02736nas a2200265   4500008004600000245010700046210006900153300001400222490000800236520184900244653001602093653003202109653001202141653002302153653001602176653002402192653001802216653004502234653003602279653003902315100002302354700002102377700002202398856005002420       Submitted                   eng d     00aDesiccation and rehydration elicit distinct heat shock protein transcript responses in flesh fly pupae0 aDesiccation and rehydration elicit distinct heat shock protein t  a963 - 9710 v2073 aHeat shock proteins (Hsps) are a ubiquitous component of the cellular response to stress in both prokaryotic and eukaryotic organisms, but their role and function during desiccation stress in terrestrial arthropods has received limited attention. Molecular responses to rehydration are arguably as important as those to desiccation in maintaining cellular integrity and enzyme activity, but the role of Hsps during stress recovery is poorly understood and has never been addressed with respect to rehydration in insects. This study identifies distinct differences in the Hsp response to desiccation and rehydration in the flesh fly Sarcophaga crassipalpis, as well as differences in the desiccation responses of diapausing and nondiapausing pupae. In nondiapausing pupae, the expression of two inducible Hsps (Hsp23 and Hsp70) is upregulated by desiccation, but the water loss threshold for Hsp expression changes at different rates of dehydration. Continued desiccation results in the prolonged expression of both Hsp23 and Hsp70, which may contribute to the delayed adult eclosion noted in samples desiccated for more than 3 days at &lt;5% relative humidity/25 degrees C. In diapausing pupae, hsp23 and hsp70 transcripts are already highly expressed and are not further upregulated by desiccation stress. Both of the constitutive Hsps investigated, Hsp90 and Hsc70, were unresponsive to desiccation in both nondiapausing and diapausing pupae. However, both Hsp90 and Hsc70 were upregulated upon rehydration in nondiapausing and diapausing pupae. These results indicate distinct roles for the different Hsps during desiccation stress and rehydration/stress recovery. The response to desiccation recovery (rehydration) is similar to the Hsp response to cold recovery identified in S. crassipalpis: Hsp90 and Hsc70 are upregulated in both cases.10a*Body Water10a*Gene Expression Regulation10aAnimals10aBlotting, Northern10aDesiccation10aDiptera/*physiology10aFluid Therapy10aHeat-Shock Proteins/genetics/*metabolism10aHibernation/genetics/physiology10aRNA, Messenger/genetics/metabolism1 aHayward, S., A. L.1 aRinehart, J., P.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/76402638nas a2200313   4500008004600000245014100046210006900187300001400256490000700270520155500277653001201832653002101844653002401865653002001889653005801909653002401967653003801991653003402029653005102063653002002114653001202134653001702146100002302163700002102186700002402207700002102231700002202252856005002274       Submitted                   eng d     00aTemporal expression patterns of diapause-associated genes in flesh fly pupae from the onset of diapause through post-diapause quiescence0 aTemporal expression patterns of diapauseassociated genes in fles  a631 - 6400 v513 aDistinct differences in the temporal expression patterns of genes associated with pupal diapause were noted in the flesh fly, Sarcophaga crassipalpis. The first change observed was a decline in expression of the gene encoding heat shock protein 90 (hsp90) 2 days after pupariation (1 day before the pupa reaches the phanerocephalic stage characteristic of diapause). In contrast, hsp23 and hsp70 transcripts were undetectable in nondiapause samples and d1-d4 diapause-programmed pupae, but were up-regulated just after the start of diapause, 5 days after pupariation. An increase of glycerol content in diapausing pupae was also noted at the start of diapause. The gene encoding proliferating cell nuclear antigen (pcna) was diapause down-regulated, and this occurred in two phases, with the first decline in expression 7 days after pupariation and a second decline in the level of expression on day 14. For pupae held at 20 degrees C for 20 days and transferred to 10 degrees C, diapause ended after 90-100 days at the