Drosophila melanogaster CHCs have been studied extensively, but the genetic basis for individual variation in CHC composition is largely unknown. We quantified variation in CHC profiles in the D. melanogaster Genetic Reference Panel (DGRP) and identified novel CHCs. We used principal component (PC) analysis to extract PCs that explain the majority of CHC variation and identified polymorphisms in or near 305 and 173 genes in females and males, respectively, associated with variation in these PCs. In addition, 17 DGRP lines contain the functional Desat2 allele characteristic of African and Caribbean D. melanogaster females . Disruption of expression of 24 candidate genes affected CHC composition in at least one sex. These genes are associated with fatty acid metabolism and represent mechanistic targets for individual variation in CHC composition.Insect cuticular hydrocarbons (CHCs) prevent desiccation and serve as chemical signals that mediate social interactions. DOI:http://dx.doi.org/10.7554/eLife.09861.001 The outermost layer of an insect’s body is called the epicuticle and is made of a blend of fat molecules. “Cuticular hydrocarbons” (or CHCs) are the most common fat molecules in the epicuticle, and play an important role in protecting the insect’s body from harsh, dry habitats. CHCs also have other roles in insect behavior. For example, these molecules act as chemical cues when insects search for mates (i.e. pheromones), and they can even contribute to camouflage.Insects are amongst the most diverse groups of animals on Earth, and different species have different blends of CHC molecules in their epicuticles. Fruit flies are a useful model to understand the genetics of CHC production, including CHCs that act as sex pheromones. Previous research has analyzed the CHCs made by both sexes in several fruit fly strains. However this work was unable to uncover which genes influence how much of a given CHC an individual fly will make.Dembeck et al. have now looked into CHC production in a collection of 205 different fly strains, all of which have already had their total genetic material sequenced and studied. Comparing these known sequences and looking for associations between genetic differences and particular CHCs uncovered 24 genes that may be involved in CHC manufacture. Only six of the genes had been identified previously. Dembeck et al. found that interfering with the activity of any of the 24 genes had a knock-on effect on many other CHCs present in the flies’ epicuticle.These 24 genes could to be pieced together in a network that is needed to make and recycle CHCs. The complexity and flexibility of this network can explain in part how insects have been able to build epicuticles for almost every environment. These data set the stage for future work directed towards understanding the evolutionary significance of variation in CHC composition in many fruit fly populations.DOI:http://dx.doi.org/10.7554/eLife.09861.002 Insects comprise the most species-rich class in the animal kingdom. They evolved about 480 million years ago and their fecundity and rapid evolutionary adaptations have made them successful in populating almost every ecological niche on our planet. The evolution of mechanisms for desiccation resistance was critical for insects to colonize dry land. To prevent desiccation insects evolved the ability to produce and accumulate species-specific blends of fatty acid-derived apolar lipids on the epicuticle; the most prominent of these are cuticular hydrocarbons (CHCs) . CHCs arAdaptive evolution acts through selection on phenotypic variation within a population. Thus, understanding the genetic basis for individual variation in CHC composition will shed light on evolutionary mechanisms of assortative mating  and evolDrosophila melanogaster make it a valuable model for studying the genetic basis of CHC production and natural variation in CHC composition. Mature D. melanogaster have sexually dimorphic CHCs ranging from chain lengths of 21 to 31 carbons (C21–C31) (The extensive genetic resources available for C21–C31) . Males pC21–C31) .ACC in the oenocytes completely eliminates CHCs in both male and female D. melanogaster . Members of a family of tissue-specific elongases (ELOVL) catalyze the incorporation of malonyl-CoA units to form very long chain fatty acids (VLCFA). After condensation of the malonyl-CoA with the fatty acyl-CoA the next three steps in each elongation cycle include reduction of a carbonyl to an alcohol , dehydration , and reduction of the carbon-carbon double bond by a trans-enoyl-CoA-reductase (TER). The VLCA as the CoA derivative is readuced to an aldehyde by a fatty acyl-CoA reductase (FAR). The one-carbon chain-shortening conversion of aldehydes to hydrocarbons is catalyzed by a cytochrome P450 enzyme, Cyp4G1  then catalyzes the synthesis of malonyl-CoA, and the multifunctional protein fatty acid synthase (FASN) successively incorporates malonyl-CoA units onto the acetyl-CoA, elongating the chain by two carbons each time and forming long chain fatty acids (LCFA). RNAi-knockdown of nogaster . Product, Cyp4G1 . Desatursmall monoene quantities (smoq) and seven pentacosene (sept), respectively, associated with variation in the proportions of 7-C23:1 and 7-C25:1 in males  (FASN2 (CG3524) which is involved in the synthesis of precursors of 2-methylalkanes (FASN1 (CG3523) which is expressed in the fat body but nonetheless contributes to the CHC pool (KAR (CG1444), TER (CG10849) and HADC (CG6746) whose knockdown eliminates or reduces CHC synthesis  in females and 15 in males contributing to variation in CHCs , but thiin males . In a mubehavior . Howeverse (ACC) ; two fatlalkanes , and FASCHC pool ; an elonCHC pool ; KAR    to perfon-Cx, where ‘‘x’’ is the total carbon number. For example, tricosane is designated n-C23. Methyl-branched alkanes are referred to with a y-Me-Cx prefix, where 'y' is the carbon onto which the methyl group is bound. For example, 9-Me-C23 is a 23 carbon chain with a methyl on the 9th carbon. For monoenes (z-Cx:1) and dienes  the number of double bonds is indicated after the colon and the double bond position/s are 'z' or 'z,z"). For example, 7-C23:1 has one double bond between the 7th and 8th carbons. We identified and quantified 71 female CHCs and 42 male CHCs in 169 and 157 DGRP lines, respectively  showed significant among-line variation in a univariate ANOVA . Broad sDesat locus was the first desaturase gene sequence described in insects  is located within the common African inversion In3R(K) , CHC phenotype and inversion status to perfectly co-segregate in the DGRP lines. However, this was not the case. A total of 17 DGRP lines with female CHC phenotypes contained the ancestral, functional Desat2 allele  was not perfect: five of the lines with the ancestral Desat2 sequence were homozygous for the standard karyotype , one is a deletion causing a frameshift (3R_8263023_DEL), and two are nonsynonymous coding (3R_8263031_MNP and 3R_8263048_SNP). The frameshift and nonsynonymous variants are potentially damaging and could explain why this line produced the Cosmopolitan phenotype despite having the functional Desat2 allele. We did not find such evidence for DGRP_551, suggesting this line may contain unknown genetic variants that inhibit the production of 5,9-C27:2.We next checked variants . We idenρ). As expected, some CHCs were highly correlated within each sex  that strongly negatively correlated with those dienes and the shorter-chain (≤ C25) alkanes of module 3 . SimilarWe found similar trends in the male CHCs . Module The correlations among CHCs are plausible given the biology of CHC production. We computed PCs of genetically variable CHCs within each sex to reduce the dimensionality of the data to orthogonal PCs. The first seven and five PCs accounted for 98.00% and 98.12% of the total variation among the DGRP lines for female and male CHCs, respectively . We hypoD. melanogaster. None of the female or male PCs were affected by the presence of the endosymbiont Wolbachia pipientis in some of the DGRP lines (In(2L)t inversion; female PC1, PC2 and PC3, and male PC5 were affected by the In(3R)K inversion; and the In(3R)P inversion only affected male PCs   . We corrC3, PC5)  and are P≤10–5) associated with female  PCs  genes nominally  PCs . Althougositives .10.7554/Lipase2 (Lip2) associated with variation in PC1; a SNP 641 bp upstream of the cytochrome P450 gene, Cyp49a1, associated with PC2; SNPs in and near Desiccate (Desi), a gene previously shown to contribute to desiccation resistance in D. melanogaster, associated with PC4 and PC6; variants in and near two peroxidase genes, Immune-regulated catalase (Irc) and Peroxidase (Pxd), associated with PC5; and variants in three fatty acid metabolism genes  associated with variation in PC6. Several notable candidate genes were also implicated by variants associated with PC7; CG9801 contained the 3 most significant SNPs. One of the top associated SNPs causes a missense mutation in Cyp6w1. Cyp4s3 and a predicted dihydrolipoamide branched chain acyltransferase  did not reach the MAF cutoff (0.05) for evaluation. Further, any effect of this variant would have been minimized by correcting for the effect of the In(3R)K inversion.Several of the top variants associated with each PC are in or near candidate genes with plausible roles in CHC metabolism . In femansferase , CG5599,CG13091, a putative fatty acyl-CoA reductase, of which one, a nonsynonymous coding variant (2L_8521314_SNP), was the most significant variant in this study (P = 2.19E-11) and passes the Bonferroni-correction for multiple tests . Variants in CG13091 were also associated with variation in PC4 and PC5. Genes tagged by variants associated with PC2 include approximated (app), a palmitoyltransferase; PHGPx, a peroxidase; and CG16979, a thiolester hydrolase. Top variants in the PC3 GWA analysis included two genes predicted to be fatty acid elongases  and an NADH dehydrogenase (CG8680). The most significant variant in the PC5 GWA analysis was a nonsynonymous SNP  in CG10097, which also encodes a fatty acyl-CoA reductase. Interestingly, there are two cytochrome P450 genes located upstream of