Longissimus dorsi) obtained from sheep of three polish breeds—Wrzosówka, Uhruska, and Świniarka—and fermented products were tested. The fermented loins obtained after 14 days of processing were characterized by pH and water activity values in the ranges, respectively, 4.76–5.12 and 0.902–0.915. The maceration of meat in a fermented milk has caused greater acidification of the meat during fermentation. Statistical analysis indicated that treatment was the factor with significant effect on peptide content; no effect of animal breed was found. The peptide content isolated from raw meat ranged from 2.90 to 4.31 mg g−1 of sample, while in fermented meat products it was significant higher (11.59–16.37 mg g−1 of product). The antioxidant properties of peptides were positively correlated with peptides content. The maceration in fermented milk resulted in a statistically significant increase of ABTS value in case of fermented lamb loin of Świniarka breed. The raw meat and fermented meat products form the Świniarka lamb breed were characterized by the highest content of the total CLA isomers. The main CLA isomer found was cis9-trans11 (rumenic acid), followed by cis9-cis11, trans9-trans11, and trans10-cis12. The rumenic acid content was higher than, respectively, 87% and 80–88% of total CLA isomers in case of raw meat and fermented lamb loins of three breeds.This study evaluated the impact of fermented milk maceration on fermented lamb loin without nitrate to obtain peptides with high activity against oxidative changes  as well as a favorable fatty acid profile, including CLA content. Additionally, an attempt was made to evaluate the influence of the lamb breed on the assessed properties. Raw loins ( Fermented meat products have a far-reaching tradition and are preferentially produced and eaten in the Western world . During longissimus lumborum muscle of Polish native breeds including Uhruska, Wrzosówka, Świniarka that are the valuable local sheep breeds in Poland [Fermented meat products are unique in so far as high-quality raw meat is used and synthetic additives are reduced or eliminated in the direction of the “clean label” trend. Such products are much preferred by modern consumers than those produced traditionally with synthetic additives, including nitrates . Fermentn Poland .Additives are another important factor influencing the quality of fermented meat products. To obtain clean label products, natural raw materials of plant or animal origin are used, which show similar properties to synthetic additives . EliminaTo the best of our knowledge, no studies have yet been published on the effect of fermented milk on the content and antioxidant properties of peptides isolated form fermented lamb loin. In this context, this study aims to assess the impact of fermented milk maceration on fermented lamb loin without nitrate in order to obtain peptides with high activity against oxidative changes  as well as a favorable fatty acid profile, including CLA content. Additionally, an attempt was made to evaluate the influence of the lamb breed on the assessed properties.The raw materials for the production of fermented meats were loins (m. Longissimus dorsi), obtained from lambs of three polish breeds: Wrzosówka, Uhruska, and Świniarka. The animals were kept in farm conditions that met the requirements of organic farming. Uhruska lambs were slaughtered at the age of 5–6 months , while the lambs of the Wrzosówka and Świniarka breed were slaughtered at the age of 7–8 months and weigh 27–29 kg. Sheep for the experiment were from husbandry under the supervision of national veterinary inspection. The breeding met all the conditions required by European law. All animal rights under European law were controlled and respected at all stages from breeding, through transport to slaughter. The meat selected for experiment was purchased at butcher shop of the slaughterhouse. The meat was vacuum packed and delivered in cooling conditions to the laboratory was stored at 4 ± 1 °C for 48 h. After this time, the meat was prepared by cleaning the surface of the fascia, remaining tendons, giving the spindle shape a portion of about 1.0 kg. The meat samples prepared in this way were subjected to technological procedures, including salting (2.8%) with sea salt or curing salt , and maceration in a natural fermented milk for 48 h. Sea salt (non-iodinated and without anti-caking agents)  and glucose  were purchased from local supermarkets . Sodium nitrate (without anti-caking agents) was obtained from StanLab . Fermented milk obtained from cow’s milk was bought fresh from certified diary product plant . In the next step, pieces of meat have been dried in ripening chamber at 18 °C for 24 h. The next day, the cement has been done, consisting of mustard seed, garlic, fresh red pepper paprika, black pepper, cumin, coriander and water. All spices have been placed in a blender and mixed until they form a cohesive dough. Then, pieces of meat have been covered by a paste of spices from all sides properly without leaving a gap to maintain the product. Batch variants were prepared by aging in fermentation chambers for 14 days under controlled humidity (75 ± 5%) and temperature (18 ± 1 °C) conditions. As