down-regulated in diapause-type pupae: pleomorphic adenoma gene is a transcription factor whose activation results in up-regulation of target genes, such as insulin-like growth factor. elongation factor delta facilitates the events of translational elongation, resulting in promotion of protein biosynthesis. oocyte zinc finger protein xlcof functions in transcriptional regulation. reptin acts as a transcriptional activator, and also as an essential cofactor for the normal function of myc, so it is required for cellular proliferation and growth. discussion the insect brain is the center of developmental control and serves as the repository of the diapause program. in pupal diapause species, photoperiodic signal is perceived by larval brain during diapause induction. then gene expression changes affected by photoperiod are first present in diapause preparation phase which follows diapause induction to regulate specific metabolism for diapause. it is well known that after pupation, a shut-down of prothoracicotropic hormone in the brain and ecdysteroids in the prothoracic gland cause diapause initiation. meola and adkisson demonstrated that the shut-down of ptth is found in day of pupal brain of helicoverpa zea, a closely related species to h. armigera. thus, these differentially expressed genes isolated from the two libraries in day 1- pupal brain of h. armigera for diapause initiation are in response to hormones, but not photoperiodic signal. in h. armigera, the photosensitive stage for diapsuse induction is from 5th instar to early stage of 6th instar. this is little different compared to h. armigera population from okayama, whose photosensitive stage for diapause induction is the early fifth instar. after pupation, h. armigera diapause-type pupae are transferred into l14:10d photoperiod, all pupae will enter diapause, and all pupae will develop without diapause even if nondiapause-type pupae are transferred into l10:14d photoperiod. apparently, photoperiod regime does not affect pupal diapause or development. the most remarkable characteristic of insect diapause is strong metabolic suppression. for example, in diapausing pupae of the flesh fly, sarcophaga argyrostoma, the metabolic rate is approximately 90% lower than in nondiapause counterparts. therefore, diapause was thought to represent a shutdown in gene expression. however, joplin et al. and flannagan et al. demonstrated that diapause should be a unique developmental pathway rather than a simple shutdown of gene expression. recently, the proteomic analysis of the brain at diapause initiation has been reported, suggesting that the expression of many diapause-specific genes in the brain accompanies certain down-regulated genes. thus, identification of diapause-associated genes at diapause initiation is the first step to understand the complex process of diapause. in the present paper, we isolated diapause-specific mrnas from h. armigera brain using ssh, and the subset of these genes with sequences similar to known genes in genbank were classified according to their functions. furthermore, we evaluated their mrna expression at diapause initiation by rt-pcr and northern blot analysis, and investigated the expression patterns of four important genes by rt-pcr and western blot analysis, showing that these genes may be associated with diapause initiation. from the ssh f library, we found a high percentage of undescribed sequences. some sequences may correspond to 3' or 5' untranslated regions, so it is impossible to find their homologues in protein databases. however, most of these undescribed sequences can be classified as novel genes related to h. armigera pupal diapause initiation, because only a few genes related to developmental arrest have been identified. the large percentage of unknown genes in the f library shows that diapause is a complex physiological process involving a number of unknown genes in the regulation of developmental arrest. we also constructed an r library to identify specific genes expressed in nondiapause individuals. the up-regulated gene expression in nondiapause pupae identified from the r library usually corresponded to down-regulated expression in diapause-type pupae, so these genes from the r library will help us to identify the genes associated with insect diapause if these differentially expressed genes in diapause-destined pupae are further characterized. a total of sequences from the two libraries that were homologous to known genes were obtained. according to gene ontology analysis, most genes belonged to cellular process and metabolic process in the category of "biological process"; this implies that the insect brain at diapause initiation focuses on alteration of cellular and metabolic state. signaling and transcriptional regulator activity also showed significant differences between the two libraries. up-regulation of signaling genes and down-regulation of transcriptional regulators at diapause initiation indicate that signaling pathways are changed, global transcription levels are down-regulated, and