Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important transmission vector of the citrus greening disease Candidatus Liberibacter asiaticus (CLas). The D. citri midgut exhibits an important tissue barrier against CLas infection. However, the molecular mechanism of the midgut response to CLas infection has not been comprehensively elucidated. In this study, we identified 778 differentially expressed genes (DEGs) in the midgut upon CLas infection, by comparative transcriptome analyses, including 499 upregulated DEGs and 279 downregulated DEGs. Functional annotation analysis showed that these DEGs were associated with ubiquitination, the immune response, the ribosome, endocytosis, the cytoskeleton and insecticide resistance. KEGG enrichment analysis revealed that most of the DEGs were primarily involved in endocytosis and the ribosome. A total of fourteen DEG functions were further validated by reverse transcription quantitative PCR (RT-qPCR). This study will contribute to our understanding of the molecular interaction between CLas and D. citri.The Asian citrus psyllid (ACP),  Candidatus Liberibacter spp” (CLas). HLB nearly destroyed the citrus industry in Florida (USA) and most citrus-producing regions of the world [CLas can decrease plant growth vigor, and ultimately result in the death of the infected citrus tree [CLas bacteria; examples include the trunk injection of penicillin, streptomycin and oxytetracycline hydrochloride [Huanglongbing (HLB) is a destructive disease of citrus that represents a major threat to the world’s citrus industry. HLB is caused by the phloem-limited, Gram-negative bacterium “he world ,2. CLas rus tree . There achloride ,4. Howevchloride .Diaphorina citri Kuwayama, is the principal transmission vector of HLB [D. citri is recognized as a key approach to preventing the spread of HLB. The application of insecticides is the most widely employed option for reducing D. citri populations in some citrus-growing regions [D. citri, revealing a suite of odorants that can be used to develop affordable and safe odor-based surveillance for D. citri control [D. citri muscle protein 20 (DcMP20) resulted in significant mortality and reduced the body weight of D. citri [D. citri tropomyosin1-X1 (DcTm1-X1) significantly increased the mortality rate of nymphs [D. citri has been achieved, the threats caused by HLB are going to continue. It is essential to investigate the molecular mechanisms of HLB transmission in D. citri. The Asian citrus psyllid (ACP), r of HLB . Vector  regions . However regions ,9. Some  control . Yu et aD. citri . By RNA f nymphs . AlthougD. citri adults and nymphs can transmit CLas. Recent studies suggested that the pathogen multiplies in D. citri after acquisition and becomes distributed among various internal tissues, including the alimentary canal, salivary glands, hemolymph, filter chamber, midgut, fat body, muscle tissues and ovaries [CLas infection [Bombyx mori larval midgut via comparative transcriptome analysis. The results showed that many of the DEGs were associated with protein metabolism, the cytoskeleton and apoptosis [Nilaparvata lugens intestine, and a total of 33 digestion-related genes, 25 immune response genes and 27 detoxification-related genes were identified [D. citri and CLas have become a heavily researched topic regarding the molecular mechanisms of HLB transmission. In recent years, high-throughput omics technologies have been widely used to study the interactions of vector-pathogens. Combining transcriptomics with proteomics methods, Kruse et al. identified many DEGs involved in the TCA cycle, iron metabolism, insecticide resistance and the insect immune system in the D. citri gut after exposure to CLas [CLas-infected adult D. citri, using two-dimensional gel electrophoresis. These DEPs were associated with energy metabolism, host detoxification processes and the cytoskeleton [D. citri immune system, where it coordinates the insect’s immune system activity. Kruse et al. investigated the effect of CLas exposure on the D. citri hemolymph, using high-resolution quantitative mass spectrometry, and the results revealed that proteins associated with fatty acid synthesis and energy metabolism were upregulated [CLas entry, multiplication and dissemination in the midgut of D. citri. Both  ovaries ,14,15. Anfection . Pathogenfection ,18. In tpoptosis . Bao et entified . The int to CLas . Lu et askeleton . The hemegulated . HoweverD. citri to CLas infection in the midgut at the mRNA level. A total of 778 DEGs were identified, and these genes were mainly involved in ubiquitination, the immune response, the ribosome, endocytosis, the cytoskeleton and insecticide resistance. This study established a foundation for further understanding the interaction mechanisms between D. citri and CLas.In this study, we performed a comparative transcriptome analysis to determine the specific responses of D. citri was reared in mesh cages on Murraya exotica at 27 ± 1 °C, 70% ± 5% relative humidity, and a 14:10 (light–dark) photocycle. Healthy D. citri adults were transferred to CLas-infected Newhall Navel oranges and maintained under the same rearing conditions. One hundred D. citri from CLas-infected Newhall Navel oranges were randomly collected for calculating the infection rate. In general, the head of D. citri was cut off using a razor blade, and the genome DNA was extracted using the TIANamp Micro DNA kit . Afterwards, a PCR reaction was performed to amplify the 16S ribosomal DNA fragment using the HLB pathogen specific primer set OI1/OI2c (forward primer 5′-GCGCGTATGCAATACGAGCGGCA-3′ and reverse primer 5′-GCCTCGCGACTTCGCAACCCAT-3′) based on a previous protocol [CLas infection rate. When the CLas-infected percentage reached above 80%, the infected D. citri were selected. The uninfected and CLas-infected D. citri were collected and dissected to obtain the midgut, which was then washed with precooled DEPC-water to remove the remaining tissues. Genomic DNA was isolated from the D. citri midgut, and a PCR was performed to confirm the CLas infection rate.protocol . Then, aCLas-infected or uninfected D. citri, were dissected for RNA extraction. In order to avoid RNA degradation, the collected midgut samples were stored in RNAlater and frozen at −80 °C. Total RNA was extracted from the midgut of uninfected and CLas-infected D. citri using an animal tissue total RNA kit . RNA concentration and purity were assayed using a NanoDrop 2000 spectrophotometer  at absorbance ratios of A260/280 and A260/230. The integrity of total RNA was confirmed using agarose gel electrophoresis.For transcriptome sequencing, three groups, each consisting of three hundred Transcriptome sequencing was conducted on the Illumina HiSeq platform . Sequencing libraries were generated using the NEBNext1Ultra™ RNA Library Prep Kit for Illumina1 . The library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kitv3-cBot-HS . After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform, and 125 bp paired-end reads were generated. The sequencing fragments were translated into fastq format raw reads using the CASAVA software. The raw reads of fastq format were firstly processed through in-house Perl scripts. The clean reads were obtained by removing reads containing adapter, reads containing poly-N, and low quality reads in which a base number of Qphred less than or equal to twenty accounted for more than fifty percent of the entire read length. At the same time, Q20, Q30 and the GC content of the clean data were calculated. ftp://ftp.citrusgreening.org/annotation/OGSv2.0/) [https://anaconda.org/biobuilds/hisat2) was used to build the index of the reference genome and align the paired-end clean reads with the reference genome [CLas-free groups and CLas-infected groups were performed using the DESeq2 R package (1.16.1). DESeq2 provides statistical routines for determining differential expression according to the negative binomial distribution. The resulting P-values were adjusted using the Benjamini–Hochberg method. A Corrected Genes P-value of 0.05 and an absolute |log2 (fold change)| of 0 were set as the threshold for significantly differential expression. The hierarchical cluster analysis of differentially expressed genes (DEGs) was conducted using Genesis software (http://genome.tugraz.at/genesisclient_download.shtml).The reference genome and gene model annotation files were downloaded from the genome website (GSv2.0/) . Hisat2 e genome . This geP-value of < 0.01 was set as the threshold. Gene Ontology (GO) is a tool used for gene annotation by collecting defined, structured, controlled vocabulary . KEGG . The relative expression level of each gene was calculated using the 2−∆∆Ct method. Three biological replicates were conducted for each sample. D. citri glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The expression patterns were compared by ANOVAs, followed by Fisher’s protected least significant difference (LSD) tests [In order to verify the reliability of the transcriptome data, the expression levels of 14 randomly selected DEGs were examined by RT-qPCR. All of the primers used are listed in D) tests .D. citri midguts were dissected in DEPC-water using a dissecting needle. The results showed that the detached midguts were relatively intact  and Q20 (sequencing error rate < 1%) were more than 92% and 97%, respectively. The GC contents were 40.57%, 40.36%, 40.91%, 37.74%, 39.94% and 39.39%, respectively  values of the two groups, we performed hierarchical clustering of all the DEGs to determine the expression patterns of the identified genes  were upregulated except for Toll-like 8B and Beat protein. For the ribosome analysis, a total of 15 DEGs were identified, and most of the genes were upregulated, except for 28S ribosomal protein S27. Based on the transcriptome analysis, many DEGs associated with ubiquitination, the immune response and ribosomes were altered in the uninfected groups and the d groups . For theCLas infection. However, tyrosine protein kinase receptor torso and dynein heavy chain were downregulated. For insecticide resistance, a total of 19 DEGs were found; of these DEGs, 13 (68.4%) were upregulated and six (31.6%) were upregulated, in the midgut after CLas infection.Based on the GO and KEGG enrichment analyses, the DEGs involved in endocytosis, the cytoskeleton and insecticide resistance were identified . For endD. citri. Chemical insecticides are currently employed as the primary management strategy, but the widespread use of insecticides leads to serious resistance in D. citri [CLas and D. citri is promising for the control of HLB. The transmission of CLas in D. citri is a long-term process that is composed of an acquisition access period (APP), a period of latency and an inoculation access period (IAP) [CLas entry into the D. citri, the midgut is an important immune barrier for defense against bacterial infection. In a previous report, draft genome sequencing revealed that