a result, three types of treatment were obtained: P1—treatment with curing salt , P2—treatment with sea salt, and P3—treatment with sea salt and macerated in natural fermented milk.Samples were taken from each variant at the end of processing. In addition, analyses were carried out for the raw material at the beginning of the experiment. The variants were replicated twice by producing two different batches on separate days. Eighteen fermented loins were produced in each batch. Both the raw material and the fermented product were tested for their physicochemical properties, peptides content and their antioxidant properties and fatty acids profile including CLA acids. All measurements were made in triplicate for each sample.w). The device was calibrated with Novasina SAL-T humidity standards based on saturated salt solutions .Water activity analyser , which gives temperature-controlled measurements, was used to measure water activity . A digital pH meter  equipped with pH electrode  and a temperature sensor was used.The pH value was measured in a slurry obtained by homogenization 10 g of a minced sample with 50 mL of de-ionized water for 1 min using a homogenizer IKA T25  method according to procedure described by Re et al. . The scaAntioxidative properties of peptides were also determined as DPPH radical scavenging activity according to the procedure described by Zhu et al. . The abi3+ (ferric) oxidation state to the Fe2+ (ferrous) oxidation state was calculated in reference to the results obtained for ascorbic acid standard curve. The results were expressed as equivalent mg ascorbic acid per mg of peptides.Ferric-reducing antioxidant power (RP) of the peptides was determined according to Mora et al. . The abiv/v) was used as the extraction solvent according to the method of Folch et al. [−1. The final temperature was held for 4 min. Reference standard methyl esters of fatty acids were used to identify the peaks.To extract fat from the sample the mixture of chloroform and methanol at a ratio of 2:1  and interactions between means were calculated using factorial ANOVA. Post hoc comparisons were made using Tukey’s test. All differences were significant at w in the range of 5.51–5.59 and 0.972–0.974, respectively) were typical for good-quality fresh meat. As reported by Malva et al. [a et al. , the vara et al.  obtaineda et al. .−1 of sample while in fermented meat products it was in the range of 11.59 to 16.37 mg g−1 of product). These results are not surprising as peptides are the results of muscle protein degradation because of proteolytic activity of endogenous enzymes together with lactic acid bacteria. In fermented meat products, the protein degradation is influenced by different variables including product formulation and processing conditions, which also influence the obtained acidity of the products [−1 of product) compared to the sample with nitrate (P1) (14.08–15.56 mg g−1 of product). The inverse relationship was found for Uhruska lamb meat products, the lowest peptide content was observed for the sample macerated with fermented milk. In the case of lamb meat products of the Świniarka breed, no statistically significant differences in the peptide content were found between the samples.products . Kato etproducts  point ouproducts ,29. As rproducts , peptideThe mechanism of the antioxidant activity of bioactive peptides is not sufficiently elucidated; however, the commonly accepted method for testing antioxidant activity focuses on donating a hydrogen atom to a free radical as well as chelating metals to prevent or remove excess free radicals. In the present study, three radical scavenging capacity assays were applied to investigate antioxidant properties of peptides isolated from fermented lamb loins samples . Based on the results of statistical analysis presented in The correlation shown  in this In our study, linoleic acid was the main polyunsaturated fatty acid, ranging from 3.57% in fatty acid profile for raw loin of Świniarka breed to 6.74% in fatty acid profile for fermented loin of Świniarka breed. The content of total polyunsaturated fatty acids reached lower values than those reported by Kawecka et al.  for Wrzolongissimus lumborum muscle of lambs fed grain-based diets with moderate level of antioxidant, supra-nutritional level of antioxidant or lucerne hay-based diet. They got n−6/n−3 values in the range 2.47–3.30 and for PUFA/SFA ratio values from 0.16 to 0.24. It is less than the 0.4 minimum PUFA/SFA nutritional recommendations for ruminant meats [In terms of nutritional important ratios, the fermented lamb loins were observed to be beneficially lower than the maximum 4:1 ratio for n6/n3 in case of Świniarka breed, but higher for Wrzosówka and Uhruska breed. Additionally, the values of 0.13–0.32 for the ratio of PUFA/SFA obtained in our study was analogous to the value 0.28–0.37) for male suckling Massese lamb meat as presented by Serra et al. . Similar for malent meats .Longissimus dorsi, Triceps brachii and Semimembranosus muscles for lambs slaughtered at 11–17 kg live weight. The present study indicated that the production process and fermentation reduced the content of this acid to such an extent that the fermented lamb loins