CG10097, one functional (Cyp9f2) and one pseudogenized (Cyp9f3Psi).In males, nearly all of the variants associated with variation in PC1 were in or near le tests . Many ofUAS-RNAi lines -GAL4 -GAL4 , to restposition  (Thibaulposition .app), fatty acyl reductases (CG13091 and CG10097), a thiol hydrolase (CG16979), thioredoxin peroxidases (PHGPx and Prx6005), fatty acid elongases , cytochrome P450s , peroxidases , an NADH dehydrogenase (CG8680), and a dihydrolipoamide branched chain acyltransferase (CG5599), which is involved in the metabolism of branched chain amino acids leucine, isoleucine and valine, which serve as precursors for methyl-branched alkanes . The effect was less severe in females, but the overall trend was also towards reduced CHCs  all affected overall amounts of CHCs. RNAi knockdown of Cyp49a1 and Cyp9f2 in males led to general increases in CHCs, with a few exceptions. The increase in CHCs in males with compromised Cyp49a1 function was accompanied by a decrease in many female CHCs. Reduced expression of Cyp4s3 resulted in increased CHC abundance in both sexes. The link between these CYPs and CHC production and maintenance is unclear. However, we speculate that oxidation reactions mediated by these CYPs may regulate CHC degradation and turnover. The specific functions of most insect CYPs are still unknown  and 4-fold (~5.5 µg/fly) increase for females and males, respectively. Thus, it is possible that these genes play roles in catabolic pathways thus resulting in an increase in CHCs. Alternatively, RNAi knockdown of these genes may lead to reduced or abnormal grooming behavior leading to an accumulation of CHCs  had the functional Desat2 allele, yet exhibited the Cosmopolitan phenotype. These results are in contrast to previous reports of complete association of the ancestral allele with the African female CHC phenotype K inversion at low frequency and individuals with the ST/INV karyotype were not detected, but the lack of correspondence between the functional allele and CHC status remains unexplained since Desat2 itself does not harbor a potentially damaging mutation in this line. Perhaps a polymorphism in an unknown gene in DGRP_551 interacts with the Desat2 functional allele to suppress its effects. Finally, there were four DGRP lines that exhibited the African phenotype and were homozygous for the ancestral allele, but they were ST/ST in karyotype. One possible explanation for this 'mismatching' of phenotype and genotype with the karyotype is that the Desat2 16-bp deletion occurred prior to the inversion event. This would mean that the inversion may be segregating for the Desat2 16-bp allele.While 17 DGRP lines contain the ancestral allele that confers a functional henotype . FurtherFASN1, Desat1, eloF, DesatF, Cyp4G1; and several genes reported in We used principal component analysis to reduce dimensionality of the data, which was motivated by the high co-linearity among the CHCs. However, PCA may cause genetic variation for a certain CHC to be distributed among multiple PCs and therefore dilute its association with QTLs. Surprisingly, we did not detect variants in the DGRP in previously identified CHC biosynthesis genes  lines were reared in vials containing cornmeal-molasses-agar medium at 25°C, 75% relative humidity, a 12:12-h light-dark cycle, and a controlled adult density of 10 males and 10 females. The parental generation was allowed to lay eggs for three days. Upon eclosion, virgin males and females were separated and placed into new vials containing the same medium and aged for four days.The flies from each line were separated into at least two samples of five flies each per sex. On average, three samples were collected for each. To avoid cross-contamination of cuticular lipids a fresh tissue paper was placed on the carbon dioxide pad and the flies were handled with acetone-washed titanium forceps at each round of sorting. Flies were placed in 2 mL glass auto-injection vials with a Teflon cap and were flash frozen. All samples were stored at -30°C until CHC extraction. We collected samples from 169 and 157 DGRP lines for females and males, respectively . All lines were reared simultaneously and DGRP lines that did not produce sufficient offspring for CHC analysis were excluded to avoid any block effects of rearing. For the two African lines, Z30 and Z53, we reared and collected 5 samples for each sex.n-C32) with gentle swirling for five minutes. The flies were briefly extracted a second time with 100 µl of hexane . After each wash the extract was transferred to a 300 µl conical glass insert. The extract was dried using a gentle stream of high-purity N2 and re-suspended in 50 µl of hexane. The samples were immediately processed using gas chromatography or stored at 4°C (no longer than one day) until processing.Cuticular lipids were extracted from each sample using 200 µl of hexane containing an internal standard  and a flame ionization detector (FID) for quantification. We introduced 1 µl of sample using an Agilent 7683B auto injector into a 290°C inlet operated in splitless mode. The split valve was turned on after 1 min. The oven temperature program was as follows: 50°C for one min, increased at 20°C/min to 150°C, and increased at 5°C/min to 300°C followed by a 10 min hold. Hydrogen was used as the carrier gas at constant flow (average linear velocity = 35 cm/sec) and the FID was set at 300°C.m/z at 1.23 scan/s scan rate. Compounds were identified based on their mass spectra in comparison to those in the reference library (Wiley 7th/NIST 05) and based on comparison of their retention indices and fragmentation patterns to already published Drosophila CHCs (D. melanogaster CHCs was deduced (based on mass spectra and retention index data) are given in Selected samples were analyzed for chemical identification in a 6890N GC system (Agilent) coupled with a 5975 mass selective detector (MSD) (Agilent) and equipped with a DB-5 (20 m x 0.18 mm x 0.18 μm) column (Agilent). Helium was used as carrier gas at 33 cm/s average linear velocity. Injection and temperature settings were identical to the settings described above, and the transfer line was maintained at 300°C. Positive electron ionization at 70 eV with default temperature settings  were used for the MSD. Ions were detected in scan mode in the range of 33–650 ila CHCs .The posiAll chromatograms were analyzed using Agilent ChemStation software. For quantification individual peak areas were obtained for 42 and 60 male and female CHC containing peaks, respectively (some of the peaks contained multiple CHCs). Response factors were not determined for individual components. To account for natural variation in body size and absolute amounts of CHCs between the lines, the data were represented as proportions by dividing each peak area by the sum of all integrated peaks.Y = µ + L + ε, where Y is phenotype, µ is the overall mean, L is the random effect of line, and ε is the residual. We estimated variance components using restricted maximum likelihood and computed the broad-sense heritability (H2) of each CHC peak as H2 = σ2L/(σ2L + σ2ε), where σ2L is the among-line variance component and σ2ε is the error variance. All analyses were performed with version 9.3 of the SAS System for Windows (2013 SAS Institute Inc.).We partitioned variation of each CHC peak into genetic and environmental components using an ANOVA model of form H2 ≥ 0.25. We retained PCs explaining greater than 1% of the variation for subsequent GWA analysis. PCA was conducted in JMP Pro10 (2013 SAS Institute Inc.).A majority of CHCs belong to homologous series in which the chain length increases by two carbons; thus these compounds may be genetically correlated due to shared biosynthetic pathways and the data may be confounded with multi-co-linearity . We visuIn(2L)t, In(2R)NS, In(3R)P, In(3R)K, and In(3R)Mo. We performed GWA studies in two stages. In the first stage, we adjusted the line means for the effects of Wolbachia infection and major inversions. We then used the adjusted line means to fit a linear mixed model in the form of Y = Xb + Zu + ε, where Y is the adjusted phenotypic value, X is the design matrix for the fixed SNP effect b, Z is the incidence matrix for the random polygenic effect u, and ε is the residual. The vector of polygenic effects u has a covariance matrix in the form of Aσ2, where σ2 is the polygenic variance component. We fitted this linear mixed model using the FastLMM program (version 1.09)  . We perfon 1.09) . All segDrosophila RNAi Center (VDRC) and crossed them to the oenocyte-specific GAL4 driver, PromE(800)-GAL4 (PromE(800)-GAL4) for 23 genes  with the assumption that body sizes between the control and RNAi knockdown or mutant were not significantly different. These measures provide a more quantitative measure of CHCs and capture differences that proportion data may not resolve. PC analyses and t-tests pairing the test lines with the controls were conducted on these data. We also calculated the mean total amount of CHCs (ng/fly); we used the more conservative Cochran and Cox test, which assumes unequal group variances to assess statistically significant effects on CHC composition. We also present these data as µg/fly in t-tests were conducted in SAS v. 9.4 (2013 SAS Institute Inc.).We selected candidate genes with available mutations and RNAi knockdown constructs to test for effects on CHC production based on FlyBase annotations. We obtained lines with RNAi knockdown constructs from the Vienna 00)-GAL4 . We test23 genes . Since nllection  along wi review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom, Daniel J Kliebenstein, is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "Genetic architecture of natural variation in cuticular hydrocarbon composition in The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.We felt that the manuscript was very interesting but that some key factors need amendment to reflect insect biochemistry and editorial changes to increase the potential audience of this nice genetic analysis. We therefore ask you to do the following:1) Rework the manuscript to properly reflect the specifics of FA biosynthesis in insects rather than rely upon the