D. citri contained the Toll and JAK/STAT pathways, but lacked genes for the IMD pathway response to Gram-negative bacterial infection [D. citri transmits CLas, beginning from a few days to a week after acquisition, and for the lifetime of the vector, suggesting that CLas have developed mechanisms to avoid psyllid cellular and humoral immune defenses. In Galleria mellonella, Bacillus thuringiensis infection significantly influenced antioxidant activity and the level of lipid peroxidation in the larval midgut [D. citri midgut would play critical roles in the interaction between CLas and D. citri. In this study, we performed RNA-pooling sequencing, and a total of 62,582,728, 58,399,847 and 56,575,636 raw reads were obtained from CLas-free groups, and 57,180,938, 63,335,414 and 54,887,010 raw reads were obtained from CLas-infected groups. These results showed that the number of raw reads for CLas-free groups and CLas-infected groups were similar. Previous studies revealed that pooling sequencing did not represent the population variations in gene expression levels, but it could save costs and limit starting material. However, stringent false discovery rates (FDRs) and the high-throughput validation of DEGs should be considered [CLas infection according to comparative transcriptome analysis. Many pathogens can induce the upregulation of host gene expression. Xiong et al. identified many immunity-related genes encoding pattern recognition receptors, signal modulators and immune effectors after the injection of the fungal pathogen Beauveria bassiana and the Gram-negative bacterium Enterobacter cloacae [Musca domestica larvae inoculated with a mixture of Escherichia coli and Staphylococcus aureus. The results showed that many genes involved in innate immunity were induced following infection [HLB is devastating citrus production worldwide and belongs to a phloem-limited α-proteobacterium. However, the study of this bacterium is highly difficult due to the difficulties in culturing it in vitro . The manD. citri . The stuod (IAP) . During nfection . D. citrl midgut . Therefonfection . In thisCLas infection. E3 ubiquitin-protein ligase was upregulated in the D. citri midgut following CLas infection. Zhang et al. revealed that zebrafish bloodthirsty member 20 with E3 ubiquitin ligase activity was involved in the immune response against bacterial infection [CLas infection. Many ubiquitinated proteins are recognized by the proteasome and are then further degraded [B. mori midgut after B. mori nucleopolyhedrovirus (BmNPV) infection [CLas infection, including proteasome activator complex subunit 3-like, proteasome subunit beta type, proteasome subunit alpha type and 26S proteasome non-ATPase regulatory subunit 12. We speculated that CLas bacteria invading the midgut might be recognized by the D. citri immune system. Meanwhile, CLas could activate host ubiquitination to eliminate immune-related proteins. Ubiquitination occurs through a series of reactions catalyzed by different enzymes, including Ub-activating E1, Ub-conjugating E2 and Ub-ligase E3 . Ubiquitnfection . Therefodegraded . The ubidegraded . Yu et anfection . In thisCLas, as Gram-negative bacteria, activate the host immune system after invading the midgut [CLas-infected groups. Serine proteases (SPs) are involved in both the prophenoloxidase (PPO) activation cascade and the Toll immune signaling pathway, especially those with a clip domain. The proteases are secreted into the hemolymph as inactive precursors and transform into the active protein involved in the melanization reaction requiring specific proteolytic cleavage [CLas infection, including CLIP domain-containing serine protease 2-like, CLIBP-serine protease 5 and CLIBP-serine protease 4, partial. In insects, PPO is activated through a serine protease cascade upon the recognition of pathogen-associated molecular patterns. In previous research, two PPOs were identified from the genome database of D. citri. We considered that CLIP domain-containing serine protease 2-like might regulate D. citri PPOs to activate melanization to defend against CLas infection [e midgut . Thus, te midgut . In totacleavage . In thisnfection . CLas-infected group to the CLas-free group. Among these genes, 14 were upregulated after CLas infection, except for 28S ribosomal protein S27 . Yu et al. also found that some ribosome-associated proteins were upregulated after BmNPV infection [CLas infection. In the process of host invasion by pathogens, many proteins are utilized to achieve replication. Ribosomal proteins, in conjunction with rRNA, make up the ribosomal subunits involved in the cellular process of translation . In thisnfection . These rCLas infection, except for heat shock protein 70. During CLso infection of the potato psyllid, CLso cells were observed between the basal lamina and the basal epithelial cell membranes [CLas are Gram-negative bacteria, but they are not pathogenic to D. citri. In addition, many studies revealed that CLas could be detected in the hemolymph from CLas-infected D. citri [CLas invading, D. citri might inhibit the expression of endocytosis-related genes in the midgut to prevent the further transmission of CLas. Moreover, CLas might avoid the host immune system by endocytosis. Clathrin-mediated endocytosis is mainly involved in the selective and facilitated internalization of cell surface receptors . Many paembranes . For exaD. citri . TherefoCLas-infected groups compared to the CLas-free groups. As a major protein constituent of cytoskeletal filaments, tubulin is involved in many vital cellular processes, including cell motility, cellular division and cytokinesis [CLas infection in D. citri. The cytoskeleton is critical for the maintenance of cell shape, cell motility and intracellular transport, and bacterial and viral infections require the cytoskeleton . Bacteriokinesis . These rCLas-infected groups and CLas-free groups. KEGG and GO enrichment analyses revealed that 80 DEGs were associated with ubiquitination, the immune response, ribosomes, endocytosis, the cytoskeleton and insecticide resistance. This study provides a foundation for further research to investigate the mechanisms of CLas invasion of the D. citri midgut.By using transcriptome sequencing, we identified 778 genes differentially expressed in the midgut between the


The evolutionary radiation of birds has produced incredible morphological variation, including a huge range of skull form and function. Investigating how this variation arose with respect to non-avian dinosaurs is key to understanding how birds achieved their remarkable success after the Cretaceous–Paleogene extinction event. Using a high-dimensional geometric morphometric approach, we quantified the shape of the skull in unprecedented detail across 354 extant and 37 extinct avian and non-avian dinosaurs. Comparative analyses reveal fundamental differences in how skull shape evolved in birds and non-avian dinosaurs. We find that the overall skull shape evolved faster in non-avian dinosaurs than in birds across all regions of the cranium. In birds, the anterior rostrum is the most rapidly evolving skull region, whereas more posterior regions—such as the parietal, squamosal, and quadrate—exhibited high rates in non-avian dinosaurs. These fast-evolving elements in dinosaurs are strongly associated with feeding biomechanics, forming the jaw joint and supporting the jaw adductor muscles. Rapid pulses of skull evolution coincide with changes to food acquisition strategies and diets, as well as the proliferation of bony skull ornaments. In contrast to the appendicular skeleton, which has been shown to evolve more rapidly in birds, avian cranial morphology is characterised by a striking deceleration in morphological evolution relative to non-avian dinosaurs. These results may be due to the reorganisation of skull structure in birds—including loss of a separate postorbital bone in adults and the emergence of new trade-offs with development and neurosensory demands. Taken together, the remarkable cranial shape diversity in birds was not a product of accelerated evolution from their non-avian relatives, despite their frequent portrayal as an icon of adaptive radiations. Using high-density morphometrics to quantify skull shape in birds and non-avian dinosaurs, this study reveals that the skull evolves at significantly slower rate in birds compared to other dinosaurs. The macroevolutionary history of the dinosaur skull reflects the mosaic functional, structural, and developmental changes in the skull across the origin of birds. Among tetrapods, extant birds exhibit incredible taxonomic and ecomorphological diversity, comprising over 10,000 extant species that occupy myriad niches on 7 continents . They poPrevious research comparing macroevolutionary patterns between avian and non-avian dinosaurs has focused on the post-cranial skeleton and/or body size ,20,22–24Fewer studies have focused on the tempo and mode of craniofacial evolution specifically, despite many of the key ‘avian’ features being localised to the skull . These studies have primarily been limited to the use of linear or two-dimensional (2D) morphometric data. These analyses support the idea that avian cranial diversity is aligned along a similar axis of variation as in non-avian dinosaurs, with similar trends in braincase to face proportions . HoweverAnalysis of avian cranial skeletal evolution using three-dimensional (3D) morphometric data has revealed a mosaic pattern of evolution . The comn = 354) and non-avialan dinosaurs (n = 36), as well as the Cretaceous ornithuran bird Hesperornis. Using semiautomated placement of surface semi-landmarks [n = 22 [n = 36 [n = 41 [n = 108 [Here, we test whether the origin of birds is marked with a distinct shift in cranial evolutionary dynamics. Specifically, we assess how the macroevolutionary patterns generating avian cranial diversity compares to those giving rise to the diversity of cranial morphology across Dinosauria as a whole. We address 2 main questions. First, did the same regions of the skull evolve at high rates and exhibit high disparity in non-avian dinosaurs as in birds? We hypothesise that because of the major differences in cranial anatomy, function, development, and phenotypic integration, non-avian and avian dinosaurs exhibit distinct patterns of regionalisation of variability within the skull. Second, do birds and non-avian dinosaurs evolve at different rates? Previous studies quantifying cranial form across dinosaurs and birds have relied on limited length measurements or a small set of 2D landmarks because of limited points of clear homology ,26–28. Tandmarks , we projandmarks . Becauseandmarks –37. Alth [n = 22 , n = 36  [n = 36 , n = 41 n = 28) and composed of 9 anatomical modules (n = 19). We then used modern comparative methods to estimate phenotypic rates of evolution across time, taxa, and cranial regions.Because of the variable completeness of the