diapause does require a unique gene expression regulatory mechanism. the quality and reliability of the two ssh libraries were validated by investigating gene expression difference between diapause- and nondiapause-destined pupae. the two libraries were quite reliable, so the ssh method was useful to search for genes related to pupal diapause initiation. subsequently, the expression patterns of four genes were detected by rt-pcr and western blot analysis. all four genes were expressed higher at both the mrna and protein levels during early pupal development in diapause-destined individuals than their nondiapause-destined counterparts. apparently, these genes from the ssh library may reflect differential expression between diapause- and nondiapause-destined pupae for promoting diapause initiation. based on the functions of the putatively up- and down-regulated genes, we have proposed a possible mechanism for diapause initiation. changes in metabolism and energy the brain of early diapause-destined pupae releases instructions to switch from development to diapause, so changes in metabolism and energy must be involved in the process. at diapause initiation, insects need to store energy and synthesize some specific compounds for cold hardiness, such as cryoprotectants. from the two ssh libraries, some transcripts function in metabolism and energy. aldolase catalyzes the fourth step in glycolysis, which cleaves fructose 6-bisphosphate and generates dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. dihydroxyacetone phosphate is synthesized into glycerol, and glyceraldehyde 3-phosphate enters the glycolytic pathway to generate energy. enolase is an enzyme that catalyzes the ninth step of the glycolytic pathway, resulting in the formation of phosphoenolpyruvate and pyruvate. fbp catalyzes the conversion of fructose- -bisphosphate to fructose-6-phosphate, a key step between glycolysis and gluconeogenesis. to the best of our knowledge, gluconeogenesis and glycolysis are coordinated so that one way is relatively inactive while the other is highly active. as shown in figure 5a, the down-regulation of fbp and up-regulation of aldolase and enolase suggest that gluconeogenesis diminished at diapause initiation, and glycerol biosynthesis is accelerated by glycolysis. glycerol protects insects from cold stress. meanwhile, the possible up-regulation of aldolase and enolase are responsible for generating pyruvate, which is also elevated in s. crassipalpis during pupal diapause, and pyruvate enters the glycolytic pathway to generate energy. aconitase and malate synthase, which participate in the tca cycle, are down-regulated. this result implies that the down-regulated aconitase and malate synthase may directly repress the tca cycle. in diapause pupae of the flesh fly, s. crassipalpis, the tca cycle is suppressed, and the metabolic intermediates from the tca cycle are also reduced. therefore, inhibition of the tca cycle and enhancement of glycolysis indicate that anaerobic metabolism is predominant at diapause initiation. in fact, respiration in diapause individuals is significantly lower than in nondiapause individuals, which is consistent with the decreased metabolic rate in diapause-destined individuals, and inhibition of the tca cycle in the brain helps diapause individuals save energy. enhancement of anaerobic metabolism has also been reported in recent studies of larval diapauses in the pitcher plant mosquito, wyeomyia smitbii, and embryonic diapauses in the cricket, allonemobius socius. additionally, three transcripts for atp generation were up-regulated at diapause initiation. atp synthase f subunit plays a role in the production of atp from adp. cytochrome c oxidase is a component of the respiratory chain in mitochondria. cytochrome c oxidase subunit transfers electrons from cytochrome c to the bimetallic center of the catalytic subunit cytochrome c oxidase subunit 7c is one of the nuclear-coded polypeptide chains of cytochrome c oxidase, the terminal oxidase in mitochondrial electron transport. such a change of cytochrome c oxidase subunits during diapause has been reported in c. pipiens. these observations suggest that energy demand still high during pupal diapause initiation. as shown in table 1a, the transcripts associated with lipid metabolism are down-regulated in diapause-destined pupal brain. down-regulation of lipase has also been reported in early stage of diapause c. pipiens, but it is up-regulated in late diapause, suggesting that fatty acid-oxidation is suppressed in early diapause. the down-regulation of apolipoprotein d and lipase implies that diapause individuals first utilize sugar as energy and store lipid for use during long diapause periods. stress resistance during the long overwintering phase, diapause pupae must encounter various stress challenges. the expressions of some specific genes are evoked in response to environmental stress, and stress resistance is important for the survival of diapause individuals. hsp functions as a molecular chaperone to protect cellular proteins