plant model of the pathway.Drosophila.2) Strive to present the broader picture and impacts of the results found in this study beyond CHC metabolism in 3) Clarify that the candidate genes found may influence the CHC accumulation but further work needs to be done to test the specific enzymatic activities.4) Discuss issues on chemical identification and quantification.Explicit reviews can be found below.Reviewer #1:Drosophila using a GWA approach and identify a highly polygenic underpinning to this trait. They then validate the potential role of 24 candidate genes in altering this trait. This analysis was very nicely conducted but the writing was solely built around CHC composition in Drosophila which will make it difficult for this paper to attract the potential readership that it should obtain. From my reading, there are two broad messages that all researchers using GWA should be able to obtain from this manuscript.The authors investigate the quantitative genetic variation in CHC hydrocarbons within 1) It is possible to being dissecting highly polygenic GWA traits that have 100s or more potential candidate genes down to the functional level. There is no need to assume that there are few causal genes for potentially adaptive traits.2) Polygenic adaptive traits may be structured in a manner that will allow data decomposition approaches to allow significant progress in understanding complex traits like metabolic mixtures.The manuscript would likely get broader visibility if there was more general information about the bigger picture presented in the manuscript that could pull in researchers studying GWA or metabolic traits in other systems as this manuscript has information for those researchers even if they are studying plants or fungi or humans. While motivated researchers will see the broader impacts of this manuscript on the GWA/metabolite field it would help to work and bring them into the research without this effort by presenting the broader ramifications of the research in the Introduction and Discussion.Arabidopsis wherein the PC approach utilized by Bergelson and colleagues was unable to identify all of the key biosynthetic enzymes that could be identified on a more metabolite by specific metabolite analysis or alternatively using ratios of precursors to products . While I don't feel this is necessary to conduct, it would be beneficial to discuss this difficulty that is imparted by the use of linear trait decomposition for GWA.While I appreciate the use of PCs to reduce the dimensionality, this is an inherently linear approach that can have trouble with non-linear processes such as epistatic variation in biosynthetic pathways. This can be illustrated by Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 112, 4032-4037.Brachi, B. et al.(2015). Coselected genes determine adaptive variation in herbivore resistance throughout the native range of Arabidopsis thaliana. PLoS Biol 9, e1001125.Chan, E.K. et al. (2011). Combining genome-wide association mapping and transcriptional networks to identify novel genes controlling glucosinolates in Wentzell, A.M. et al. (2007). Linking metabolic QTLs with network and cis-eQTLs controlling biosynthetic pathways. PLoS Genet 3, 1687-1701.Reviewer #2:This manuscript represents a great deal of very good work and can be made into an excellent publication. However, much of the biochemistry upon which it is based needs extensive correcting and rewriting.The identification of new components in this much studied species is well done and important.Drosophila FAS, can be 14, 16 or 18 carbons. A thioesterase that is part of the multienzyme FAS removes the elongated chain as a free fatty acid. I do not know what is meant by conjugating and the acyl group is never removed by a thioreductase. In all cases studied in insects, desaturation and elongation take place in a microsomal fraction (endoplasmic reticulum) and not the mitochondrial fraction (many references).In the subsection “Functional validation of candidate genes”, second paragraph: Fatty acid biosynthesis begins with an acetyl-CoA and then elongation occurs with a malonyl-CoA in which the energy is supplies by decarboxylation of the malonyl-CoA as two carbons incorporated into the growing chain. The next three steps are a reduction of a carbonyl to an alcohol, dehydration, and reduction of the carbon-carbon double bond. The authors are making too extensive use of plant lipid metabolism data. The FAS in animals, including flies, occurs on the multifunctional protein fatty acid synthetase (FAS) in which the growing chain is attached to an acyl carrier type unit that is part of the multifunctional . It is not typically called an acyl carrier protein complex. Products of insect FASs, particularly Cyp4G1 in Drosophila . While it is possible that the elongated fatty acyl-CoA is reduced from acyl-CoA to aldehyde to alcohol and then re-oxidized to aldehyde before oxidative decarbonylation, there is no evidence for this to date in insects. In the subsection “Functional validation of candidate genes”, fourth paragraph: Fatty acids are elongated and desaturated attached to an ACP in plants. In insects, the parallel reactions occur with the acyl group attached to CoASH. The evidence to date suggests that the elongated fatty acyl-CoA is reduced to an aldehyde prior to oxidative decarbonylation, the latter step catalyzed by In the subsection “Functional validation of candidate genes”, end of fourth paragraph: An esterase cleaves an ester bond and does not reduce the carboxylic ester group on the fatty acyl-CoA (not ACP) to an alcohol. A FAR (fatty acyl-CoA reductase) does the reduction.In the subsection “Functional validation of candidate genes”, sixth paragraph: I am not convinced the CYPs studied here are involved in CHC turnover.In the subsection “Functional validation of candidate genes”, last paragraph: The authors are comparing 3-methyl fatty acids with a 3-methyl hydrocarbon derived from a n-3 methyl fatty acid.When stating “Our results provide a new perspective and highlight the complexity of the biosynthetic and catabolic pathways that regulate the dynamics of CHC composition”, the authors are blithely referring to the biosynthetic pathways of hydrocarbons (which are somewhat known) with the catabolic pathways . This sentence is illustrative of several times in the manuscript where the authors make sweeping statements of biochemistry for which there is no basis.Reviewer #3:Drosophila melanogaster. This manuscript reports three main findings: 1) the discovery of 16 previously unidentified CHCs in D. melanogaster, 2) that reported Desat2 alleles do not match reported female CHC phenotypes; and 3) the identification of previously unidentified candidate genes in the biosynthesis of CHCs .This manuscript by Dembeck et al. is the latest in a series of fine papers from the Mackay lab using the sequenced inbred lines from the DGRP to understand the genetic architecture of phenotypic trait variation, in this case, the variation in cuticular hydrocarbons (CHC) in The main strengths of this manuscript are its systematic approach to gene identification and functional validation. Previously, much work in the area of CHC biosynthesis has relied on biochemical approaches and the field has largely not taken advantage of potential genetic approaches as demonstrated here. The results here provide concrete functional evidence of the roles of numerous genes in CHC biosynthesis. This paper is the product of a tremendous amount of work that will provide good leads for future biological studies. The main weakness of the manuscript is that it does not provide new mechanistic insights into any compelling biological question.Detailed comments are as follows:Drosophila melanogaster, including dienes with double bonds in the even-numbered configurations as well as methyl-branched CHCs which are not 2-methyl-branched. The authors stated that they were able to identify these compounds because they use a thin high res column (DB-5) and a relatively long temperature program. I am concerned about the use of this column for the separation of D. melanogaster CHCs as this column cannot separate two major compounds in female Drosophila. Peak 46 contains both 2Me-C26 and 7,11-C27:2. This is a concern for the second part of the paper  because 2Me-C26 is a major compound. If they cannot separate these two compounds, then it would be impossible to quantify accurately the amount of 7,11-C27:2 in the fly. Another recent paper  also used a different method to identify CHC in D. melanogaster. While they identified some methyl-branched CHCs which are not 2-methyl-branched, they did not identify the dienes with the even double bonds presented in this study. The authors stated that these compounds are identified based on comparison to the reference library and to already published D. melanogaster CHCs and the position of the double bond was not confirmed for any of the CHCs. While most of these compounds exist in low quantities, I suggest the authors proceed with caution on claiming that they have identified new  compounds that many other groups using different methods over the years have not been able to identify.In the first section of the paper, Dembeck et al. claim to have detected 16 previously unidentified CHCs from Desat2 and female CHC phenotype. Two DGRP lines, DGRP_105 and DGRP_551 have the African allele  but the cosmopolitan phenotype. This analysis is based on protein sequence alone and does not take into account the possibility that unique polymorphisms in the Desat2 regulatory region of these lines changed the expression of Desat2. Therefore, without properly quantifying whether Desat2 is expressed or not in these lines, the authors should be careful in determining which alleles are functional based on protein sequence analysis alone (and a 16-bp deletion in the promoter that was not properly characterized previously).In the second part of the manuscript, Dembeck et al. report that the African CHC phenotype  is segregating in the DGRP. This section reports the mismatch between what is called the "16-bp deletion / ancestral alleles" of D. melanogaster. The same authors have used similar methods