fossil specimens, we conducted all analyses concerning rate and disparity of a region  using all specimens that preserve that region or element. For analyses concerning rate and disparity across the entire skull, we performed tests using 2 subsets of the total sample: one consisting of those taxa that preserve the dorsal and lateral sides of the skull ( modules  and the mult) rate statistic [Under a variable-rates Brownian Motion (BM) model , all cratatistic  that assDiplodocus and Lambeosaurus, which convergently have distinct cranial configurations with posterodorsally positioned nasals and elongate premaxillae compared to their closest relatives in this analysis . Along the early avialan lineages (including Hesperornis), rates of rostrum evolution are slightly higher, but the rates along the branch leading to crown Aves and basal branches within the crown group are relatively slow compared to non-avian dinosaurs.For each skull region, variable-rates evolutionary models illustrate which subclades are driving the differences in rates between non-avian dinosaurs and birds . In the analysis –S4 Figs.Tyrannosaurus and Allosaurus, although these structures are related to muscle attachment rather than display [Ornithomimus, Struthiomimus, and Incisivosaurus , and theropods with fast-evolving parietals are tyrannosaurids with parietal  crests . As with the parietal, the squamosal generally evolved fastest in ornithischians and slowly in saurischians —evolves approximately 2.7 times faster than the slowest-evolving modules: the frontal and the lacrimal . This difference reflects the inclusion of a larger sample of ceratopsians in that dataset, which possess elaborate frills formed by the parietal and squamosal  the parietal and quadratojugal retain high evolutionary rates , as obseCitipati, Tyrannosaurus, Allosaurus, and Majungasaurus), the quadrate and quadratojugal are also very different from the average non-avian dinosaur skull shape. In ornithischians, the largest amount of phenotypic change is typically localised to the circumorbital bones and the cranial vault (Pawpawsaurus and Panoplosaurus) and Stegosaurus, like non-avian theropods, exhibit a large amount of phenotypic change in the premaxilla. Unlike theropods, however, the maxilla has low Procrustes distance from the mean in these taxa. Due to its prominent frill, the region with the highest magnitude of change in Protoceratops is the posterior parietal. Unexpectedly, the prosauropod Plateosaurus most closely resembles the pattern of cranial variation typical of Theropoda, with the greatest deviations from the average dinosaur skull observed in the face and jaw joint. Sauropods, in contrast, exhibit a different pattern. Camarasaurus is characterised by a large amount of phenotypic change in the vault, lacrimal, and ventrolateral margin of the skull. In contrast, Diplodocus, with its distinctive anteroposteriorly elongate face, has the ventral margin of the premaxilla, maxilla, and jugal as regions of greatest change.To further illustrate the evolutionary mosaicism across the cranial regions and taxa, we calculated the distance from the mean skull shape for each specimen in the 11-module dataset for each landmark . In non-al vault . NotablyThe patterns of cranial shape evolution exhibited by birds do not reflect those of the other dinosaur groups. Despite their ecomorphological diversification, birds display slower rates of evolution than non-avian dinosaurs in almost all cranial regions. The regions of the dinosaur skull that have experienced the highest rates of evolution and achieved the highest disparity differ across clades and largely reflect clade-specific anatomical specialisations, including elaborate skull ornamentations. Whereas the fastest evolving part of the avian skull is the anterior rostrum ,46, thisWe propose that these regions have experienced rapid evolutionary change in non-avian dinosaurs, but not birds, for several reasons. First, rapid evolution in the parietal and squamosal are attributed to the diverse cranial ornaments that occur across theropods and marginocephalians. Bony ornamental structures are uncommon in extant birds, with most cranial display in birds being achieved through soft tissue and feathers . Howeverm. adductor mandibulae externus profundus, medialis, and superficialis, jaw closing muscles that originate on the temporal fossa and temporal bar [pterygoideus ventralis muscle attaches to the jugal, further emphasising the importance of shape variation in this region for jaw function [Another possible driver of phenotypic variation and rapid evolutionary change in the vault in non-avian dinosaurs is the structure and function of the adductor chamber. The squamosal and parietal contribute to the borders of the superior temporal fenestra, through which several major jaw muscles pass. These include the oral bar . The shafunction .In non-avian dinosaurs, the quadrate exhibits the highest evolutionary rate in the 11-module dataset  in contrLambeosaurus or contributes to unusually positioned nares as in sauropods such as Diplodocus. However, rapid rostrum evolution in non-theropod dinosaurs is primarily driven by the premaxilla, which evolves slowly in most non-avian theropods but much faster in most ornithischians. The non-avian theropods that do achieve high rates of premaxilla evolution are those with edentulous premaxillae and/or rhamphotheca, including ornithomimosaurs and Citipati , starkly contrasts with previous studies comparing disparity and evolutionary rates in the postcranial skeleton of birds and non-avian dinosaurs. Birds have significantly greater disparity in limb proportions ,19 and hAs birds were experiencing high rates of evolution in the postcranial skeleton and using their newfound variation in limb morphology to exploit a variety of habitats, their cranial evolution had slowed down relative to other dinosaurs. Whether this is related to the biomechanical demands of aerial locomotion or encephalisation, developmental factors caused by reorganisation of the palate and vault, or phylogenetic inertia, birds failed to achieve the high rates of cranial evolution observed in non-avian dinosaurs. While the wings ,63, hindSampled taxa include 354 extant birds, 1 extinct bird, and 36 extinct non-avian dinosaurs. Extant taxa were selected to represent the breadth of cranial diversity and over 80% of extant families. Fossil specimens were selected on the quality of their preservation, with a mostly complete, articulated, and 3D skull. Repositories for each specimen, along with specimen ID numbers, are listed in Digital 3D models of specimens were generated via CT scanning and surface scanning. We digitised anatomical landmarks and semi-landmark curves on the surface of each specimen using IDAV Landmark , defininBecause of the fusion of cranial elements in birds, not all anatomical landmarks and semi-landmark curves can be digitised in all taxa. For example, the parietal, frontal, and squamosal are distinct elements in non-avian dinosaurs, but their borders are typically indistinguishable in adult birds. To enable comparison between birds and non-avian dinosaurs, we projected the same surface semi-landmarks onto birds and non-avian dinosaurs using two separate templates. The initial template with 16 anatomical regions was generated based on the bones and sutures present in non-avian dinosaurs . We thenEven though we selected only the most complete fossil skulls, only 9 specimens preserved every element of interest. We analysed 3 subsets in order to maximise taxonomic and anatomical breath:n = 27); this dataset includes maxilla, premaxilla, nasal, frontal, parietal, squamosal, jugal + quadratojugal, postorbital, and lacrimal + prefrontal and is referred to hereafter as the “9-module dataset”The non-avian dinosaur specimens that adequately preserve lateral and dorsal sides of the skull (n = 19), referred to hereafter as the 11-module datasetThe non-avian dinosaur specimens that preserve all the regions in the 9-module dataset, as well as the occipital region and the articular surface of the quadrate , palate , cranial vault , occipital region, basisphenoid, and quadrate + pterygoid . We alsoThe major phylogenetic relationships among dinosaurs are currently debated. The traditional view on dinosaur relationships has split the group into 2 major groups, Saurischia (Theropoda and Sauropoda) and Ornithischia. However, some recent analyses have supported an alternative hypothesis, with Theropoda and Ornithischia forming a monophyletic clade (Ornithoscelida) that is sister to Saurischia + Herrerasauridae ,70. As sDiplodocus occupies a region or morphospace closer to birds, unlike theropod taxa that are more closely related to birds. This is a result of the elongate face and more posteriorly positioned nares in Diplodocus superficially resembling birds. Similarly, the highly domed cranial vault of pachycephalosaurs generates similarities to the highly encephalised birds.We generated morphospace plots  to inveshttp://www.evolution.rdg.ac.uk/). Preliminary BayesTraits analyses confirmed that variable-rates models [We quantified rates of skull shape evolution across Dinosauria using BayesTraitsV3 (s models  were favs models . Becauses models . The runhttp://github.com/hferg/BTRTools). We then extracted the subtrees for birds, non-avian theropods, and non-theropod dinosaurs and calculated the mean rate scalar for that subtree, then divided this value by total subtree height to produce time-corrected mean rate scalar (t tests (Wilcoxon tests).To compare evolutionary rates between birds and other dinosaur groups, we calculated the mean rate scalar per branch for each group and each module . For eace scalar . To testBecause the number of bird species is much greater than the number of non-avian dinosaurs, we performed a subsampling analysis to confirm that the rate of evolution in each group was robust to sample size and to the effects of individual branches with extremely high or low rates. We randomly selected one species per avian order and removed all other birds from the dataset, resulting in 40 extant birds and up to 37 extinct dinosaurs. Using this subsampled dataset, we generated a new phylogenetic principal components analysis and again modelled variable-rate evolution using BayesTraits with the same parameters as with the full dataset. This procedure was repeated for 100 iterations for each skull region. To summarise these results, we extracted the mean branch rate scalars from the posterior distribution from each iteration . BecauseAs noted earlier, separate Procrustes alignments for each module were applied so that every specimen that preserved each structure could be used, regardless of the preservation of the rest of the skull. Therefore, only the shape of each structure was considered and not its relative position in the skull. However, for some structures , position was hypothesised to be a significant aspect