from denaturation in many species, including diptera, lepidoptera, coleoptera and hymenoptera. in addition, a small hsp, hsp identified by proteomic analysis, is more abundant in the brain of h. armigera pupae at diapause initiation. thus, the up-regulated hsp at diapause initiation plays a role in cold-hardiness for overwintering. moreover, up-regulation of hsp has also been reported under short day-length conditions, and hsp up-regulation could represent a molecular exaptation to diapause. ferritin is the primary iron storage protein, and it functions in scavenging oxygen radicals. ferritin and ferritin light chain are up-regulated at diapause initiation, as reported in nasonia and in s. crassipalpis. mnsod is also up-regulated at diapause initiation. in caenorhabditis elegans, mnsod participates in the regulation of both longevity and dauer formation as a physiological redox signaling modulators. gst and bombyrin also have antioxidant function and are up-regulated in the brain, as reported in proteomic analysis of h. armigera. oxidative stress can damage tissues and cellular components during diapause. therefore, the up-regulation of transcripts of antioxidant proteins will protect diapause individuals from oxidative stress. rad is a nucleotide excision repair gene that, functions in dna repair and protein degradation. integrator complex subunit which is also called soss-a, is involved in sensing ssdna and maintaining genome stability. up-regulation of genes related to dna repair in diapause has not been reported previously. however, it is possible that dna lesions occur under extreme environmental conditions during diapause, and the integrity of dna is crucial for re-starting the development into an adult when diapause is terminated. therefore, these up-regulated genes at diapause initiation mainly respond to stress resistance for insect survival in rigorous environmental conditions. signaling pathways genes involved in signaling pathway were also found in the ssh library. akt is an essential component of the insulin signaling pathway for glucose uptake to synthesize sugar and also as an activator of the target of rapamycin pathway to increase protein synthesis. insect organs and tissues need to accumulate a large store of sugar as energy and other substances, such as antifreeze agents glycerol and sorbitol, for use during a long diapause phase. therefore, enhanced akt transcription reflects increased sugar metabolism in diapause-destined pupal brain, and akt participates in the regulation of energy reserves and in response to environmental stress at the onset of diapause. calmodulin signaling, which is involved in the regulation of neuronal development and plasticity, is down-regulated at diapause initiation in h. armigera. in this study, camk ii, which modulates synaptic plasticity, learning, and memory, was down-regulated. argk was also down-regulated at diapause initiation, and high expression of argk, which is a developmental signal, was closely correlated with pupal development. thus, down-regulation of camk ii and argk may cause developmental arrest at diapause initiation. cell cycle during diapause, the cell cycle is arrested in the embryo of b. mori and in the brains of s. crassipalpis and chymomyza costata. cyclin-dependent kinase is a kinase partner of cyclin c, interacts with the large subunit of rna polymerase ii, and then participates in the regulation of the g1/s transition of mitosis. more than 97% of the brain cells become arrested in the g0/g phase in the diapause pupae of s. crassipalpis. proteomic analysis of sitodiplosis mosellana has found a strong up-regulation of inhibitor of nuclear factor kappa-b kinase interacting protein isoform during diapause, which contributes to inhibiting cell division during diapause. therefore, cyclin-dependent kinase and five other transcripts down-regulated in the brain at diapause initiation may cause cell cycle arrest, inducing the insect to enter diapause. transcription and translation transcription and translation are two major energetic costs in cellular development. to reduce energy consumption, many genes are silenced during diapause. in this study, several genes involved in the regulation of transcription and translation were identified. the down-regulation of transcription factor plag may result in the modulation of downstream target genes. the down-regulation of elongation factor delta indicates that translation is also suppressed at diapause initiation. in addition, some transcripts of proteins involved in transcription were up-regulated at diapause initiation: hardp-c is homologous to drosophila cg which contains the conserved mynd and set domains found in human smyd homologues. drosophila smyd represses transcription. smt is a reversible post-translational protein modifier that usually represses the activity of transcriptional activators. thus, we conclude that the down-regulation of plag and elongation factor delta and the up-regulation of transcriptional repressors and sumo lead to the global down-regulation of transcription and translation at diapause initiation.  