lower temperature. However, pupae remained in a state of post-diapause quiescence (d100-d150) and sustained diapause-like hsp and pcna expression patterns until adult development was initiated. Glycerol concentrations and survival declined during the post-diapause phase. This study suggests a distinct sequence in the pattern of gene expression at the onset of diapause, but the genes we have monitored do not contribute to the switch to covert developmental potential at the transition from diapause to post-diapause quiescence.10aAnimals10aCold Temperature10aDiptera/*physiology10aDown-Regulation10aGene Expression Regulation, Developmental/*physiology10aGlycerol/metabolism10aHeat-Shock Proteins/*biosynthesis10aInsect Proteins/*biosynthesis10aProliferating Cell Nuclear Antigen/*metabolism10aPupa/physiology10aSeasons10aTime Factors1 aHayward, S., A. L.1 aPavlides, S., C.1 aTammariello, S., P.1 aRinehart, J., P.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/64702229nas a2200265   4500008004600000245013400046210006900180300001600249490000700265520128500272653002201557653003201579653002501611653001201636653003001648653003101678653003501709653004101744653002801785653002701813653003101840100002001871700002201891856005001913       Submitted                   eng d     00aOleic acid is elevated in cell membranes during rapid cold-hardening and pupal diapause in the flesh fly, Sarcophaga crassipalpis0 aOleic acid is elevated in cell membranes during rapid coldharden  a1073 - 10820 v523 aThe integrity of cellular membranes is critical to the survival of insects at low temperatures, thus an advantage is conferred to insects that can adjust their composition of membrane fatty acids (FAs). Such changes contribute to homeoviscous adaption, a process that allows cellular membranes to maintain a liquid-crystalline state at temperatures that are potentially low enough to cause the membrane to enter the gel state and thereby lose its ability to maintain homeostasis. Flesh flies (Sarcophaga crassipalpis) were subjected to two experimental conditions that elicit low temperature tolerance: rapid cold-hardening and diapause. FAs were isolated and analyzed using gas chromatography-mass spectrometry. FAs changed in response to both rapid cold-hardening and diapause. In response to rapid cold-hardening (8 h at 4 degrees C), the proportion of oleic acid (18:1n-9) in pharate adults increased from 30% to 47% of the total FA pool. The proportion of almost every other FA was reduced. By entering diapause, pupae experienced an even greater increase in oleic acid proportion, to 58% of the total FA pool. Oleic acid not only promotes membrane fluidity at low temperature but also allows the cell membrane to maintain a liquid crystalline state if temperatures increase.10a*Cold Temperature10aAcclimatization/*physiology10aAnalysis of Variance10aAnimals10aCell Membrane/*metabolism10aChromatography, Thin Layer10aDiptera/metabolism/*physiology10aGas Chromatography-Mass Spectrometry10aHibernation/*physiology10aOleic Acid/*metabolism10aPupa/metabolism/physiology1 aMichaud, M., R.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/54502232nas a2200313   4500008004600000245017700046210006900223300001600292490000700308520100200315653001201317653004201329653003301371653002601404653005501430653006701485653005201552653000901604653002801613653005201641653006301693653002801756653001601784653001701800100001701817700001201834700002201846856005001868       Submitted                   eng d     00aA nondiapausing variant of the flesh fly, Sarcophaga bullata, that shows arrhythmic adult eclosion and elevated expression of two circadian clock genes, period and timeless0 anondiapausing variant of the flesh fly Sarcophaga bullata that s  a1213 - 12180 v523 aWe describe a variant of the flesh fly, Sarcophaga bullata, which fails to enter pupal diapause in response to short daylength and low temperatures. This fly also has an arrhythmic adult eclosion pattern: rather than eclosing in early photophase, the variant ecloses arrhythmically throughout the photophase and scotophase. The loss of both diapause (photoperiodic response) and the gating of adult eclosion (presumably a circadian response) suggests that the same clock system is involved in these two responses. An