of variation. To quantify this, we also estimated variable-rates models on the rostrum, vault, occipital region, and quadrate with a global Procrustes alignment for all of these regions , thus camult metric implemented in the “geomorph” R package [mult tests with these data are robust to this violation of the assumptions of the method, we calculated per-group rates for each region using equal rates with σmult . The relative branch lengths in these topologies reveals that the MBL-dated tree reconstructs shorter branch lengths at the root of Coelurosauria, Maniraptora, and Ornithomimosauria compared to the FBD-dated tree, resulting in faster rates of evolution in non-avian theropods using the FBD-dated tree than in the MBL-dated tree. Taken together, the consistency between the results of clade-wise comparisons of rates with σmult and BayesTraits variable-rates models demonstrates that these findings are robust to model choice and method.Whereas these evolutionary models were estimated using phylogenetic principal components scores of shape, we also sought to analyses rates of evolution from the landmark configurations directly. This is possible using the σ package ,78. Thisth σmult  and compth σmult . Using tth σmult . As singth σmult  for bothth σmult . We alsotic tree  and globtic tree . Again, mult for each landmark in the 9-module and 11-module datasets using the functions “per_lm_rates” and “per_lm_variance” in the R package “hot.dots” [We further visualised fine-scale regional variation in evolutionary rates by quantifying σot.dots” . This isot.dots” . As withot.dots” . For eact test: t = −4.3918, df = 73.732, p = 3.6 × 10−5). However, when the avian phylogeny is subsampled to include the same number of taxa as our sampling of non-avian dinosaurs, the branch lengths are not significantly different between these two groups . Because the results of σmult analyses with and without subsampling are largely congruent, these findings imply that branch length distributions are not skewing rate calculations. To test whether the evolutionary rates estimated using BayesTraits were biased by the estimated branch lengths, we regressed log-transformed mean rate from variable-rates BM model onto the lengths of each branch in the 3 phylogenetic hypotheses , further supporting the conclusion that rate heterogeneity is not primarily driven by differences in branch lengths.To validate our results, we performed 2 post hoc tests to examine whether rate estimations is strongly influenced by artefacts related to sampling and tip-dating strategies. First, we tested whether the bird and non-avian dinosaurs show different distributions of branch lengths in the time-calibrated phylogeny, as disproportionately long branches could artificially decrease mean rates. With our interspecific sampling, birds indeed have shorter branch lengths than non-avian dinosaurs . Data and code archived at www.github.com/rnfelice/Dinosaur_Skulls.Anatomical landmarks and semi-landmark curves were digitised onto 3D surface meshes, and surface semi-landmarks were projected onto the skull using the Morpho R package . Landmark configurations for dinosaurs were partitioned into a total of 16 anatomical regions . Non-avian dinosaurs were compared to non-avian dinosaurs by re-partitioning landmarks into regions that can be distinguished in both birds and non-avian dinosaurs . Landmark configuration illustrated on (PDF)Click here for additional data file.S2 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S3 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S4 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S5 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S6 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S7 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S8 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S9 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S10 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S11 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S12 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S13 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S14 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S15 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S16 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S17 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S18 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S19 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S20 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S21 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S22 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S23 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S24 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S25 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S26 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S27 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S28 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S29 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S30 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S31 Figwww.github.com/rnfelice/Dinosaur_Skulls.Estimation of rates of jugal and quadratojugal bone evolution. Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S32 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S33 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S34 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S35 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S36 Figwww.github.com/rnfelice/Dinosaur_Skulls.Modelled using the (A) traditional phylogenetic hypothesis and (B) Ornithoscelida hypothesis under a variable-rates BM model of evolution. Data and code archived at (PDF)Click here for additional data file.S37 Figt tests; significantly different distributions are indicated with ****p < 0.00005. Data and code archived at www.github.com/rnfelice/Dinosaur_Skulls.Birds were randomly subsampled to 1 species per extant order for 100 iterations. Subsampled data were used to estimate variable-rates BM models, and then mean rate was calculated for each group and for each iteration and scaled by dividing by the sum of the branch lengths in the corresponding time-scaled subtree. Models used the traditional Dinosauria phylogenetic topology (Saurischia and Ornithischia as sister clades). Mean rate scalars were compared between groups modelled using nonparametric (PDF)Click here for additional data file.S38 Figt tests; significantly different distributions are indicated with ****p < 0.00005. Data and code archived at www.github.com/rnfelice/Dinosaur_Skulls.Mean rate scalar was calculated from the mean of the rate scalars for the indicated group in the post-burn-in posterior distribution of the variable-rates evolutionary model estimated modelled using BayesTraits. Mean rates were scaled by dividing by the sum of the branch lengths in the corresponding time-scaled subtree. Models used the traditional Dinosauria phylogenetic topology (Saurischia and Ornithischia as sister clades). Mean rate scalars were compared between groups Modelled using nonparametric (PDF)Click here for additional data file.S39 Figmult metric [t tests; significantly different distributions are indicated with ****p < 0.00005. Data and code archived at www.github.com/rnfelice/Dinosaur_Skulls.Birds do not have the highest rates of evolution in any cranial region. Rate of evolution was calculated using the σt metric . Because(PDF)Click here for additional data file.S40 Figmult metric [t tests; significantly different distributions are indicated with ****p < 0.00005). Data and code archived at www.github.com/rnfelice/Dinosaur_Skulls.Birds do not have the highest rates of evolution in any cranial region. Rate of evolution was calculated using the σt metric . Because(PDF)Click here for additional data file.S41 Figmult metric [t tests; significantly different distributions are indicated with ****p < 0.00005). Data and code archived at www.github.com/rnfelice/Dinosaur_Skulls.Rate of evolution was calculated using the σt metric . In addi(PDF)Click here for additional data file.S42 Figt tests; significantly different distributions are indicated with ****p < 0.00005. Data and code archived at www.github.com/rnfelice/Dinosaur_Skulls.Rates calculated using the FBD-dated phylogenetic hypothesis. The high rates of evolution in the non-avian dinosaur quadrate observed in (PDF)Click here for additional data file.S43 Figwww.github.com/rnfelice/Dinosaur_Skulls.Bivariate plots of disparity (Procrustes variance), mean evolutionary rate, and within-partition trait correlation (integration). Values were calculated for the 9-module dataset (A–C) and the 11-module dataset (D–F). Disparity is corrected by the number of landmarks and sliding semi-landmarks in the region. Within-module correlation values derived from ref . Rates c(PDF)Click here for additional data file.S44 Figwww.github.com/rnfelice/Dinosaur_Skulls.Calculated using the morphol.disparity function in the geomorph R package. Each cranial region except the quadrate is less disparate in birds than in non-avian groups. Data and code archived at (PDF)Click here for additional data file.S45 Figwww.github.com/rnfelice/Dinosaur_Skulls.The extant bird portion of the phylogeny has shorter branch lengths due to denser sampling, but this effect is removed by the subsampling procedure. Data and code archived at (PDF)Click here for additional data file.S46 FigR2 = 0.003, p = 0.07). Data and code archived at www.github.com/rnfelice/Dinosaur_Skulls.Linear regression reveals that the relationship between rate and branch length is not significant ((PDF)Click here for additional data file.S47 FigDiplodocus, with its elongate rostrum and posteriorly positioned nares, is convergent with birds with high PC axis 1 and moderate PC axis 2 scores. The domed skulls of pachycephalosaurs cause them to group with birds with high negative PC 1 scores Data and code archived at www.github.com/rnfelice/Dinosaur_Skulls. PC, principal componentThe first 4 principal components axes explain a cumulative 66% of the total variance. Grey silhouettes represent the reconstructed shape at the corresponding region of morphospace. Birds and non-avian dinosaurs largely occupy distinct regions of morphospace. (PDF)Click here for additional data file.S1 TableCalculated with the full dataset using the traditional dinosaur phylogenetic topology dated with the MBL method. Shaded cells indicate significantly faster rate than birds.(PDF)Click here for additional data file.S2 TableCalculated using the morphol.disparity function in the geomorph R package.(PDF)Click here for additional data file.S3 TableCalculated using the morphol.disparity function in the geomorph R package.(PDF)Click here for additional data file.S4 TableSpecimen number and museum collection data for all sampled taxa.(XLSX)Click here for additional data file.S5 TableAnatomical descriptions of landmark locations.(DOCX)Click here for additional data file.