were characterized by the rumenic acid content in the range of 80–88% of the total CLA isomers. As in the previous study [Ruminants’ meat including lamb is an important source of conjugated linoleic acid  showing us study , in whicThe results of this study demonstrated that fermented lamb loins, in which nitrate was eliminated during the production and fermented milk maceration was applied, are capable of retaining and delivering the nutritionally beneficial bioactive peptides and conjugated linoleic acid isomers. The study showed that the use of technological treatments affects the peptides content and its antioxidant properties. It seems that the meat of each of the studied breeds of lambs is valuable in the aspect of obtaining fermented products that are a rich source of peptides showing antioxidant properties. However, the most beneficial profile of fatty acids and the highest antioxidant activity of peptide extracts were obtained in the case of products made of the meat of the Świniarka breed.


Melospiza melodia) show that mean breeding value for local juvenile survival to adulthood, a major component of fitness, increased across cohorts more than expected solely due to drift. Such micro‐evolutionary change should be expected given nonzero additive genetic variance and consistent directional selection. However, this evolutionary increase was counteracted by negative additive genetic effects of recent immigrants, which increased total additive genetic variance but prevented a net directional evolutionary increase in total additive genetic value. These analyses imply an approximate quantitative genetic migration‐selection balance in a major fitness component, and hence demonstrate a key mechanism by which substantial additive genetic variation can be maintained yet decoupled from local adaptive evolutionary change.Ongoing adaptive evolution, and resulting “evolutionary rescue” of declining populations, requires additive genetic variation in fitness. Such variation can be increased by gene flow resulting from immigration, potentially facilitating evolution. But, gene flow could in fact constrain rather than facilitate local adaptive evolution if immigrants have low additive genetic values for local fitness. Local migration‐selection balance and micro‐evolutionary stasis could then result. However, key quantitative genetic effects of natural immigration, comprising the degrees to which gene flow increases the total local additive genetic variance yet counteracts local adaptive evolutionary change, have not been explicitly quantified in wild populations. Key implications of gene flow for population and evolutionary dynamics consequently remain unclear. Our quantitative genetic analyses of long‐term data from free‐living song sparrows ( How fast can evolution occur in wild populations? What processes can facilitate rapid evolution, or else prevent evolution from occurring? Answering these questions is central to understanding how species might adapt to current rapid environmental changes.Evolutionary change in any population results from a combination of genetic variation and natural selection, following Darwin's basic principle. Yet, the pace of local evolution could be dramatically altered by the arrival of immigrants from other populations, which could be better or less well adapted to local conditions. Immigrants might facilitate local adaptive evolution by importing beneficial genetic variants, or might impede or even reverse local adaptive evolution by importing detrimental genetic variants that negate local evolutionary advances. We need to distinguish between these possibilities in order to predict how evolution will proceed; yet we still know surprisingly little about the impacts of natural immigration on the critical genetic variation that underlies adaptive evolution in nature.We applied advanced “quantitative genetic” analyses to >25 years of pedigree  data from a Canadian population of song sparrows to quantify the effects of recent immigration on local adaptive evolution. We demonstrate that, during the past quarter century, there has been a substantial local evolutionary increase in the probability that a juvenile sparrow will survive to adulthood. However, we show that this recent evolutionary advance has been wiped out by detrimental genetic effects imported by recent immigrants, that reduce local survival. Song sparrows are therefore caught in a balance between the conflicting evolutionary forces of local natural selection and gene flow resulting from immigration.These results illustrate an important route by which rapid adaptive evolution can be constrained, despite the presence of substantial genetic variation and strong natural selection. Predictions of the rate of contemporary evolution in nature must consequently consider the complex genetic consequences of immigration.A) in wild populations  can then be specified as a function of introgression between defined groups of recent immigrants versus preexisting local natives (termed “genetic groups”). Specifically, ui can be partitioned into a breeding value , plus the difference in mean value between the defined immigrant and native genetic groups (the genetic group effect