examination of the expression patterns of the clock genes period and timeless demonstrates that both genes are present in the nondiapausing variant, but surprisingly, both genes are expressed at higher levels. This abnormality we observe, possibly the consequence of an upstream clock gene malfunction or a malfunction of the autoregulatory loop, results in disruption of a component of the clock system that is apparently needed for both photoperiodism and circadian rhythmicity.10aAnimals10aCircadian Rhythm/genetics/*physiology10aDiptera/genetics/*physiology10aDNA Primers/chemistry10aDrosophila Proteins/analysis/biosynthesis/genetics10aGene Expression Regulation, Developmental/genetics/*physiology10aInsect Proteins/analysis/*biosynthesis/genetics10aMale10aMolecular Sequence Data10aNuclear Proteins/analysis/biosynthesis/genetics10aReverse Transcriptase Polymerase Chain Reaction/veterinary10aRNA, Messenger/analysis10aTemperature10aTime Factors1 aGoto, S., G.1 aHan, B.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/54702479nas a2200253   4500008004600000245019500046210006900241300001400310490000800324520153600332653002901868653002201897653002801919653001201947653003501959653004101994653002302035653002502058653003302083653001702116100002002133700002202153856005002175       Submitted                   eng d     00aShifts in the carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in flesh flies (Sarcophaga crassipalpis): a metabolomic comparison0 aShifts in the carbohydrate polyol and amino acid pools during ra  a753 - 7630 v1773 aFlesh flies can enhance their cold hardiness by entering a photoperiod-induced pupal diapause or by a temperature-induced rapid cold-hardening process. To determine whether the same or different metabolites are involved in these two responses, derivatized polar extracts from flesh flies subjected to these treatments were examined using gas chromatography-mass spectrophotometry (GC-MS). This metabolomic approach demonstrated that levels of metabolites involved in glycolysis (glycerol, glucose, alanine, pyruvate) were elevated by both treatments. Metabolites elevated uniquely in response to rapid cold-hardening include glutamine, cystathionine, sorbitol, and urea while levels of beta-alanine, ornithine, trehalose, and mannose levels were reduced. Rapid cold-hardening also uniquely perturbed the urea cycle. In addition to the elevated metabolites shared with rapid cold-hardening, leucine concentrations were uniquely elevated during diapause while levels of a number of other amino acids were reduced. Pools of two aerobic metabolic intermediates, fumarate and citrate, were reduced during diapause, indicating a reduction of Krebs cycle activity. Principal component analysis demonstrated that rapid cold-hardening and diapause are metabolically distinct from their untreated, non-diapausing counterparts. We discuss the possible contribution of each altered metabolite in enhancing the overall cold hardiness of the organism, as well as the efficacy of GC-MS metabolomics for investigating insect physiological systems.10a*Carbohydrate Metabolism10a*Cold Temperature10aAmino Acids/*metabolism10aAnimals10aDiptera/metabolism/*physiology10aGas Chromatography-Mass Spectrometry10aModels, Biological10aPolymers/*metabolism10aPrincipal Component Analysis10aTime Factors1 aMichaud, M., R.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/46702445nas a2200313   4500008004600000245011900046210006900165300001600234490000700250520130100257653002101558653001201579653002301591653003701614653003201651653003701683653002901720653003501749653006801784653003101852653002601883653002801909653003701937653003301974653003502007100001702042700002202059856005002081       Submitted                   eng d     00aHigh temperature and hexane break pupal diapause in the flesh fly, Sarcophaga crassipalpis, by activating ERK/MAPK0 aHigh temperature and hexane break pupal diapause in the flesh fl  a1276 - 12820 v533 aPupal diapause in the flesh fly, Sarcophaga crassipalpis, can be terminated by exposure to high temperatures or, artificially, with a topical application of organic solvents. To analyze the molecular mechanisms involved in diapause termination we explored the possibility that the mitogen-activated protein kinases (MAPK) are involved in this response. Levels of phospho-ERK increased within 10 min after hexane application. Extracellular signal-regulated kinase (ERK) was also activated when pupae were transferred from 20 to 25 degrees C, thus suggesting that ERK activation is a likely component of the signal transduction pathway used to initiate development in response to diapause-terminating signals. 