Actinidia chinensis) when CPPU was sprayed on fruit 25 days after anthesis. The CPPU application did not significantly influence the dry matter, soluble solids, starch, vitamin C or protein concentrations. However, the fresh weight, length and maximum diameter were significantly increased compared with the control. Moreover, in fruit of the same developmental stage, the fructose, glucose and soluble sugar levels increased after the CPPU treatment, compared with the control. On the contrary, citric, quinic and titratable acid concentrations decreased. However, a correlation analysis between fresh weight and the nutritional contents revealed that CPPU did not affect the concentrations of the most abundant organic acids (quinic and citric) and sugars , compared with control fruit of the same weight. Therefore, CPPU applications enhance ‘Hongyang’ kiwifruit weight/size. However, there were no significant differences in the nutritional qualities of treated and untreated fruit having the same weights.Forchlorfenuron, N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU), is often used to promote fruit growth and improve production. The role of CPPU in kiwifruit growth has been established. However, the correlation between fruit weight and nutritional metabolism during development after CPPU treatments remains largely undetermined. Here, we surveyed the variations in weight and nutrient components of the ‘Hongyang’ kiwifruit ( Actinidia deliciosa (A. Chev.) C.F. Liang and A.R. Ferguson.) bred in New Zealand, was the first globally traded kiwifruit cultivar. Subsequently, the yellow-fleshed ‘Hort16A’ (A. chinensis Planch. var. chinensis) cultivar was selected and stimulated the development of the world kiwifruit industry. The success of ‘Hort16A’ kiwifruit is ascribed largely to the tropical-like flavors and sweeter yellow-flesh compared with ‘Hayward’ kiwifruit (A. chinensis Planch. var. chinensis), cultivated by the Sichuan Provincial Nature Resources Institute, is the first red-fleshed kiwifruit cultivar to be grown on a commercial scale  Planch. ex Miq. ‘Mitsuko’ were lower than those of untreated fruit . The grafted kiwifruit vines were trained by a Pergola system with 3-m spacings within rows and 4-m spacings between rows. In total, 40 representative vines were randomly selected and labeled for the subsequent experiments. At 25 days after anthesis (DAA), half of the vines were treated with 5 ppm CPPU  for 10 sFruit samples were collected from three biological replicates, snap-frozen in liquid nitrogen, and then stored at −80 °C for subsequent analyses.Fruit fresh weight and dry matter were measured as reported by Total soluble sugars was extracted using the method of 4H2PO4  and centrifuged at 12,000×g at 4 °C for 15 min. The supernatant was collected and subsequently filtered. An HPLC analysis was performed using an Agilent 1,200 Series instrument with a C18 column  at 210 nm. The mobile phase was NH4H2PO4 . The sugar concentration was calculated as described in Organic acids were detected according to p < 0.05. In the early stage of data collection, each sample was repeated three times. In the analysis process, taking time as a factor, whether the fruit was treated was correlated with the change in nutritional index content within equal time periods was considered. The software R 3.0.0  was used to make comparisons among groups. A difference was considered statistically significant at  R 3.0.0  and SPSSA. chinensis ‘Hongyang’ fruit weight and size at harvest -N-phenylurea applications significantly increased fructose and glucose concentrations. Differences in fructose levels between treated and untreated fruit were first detectable at 70 DAA and continued until harvest. In addition, from 85 to 130 DAA, the glucose concentration of the CPPU-treated fruit was much greater than that of the untreated fruit. By harvest time, the CPPU applications had significantly increased the fructose and glucose concentrations in fruit by 75.2% and 43.1%, respectively, compared with untreated fruit  and 4B. The quinic acid concentration of fruit declined after CPPU applications. There was a difference in the quinic acid concentration at 70 DAA until final harvest. Finally, the CPPU application caused a decrease of ~19.5% in quinic acid compared with the untreated fruit . The citR2 > 0.8). For sugars, there were no significant differences in the slope and intercept between CPPU-treated and control fruit. Using the same statistical analysis method, quinic acid and citric acid had values greater than 0.05 for both the slopes and intercepts.For the fruit sample data, the log function was used to linearize the glucose concentration distribution and a linear regression equations of this fruit nutrient as well as the others and weight were obtained. The regression results are shown in Fruit weight/size and dry matter are important integrated indices used to evaluate fruit quality . In thisThe fruit length and diameter define the shape of the fruit. CPPU applications promote cell division and/or expansion to increase fruit size . The lenSoluble solids concentration is a crucial factor for evaluating the fruit ripening time . No signSugars and organic acids are the parameters of greatest concern regarding the flavor of fruit . CompareA. argute kiwifruit was less, than that of untreated fruit (The vitamin C concentration is a preferential factor for the assessment of the quality levels of many fruit. Kiwifruit contains more ascorbic acid than oranges, strawberries, lemons and grapefruit . Here, ted fruit .The changes that occur in protein levels during kiwifruit development are unknown. In the present study, the protein concentration showed similar values between CPPU-treated and untreated fruit . Qian etActinidia deliciosa, while the starch content was similar to the control at harvest (The accumulation of starch was similar to that of the ‘Hayward’ kiwifruit . However harvest .The most abundant sugars in ‘Hongyang’ kiwifruit are glucose, fructose and sucrose. Citric, quinic and malic acids are the main organic acids . CompareAccording to the regression analysis between weight and major organic acid/sugar concentrations , there wA CPPU application significantly altered ‘Hongyang’ kiwifruit development, leading to increases in fresh weight and sugar concentrations, as well as a decline in organic acid concentrations at the same developmental stage as the untreated kiwifruit. However, the impacts on ‘Hongyang’ were lesser compared with other kiwifruit cultivars treated with CPPU. In contrast, from the regression analysis between weight and nutritional indices, the CPPU treatment did not affect major organic acid and sugar concentrations considering a constant fruit weight. These results indicated that CPPU treatments have the potential to increase ‘Hongyang’ kiwifruit production with limited negative effects on quality.10.7717/peerj.9724/supp-1Supplemental Information 1The HY represents untreated fruit, the HYP represents CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-2Supplemental Information 2The HY represents untreated fruit, the HYP represents CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-3Supplemental Information 3The raw data of dry matter. The HY represent untreated fruit, the HYP represent CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-4Supplemental Information 4The HY represents untreated fruit, the HYP represents CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-5Supplemental Information 5The HY represents untreated fruit, the HYP represents CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-6Supplemental Information 6The raw data of titratable acid. The HY represent untreated fruit, the HYP represent CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-7Supplemental Information 7The HY represents untreated fruit, the HYP represents CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-8Supplemental Information 8The HY represents untreated fruit, the HYP represents CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-9Supplemental Information 9The HY represents untreated fruit, the HYP represents CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-10Supplemental Information 10The HY represents untreated fruit, the HYP represents CPPU-treated fruit.Click here for additional data file.10.7717/peerj.9724/supp-11Supplemental Information 11The HY represents untreated fruit, the HYP represents CPPU-treated fruit.Click here for additional data file.