in this study, differentially expressed genes at the early pupal stage of diapause- and nondiapause-destined individuals were isolated and identified that may be involved in regulation of diapause initiation. diapause initiation is an intriguing developmental process with a complex molecular mechanism. based on the above results, we suggest a possible molecular mechanism for diapause initiation. transition of metabolism and energy utilization. in addition to a decrease of metabolic activity, metabolic pathways are also changed in diapause-destined pupae at diapause initiation. anaerobic metabolism predominates, and sugars and polylols accumulate in the brain. enhancement of stress resistance. the antifreeze agents glycerol and sorbitol as well as hsp, gst, and others are heavily synthesized to protect the insect from rigorous environmental conditions. regulation of cellular development. the cell cycle is arrested, resulting in repression of pupal development toward adulthood. repression of transcription and translation. the up-regulation of transcriptional repressors, down-regulation of translational activators, and increased protein sumoylation result in decreases of both gene transcription and protein translation at diapause initiation. this idea awaits detailed experimental investigation in the future. materials and methods animals h. armigera larvae were reared on an artificial diet at 20°c with a l14:d and a l10:d photoperiod. after pupation, the two types of pupae were moved to the same conditions. under these conditions, all nondiapause pupae developed toward adults, and more than 95% of diapause type pupae entered diapause. the developmental stages were synchronized at each molt by collecting new larvae or pupae. all tissues were dissected in insect saline containing % nacl, and stored at -80°c until use. suppression subtractive hybridization we constructed two subtracted cdna libraries to detect high gene expression in diapause- and nondiapause-destined individuals at the early pupal stage using the pcr-select™ cdna subtraction kit. in the f library, diapause type pupae were used as the "tester", and nondiapause pupa as the "driver". in the r library, the "tester" and "driver" were reversed. after pupation, diapause- and nondiapause-destined pupae were incubated with the same condition 20°c and a short daylength for 2- days before dissection. total rna from day 1- brains of diapause- and nondiapause-destined pupae was isolated using a guanidinium thiocyanate-chloroform method. the mrna was obtained according to the manufacturer's protocols of quickprep micro mrna purification kit. double-stranded cdnas were synthesized from μg of polya+ mrna and digested with rsai to obtain shorter blunt-ended cdna. the "tester" cdna was subdivided into two populations, which were ligated to adaptor and adaptor 2r, respectively. two hybridizations were performed with the tester and driver cdnas. in the first hybridization, the amount of driver cdna was times more than the tester cdna. as a result, cdnas that were not up-regulated were hybridized by driver cdnas, only the up-regulated tester cdnas were left as single strand. in the second hybridization, the amount of driver cdna was times more than the tester cdna. as a result, the hybridized cdnas were eliminated, leaving only the unhybridized cdnas. the entire population of unhybridized molecules was then subjected to pcr to amplify target cdna fragments. only the molecules of the tester sample, which were ligated to the two different adaptors, could be amplified exponentially. a second pcr amplification was performed using nested primers to get a low-background, high-level enrichment of the differentially expressed sequences. the pcr products were analyzed by 2% agarose gel electrophoresis. products from the secondary pcrs were inserted into pmd18-t by t/a cloning. the recombinant plasmid dnas were transformed into xl- blue competent cells. the dna from recombinant clones was isolated and sequenced. bioinformatics analysis all contigs and singlets were annotated according to the go classification and the hierarchical structure using the blast2go suite. the blast2go program, which assigns the go terms based on the blast definitions, was applied with an e-value < 10- if a transcript was annotated with more than one go category, it was split equally among them. rna extraction and rt-pcr total rna was extracted from the brain using the acid-guanidine method. first-strand cdna was synthesized using μg of total rna at 37°c for h, with an m-mlv reverse transcription system. the primers used to identify of differentially expressed transcripts by rt-pcr are presented in additional file the pcr reactions were subjected to 22-- cycles consisting of 94°c for s; 55°c for s; 72°c for min. actin was used as an internal standard. northern blot hybridization total rna from the brain of day 1-- diapause- and nondiapause-destined pupae was separated on a % agarose gel containing mol/l formaldehyde, and transferred to a nylon