20-Hydroxyecdysone and cyclic GMP terminate diapause in this fly, and the juvenile hormone analog methoprene shortens the diapause, but none of these agents activated ERK. ERK was readily activated in isolated abdomens treated with hexane, thus we conclude that ERK is directly activated by the hexane treatment. ERK activation was evident in the brain, epidermis, midgut and fat body, but not in the ventral nerve mass or ring gland, thus suggesting that ERK does not act directly on the ring gland to promote ecdysteroid synthesis but exerts its effect through stimulation of the brain.10a*Hot Temperature10aAnimals10aCloning, Molecular10aDiptera/drug effects/*physiology10aDNA, Complementary/genetics10aDose-Response Relationship, Drug10aEcdysterone/pharmacology10aEnzyme Activation/drug effects10aExtracellular Signal-Regulated MAP Kinases/genetics/*metabolism10aGene Expression Regulation10aHexanes/*pharmacology10aMethoprene/pharmacology10aNucleotides, Cyclic/pharmacology10aPupa/drug effects/physiology10aSignal Transduction/physiology1 aFujiwara, Y.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/46902476nas a2200277   4500008004600000245014300046210006900189300001600258490000800274520150900282653002201791653003201813653002401845653001201869653002301881653002801904653003601932653002201968653005401990653002002044653000902064653003602073100001702109700002202126856005002148       Submitted                   eng d     00ap38 MAPK is a likely component of the signal transduction pathway triggering rapid cold hardening in the flesh fly Sarcophaga crassipalpis0 ap38 MAPK is a likely component of the signal transduction pathwa  a3295 - 33000 v2103 aRapid cold hardening (RCH) is an adaptation enabling insects to quickly respond to low temperature, but little is known about the molecular events that trigger this response. In this study of the flesh fly Sarcophaga crassipalpis, we explore a possible role for mitogen-activated protein kinases (MAPKs) in the low temperature signaling that elicits RCH. We report that p38 MAPK from S. crassipalpis, which shows high cDNA sequence homology to p38 MAPKs from other insects and mammals, is rapidly activated at temperatures around 0 degrees C, temperatures that are most effective for inducing RCH. By contrast, low temperature does not activate either extracellular signal-regulated kinase (ERK) or Jun N-terminal kinase (JNK). An increase in phospho-p38 MAPK was observed within 10 min following exposure to 0 degrees C and reached its maximum level in 2 h. When flies were transferred from 0 to 25 degrees C, the level of phospho-p38 MAPK decreased immediately and reached trace levels by 3 h. Nondiapausing flies were much more responsive to p38 MAPK activation than cold-hardy diapausing pupae. Thus, p38 MAPK activation and RCH both show the same narrow ranges of temperature sensitivity, temporal profiles of activation and decay, and developmental specificity. These correlations suggest that p38 MAPK plays a potential role in regulating the induction of RCH. The p38 MAPK response was not dependent upon the brain, as evidenced by high activation in isolated abdomens exposed to low temperature.10a*Cold Temperature10aAcclimatization/*physiology10aAmino Acid Sequence10aAnimals10aCloning, Molecular10adevelopment/*physiology10aDiptera/*enzymology/growth &amp10aEnzyme Activation10ap38 Mitogen-Activated Protein Kinases/*metabolism10aPhosphorylation10aPupa10aSignal Transduction/*physiology1 aFujiwara, Y.