g) multiplied by the expected contribution of the immigrant genetic group to individual i's genome  above VA  as long as g≠0 and there is some among‐individual variance in qi  between generations due to local selection. For example, if g<0, meaning that immigrants have a lower mean value than preexisting natives, and the mean genetic contribution of immigrants  increased between generations  then the product g.qi would become more negative. There might then be little or no change in mean ui despite the underlying micro‐evolutionary increase in mean ai.Equation w of qi . FurtherMelospiza melodia) revealed a negative additive genetic effect of recent immigrants  on local juvenile survival to adulthood, and consequently on overall individual fitness measured as total local lifetime reproduction from hatch  for local juvenile survival increased over an observed 26‐year timeframe, representing micro‐evolutionary change that should be expected given non‐zero VA in a major fitness component. Second, we tested whether the overall additive genetic effect of recent immigrants  became increasingly negative over the same timeframe, generating an additive genetic counter‐gradient. Third, we tested whether mean total additive genetic value  changed across years, or whether the negative effect of immigration counteracted local micro‐evolution in mean ai to generate approximate migration‐selection balance. Finally, we quantified the degree to which the variance and skew in total additive genetic value ui exceeded those in ai, and hence to which natural immigration reshaped the total local additive genetic variation that is potentially available to generate further micro‐evolutionary responses to selection.Accordingly, we used the song sparrow data to quantify four key effects that determine how local micro‐evolution and immigration combine to shape local evolutionary outcomes. First, we tested whether mean breeding value  since 1975, allowing advanced quantitative genetic analyses to test key evolutionary hypotheses , and estimate ai for all individuals, by fitting random individual effects with variance‐covariance structure defined by pedigree‐derived relatedness. The model also included random natal brood and year effects to estimate variances associated with common environmental effects within broods and years  to account for known inbreeding depression  to “immigrant” and “native” groups, and estimate effects for the respective defined base populations . One individual that was apparently distantly related contributed very little to variation in qi, meaning that alternative assumptions regarding its ancestry and hence genetic group membership did not perceptibly alter the estimate of g.To minimize any bias due to remaining paternity error in the pre‐1993 pedigree, phenotypic data  were restricted to 26 cohorts hatched on Mandarte during 1993–2018. This comprises all individuals whose parents were genetically verified, with local survival to adulthood observed by April 2019. Since mean generation time is approximately 2.5 years , and hence largely uninformative. To facilitate mixing, priors on variance components were parameter‐expanded, giving proper Cauchy priors for the standard deviations. Posterior means, modes, and 95% highest posterior density credible intervals (CIs) for variance components and fixed effects were summarized across 3000 posterior samples with autocorrelation <0.05 , immigrant genetic effect (g.qi), and total additive genetic value (ui) on year across the focal cohorts . Full posterior distributions of the cohort population variance and skew in ai and ui, the differences between the variance and skew in ui versus ai, and the regressions of these quantities on year, were also computed and summarized. These statistics should be unbiased given that selection is ignorable , we simulated values of ai on the pedigree with founder values (for both natives and immigrants) drawn from a normal distribution with mean zero and variance equal to each posterior sample of VA . The proportion of positive differences gives the posterior probability that an observed increase in ai exceeds that which could arise by chance given the observed pedigree .Since individuals that do not locally survive to adulthood by definition leave zero local offspring, while most individuals that survive to adulthood do leave some offspring, local phenotypic selection gradients on local juvenile survival must be consistently positive. Nevertheless, to illustrate the magnitude of selection, we calculated selection gradients as the slopes of regressions of individual reproductive success on juvenile survival within each cohort  scale estimates for these years were back‐transformed onto the phenotypic scale taking mean values of all other modeled fixed effect variables. As an additional summary statistic, the posterior distribution of the latent‐scale heritability of juvenile survival was calculated as the ratio of VA to the sum of all estimated variance components . The grand mean standardized phenotypic selection gradient on juvenile survival across cohorts was 1.8 . There were 15 defined native founders, and 33 informative immigrants  that arrived during the full study period Fig. . The