Bactrocera dorsalis is a destructive polyphagous and highly invasive insect pest of tropical and subtropical species of fruit and vegetable crops. The sterile insect technique (SIT) has been used for decades to control insect pests of agricultural, veterinary, and human health importance. Irradiation of pupae in SIT can reduce the ecological fitness of the sterile insects. Our previous study has shown that a gut bacterial strain BD177 that could restore ecological fitness by promoting host food intake and metabolic activities.K. michiganensis BD177 strain. The complete genome of K. michiganensis BD177 comprises one circular chromosome and four plasmids with a GC content of 55.03%. The pan-genome analysis was performed on 119 genomes (strain BD177 genome and 118 out of 128 published Klebsiella sp. genomes since ten were discarded). The pan-genome includes a total of 49305 gene clusters, a small number of 858 core genes, and a high number of accessory (10566) genes. Pan-genome and average nucleotide identity (ANI) analysis showed that BD177 is more similar to the type strain K. michiganensis DSM2544, while away from the type strain K. oxytoca ATCC13182. Comparative genome analysis with 21 K. oxytoca and 12 K. michiganensis strains, identified 213 unique genes, several of them related to amino acid metabolism, metabolism of cofactors and vitamins, and xenobiotics biodegradation and metabolism in BD177 genome.Using long-read sequence technologies, we assembled the complete genome of K. michiganensis. Comparative genome analysis suggested that K. michiganensis BD177 has the strain-specific ability to provide three essential amino acids  and two vitamins B (folate and riboflavin) to B. dorsalis. The clear classification status of BD177 strain and identification of unique genetic characteristics may contribute to expanding our understanding of the symbiotic relationship of gut microbiota and B. dorsalis.Phylogenomics analysis reclassified strain BD177 as a member of the species The online version contains supplementary material available at 10.1186/s12863-020-00945-0. Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is a destructive polyphagous and highly invasive insect pest of tropical and subtropical species of fruit and vegetable crops. While insecticides have been used to control this pest, insecticide resistance, and environmental pollution by chemical pesticides have been severely limiting this type of control [B. dorsalis has a powerful biological invasion ability. It is its invasion, spread, and establishment in sub-Saharan Africa and has caused about $2 billion in economic losses in the horticultural export markets of Africa [B. dorsalis into Central China, without any apparent intense selective pressure, must pose a deep concern for other temperate regions of the world, especially Europe and North America [ control . Moreove America .B. dorsalis. Previously, SIT has been used to control pest fruit fly species, including Ceratitis capitata [B. tryoni [B. cucurbitae [B. dorsalis [The sterile insect technique (SIT) has been used for decades to control insect pests of agricultural, veterinary and human health importance , 5. Compcapitata , B. tryo. tryoni , B. cucucurbitae  and B. ddorsalis . Howeverdorsalis . These pdorsalis , 13. Thudorsalis , 14.Gut microbiota is strongly connected with the biology of the host and contributes to its health . Gut micB. dorsalis has been investigated [B. dorsalis populations and different developmental stages from laboratory-reared and field-collected samples [B. dorsalis fitness. The here presented study aims to elucidate the genomic basis of strain BD177 its beneficial impacts on the sterile males of B. dorsalis. An insight into strain BD177 genome feature helps us make better use of the probiotics or manipulation of the gut microbiota as an important strategy to improve the production of high performing B. dorsalis in SIT programs.Gut symbiotic bacteria community of stigated , 27, 29. samples . We succ samples . HoweverK.michiganensis BD177 was sequenced using the Illumina HiSeq and Pacbio technology. Raw reads (~ 1.3 Gbp) were processed to remove SMRT bell adapters, short and low-quality reads (< 80% accuracy) using SMRT Analysis version 2.3 . A total of 155,828 filtered reads  were used for de novo assembly using Celera Assembler [K.michiganensis BD177 complete genome of 6,812,698 bp with GC content 55.03% in a single chromosome and four plasmids. The annotated genome contains 6714 genes, 25rRNA genes, and 86 tRNA genes  among all sequences. Biochemical tests were performed on BD177 by API20E to confirm its position in the K. oxytoca and K. michiganensis. Biochemical characteristics of K. oxytoca ATCC13182(T)(=NCTC13727) and K. michiganensis W14(T)(= DSM2544) were obtained from BacDive Webservices [K. oxytoca ATCC13182(T) and K. michiganensis W14(T), BD177 is the same with W14 in the β-galactosidase enzyme positive and urease negative, same with ATCC13182(T) in arginine dihydrolase positive. However, BD177 does not have the utilization ability of citrate as only carbon source, different from both type strains  by quast and checkm  and a small group of two genomes (57.02 and 57.16%) showing a much greater GC content, similar to that of the K. pneumoniae, K.variicola and K. quasipneumoniae genomes . Based on GC content of the genome, K. pneumoniae, K.variicola, K. quasivariicola, and K. quasipneumoniae were considered as a high GC group content, and K. michiganensis and K. oxytoca were considered as a low GC content group. The genome sequence of our new isolate, which we designated K. michiganensis BD177 with 55.03% GC content  that are distinct from the other clades  of genome-wide comparison has been recommended as a standard method to improve the accuracy of taxonomic identification of prokaryotic genomes . Figure Klebsiella sp. strains, we focused on the genomic relatedness of K. michiganensis BD177, 21 K. oxytoca, and 12 K. michiganensis strains. In total, 201,774 genes were present in their genomes, with an average number of 5934 genes per genome. These genes were clustered into 9643 orthogroups by OrthoFinder [K. michiganensis BD177, 213 strain-specific orthogroups were identified. Mapping of these orthogroups to the eggNOG database (v4.5) [K. michiganensis BD177 were also investigated by cumulatively mapping the unique functional KEGG genes of the K. michiganensis BD177 to the KEGG pathways   revealed.5  reveaKlebsiella sp. strains are among the dominant symbiotic bacteria in the gut of Tephritidae, with the ability to play diverse roles [K. michichanensis BD177, isolated from wild adult B. dorsalis. We found that the taxonomic status of the B. dorsalis gut bacterial strain BD177 could not be identified only based on the 16S rRNA gene phylogenetic tree and biochemical analysis of the strain. Hence, we used the Illumina HiSeq and Pacbio technology to assemble a complete 6.8 Mbp length genome of K. michiganensis BD177. Using the genome-wide information, we identified the taxonomic status and strain-specific genome features of the BD177 strain to explore the symbiotic relationship with B. dorsalis.se roles . In thisK. oxytoca and K. michiganensis  differences in whole-genome GC content  and a high GC content group . Strain BD177 with 55.03% GC content belongs to the low GC genome group. The GC content of complex microbial communities seems to be globally and actively influenced by the environment. Similar environments tend to have similar GC-content patterns [Klebsiella sp. strains. The K. michiganensis group, including type strain K. michiganensis DSM25444, have the unique gene cluster 2. K. oxytoca group, including type strain K. oxytoca NCTC13727, has the unique cluster 1. Non-type strains K. michiganensis and K. oxytoca are distinguishable based on genes cluster 1 and cluster 2 from the pan-genome analysis. Whole-genome sequence data as a basis for taxonomic assignment display greater discriminatory power than 16S rRNA gene sequence analysis alone [K. michiganensis and K. oxytoca similarity, BD177 belongs to K. michiganensis species rather than K. oxytoca species  and folate (B9). In humans, gut microbiota can synthesize and supply vitamins B to their hosts, which lack the biosynthetic capacity for most vitamins [Drosophila microbiota in supplying folate [K. michiganensis BD177 suggests these B-vitamins may be of particular importance, especially in adult life stages fed on undernourished nectar and dew [D. melanogaster, Acetobacter pomorum provides thiamine to its host to promote larval development [Wigglesworthia glossinidia plays a role in Glossina morsitans maturation and reproduction [K. michiganensis BD177 improved the mating competitiveness and lifespan of sterile male B. dorsalis [K. oxytoca could affect the foraging decision [B. dorsalis. It is suggested that riboflavin and folate synthetic ability of K. michiganensis BD177 may contribute to the sexual performance and lifespan of B. dorsalis.Additionally, vitamins . Recent g folate , riboflag folate  and thiag folate . The rib and dew . In D. melopment . Folate oduction . Our predorsalis . Additiodecision  and matedecision  of B. doKlebsiella sp. genomes, some strain-specific genes from xenobiotics biodegradation and metabolism pathway were also identified in the K. michiganensis BD177 genome. Recent research shows that gut bacteria of insects significantly contribute to resistance against xenobiotics, including phytotoxins and pesticides [Pseudomonas fulva can assist with digestion and detoxification of caffeine as alkaloid allelochemical in the coffee berry borer [Burkholderia of Riptortus pedestrians has gained the ability to hydrolyze insecticide fenitrothion [Citrobacter sp. can degrade trichlorphon and conferred host insecticide resistance in B. dorsalis [Lactobacillus plantarum of D. melanogaster can significantly enhance the toxicity of insecticide chlorpyrifos [K. michiganensis BD177 may play a role in insect resistance against pesticides in the wild field. However, the potential mechanisms of resistance or sensitivity of insect to insecticide are unclear.Compared with other 33 sticides . Pseudomry borer . Gut symtrothion . Gut symdorsalis . Interesrpyrifos . It is hK. michiganensis BD177 strain. The comparison