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/47002802nas a2200313   4500008004600000245017800046210006900224300001400293490000700307520167000314653001701984653002802001653001202029653001602041653002502057653003702082653003502119653002902154653003602183653004202219653003502261653003402296653003602330100001402366700001602380700002002396700002202416856005002438       Submitted                   eng d     00aDiapause hormone in the corn earworm, Helicoverpa zea: optimum temperature for activity, structure-activity relationships, and efficacy in accelerating flesh fly pupariation0 aDiapause hormone in the corn earworm Helicoverpa zea optimum tem  a196 - 2050 v293 aDiapause hormone (DH) effectively terminated pupal diapause in Helicoverpa zea. This effect was temperature-dependent, with an optimum of 21 degrees C. The dose-response curve indicated an ED50 of DH for diapause termination of approximately 100 pmol. The core sequence and essential amino acids were determined by bioassays using modified and truncated DH analogs. A C-terminal hepta-peptide, LWFGPRLa, was the core sequence required for diapause termination. Activity was lost when Alanine was substituted for any of the amino acids in the hepta-peptide, with the exception of Glycine. A fragment series of analogs suggested that the amide and Arginine were the most important components needed for terminating diapause. Leucine, Tryptophan, and Phenylalanine at the N-terminus of the hepta-peptide were also critical for activity. The C-terminal Leucine was less important: deletion resulted in decreased activity, although it could not be substituted by Alanine. The fact that a portion of the DH sequence is similar to the pyrokinin that accelerates fly pupariation prompted us to also evaluate the capability of DH to accelerate development in the flesh fly, Sarcophaga bullata. The threshold dose of DH essential to accelerate fly pupariation was 5 pmol for immobilization/retraction and longitudinal contraction and 10 pmol for tanning, approximately one or two orders of magnitude lower than the effective dose required for diapause termination in H. zea. Tensiometric measurements revealed that DH affected neuromuscular patterns of pupariation behavior and associated cuticular changes in a manner similar to that of the fly pyrokinins and their analogs.10a*Temperature10aAmino Acid Substitution10aAnimals10adevelopment10aDiptera/*growth &amp10aDose-Response Relationship, Drug10aLarva/drug effects/growth &amp10aLepidoptera/*growth &amp10aMuscle Contraction/drug effects10aNeuropeptides/chemistry/*pharmacology10aPeptide Fragments/pharmacology10aPupa/drug effects/growth &amp10aStructure-Activity Relationship1 aZhang, Q.1 aZd'arek, J.1 aNachman, R., J.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/36301567nas a2200253   4500008004600000245008100046210006900127300001400196490000800210520077800218653001200996653002101008653001601029653003501045653001101080653003501091653002301126653000901149653005101158653002001209100001201229700002201241856005001263       Submitted                   eng d     00aMendelian inheritance of pupal diapause in the flesh fly, Sarcophaga bullata0 aMendelian inheritance of pupal diapause in the flesh fly Sarcoph  a251 - 2550 v1003 aPupal diapause (dormancy) in the flesh fly, Sarcophaga bullata, is induced by short-day photoperiods and low temperature. In this study, the inheritance mode of diapause was investigated by crossing a nondiapausing (nd) strain of S. bullata with 2 diapausing strains having different diapause capacities. The results consistently indicated that diapause incidence is inherited in a simple Mendelian pattern, thus a single gene or a small gene cluster linked to the photoperiodic clock controls the seasonal response of diapause. The fact that the nd strain lacked daily rhythmicity in adult eclosion and showed altered expression of 2 circadian clock genes suggests that the photoperiodic and circadian clocks are related through a shared molecular component in S. bullata.10aAnimals10aCrosses, Genetic10adevelopment10aDiptera/*genetics/*growth &amp10aFemale10aInheritance Patterns/*genetics10aLinkage (Genetics)10aMale10aMetamorphosis, Biological/*genetics/physiology10aModels, Genetic1 aHan, B.