totqi) across all 2478 focal individuals hatched during 1993–2018 was 0.56 ± 0.14 SD. Hence, these individuals are on average expected to have inherited approximately half their genome from recent (post‐1975) immigrant ancestors. Mean qi was 0.42 ± 0.16 SD in 1993, and increased across cohorts to 0.69 ± 0.07 SD in 2018  was negative, and the 95%CI did not span zero  for juvenile survival increased during 1993–2018  and 0.19 (95%CI 0.02‐0.42) during 1993–2018 for females and males given constant mean values of all other modeled variables.As expected given nonzero Vqi) increased across years  for local juvenile survival (i.e. g.qi) became increasingly negative during 1993–2018  for local juvenile survival was relatively small  for local juvenile survival increased across years, representing a micro‐evolutionary increase that should be  expected given moderate VA and consistent directional selection. However, this increase was substantially counteracted by introgression from recent immigrants with low mean breeding values . Introgression increased the total additive genetic variance (VU), but greatly reduced the increase in mean total additive genetic value  across cohorts that would otherwise have occurred, generating approximate migration‐selection balance. Since VA in juvenile survival accounts for most VA in lifetime reproductive success in song sparrows, particularly in females . Posterior mean VA in local juvenile survival is then approximately 47% greater, resulting in greater estimated micro‐evolutionary increases across cohorts and a stronger negative environmental counter‐gradient . Such negative g could reflect some combination of local adaptation and/or non‐random immigration or heritable dispersal. These mechanisms have been shown or suggested to occur in diverse systems (Lenormand It is also useful to consider why immigrants’ expected genetic contributions to the focal population are high and increasing further Fig. , even thJ.M.R. conceived and undertook the analyses and drafted the manuscript. P.A. oversaw long‐term data collection. P.N. and L.F.K. undertook pedigree construction, and contributed to fieldwork along with J.M.R. S.M. and M.E.W. contributed to analyses. All authors contributed substantially to conceptual development and manuscript editing.https://doi.org/10.5061/dryad.rjdfn2z95Data are available from the Dryad digital repository: Associate Editor: A. CharmantierAppendix S1. Variance and skew in total additive genetic value.Appendix S2. Summary of local survival probabilities.Appendix S3. Further details of pedigree and covariate data.Appendix S4. Further details of quantitative genetic analyses.Appendix S5. Multi‐collinearity, sampling correlations and data structure.Appendix S6. Phenotypic selection on local juvenile survival.Appendix S7. Models without immigrant genetic effects.Click here for additional data file.


Aim of the study: Genetic testing is becoming increasingly common in clinical practice and health management; nonetheless, little is known about how the population approaches genetic services through private companies. Our study aims to describe socio-demographic aspects, health-related habits, and overall beliefs and knowledge about genetic risk and testing in a population of Italian citizens who decided to undergo a genetic examination through a private genetic company.Study design: A sample of 152 clients from an Italian private genetic company completed an ad-hoc survey from September 2016 to February 2018, addressing socio-demographic data, health habits, psycho-physic condition, perceived utility of genetic results, decision purposes about data sharing, and behavioral changes after results.Results: Participants (mean age 42.4) were predominantly female (82.2%) and were overall well-educated. Their main source of information were physicians (77%), and 41.1% entrusted the management of results to the same. Thirty-eight percentage underwent genetic analysis for cancer predisposition, 31.3% for fertility problems, 24% for dietary or intolerance issues in the period of enrolment. More than half of them (62.7%) reported a family history of the disease, and overall 69% had a current or past experience with a disease. Clients perceived the genetic screening as useful to adopt behaviors that may prevent disease onset (37.7%), to know their “real health status” (27.4%), and to adopt health-related behaviors (23.3%). 62.8% claimed they were motivated to change behaviors after results , and they wanted to share results with their physician and family members.Discussion/Conclusion: The overview of consumers' profiles in Italy and other European countries can contribute to tailoring and regulating genetic services in a way that could be efficient in terms of healthy choices, behaviors, and health resource expenditures for the general public. Before the sequencing of the human genome, which refers to the individual's entire genetic makeup, the general public had little reason to understand human genetics research and freely decide to undergo genetic testing for health-related purposes, since the science was limited and had little impact on most people's daily lives. That situation changed, since modern research has