of the genome sequence against other Klebsiella species showed a percentage of genes that are unique to the BD177 strain, including metabolic key enzymes of amino acid, vitamins, and xenobiotics that could play an important role in the resistance and fitness of B. dorsalis. These findings extend our previous work [B. dorsalis. In the future, we can engineer the gut bacteria strain symbionts by strengthening the probiotic genetic elements of bacteria. It will improve the application efficiency of gut microbiota in pest management programs incorporating SIT.Supplement of gut symbiotic bacteria as probiotic to the larval or adult diet are encouraging for their potential application in SIT programs to produce high-quality insects. However, understanding of the bacterial probiotic mechanism is important for the selection and application of different insect species probiotics. Using long-read sequence technologies, we assembled the complete genome of ous work  to improKlebsiella michiganensis BD177 reported in this study was isolated in a previous study from the gut of B. dorsalis male adult, collected from the Institute of Urban and Horticultural Pests of Huazhong Agricultural University [Klebsiella michiganensis type strain W14 and Klebsiella oxytoca type strain JCM 1665 was 99.25 and 99.15%, respectively. In addition, 16S rRNA gene sequences longer than 1300 nucleotides of type strains K. oxytoca, K. michiganensis, K. pneumonia, K. quasipneumoniae, K. aerogenes, Klebsiella variicola, and Pseudomonas aeruginosa were downloaded from EzBioCoud database [The iversity . The baciversity . Subsequiversity . The simdatabase . Alignmedatabase . A neighdatabase , using tdatabase .K. michiganensis BD177 strain was grown on LB, with incubation at 37 °C in aerobic conditions. For DNA extraction, the strain was grown in 8 ml of medium for overnight, followed by pelleting at 6000 g for 10 min, and genomic DNA was obtained using E.Z.N.A.® Bacterial DNA Kit (OMEGA). The bacterial DNA extraction was followed by quantity and quality estimation using a NanoDrop (Thermo Scientific) and also visualization of aliquots onto a 1.2% agarose gel stained with ethidium bromide to verify DNA integrity. Whole-genome sequencing of Klebsiella michiganensis BD177 was performed on the PacBio RS II platform and Illumina HiSeq 4000 platform at the Beijing Genomics Institute . For Illumina sequencing, genomic DNA was sheared randomly to construct three read libraries with lengths 500 bp by a Bioruptor ultrasonicator  and physicochemical methods. DNA was sequenced on a HiSeq sequencer (Illumina) with pair-end 125 bp reads. Low-quality trimming and adapter removal for the Illumina reads was performed using Trimmomatic [https://github.com/PacificBiosciences/pbdagcon) was used for self-correction. Then these reads were filtered using the RS_Subreads protocol , resulting in a total of 1394 Mb useable data .mmomatic , resultihttps://www.broadinstitute.org/gatk/), and SOAP tool packages  were used to make single-base corrections [Draft genomic unitigs, which are uncontested groups of fragments, were assembled using the Celera Assembler  against rections . To tracrections  to the brections .K. michiganensis BD177 genome assembly by glimmer3 [Gene prediction was performed on the glimmer3  with Hidglimmer3 , RNAmmerglimmer3  and the glimmer3 . The tanglimmer3 , and theglimmer3  with Islglimmer3  and CRISglimmer3 .Seven databases, which are KEGG (Kyoto Encyclopedia of Genes and Genomes) , COG (ClK. oxytoca and K. michiganensis were downloaded from the NCBI database on 19 March 2019 with NCBI-Genome-Download scripts (https://github.com/kblin/ncbi-genome-download). Genomic assemblies of K. pneumonia, K. quasipneumoniae, K. quasivariicola, K. aerogenes, and Klebsiella variicola type strains also were manually obtained from the NCBI database. The quality of the genomic assemblies was evaluated by QUAST [Klebsiella strains was inferred with Roary [Klebsiella genomes was constructed with FastTree [All complete genomic assemblies classified as by QUAST  and Checby QUAST . Genomesby QUAST . All paiby QUAST . To avoiby QUAST . The panth Roary . The genth Roary . The phyFastTree  using thKlebsiella sp. (K. michiganensis and K. oxytoca) genome assemblies were inferred with OrthoFinder [Orthogroups of BD177 and 33 hoFinder . All prohoFinder  all-agaihoFinder . KEGG pahoFinder .Additional file 1 Figure S1. Phylogenetic tree based on 16S rRNA gene sequences indicating the relationship between isolate BD177 with other type strains of the family Enterobacteriaceae, constructed using MUSCLE and MEGAX. (a) A maximum likelihood phylogenetic tree based on 16S rRNA gene sequences was assessed using bootstrap with 1000 replicates. Pseudomonas aeruginosa JCM5962 was selected as the outgroup. (b) A maximum parsimony phylogenetic tree based on 16S rRNA gene sequences was assessed using bootstrap with 1000 replicates.Additional file 2 Table S1. Distance matrix of evolutionary divergence between 16 s rRNA gene sequences. This distance matrix was obtained by a bootstrap procedure (1000 replicates) and the Kimura 2-parameter model.Additional file 3 Table S2. RefSeq assembly accession numbers and strain name of the 128 Klebsiella sp. genomes from the NCBI database used in this study.Additional file 4 Table S3. Overview of the various metrics employed to evaluate strain BD177 and 128 Klebsiella sp. genomes using QUAST and CheckM.Additional file 5 Fig. S2. Diagram of conserved genes per number of genomes from the pan-genome analysis by Roary.Additional file 6 Table S4. ANI values (%) for each pairwise genome comparison of 119 genomes passed strict quality control.Additional file 7 Table S5. General statistics about orthogroup sizes and the proportion of genes assigned to orthogroups by OrthoFinder.Additional file 8 Table S6. Annotate results of the unique orthogroups of BD177 from comparative genomics by eggNOGmapper.


Staphylococcus aureus demonstrates considerable membrane lipid plasticity in response to different growth environments, which is of potential relevance to response and resistance to various antimicrobial agents. This information is not available for various species of coagulase-negative staphylococci, which are common skin inhabitants, can be significant human pathogens, and are resistant to multiple antibiotics. We determined the total fatty acid compositions of Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, and Staphylococcus aureus for comparison purposes. Different proportions of branched-chain and straight-chain fatty acids were observed amongst the different species. However, growth in cation-supplemented Mueller–Hinton broth significantly increased the proportion of branched-chain fatty acids, and membrane fluidities as measured by fluorescence anisotropy. Cation-supplemented Mueller–Hinton broth is used for routine determination of antimicrobial susceptibilities. Growth in serum led to significant increases in straight-chain unsaturated fatty acids in the total fatty acid profiles, and decreases in branched-chain fatty acids. This indicates preformed fatty acids can replace biosynthesized fatty acids in the glycerolipids of coagulase-negative staphylococci, and indicates that bacterial fatty acid biosynthesis system II may not be a good target for antimicrobial agents in these organisms. Even though the different species are expected to be exposed to skin antimicrobial fatty acids, they were susceptible to the major skin antimicrobial fatty acid sapienic acid (C16:1Δ6). Certain species were not susceptible to linoleic acid , but no obvious relationship to fatty acid composition could be discerned. Staphylococcus are divided into two major groups based upon their ability to coagulate plasma. By far the best-known coagulase-positive species is Staphylococcus aureus, which is a virulent pathogen that is often resistant to multiple antibiotics, including β-lactam antibiotics, often through presence of the mecA determinant. In contrast the coagulase-negative staphylococci (CONS) are a large heterogeneous group of fifty-three validly described species, as of 2019 [Species of members of the genus S. aureus infections given resistance to multiple anti-staphylococcal agents exhibited by the organism [S. aureus cytoplasmic membrane plays an important role in the susceptibility and resistance to various antimicrobial agents of the bacterium. The cytoplasmic membrane of S. aureus is composed of the phospholipids phosphatidyl glycerol, lysyl-phosphatidyl glycerol and cardiolipin, and the glycolipids monoglucosyldiglyceride and diglucosyldiglyceride [S. aureus, namely iso fatty acids where the methyl branch is on the penultimate carbon of the fatty acid chain, and anteiso fatty acids where the methyl branch is on the antepenultimate carbon. Even-numbered iso fatty acids, odd-numbered iso fatty acids, and odd-numbered anteiso fatty acids are biosynthesized from the branched-chain amino acids valine, leucine, and isoleucine, respectively [S. aureus membrane lipid component is the golden yellow carotenoid known as staphyloxanthin [S. aureus susceptibility to various antimicrobial peptides and fatty acids [S. aureus strains [Novel antimicrobial agents are needed for the therapy of organism . The S. lyceride ,5. Decrelyceride . The fatlyceride ,8. Thereectively . BCFAs iectively . Anteisoectively . The groectively . Interesectively . The othoxanthin . Staphylty acids ,14. For  strains . In a ca strains .S. aureus BCFAs and SCFAs are biosynthesized by the fatty acid biosynthesis system II (FASII) [S. aureus grown in laboratory media, characterized by a mixture of BCFAs and SCFAs, is unlikely to mimic that of the organism growing in vivo in an infection. S. aureus has the ability, like many pathogenic bacteria, to utilize preformed host fatty acids, typically straight-chain unsaturated fatty acids (SCUFAs) and SCFAs, in what is thought to be a means of saving energy and carbon that would otherwise be devoted to fatty acid biosynthesis [S. aureus is grown in the presence of complex host biological materials such as serum [sn-2 portion of S. aureus glycerolipids, and hence a requirement for operation of the FASII system when S. aureus is growing in the host. However, demonstration of phosphatidyl glycerol