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/28002125nas a2200253   4500008004600000245009500046210006900141300001400210490000700224520130100231653002401532653001201556653002101568653004601589653002801635653002801663653003301691653002001724653002301744100001101767700002101778700002201799856005001821       Submitted                   eng d     00aNeuropeptide-like precursor 4 is uniquely expressed during pupal diapause in the flesh fly0 aNeuropeptidelike precursor 4 is uniquely expressed during pupal   a518 - 5210 v303 aSuppression subtractive hybridization comparing brains from diapausing and nondiapausing pupae of the flesh fly, Sarcophaga crassipalpis, suggested that the gene encoding neuropeptide-like precursor 4 (Nplp4) was uniquely expressed during diapause. We have sequenced the full-length cDNA encoding Nplp4 and used Northern blots to further evaluate linkage to diapause. The open reading frame of this cDNA encodes a 61-amino acid residue precursor protein containing a predicted 18 residue signal peptide, one 22-amino acid and one 2-amino acid propeptides, and a 19-amino acid neuropeptide. The amino acid sequence of the precursor protein shows 64% identity to Drosophila melanogaster Nplp4; homologues of this precursor protein are not known from species other than these two flies. Nplp4 mRNA levels were quite low in nondiapausing (long day) pupae, but in contrast the gene was highly upregulated in diapausing (short day) pupae. Expression increased at the onset of diapause, remained high throughout diapause, and then decreased 2 days after diapause was terminated. Although the function of this precursor protein and the neuropeptide it yields remain unknown, this close association with diapause suggests a potential role for Nplp4 in initiating and maintaining diapause in the flesh fly.10aAmino Acid Sequence10aAnimals10aDiptera/genetics10aGene Expression Regulation, Developmental10aMolecular Sequence Data10aNeuropeptides/*genetics10aProtein Precursors/*genetics10aPupa/metabolism10aSequence Alignment1 aLi, A.1 aRinehart, J., P.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/28202350nas a2200313   4500008004600000245014400046210006900190300001400259490000900273520122600282653003001508653001201538653002101550653002301571653003901594653003501633653004201668653003001710653003101740653003701771653003201808653002201840653005001862653002101912100001101933700002001944700002201964856005001986       Submitted                   eng d     00aRapid elevation of Inos and decreases in abundance of other proteins at pupal diapause termination in the flesh fly Sarcophaga crassipalpis0 aRapid elevation of Inos and decreases in abundance of other prot  a663 - 6680 v17943 aWe analyzed changes in brain proteins 24 h after pupal diapause termination in Sarcophaga crassipalpis by a combination of 2-dimensional gel electrophoresis and mass spectrometry. The proteome analysis revealed significant changes in 20 proteins, 11 of which represented &gt;or=2.5-fold changes. Three proteins were present only in the brains of diapausing pupae. Among the most abundant proteins that showed a change, 1 was more abundant, 7 were less abundant, and 2 were absent following diapause termination. The protein that increased in abundance following diapause termination showed highest identity to myo-inositol-1-phosphate synthase (Inos). Proteins that decreased at diapause termination included those showing highest identities to fatty acid binding protein, CG2331-PA, twinstar, catalase, and a histone. Proteins absent at diapause termination included ribosomal protein L17A and one unnamed protein. An increase of Inos protein level was confirmed using Western blot analysis. Attempts to terminate diapause by injection of several Inos-related metabolites failed, thus suggesting that the elevation of Inos at diapause termination is downstream of the physiological regulation that initiates development.10aAdaptation, Physiological10aAnimals10aBrain/metabolism10aDatabases, Protein10adevelopment/*metabolism/physiology10aDiptera/*metabolism/physiology10aElectrophoresis, Gel, Two-Dimensional10aGene Expression Profiling10aGene Expression Regulation10aInositol/metabolism/pharmacology10aInsect Proteins/*metabolism10aMass Spectrometry10aMyo-Inositol-1-Phosphate Synthase/*metabolism10aPupa/growth &amp1 aLi, A.1 aMichaud, M., R.