revealed genetic associations with the leading causes of mortality, including cancer, diabetes, and heart disease , 2, makiDifferently from the US social and cultural context, the DTC-GT was rather unknown in Italy until the first decade of the 2000s, since genetic tests have stayed mainly in the hands of experts. The Italian population seems to be far behind in terms of awareness of the opportunity to know their genetic profile for health purposes, and the implication of such information . ResultsVayena et al.  reportedRecently, Wöhlke et al.  contribuIn Belgium, Italy, and the UK there is no specific legislation that forbids or regulates the provision of DTC-GT, while in France, Germany, Portugal, and Switzerland there is a specific legislation that dictates that genetic tests can only be carried out by a medical doctor after the provision of sufficient information and appropriate genetic counseling , 23. NevThis contribution aims to provide a preliminary description of GT consumers' in Italy, sounding out their socio-demographic aspects, health-related habits, beliefs, and knowledge about genetic risks and testing.Participants were recruited via GenomaLab–Molecular Genetics Laboratory, in Rome and Milan. GenomaLab is a private genetic company, that offers a wide range of panels of genetic analysis for disease susceptibility/risk. It provides their clients with a focused and personalized counseling. Clients who underwent genetic testing for disease carriers, infertility problems, disease risk susceptibility, food intolerances, or wellness were included in our survey. Clients who required genetic testing during pregnancy or for Medically Assisted Procreation (MAP)  were excluded since they belong to an area accountable for being treated in a dedicated study. All participants signed informed consent forms and completed the survey before undergoing GT, in the waiting room of the Lab, through a paper-and-pencil version administered by the main experimenter of this study, or before going to the Lab for blood sampling, through the Survey Monkey platform online. Recruitment started in February 2016 and ended in September 2018. As an incentive, participants were given a discount on the cost of their genetic tests. The price range for testing in this Lab varies from a minimum of 80–100 euros for genetic lifestyle analysis, specific intolerances, or one specific disease, to 450–500 euros for a predictive package analysis for a series of diseases . A total of 152 participants agreed to complete the questionnaire. The response rate was 48% of 317 clients who underwent genetic testing in the period of recruitment.The research protocol was approved by the Institutional Review Board of the University of Milan, the principal coordination center of the survey, and by the Center for Research Ethics and Bioethics, University of Uppsala, coordinator of the Mind the Risk project (see funding declaration). The study was conducted according to the Helsinki declaration.Materials. A structured and self-administered ad hoc questionnaire  was created to assess the domains described in The questionnaire took around 45 min to complete.Descriptive statistics (frequencies and percentages) were calculated on raw data to report participants' socio-demographic characteristics, their health-related habits, their psycho-physic condition, and their motivation toward genetic testing. Contingency tables and Chi-Square tests were then performed to compare subgroups distinguished based on of the above-mentioned variables and the level of consumers' perceived knowledge about genetics. Expected values and residuals in every box were calculated to verify if a specific group gave a significantly higher or lower rate of response  to certain items, compared to the percentage expected and calculated on the number of subjects recruited. Analyses were performed with SPSS .Participant's mean age was 42.4 years  and most of them were female (82.2%). More than half of the population was married (58.9%) (followed by co-habitants 16.5% and single 13.9%); 49.3% did not have any child whereas 50.7% had one or more than one child. The overall sample was well-educated: 42.8% completed high-school while more than 51% had an academic education . They were white-collar workers (42%) or freelance workers (27%) mainly , 44.7% declared to usually practice moderate physical activity, 32.2% light physical activity, and 23% vigorous physical activity. Their sleep quality was generally poor: 44.3% answered that they woke up very early in the morning and could not continue to sleep during the night, or they fall asleep often even during the day and wake up often during the night. Just 22.8% of the sample fell asleep easily and had restorative sleep.Among participants, 40.7% had past or present psychological problems . They were also asked about the frequency of their medical checkups: the majority underwent medical checkups regularly (63.2%), as described in As described in Thirty-eight percentage of participants underwent the genetic test to know about their predisposition to cancer ; 31.3% required analysis for fertility problems or to prevent genetic transmission to the offspring ; 24% for dietary or intolerance