to identify novel pigmentation genes in Drosophila . In this section, using GWA, the authors identified genetic variants in hundreds of genes which could potentially affect CHC composition. The authors selected 24 candidate genes for functional studies. They used an oenocyte specific driver  to knockdown candidate genes in the oenocytes, where CHCs are synthesized. This is a very comprehensive section that entails a tremendous amount of work in both genomic analysis and functional characterization. The authors found that oenocyte-driven RNAi knockdown of 23 candidate genes individually showed quantitative changes in CHC profiles. These results provide very strong evidence that these candidate genes are involved in CHC biosynthesis, but there are two caveats to consider.In the third section which forms the bulk of the paper, the authors identify numerous genes potentially involved in CHC synthesis in First, some of these genes might not be expressed in adult oenocytes. A brief look on the FlyAtlas website showed that the transcripts of some of these genes are not present or expressed at very low levels in the "adult carcass" where the oenocytes are located. Therefore, it is not known whether the phenotypes that the authors observed are off target effects of RNAi or altered physiology of the whole fly due to the oenocyte driver that they use, which may be driving GAL4 in other parts of the fly.Cyp4g1 in CHC synthesis). How would a P450 (Cyp9f2) be involved in elongation of CHCs? As far as is known, that is not one of the reactions that a P450 could catalyze.Second, while some of the effects of the candidate genes can be explained by the biological/molecular functions of the gene (i.e. fatty acid reductase influencing chain length), others are not  Rework the manuscript to properly reflect the specifics of FA biosynthesis in insects rather than rely upon the plant model of the pathway.We have addressed these points in the revised manuscript.2) Strive to present the broader picture and impacts of the results found in this study beyond CHC metabolism in Drosophila.We have revised the manuscript to address this comment in the Introduction and Discussion. We note that it is possible to dissect highly polygenic traits down to the functional level, in accordance with the suggestion of Reviewer 1. We also note in the Discussion that there is no need to assume that there are few causal genes for potentially adaptive traits. However, we are not the first to demonstrate that the genetic basis of high-dimensional polygenic traits can be assessed using data decomposition approaches, so we did not stress this point.3) Clarify that the candidate genes found may influence the CHC accumulation but further work needs to be done to test the specific enzymatic activities.We have added the following statement to the Discussion: “Further studies are needed to clarify the effects on CHC production of these genes and their interactions, and to test specific mechanisms and enzymatic activities through which they exert these effects.”4) Discuss issues on chemical identification and quantification.We have addressed this issue by adding a new Explicit reviews can be found below.Reviewer #1:The authors investigate the quantitative genetic variation in CHC hydrocarbons within Drosophila using a GWA approach and identify a highly polygenic underpinning to this trait. They then validate the potential role of 24 candidate genes in altering this trait. This analysis was very nicely conducted but the writing was solely built around CHC composition in Drosophila which will make it difficult for this paper to attract the potential readership that it should obtain. From my reading, there are two broad messages that all researchers using GWA should be able to obtain from this manuscript.1) It is possible to being dissecting highly polygenic GWA traits that have 100s or more potential candidate genes down to the functional level. There is no need to assume that there are few causal genes for potentially adaptive traits.2) Polygenic adaptive traits may be structured in a manner that will allow data decomposition approaches to allow significant progress in understanding complex traits like metabolic mixtures.We appreciate these suggestions and have revised the manuscript to address them in the Introduction and Discussion. We explicitly note that it is possible to dissect highly polygenic traits down to the functional level and that there is no need to assume that there are few causal genes for potentially adaptive traits.The manuscript would likely get broader visibility if there was more general information about the bigger picture presented in the manuscript that could pull in researchers studying GWA or metabolic traits in other systems as this manuscript has information for those researchers even if they are studying plants or fungi or humans. While motivated researchers will see the broader impacts of this manuscript on the GWA/metabolite field it would help to work and bring them into the research without this effort by presenting the broader ramifications of the research in the Introduction and Discussion.Again, we appreciate the suggestion but feel that in this case the broader impacts with respect to other complex traits and organisms will be readily understood by a general reader, and that our CHC analysis will be of broad interest in and of itself. Since we are not the first to demonstrate that the genetic basis of high-dimensional polygenic traits can be assessed using data decomposition approaches, we did not stress this point.While I appreciate the use of PCs to reduce the dimensionality, this is an inherently linear approach that can have trouble with non-linear processes such as epistatic variation in biosynthetic pathways. This can be illustrated by  Arabidopsis wherein the PC approach utilized by Bergelson and colleagues was unable to identify all of the key biosynthetic enzymes that could be identified on a more metabolite by specific metabolite analysis or alternatively using ratios of precursors to products . While I don't feel this is necessary to conduct, it would be beneficial to discuss this difficulty that is imparted by the use of linear trait decomposition for GWA.Brachi, B. et al.(2015). Coselected genes determine adaptive variation in herbivore resistance throughout the native range of Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 112, 4032-4037.Chan, E.K. et al. (2011). Combining genome-wide association mapping and transcriptional networks to identify novel genes controlling glucosinolates in Arabidopsis thaliana. PLoS Biol 9, e1001125.Wentzell, A.M. et al. (2007). Linking metabolic QTLs with network and cis-eQTLs controlling biosynthetic pathways. PLoS Genet 3, 1687-1701.We appreciate this insightful and thought-provoking comment from the reviewer. We added the following text to the Discussion regarding the motivation of using PCs and caveats of losing the ability to detect genes that control variation of individual CHCs: “We used principal component analysis to reduce dimensionality of the data, which was motivated by the high co-linearity among the CHCs. However, PCA may cause genetic variation for a certain CHC to be distributed among multiple PCs and therefore dilute its association with QTLs.”Regarding the specific concerns raised by the reviewer:A) PC1 and PC2 are orthogonal (uncorrelated) by definition. The appearance of certain linear relationships with subsets of lines may be a result of projecting multi(>2)-dimensional data onto the 2-D plane created by PC1 and PC2. Such patterns may or may not be biologically meaningful. However, we are not aware of a good approach that would use such patterns to synthesize a biosynthetic pathway. On the other hand, albeit incomplete and lacking hierarchical information, our approaches using individual PCs did arrive at key genes involved in the CHC biosynthetic pathways.B) We agree with the reviewer that analysis using PCs may miss genes controlling the biosynthesis of CHCs. However, given the severe co-linearity of the data, we think the gain by reducing dimensionality is greater than the loss. Following the reviewer’s suggestion, we acknowledge this limitation in the Discussion.Reviewer #2:This manuscript represents a great deal of very good work and can be made into an excellent publication. However, much of the biochemistry upon which it is based needs extensive correcting and rewriting. The identification of new components in this much studied species is well done and important. In the subsection “Functional validation of candidate genes”, second paragraph: Fatty acid biosynthesis begins with an acetyl-CoA and then elongation occurs with a malonyl-CoA in which the energy is supplies by decarboxylation of the malonyl-CoA as two carbons incorporated into the growing chain. The next three steps are a reduction of a carbonyl to an alcohol, dehydration, and reduction of the carbon-carbon double bond. The authors are making too extensive use of plant lipid metabolism data. The FAS in animals, including flies, occurs on the multifunctional protein fatty acid synthetase (FAS) in which the growing chain is attached to an acyl carrier type unit that is part of the multifunctional . It is not typically called an acyl carrier protein complex. Products of insect FASs, particularly Drosophila FAS, can be 14, 16 or 18 carbons. A thioesterase that is part of the multienzyme FAS removes the elongated chain as a free fatty acid. I do not know what is meant by conjugating and the acyl group is never removed by a thioreductase. In all cases studied in insects, desaturation and elongation take place in a microsomal fraction (endoplasmic reticulum) and not the mitochondrial fraction (many references).We appreciate the reviewer's expertise on this topic and re-wrote the entire paragraph as the reviewer suggested to accurately describe fatty acid biosynthesis in insects.In the subsection “Functional validation of candidate genes”, fourth paragraph: Fatty acids are elongated and desaturated attached to an ACP in plants. In insects, the parallel reactions occur with the acyl group attached to CoASH. The evidence to