membrane. probes for hybridization were labeled with -dctp using the random primer labeling kit. after prehybridization for h in 5× sspe containing 50% formamide, 5× denhardt's solution, % sds, and μg/ml salmon sperm dna, the radiolabeled probe was added and hybridization was conducted overnight at 42°c. after hybridization, the membrane was washed in × sspe at 42°c three times and exposed to x-ray film overnight at -70°c. polyclonal antibody generation and western blot analysis the orfs of four genes were amplified by pcr, using primers that contained restriction sites. the pcr product was digested by the appropriate restricted enzymes, then purified and subcloned into the pet28a vector. the recombinant pet plasmid was transfected into bl cells and induced by iptg. the e. coli pellet was solubilized in m urea in mm tris-hcl buffer, ph, followed by ni-nta column purification. purified recombinant proteins were used to generate polyclonal antibodies in rabbit. proteins for western blotting were extracted from the brain and sg or the brain--sg complexes of pupae, quantified by the bradford method, and stored at -80°c. protein was separated by 15% sds-page and transferred onto an immobilon-p transfer membrane. the immuno-reactivity was tested with antiserum, and then incubated with goat anti-rabbit igg, and protein was detected using the novex chemiluminescent substrates. authors' contributions whx designed the research project, bb performed experiments. bb and whx wrote the paper, and approved the final manuscript. supplementary material additional file 1 table s checklist of known genes in the two libraries. checklist of the sequences of known genes in the f library. checklist of the sequences those were homologous to known genes in the r library. all transcripts were compared to the sequences in genbank using blastx. sequence description, minimum e-value and similarity are shown in the table. click here for file additional file 2 table s comparison of the two libraries according to gene ontology criteria. click here for file additional file 3 figure s classification of ssh ests according to gene ontology criteria. classification of ssh ests according to gene ontology criteria. gene ontology analysis was carried out on the transcripts isolated from the two ssh libraries by blast2go program. the molecular function combined graph was made based on ontology level classification of ssh ests according to gene ontology criteria. the cellular component combined graph was made based on ontology level by blast2go. click here for file additional file 4 table s primer sequences used in pcr. click here for file acknowledgements this study was supported by a grant-in-aid for the natural scientific foundation from the national natural science foundation of china, and the major state basic research developmental program from the ministry of science and technology of china.
helicoverpa armigera undergoes pupal diapause, but the molecular mechanism of diapause initiation is unclear. using suppression subtractive hybridization, we investigated differentially expressed genes in diapause- and nondiapause-destined pupal brains at diapause initiation. we constructed two ssh libraries to isolate genes that are up-regulated or down-regulated at diapause initiation. finally, we classified the genes and predicted their possible roles at diapause initiation. differentially expressed genes at pupal diapause initiation are possibly involved in the regulation of metabolism, energy, stress resistance, signaling pathways, cell cycle, transcription and translation. we obtained unique sequences in the f library and unique sequences in the r library. diapause, a state of arrested development accompanied by a marked decrease of metabolic rate, helps insects to overcome unfavorable seasons. further, genes expression at the mrna and protein level in diapause- and nondiapause-destined pupal brains were confirmed by rt-pcr, northern blot or western blot analysis.
 diapause, a state of arrested development accompanied by a marked decrease of metabolic rate, helps insects to overcome unfavorable seasons. helicoverpa armigera undergoes pupal diapause, but the molecular mechanism of diapause initiation is unclear. using suppression subtractive hybridization, we investigated differentially expressed genes in diapause- and nondiapause-destined pupal brains at diapause initiation.  we constructed two ssh libraries to isolate genes that are up-regulated or down-regulated at diapause initiation. we obtained unique sequences in the f library and unique sequences in the r library. further, genes expression at the mrna and protein level in diapause- and nondiapause-destined pupal brains were confirmed by rt-pcr, northern blot or western blot analysis. finally, we classified the genes and predicted their possible roles at diapause initiation.  differentially expressed genes at pupal diapause initiation are possibly involved in the regulation of metabolism, energy, stress resistance, signaling pathways, cell cycle, transcription and translation. 