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/28401624nas a2200241   4500008004600000245015400046210006900200300001400269490000700283520073500290653002301025653002401048653001201072653002701084653004501111653005101156653002801207653004001235653002301275100001201298700002201310856005001332       Submitted                   eng d     00aLength variation in a specific region of the period gene correlates with differences in pupal diapause incidence in the flesh fly, Sarcophaga bullata0 aLength variation in a specific region of the period gene correla  a415 - 4180 v553 aWe report differences in the length of a specific region of the circadian clock gene period (per) that correlate with different capacities for pupal diapause in the flesh fly, Sarcophaga bullata. The conspicuous difference is located in a region we refer to as the putative C-terminal photoperiodic (CP) region. The length of the CP region correlates inversely with the incidence of diapause. A deletion of 33 amino acids in this region correlates with a significant increase in the incidence of diapause, from 78.1% to 93.0%, and an insertion of 9 amino acids in the same area correlates with a drop in the diapause incidence to 4.0%. This correlation suggests a possible functional role for this region of per in photoperiodism.10a*Genetic Variation10aAmino Acid Sequence10aAnimals10adevelopment/metabolism10aDiptera/chemistry/*genetics/*growth &amp10aInsect Proteins/chemistry/*genetics/metabolism10aMolecular Sequence Data10aPupa/chemistry/genetics/growth &amp10aSequence Alignment1 aHan, B.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/28603073nas a2200289   4500008004600000245011700046210006900163300000800232490000700240520212900247653002902376653002002405653001202425653002202437653003002459653001702489653002702506653002602533653002102559653003602580653003602616100001702652700002002669700002202689700002202711856005002733       Submitted                   eng d     00aGene discovery using massively parallel pyrosequencing to develop ESTs for the flesh fly Sarcophaga crassipalpis0 aGene discovery using massively parallel pyrosequencing to develo  a2340 v103 aBACKGROUND: Flesh flies in the genus Sarcophaga are important models for investigating endocrinology, diapause, cold hardiness, reproduction, and immunity. Despite the prominence of Sarcophaga flesh flies as models for insect physiology and biochemistry, and in forensic studies, little genomic or transcriptomic data are available for members of this genus. We used massively parallel pyrosequencing on the Roche 454-FLX platform to produce a substantial EST dataset for the flesh fly Sarcophaga crassipalpis. To maximize sequence diversity, we pooled RNA extracted from whole bodies of all life stages and normalized the cDNA pool after reverse transcription. RESULTS: We obtained 207,110 ESTs with an average read length of 241 bp. These reads assembled into 20,995 contigs and 31,056 singletons. Using BLAST searches of the NR and NT databases we were able to identify 11,757 unique gene elements (E&lt;0.0001) representing approximately 9,000 independent transcripts. Comparison of the distribution of S. crassipalpis unigenes among GO Biological Process functional groups with that of the Drosophila melanogaster transcriptome suggests that our ESTs are broadly representative of the flesh fly transcriptome. Insertion and deletion errors in 454 sequencing present a serious hurdle to comparative transcriptome analysis. Aided by a new approach to correcting for these errors, we performed a comparative analysis of genetic divergence across GO categories among S. crassipalpis, D. melanogaster, and Anopheles gambiae. The results suggest that non-synonymous substitutions occur at similar rates across categories, although genes related to response to stimuli may evolve slightly faster. In addition, we identified over 500 potential microsatellite loci and more than 12,000 SNPs among our ESTs. CONCLUSION: Our data provides the first large-scale EST-project for flesh flies, a much-needed resource for exploring this model species. In addition, we identified a large number of potential microsatellite and SNP markers that could be used in population and systematic studies of S. crassipalpis and other flesh flies.10a*Expressed Sequence Tags10a*Genome, Insect10aAnimals10aDiptera/*genetics10aGene Expression Profiling10aGene Library10aMicrosatellite Repeats10aOID - NLM: PMC270081710aPMC - PMC270081710aPolymorphism, Single Nucleotide10aSequence Analysis, DNA/*methods1 aHahn, D., A.1 aRagland, G., J.1 aShoemaker, D., D.1 aDenlinger, D., L.  uhttps://sarcophagidae.myspecies.info/node/290