date suggests that the elongated fatty acyl-CoA is reduced to an aldehyde prior to oxidative decarbonylation, the latter step catalyzed by Cyp4G1 in Drosophila . While it is possible that the elongated fatty acyl-CoA is reduced from acyl-CoA to aldehyde to alcohol and then re-oxidized to aldehyde before oxidative decarbonylation, there is no evidence for this to date in insects. As the reviewer suggested, we have removed the reference to plant fatty acid elongation and changed the paragraph to accurately state what is known in insects.In the subsection “Functional validation of candidate genes”, end of fourth paragraph: An esterase cleaves an ester bond and does not reduce the carboxylic ester group on the fatty acyl-CoA (not ACP) to an alcohol. A FAR (fatty acyl-CoA reductase) does the reduction.We have removed this sentence from the manuscript.In the subsection “Functional validation of candidate genes”, sixth paragraph: I am not convinced the CYPs studied here are involved in CHC turnover.We appreciate the reviewer's skepticism. This line is our speculation based on the RNAi knockdown results. We propose it merely as a hypothesis for future functional studies of these CYPs. We have modified this statement to explicitly state that it is speculation.In the subsection “Functional validation of candidate genes”, last paragraph: The authors are comparing 3-methyl fatty acids with a 3-methyl hydrocarbon derived from a n-3 methyl fatty acid.We understand the reviewer’s point that for the 3-methylhydrocarbons the methyl branch is inserted right at the beginning and then elongation happens with the methyl-branched end becoming farther and farther away from the CoA “head”, whereas in a 3-methyl fatty acid the methyl group is on carbon 3 counted from the carboxyl “head”. We have removed our previous speculation from the manuscript.When stating “Our results provide a new perspective and highlight the complexity of the biosynthetic and catabolic pathways that regulate the dynamics of CHC composition”, the authors are blithely referring to the biosynthetic pathways of hydrocarbons (which are somewhat known) with the catabolic pathways . This sentence is illustrative of several times in the manuscript where the authors make sweeping statements of biochemistry for which there is no basis.We have modified this sentence to say "Our results provide new perspective and highlight the complexity of the CHC dynamics."Reviewer #3:In the first section of the paper, Dembeck et al. claim to have detected 16 previously unidentified CHCs from Drosophila melanogaster, including dienes with double bonds in the even-numbered configurations as well as methyl-branched CHCs which are not 2-methyl-branched. The authors stated that they were able to identify these compounds because they use a thin high res column (DB-5) and a relatively long temperature program. I am concerned about the use of this column for the separation of D. melanogaster CHCs as this column cannot separate two major compounds in female Drosophila. Peak 46 contains both 2Me-C26 and 7,11-C27:2. This is a concern for the second part of the paper  because 2Me-C26 is a major compound. If they cannot separate these two compounds, then it would be impossible to quantify accurately the amount of 7,11-C27:2 in the fly. Another recent paper  also used a different method to identify CHC in D. melanogaster. While they identified some methyl-branched CHCs which are not 2-methyl-branched, they did not identify the dienes with the even double bonds presented in this study. The authors stated that these compounds are identified based on comparison to the reference library and to already published D. melanogaster CHCs and the position of the double bond was not confirmed for any of the CHCs. While most of these compounds exist in low quantities, I suggest the authors proceed with caution on claiming that they have identified new  compounds that many other groups using different methods over the years have not been able to identify.Separation of the odd-carbon 7,11-dienes and the preceding even-carbon 2-methyl-alkane: Both separate on our column  by just a couple of Kovats units. So, when the diene is absent the alkane can be readily separated and vice versa. However, when the diene is a major component , the alkane represents a tiny shoulder preceding the diene and they cannot be reliably separated. See our Desat2 allele and female CHC phenotyping) “because 2Me-C26 is a major compound”. As stated above, in normal females 2Me-C26 represents a tiny fraction of 7,11-C27:2. In the African phenotype and in females with an intact Desat2 gene we found high 5,9-C27:2 and 5,9-C29:2 and low amounts of 7,11-dienes. The reviewer is correct that 2Me-C26 now represents a larger fraction of this combined peak. See our The reviewer is rightly concerned that in the second part of the paper , namely that these compounds (as the ones with the double bond in odd positions) have characteristic fragments resulting from the fragmentation of the molecular ion where the double bonds are and from α-cleavage at the outer side of the double bonds. An example on Figure C demonstrates the above with 6,10-hexacosadiene from one of the females analyzed in this study.We have included a new In the second part of the manuscript, Dembeck et al. report that the African CHC phenotype  is segregating in the DGRP. This section reports the mismatch between what is called the "16-bp deletion / ancestral alleles" of Desat2 and female CHC phenotype. Two DGRP lines, DGRP_105 and DGRP_551 have the African allele  but the cosmopolitan phenotype. This analysis is based on protein sequence alone and does not take into account the possibility that unique polymorphisms in the Desat2 regulatory region of these lines changed the expression of Desat2. Therefore, without properly quantifying whether Desat2 is expressed or not in these lines, the authors should be careful in determining which alleles are functional based on protein sequence analysis alone (and a 16-bp deletion in the promoter that was not properly characterized previously).Desat2 expression, we found that DGRP_105 has many unique polymorphisms. We recently completed a gene expression analysis of 185 DGRP lines using Affymetrix tiling arrays – the manuscript reporting these results is in press at the Proceedings of the National Academy of Sciences of the USA . This study was done on whole young adult flies. We checked the expression of Desat2 from this study and found that it is highly genetically variable in females  but not in males . We present these results below, classifying the DGRP lines as homozygous for the derived deletion (Del/Del), the ancestral insertion (Ins/Ins) and heterozygous (Ins/Del). The expression level of DGRP_105 is shown by the red data point. It is clear that the Desat2 expression of this line overlaps that of the Del/Del genotypes, completely consistent with our CHC analysis and DNA sequence analysis showing that variants in the Desat2 gene in this line likely result in a non-functional protein. The genotype of this line indeed matches the Cosmopolitan CHC phenotype despite the ancestral ‘functional’ insertion. Unfortunately, DGRP_551 was not among the 181 DGRP lines included in the recent gene expression analysis. We feel that further characterization of the nature of the functional variation at Desat2 in these lines warrants many additional experiments and is outside the scope of the current manuscript.For the analysis of DGRP lines DGRP_105 and DGRP_551, we studied the DNA sequence data. While DGRP_551 does not have any obvious mutations that could disrupt In the third section which forms the bulk of the paper, the authors identify numerous genes potentially involved in CHC synthesis in D. melanogaster. The same authors have used similar methods to identify novel pigmentation genes in Drosophila . In this section, using GWA, the authors identified genetic variants in hundreds of genes which could potentially affect CHC composition. The authors selected 24 candidate genes for functional studies. They used an oenocyte specific driver  to knockdown candidate genes in the oenocytes, where CHCs are synthesized. This is a very comprehensive section that entails a tremendous amount of work in both genomic analysis and functional characterization. The authors found that oenocyte-driven RNAi knockdown of 23 candidate genes individually showed quantitative changes in CHC profiles. These results provide very strong evidence that these candidate genes are involved in CHC biosynthesis, but there are two caveats to consider.First, some of these genes might not be expressed in adult oenocytes. A brief look on the FlyAtlas website showed that the transcripts of some of these genes are not present or expressed at very low levels in the "adult carcass" where the oenocytes are located. Therefore, it is not known whether the phenotypes that the authors observed are off target effects of RNAi or altered physiology of the whole fly due to the oenocyte driver that they use, which may be driving GAL4 in other parts of the fly.GAL4 driver. Only one off-target effect is reported for the RNAi constructs we tested (Irc has a reported off-target effect on CG6170). We added the following text to the Discussion to acknowledge this caveat: “In addition, we note that phenotypic changes associated with knocking down expression of a target gene with RNAi may not be causal, but a consequence of off-target effects of RNAi or the GAL4 driver. Further studies are needed to clarify the effects on CHC production of these genes and their interactions, and to test specific mechanisms and enzymatic activities through which they exert these effects.”The reviewer is correct: formally, one can never disprove the potential for off-target effects of RNAi or Second, while some of the effects of the candidate genes can be explained by the biological/molecular functions of the gene (i.e. fatty acid reductase influencing chain length), others are not . How would a P450 (Cyp9f2) be involved in elongation of CHCs? As far as is known, that is not one of the reactions that a P450 could catalyze.Cyp9f2 may be involved in the elongation of precursors of longer chain n-alkanes and monoenes in females. We later state that: “The link between these CYPs and CHC production and maintenance is unclear. However, we speculate that oxidation reactions mediated by these CYPs may regulate CHC degradation and turnover.”We removed the sentence suggesting that