species with no biosynthesized fatty acids on either the sn-1 or sn-2 position of the glycerol moiety seriously undermines the viability of agents targeting the FASII system [In laboratory media, the  (FASII) . This pa (FASII) . Howeverynthesis ,18. Thusas serum , or low-as serum , BCFAs das serum . It has I system ,22.S. aureus can incorporate free fatty acids into lipids from media supplemented with them [Although ith them ,23, the ith them ,25,26. Fith them ,28. LittS. epidermidis is prevalent on moist areas and is the most prevalent staphylococcal species on skin. It was cultured from the nose of 97% of people in a recent study [S. haemolyticus and S. hominis are associated with areas high in apocrine glands. S. capitis is isolated from the forehead and scalp. S. saprophyticus colonizes the genitourinary tract and rectum, and is a common cause of urinary tract infection in women [S. auricularis is uniquely found in the external ear canal [The CONS are a heterogeneous group of organisms colonizing the skin and mucus membranes of human and other animal hosts. Based upon sequences of four loci, Lamers et al.  placed snt study . S. haemin women . S. auriar canal . As a grar canal ,30.S. aureus membrane lipid composition clearly is an important consideration in antibiotic response and resistance, lack of susceptibility to FASII inhibitors, and susceptibility to antimicrobial skin SCUFAs. In contrast, there is little information on the impact of different media on the fatty acid composition and membrane fluidity of CONS. Fatty acids in the CONS are a mixture of BCFAs and SCFAs with differences in the proportions of individual fatty acids such as anteiso C15:0, anteiso C17:0, iso C15:0, C18:0 and C20:0 amongst individual species [The well documented plasticity of  species ,34. S. aureus, growth in MHB increased the proportion of BCFAs and membrane fluidity in CONS. CONS lack staphyloxanthin, which removes a complication from the determination of membrane fluidity. The different species had the ability to incorporate SCFAs and SCUFAs from serum. In this study, we investigate the effect of growth medium on the fatty acid composition and membrane fluidity of CONS. We also investigated the response of the different species to host-derived antimicrobial SCUFAs, sapienic acid, palmitoleic acid, and linoleic acid. As in S. aureus was also used for comparative purposes. The different species were observed to have different proportions of fatty acid compositions in a given culture medium. TSB-grown S. auricularis and S. capitis had almost equal proportions of SCFAs and BCFAs  . However, the other staphylococcal species grown in TSB had noticeably higher proportions of BCFAs . The BCFA fraction was composed of almost equal proportions of anteiso and iso fatty acids. To investigate the variability of fatty acid compositions in the CONS, they were grown in TSB, MHB and the fatty acid compositions were analyzed by gas-liquid chromatography of fatty acid methyl esters. A strain of 9% BCFA) , similarS. auricularis, 63%; S. epidermidis, 78%; S. haemolyticus, 79%; S. hominis, 82%; S. saprophyticus, 82%; S. capitis, 83%; and S. aureus, 81%)  . AnteisoS. haemolyticus and S. hominis) having similar ratios to each other. S. saprophyticus had the highest proportion of BCFAs in either TSB or MHB-grown cultures. Growth in MHB increased the proportion of BCFAs in all the CONS. The differences in BCFAs to SCFAs ratios in MHB- versus TSB-grown cells have been attributed to the acquisition of branched-chain amino acids, which are the precursors of BCFAs [The different CONS showed a range of BCFAs to SCFAs ratios in TSB, with the Haemolyticus cluster group members . The ratio of anteiso to iso fatty acids varied from about 1.02:1.00 in S. epidermidis to as high as 2.59:1.00 in S. auricularis. Importantly, BCFAs are not present in serum and, hence, the BCFAs are produced by biosynthesis [CONS grown in the common laboratory culture media lacked SCUFAs because they lack the genes for their biosynthesis . HoweverS. auricularis to as high as 35% in S. epidermidis  than linoleic acid (9%). Other SCUFAs detected in low amounts were arachidonic acid , palmitoleic acid (C16:1Δ9), vaccenic acid (C18:1Δ11) and eicosenoic acid (C20:1Δ11). The serum-grown S. aureus had oleic acid  as a major SCUFA fraction and 8% eicosenoic acid, and 3% C18:1Δ11  was the most abundant component, comprising from 13% in dermidis . Oleic aC18:1Δ11 . InteresS. aureus grown in the presence of human serum showed that serum lipids become associated with the cell surface and resist washing away with saline solution, but are removeable to a significant extent with Triton X-100 [S. aureus to bypass FASII inhibitors through the incorporation of fatty acids derived from host lipids. This is likely to be the case with the CONS also. These authors [S. epidermidis and S. haemolyticus in accordance with our findings.The SCUFAs present in the total fatty acid profile of the CONS probably represent a mixture of SCUFAs truly incorporated into glycerolipids ,19,22, aon X-100  showed t authors  reportedS. saprophyticus had most fluid membrane (0.156 ± 0.013 and 0.134 ± 0.006 respectively). On the other hand, the Haemolyticus cluster group members (S. haemolyticus and S. hominis) grown in TSB and MHB had the highest anisotropy values among the CONS. TSB-grown S. hominis had slightly higher (0.245 ± 0.029) anisotropy values than S. haemolyticus (0.228 ± 0.019), while serum-grown S. hominis had slightly lower (0.146 ± 0.027) anisotropy values than S. haemolyticus (0.169 ± 0.038). The Epidermidis group members (S. epidermidis and S. capitis) grown in TSB and MHB had comparable anisotropy values; however, the serum-grown S. epidermidis had a lower anisotropy value (0.096 ± 0.033). Notably, the serum-grown S. epidermidis had the highest proportion of SCUFAs and lowest anisotropy values among the CONS.To understand how the membrane biophysical properties of the different CONS were affected when grown in the different media, membrane fluidity was measured in terms of anisotropy values for each species. The higher the anisotropy values, the more rigid (less fluid) the membrane is. As shown in S. aureus, which indicates the membrane of the CONS were more fluid than those of S. aureus. This difference may be due to the presence of staphyloxanthin in the S. aureus membrane that is believed to decrease its fluidity [S. hominis cells had a yellowish pigment , and thn in MHB . IncreasInterpretation of the anisotropy determinations in serum-grown cells is more difficult given the likely association of serum-lipids with the cell surface . In cellS. aureus also had similar susceptibilities to sapienic acid  and palmitoleic acid  as the CONS.To understand the susceptibilities of the CONS towards known host-derived antibacterial fatty acids, the minimum inhibitory concentration (MIC) values of palmitoleic acid (C16:1Δ9), sapienic acid (C16:1Δ6), and linoleic acid , which are all abundant on human skin, were determined against the different species of CONS. The CONS were more susceptible to sapienic acid  than to palmitoleic acid  . S. aureS. auricularis, S. capitis, S. saprophyticus, and S. aureus were highly susceptible to linoleic acid , whereas S. epidermidis, S. haemolyticus and S. hominis were highly resistant to this antimicrobial fatty acid . The antibacterial activities of these fatty acids involve insertion into and disruption of membrane function [S. epidermidis, S. haemolyticus and S. hominis to linoleic acid may indicate the presence of an efflux pump, such as the inducible linoleic acid and arachidonic pump in some strains of S. aureus [MICs, >10 µM. The function ,26. Ther. aureus .Staphylococcus auricularis ATCC 33757T, Staphylococcus capitis ATCC 27840T, Staphylococcus epidermidis ATCC 12228T, Staphylococcus haemolyticus ATCC 27836T, Staphylococcus hominis ATCC 27844T, Staphylococcus saprophyticus ATCC 15305T, and Staphylococcus aureus RN450 were grown in various culture media and analyzed for growth characteristics through the determination of culture turbidity [2+ and 12.5 mg/liter Mg2+. Human serum was heated to 56 °C for 30 min to inactivate complement before using. Staphylococci were grown in 50 mL of medium in a 250 mL Erlenmeyer flask with shaking at 200 rpm at 37 °C unless otherwise specified.600) of about 0.8 in TSB, MHB and 100% human serum, harvested by centrifugation at 2000× g for 5 min, followed by washing two times with cold 1x phosphate buffer saline . The samples were sent for fatty acid methyl ester analysis by gas-liquid chromatography at Microbial ID, Inc.  and identified by the MIDI microbial identification system , as described previously [The different species were grown to an optical density  as described previously . BrieflyMICs of known antimicrobial fatty acids, viz., palmitoleic acid , sapienic acid , and linoleic acid , dissolved in absolute ethanol, were determined against the staphylococcal species by the broth microdilution method as recommended by the Clinical Laboratory Standards Institute  guidelinS. aureus. The growth medium used for routine antibiotic susceptibility testing, cation-supplemented MHB dramatically increased the proportions of BCFAs and membrane fluidity compared to cells grown in TSB. Preformed SCUFAs become prominent members of the total fatty acid profile in cells grown in serum, indicating a lowered dependency on fatty acid biosynthesis. The different species were susceptible to sapienic acid but showed varying susceptibilities to palmitoleic acid and linoleic acid.The fatty acid compositions of several CONS showed a similar growth-environment dependent plasticity as previously established for Our findings suggest that agents directed against the FASII system are unlikely to be of value against CONS given the ability of these organisms to incorporate host fatty acids into their lipids. In the context of these organisms as inhabitants of the skin, their ability to incorporate major amounts of linoleic acid into their lipids may suggest that this is a means of detoxifying this antimicrobial fatty acid. Further study of the incorporation of individual fatty acids is probably warranted for the CONS in the context of the skin environment and their exposure to host lipids.