A large body of research has documented infants’ ability to classify animate and inanimate objects based on static or dynamic information. It has been shown that infants less than 1 year of age transfer animacy-specific expectations from dynamic point-light displays to static images. The present study examined whether basic motion cues that typically trigger judgments of perceptual animacy in older children and adults lead 7-month-olds to infer an ambiguous object’s identity from dynamic information. Infants were tested with a novel paradigm that required inferring the animacy status of an ambiguous moving shape. An ambiguous shape emerged from behind a screen and its identity could only be inferred from its motion. Its motion pattern varied distinctively between scenes: it either changed speed and direction in an animate way, or it moved along a straight path at a constant speed . At test, the identity of the shape was revealed and it was either consistent or inconsistent with its motion pattern. Infants looked longer on trials with the inconsistent outcome. We conclude that 7-month-olds’ representations of animates and inanimates include category-specific associations between static and dynamic attributes. Moreover, these associations seem to hold for simple dynamic cues that are considered minimal conditions for animacy perception. Among those static attributes considered relevant for identifying animates in terms of their appearance are facial features . However, infants’ sensitivity to the specific cues that trigger animacy perception in adults has not yet been studied systematically. We do not yet know whether information about the speed constancy and direction of a given object’s motion is sufficient to make infants, like adults, to form expectations regarding the animacy status of a given entity. The present study addresses this issue. For this purpose, we developed a paradigm that requires infants to infer the identity of a moving “shadow,” a gray colored silhouette whose ambiguous shape does not reveal its identity. Speed constancy, motion direction, and orientation of the shadow’s motion were distinctively varied to disambiguate the animacy status of the target entity before it was finally revealed during the test phase. We tested 7-month-olds, based on the findings of http://www.uni-heidelberg.de/universitaet/profil/wissenschaftliche_praxis/). The study complies with the APA ethical standards.All experiments reported in this manuscript were conducted at a German University where institutional review boards or committees are not mandatory, but where researchers need to follow the rules and regulations of the code of conduct for good scientific practice  before showing only one shadow moving either in an animate or an inanimate way (phase 2). At test (phase 3) the semi-opaque screen is removed to reveal one object whose motion was either consistent or inconsistent with its animacy status. We predicted that 7-month-olds would look longer at inconsistent than at consistent trials. To test this assumption, we used a within-subject design.M = 7 months, 13 days; range = 7 months, 1 day to 7 months, 29 days), 18 girls and 17 boys participated. 10 additional infants had to be excluded due to fussiness (n = 2), not looking at least 3 s at the critical motion event during phase 2 (n = 2), or failing to look during all four test trials during phase 3 (n = 6). All infants came from a White, middle-class socioeconomic background.A total of 35 7-month-old infants , and two inanimate objects , each presented as realistic 2D photographs. We chose mammals and vehicles because exemplars of both types have a comparably complex appearance as well as parts associated with movement. Perhaps most importantly, identical blurred shadows in terms of overall shape and size could be created when both objects were covered by a semi-lucent screen. The dog was always paired with the truck, and the cow was paired with the motorcycle. The two entities were presented in computer-animated scenes (created with Microsoft® Power Point®).Figure 1).Using a within-subjects design, each infant was administered four trials including both stimulus pairs . The first two trials (Part A) as well as the last two trials (Part B) ended with the same outcome object. If the first two trials ended with the animate object of the respective object pair, then the last two trials ended with the inanimate object of the respective object pair and vice versa . The type of motion pattern shown during the motion scene alternated between the four trials. Whether the first trial included an animate motion or an inanimate motion during the motion presentation phase, i.e., whether this motion pattern was consistent or inconsistent with the object shown at test, was counterbalanced across the entire sample. Each infant saw two consistent and two inconsistent test trials .Figures 2A–C). After the third covering event with the left screen, another semi-lucent screen was lowered down to cover the middle section of the screen and remained in place. Next, an opaque screen emerged from the right side of the screen and covered the right side of the display, followed by an additional opaque screen entering from the left side overlaying the left semi-lucent screen and fully covering both shadows . At the end of phase 1, which lasted a total of 25 s, all three screens (two opaque and one semi-lucent) covered the display thus hiding the two stimuli as well as their shadows.The stimuli  were presented in computer-animated scenes  each consisting of three phases. During phase 1, infants were shown both stimuli as well as three separate screens: two smaller screens covering 25% of the scene on the left and the right side of the display, respectively, and one screen covering the middle of the display (50%). At the beginning of each trial, infants first saw one pair of stimuli , placed above each other, both located on the left side of the display. During a 15-s time interval the two objects were covered and uncovered three times by a semi-lucent screen emerging from, and disappearing to the left side of the display. When covered, the two objects appeared as two identical shadows . The motion pattern was either animate or inanimate: The animate motion pattern included changes in speed constancy (4× motion changes) as well as direction changes , with the principal axis of the shadow always aligned with the direction of motion. These changes were evenly distributed across the 10-s motion phase so that infants who were attentive to the motion phase for 3 s or more saw at least one change in speed constancy and one direction change. Infants who did not meet this criterion were excluded from further analyses.During phase 2 (motion phase) that immediately followed phase 1 and lasted for 10 s, infants saw one single shadow emerging from behind the middle of the left opaque screen, moving across the middle semi-lucent screen and disappearing behind the right opaque screen . The corresponding stimulus was presented for 15 s. Depending on the motion pattern presented during phase 2 and the stimulus revealed during phase 3, the trials could either be consistent  or inconsistent .Phase 3 (test phase) started once the shadow had disappeared behind the right opaque screen. The opaque screen then slid to the right (3 s) revealing either the animate or the inanimate entity presented during phase 1  was about 3.5 min. For every given pair of stimuli , each entity was shown once in the upper position and once in the lower position during the first phase. Each entity appeared equally often as test-exemplar and was equally often preceded by an animate or an inanimate shadow-motion during the motion phase.The parent sat in a nearby chair and was asked not to interact with the infant. During the presentation, the experimenter was occluded by a curtain and monitored infants’ fixations via a video-camera. As soon as the infant’s attention was focused on the screen, the experimenter started the computer-controlled presentation. For the first 15-s period of each trial (phase 1) it was measured how long infants looked to the left 25% part of the screen, showing both objects one above the other. For each test phase (phase 3), an area of interest (AOI) was defined that covered the outcome-object . Infant’s fixation duration to that AOI during the test phase was measured. Fixation was defined as stable gaze within one visual degree for at least 200 ms e.g., .a priori preferences for one of the two object pairs shown during phase 1 . There was also no significant motion-type dependent difference in infants’ looking times during the motion phase (phase 2) with similar looking times at animate motion scenes  compared to inanimate motion scenes , t(34) = 1.35, p = 0.18.The mean looking time during the first 15 s period showing the two objects before the semi-lucent screen covered them for the third time was 4.57 s (SE = 0.16). Infants had no Congruency Type (motion pattern consistent or inconsistent with test object) and Motion Type (animate or inanimate motion) as within-subjects factors. This analysis revealed a significant main effect for Congruency Type F = 34.00, p < 0.001, η2 = 0.50, with longer looking times for inconsistent outcomes  compared to consistent ones . No other main effect or interaction was observed.For the test phase, an analysis of variance was conducted to test whether infants differentiated between test trials showing objects congruent or incongruent with the observed motion pattern of the shadow. Preliminary analyses revealed that neither the type of object (animate or inanimate objects at test), nor the order of presentation (consistent or inconsistent outcomes first) had any effect on infants’ looking responses during the test trials. A 2 × 2 repeated measures analysis of variance was conducted with 2(1) = 17.86, p < 0.001]. Figure 3 shows the mean looking times for the congruent and incongruent test trials as a function of Motion Type.An examination of individual infants’ looking times confirmed these results. 30 of the 35 infants looked on average longer at the incongruent test trials compared to the congruent test trials . Figure 4 shows the mean looking times for the congruent and incongruent test trials as a function of Motion Type.A 2 × 2 repeated measures analysis of variance was conducted with In conclusion, infants’ looking pattern in Experiment 2, presenting no static pictures of an animal and a vehicle prior to the shadow’s motion, does not differ from the results obtained in Experiment 1. On the basis of these data, it seems unlikely that the static pictures shown at the beginning of each trial in Experiment 1 played a key role in infants’ detection of category-motion incongruencies at test. This suggests that longer looking times during the inconsistent test trials in Experiment 1 reflected the recognition of category-specific motion cues without any form of priming.The main goal of the current experiments was to determine if infants have already formed specific expectations about the type of motion cues that have been found to generate animacy judgments in older children and adults. The present results suggest that 7-month-olds’ representations of animals and vehicles include category-specific associations between static and dynamic cues that are considered minimal conditions for animacy perception e.g., .These findings suggest that within their first year of life, infants have already formed some stable associations between static and dynamic attributes characterizing global categories. However, while Referring to One might speculate that it was easier for infants tested with the Such a dissociation between dynamic and static feature processing is in accordance with the fact that both types of information are processed along different neural pathways . It is wa priori static information before the start of the shadow movement. This suggests that a priori given static information is not necessary for establishing animacy-specific relations between dynamic and static features.In comparison to Previous research on infants’ knowledge of properties of animates and inanimates based on dynamic information typically focused on specific motion patterns like rigid versus non-rigid (caterpillar) motion. The present study extends this line of research by showing that in addition to motion pattern, motion path is also an important and early accessible cue to animacy status. Until today, the nature of early animacy-ascriptions to given events or entities is not fully clarified. While some authors interpret corresponding data as a sign for conceptual or higher level processes e.g., , others The present set of data cannot resolve this issue. It might be that the motion cues provided in the present task are processed within an automatic encapsulated perceptual mechanism, resulting in an impression of animacy that either does or does not match with conceptual representations activated by the outcome picture presented at test. Furthermore, the matching process at test might also be mainly perceptual in nature, reflecting perceptually based associations between animacy-specific dynamic and static features. Such an assumption conflicts with more conceptual accounts that assume that higher-level processes determine the animacy status of an entity, including various assumptions on the nature and content of higher-level mechanisms that might be involved in conceptual information processing e.g., . RegardlIn any case, we conclude that transfer performances as shown by 7-month-old infants in the present task and also by slightly older infants in the study of In sum, the current results obtained with an innovative paradigm are in line with the assumption that 7-month-old infants are able use specific dynamic cues, known to trigger animacy perception in adults, to infer the identity of morphologically ambiguous entities. Future research with this paradigm can clarify whether infants draw similar inferences about animacy from a wide range of motion cues.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Severe sepsis is associated with high levels of morbidity and mortality, placing a high burden on healthcare resources. We aimed to study outcomes in the five years after severe sepsis.This was a cohort study using data from a prospective audit in 26 adult ICUs in Scotland. Mortality was measured using clinical databases and quality of life using Short Form 36 (SF-36) at 3.5 and 5 years after severe sepsis.A total of 439 patients were recruited with a 58% mortality at 3.5 years and 61% mortality at 5 years. A total of 85 and 67 patients responded at 3.5 and 5 years follow-up, respectively. SF-36 physical component score (PCS) was low compared to population controls at 3.5 years (mean 41.8 (SD 11.8)) and at 5 years (mean 44.8 (SD 12.7)). SF-36 mental component score (MCS) was slightly lower than population controls at 3.5 years (mean 47.7 (SD 14.6)) and at 5 years after severe sepsis (mean 48.8 (SD 12.6)). The majority of patients were satisfied with their current quality of life (QOL) (80%) and all patients would be willing to be treated in an ICU again if they become critically ill despite many having unpleasant memories (19%) and recall (29%) of ICU events.Patients with severe sepsis have a high ongoing mortality after severe sepsis. They also have a significantly lower physical QOL compared to population norms but mental QOL scores were only slightly below population norms up to five years after severe sepsis. All survivors would be willing to be treated in an ICU again if critically ill. Mortality and QOL outcomes were broadly similar to other critically ill cohorts throughout the five years of follow-up. Severe sepsis is an increasingly common condition that is associated with high levels of morbidity and mortality as well as placing a large burden upon healthcare resources throughout the world -4. The mQOL is a very important outcome for patients after critical care and has been shown to be poor in all subgroups of critical illness compared to the general population at various time points -16. PoorThe aim of this study was to determine the mortality and QOL in patients in the five years after severe sepsis. As secondary outcomes of interest, we also wanted to study the patients' willingness to be treated in an ICU again if they become critically ill, the nature of their memories of the ICU, their satisfaction with their current QOL and the impact of the index illness on their place of residence and employment status.This was a prospective cohort study of patients with severe sepsis with ethics approval granted by the Scottish multi-center research ethics committee and informed consent obtained from all patients' quality of life follow-up after three years. The ethics committee approval required the research team to make contact with patients indirectly through their parent hospital team, and first and only if the patients agreed, could the research team contact the patient. Mortality follow-up used national registry databases which are known to have an accuracy of 99%.The study was of a prospective cohort of patients with severe sepsis collected between April and October 2003. Patients were identified as having: a.) evidence of three of four systemic inflammatory response syndrome (SIRS) criteria within the previous 24 hours, b.) confirmed or clinically strongly suspected infection, c.) two or more sepsis induced organ failures of less than 24 hours duration , and d.)Patients completed Short Form-36 (SF-36) questionnaires and euroQOL-5D (EQ-5D) by telephone survey at 3.5 and 5 years after ICU admission and the results were calculated using standard techniques -16,21,22Data are presented as numbers and percentages, means and standard deviations or median and interquartile ranges as appropriate. For baseline sepsis and ICU data, numbers and percentages or median and interquartile ranges were calculated as appropriate. Differences in quality of life between 3.5 years and 5 years were compared using paired-tests.The study sponsor and funding sources had no role in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.The flow of patients through the study is presented in Figure The survival data show that 267 patients (63%) survived to leave the ICU, 249 (57%) survived to leave the hospital, 185 (42%) survived to 3.5 years and 172 (39%) survived to 5 years . The equivalent comparison for the mental component at five years shows no significant difference (P = 0.453).At five years, the SF-36 PCS of 44.8 is significantly lower than the mean population norm of 50  study showed that the reduction in mortality seen at 28 days was lost after hospital discharge through to 3.6 years although most patients in this study were followed up for shorter durations [In a landmark study, Quartin controls . Other scontrols -27. A pourations ,15,24,29urations . All theurations ,25-27.et al. suggested that SF-36 QOL scores were lower than the general population and that SF-36 had a good reliability and validity in this group [et al. showed poor QOL scores at six months after an ICU stay compared to age- and sex-matched controls [et al. also found poor QOL in survivors of sepsis that was comparable to other non-septic critical illness survivors [et al. studied patients receiving activated protein C compared to unmatched controls and looked at mortality and QOL outcome up to seven months after ICU discharge [There have been a few papers looking at differences in QOL outcomes in severe sepsis patients. A study by Heyland is group . A studycontrols . A studyurvivors . A studyischarge . They foischarge . This stper se but it may confound the measurement of both mental and physical QOL scores.The current study focuses on QOL outcomes up to five years after severe sepsis. There are no studies we have identified that studied QOL in severe sepsis patients for this long a time period. The most comparable studies are ones of general ICU cohorts ,15,24 anIt was very interesting to find that all our patients would be willing to undergo treatment in an ICU again if they become critically ill despite generally poor physical QOL scores and 19 to 29% having either unpleasant recall or memories of their ICU stay ,39,40. DThis study has a number of strengths and limitations that need to be considered. Strengths include the multi-center nature of the data and the fact that all adult general ICUs in one country were included. Further strengths include the prolonged follow-up period that has not been attained in previous studies. Compared to previous studies in this field, this is a reasonable sized study, but from an epidemiological perspective the limited patient number reduces the ability to make strong conclusions about this cohort. Limitations include the loss to follow-up in this study. The cohorts were not approached for consent for long term follow-up until well after their index ICU admission, thus denying us shorter term QOL data. The ethics committee approval required the research team to make contact with patients indirectly through their parent hospital team first and only if the patients agreed could the research team contact the patient. We believe that this additional step led to a significantly lower recruitment and retention to the study than a direct approach would have achieved. This has the potential to introduce a recruitment bias into the study data collection, although we are unable to identify the magnitude of direction of such a bias. Since it is likely that poor QOL is more common in those patients who die during follow-up, there is likely excess QOL loss to follow-up in the highest risk patients and, therefore, these QOL results may, on average, be better than those actually experienced by the patients. This issue is standard to all studies using prospective cohort designs. With regard to the generalizability of the results, we recruited patients from all adult ICU patients in one country which enhances the generalizability but we only recruited patients with an APACHE II score ≥25 making this a sicker cohort than in some studies of severe sepsis. It is noted that despite the inclusion criteria for this cohort being APACHE II ≥25, some patients with lower APACHE II scores were recruited to the study. In this study we demonstrated that 35% of infections were microbiologically confirmed compared to those in European Prevalence of Infection in Intensive Care (EPIC) and 60% in Sepsis Occurrence in Acutely Ill Patients (SOAP) studies ,44. A fuPatients with severe sepsis have a high ongoing mortality after severe sepsis with only 61% surviving five years. They also have a significantly lower physical QOL compared to the population norm but mental QOL scores were only slightly below population norms up to five years after severe sepsis. Mortality and QOL outcomes were broadly similar to other critically ill cohorts throughout the five years of follow-up. These data need to be considered when evaluating longer-term outcomes and when considering the cost-effectiveness of care in this patient group.• Sepsis is known to have a high, shorter-term mortality; this high mortality seems to continue for up to five years after severe sepsis.• Quality of life is known to be poor in the years after critical care admission and we have demonstrated similar patterns of QOL deficit after severe sepsis.• The majority of severe sepsis survivors were satisfied with their current QOL and all patients would be willing to be treated in an ICU again if they become critically ill despite many having unpleasant memories and recall of ICU events.ADL, activities of daily living; APACHE II, Acute Physiology, Age And Chronic Health Evaluation; ARDS, Acute respiratory distress syndrome; EPIC, European Prevalence of Infection in Intensive Care; EQ-5D, euroQOL-5D; ICU, intensive care unit; MCS, mental component score; PCS, physical component score; QOL, quality of life; SAPS, Simplified Acute Physiology Score; SD, standard deviation; SF-36, short form 36; SIRS, systemic inflammatory response syndrome; SOAP, Sepsis Occurrence in Acutely Ill PatientsThis study was funded by the Scottish Intensive Care Society and an unrestricted research grant from Eli Lilly and Company. Eli Lilly and Company did not have input into the study design or analysis and did not approve the final results. Dr Brian Cuthbertson has received honoraria for presentations and for consultancies from Eli Lilly and Company. There are no other financial or non-financial competing interests for this manuscript.BHC, FM and SJM have participated fully in the design of this study, the collection and analysis of data and in the writing of the paper, and have seen and approved the final version of the paper. AE and GM have participated fully in analysis of the data and in the writing of the paper, and have seen and approved the final version of the paper. SH and JT have participated fully in the collection of data, analysis of the data and in the writing of the paper, and have seen and approved the final version of the paper.


Cotesia rubecula was previously shown to inhibit melanization in the host hemolymph by blocking activation of prophenoloxidase to phenoloxidase, a key enzyme in melanin formation. Similar to other SPHs, Vn50 consists of a clip and a protease-like (SPL) domain. Protein modeling demonstrated that Vn50 has a very similar structure to known SPHs and functional analysis of Vn50 domains expressed in insect cells indicated that neither of the domains on its own has an inhibitory effect on melanization.Endoparasitoid wasps inject a variety of components into their host hemocoel at oviposition to facilitate successful development of their progeny. Among these are venom proteins which have been shown to play crucial roles in host regulation. A serine protease homolog (SPH)-like venom protein from  The pt al. 200 presenteCotesia rubecula (Hymenoptera: Braconidae) which binds to proPAP, PAP, proPO and PO and inhibits activation of PO by competing with host SPHs for binding to proPO [Endoparasitoids introduce a mixture of components during oviposition, including calyx fluid proteins, venom components, and symbiotic viruses and virus-like particles such as polydnaviruses . These ato proPO ,9. Unlikto proPO . Vn50 isto proPO .H. diomphalia as a template [To investigate if Vn50 has a similar structure to SPHs, tertiary structure predictions were generated using ESyPred3D v1.0 . The modtemplate ; therefoSpodoptera frugiperda Sf9 cells according to the manufacturer's instructions (Nitrogen). The corresponding coding regions for the two domains were first amplified with PCR by incorporating the restriction sites for direct cloning into the transfer vector pFastBac-HTa. For efficient secretion of the proteins, the coding sequences for the secretion signal of the honey bee, Apis mellifera, melittin gene  were inserted into the transfer vector after which the corresponding Vn50 constructs were cloned. To confirm expression of the proteins, media were collected 72 h after infection and reduced in volume to approximately a tenth of their original volumes using a rotary evaporator and were run on a Western blot. The blot was then probed with an anti-Vn50 polyclonal antibody [To find out whether either of the Vn50 domains on their own have the PO inhibitory function, we expressed the two domains separately as well as the full-length Vn50, using the Bac-to-Bac baculovirus expression system in antibody  used as th instar Pieris rapae larvae were surface sterilized in 70% EtOH before bleeding from a proleg into 100 μL of ice-cold phosphate buffered saline (PBS). This was then centrifuged for 5 min at 800 × g and the supernatant (plasma) was transferred into a fresh tube. A 20 mM solution of L-3,4-dihydroxyphenylalanine (L-DOPA) in PBS was prepared immediately before use in order to avoid the effects of auto-oxidation. For each replicate, 10 μL of hemolymph was mixed with 10 μL of protein and 80 μL of L-DOPA. A control without hemolymph was also included to correct the data for auto-oxidation. Absorbance was measured at 485 nm each 10 min for 300 min.Following confirmation of expression of the proteins, they were affinity purified from the medium collected from cells 72 h after infection using Ni-NTA agarose beads (BioRAD) that specifically bind to the 6× His-tag. These were then used for melanization assays. To provide hemolymph for the assays, 4P < 0.0001 One-way ANOVA). The RNase III control, SPL domain only, clip domain only and a mixture of SPL and clip domains all showed increases in absorbance similar to the control (mock-infected) . The results suggested that only the full-length Vn50 was successful in inhibiting melanization activity and that the expressed domains were not able to bind to proPO in an inhibitory fashion like Vn50, but also that they were unable to increase levels of melanization. Therefore, cleavage of Vn50 into the two domains is not all that is missing in Vn50 compared to other SPHs and the close interaction of the clip and protease domains is important in the activity of SPHs.Absorbance readings for the control (mock-infected) showed a significant increase in melanization compared to the full-length Vn50 which showed only slight melanization  who published the crystal structure of PPAF-II, a clip-domain family SPH [The activation of proPO to PO in the process of melanization is of major importance in the melanization process . Usuallyt al. 200 who publ2.Protein modeling showed that Vn50 has a very similar 3D structure to a known SPH (PPAF-II) with determined crystal structure, and functional analysis of Vn50 domains (clip and SPL) indicated that neither of the domains on its own has an inhibitory effect on melanization. These results give us deeper insight into two effects. Firstly, they reinforce the findings of past studies that emphasized the importance of the close interactions between the SPL and clip domains of SPHs and that this is essential for their activity. Secondly, they show the need for this interaction for successful binding to proPO and support the past assertion that Vn50's inhibitory activity is through competing with SPHs for the binding of proPO.