investigation; 6.7% to test their predisposition to other diseases such as Thrombophilia, Huntington, and Alzheimer's disease. The genetic analysis performed by GenomaLab on their clients is represented in The main way that participants learned about private genetic testing and GenomaLab was through their family physician or specialist  (77%), followed by mass media (9.2%), relatives (7.9%), and friends (7.2%) in almost equal extent. Clients claimed that their preferred way of keeping in touch and to manage procedures and results was through their physicians (41.1%), but 36.4% of them decided to directly keep in contact with the genetic lab, handling the results without the physicians as a mediator see .X2 (N = 151) = 22.349, p = 0.000.Pearson Chi-square test showed a significant association between having learned about and having been referred to GenomaLab through a physician and the decision of handling the procedures and communication with the GenomaLab through the physician as a mediator, 19.3% of participants were current smokers at the moment of enrollment (a mean of 8.4 cigarettes per day), while 24% of the sample stated that they were former smokers. The mean scores for dietary risk behaviors were 13 .More than half of the participants gathered information and increased their knowledge concerning genetic risk and screening from physicians, genetic counselors, or other professionals (52%). More than 35% of participants declared they did not care about genetic risk/screening information or were not confident with these issues, thus referring substantial illiteracy on this topic. 10.1% collected information from the web and the remaining 2% from other sources see .Half of the sample (52.8%) had one or more consultations with specialists (gynecologists mainly), family physicians, and geneticists before undergoing genetic exams.In When asked about the intention to change their lifestyle after receiving the genetic test results, 72.3% of participants claimed they were motivated to change their behaviors. Among this population, participants reported the will to follow a healthier diet (47.4%) and undergo preventive medical check-ups (31.6%) (details in X2 (N = 148) = 24.064, p < 0.01] in deciding to undergo genetic testing for procreation purposes or carrier investigation, with participants without children more frequently requiring gene testing for these reasons (43.8% without children vs. 18.7% with children). Differently, participants who had children underwent more frequently genetic testing for cancer susceptibility (8.2% without children vs. 40% with children).We investigated the link between parenthood and motivations for taking a genetic test. Pearson Chi-square test showed a significant difference between people who had children vs. people without children . In particular, participants were convinced that genetic testing was useful to “adopt behaviors that can improve the state of my health” (81.8%).Contingency tables revealed no significant differences between groups based on knowledge/source of information with the perceived utility of genetic testing or changes in lifestyle after results. Participants who reported the intention to change lifestyle after results showed significant differences in the perceived utility of genetic testing, compared to those who declared they would not [No other significant differences emerged comparing the variables measured.Considering the recent progress in genetic analysis and the number of people asking for genetic tests, consumer profiling is becoming increasingly important as it is the first step to creating appropriate guidelines for the provision of genetic services, genetic counseling, and education campaigns, and in providing tailored genetic information useful for prevention. Recent evidence showed that public awareness of genomics and personalized medicine was not increasing in line with advancements in the industry . In thisIn our sample, participants were predominantly young adults, married and well-educated, white-collar or freelance workers. The well-educated middle class might request genetic tests in private laboratories more than the other categories because they have higher literacy rates and higher economic resources. The potential inequities in the access to genetic information and health care, in general, has been already discussed in the literature , 31. ForAmong our participants, those without children were more predisposed to require genetic testing for fertility problems, to prevent genetic transmission to future offspring, or to investigate food intolerances ; on the contrary, participants with children mainly asked to be tested for cancer predisposition. This result suggests that the condition of being a parent, or the intention to become a parent, influenced the individual's attitude toward genetic testing and the importance given to investigating a predisposition for a threatening disease. Other studies in the literature have shown that subjects who undergo a genetic test for cancer (breast and ovarian) are worried about their offspring and experience decisional conflicts toward their relatives , 34.The main resource of information on genetic tests is the family doctor or the specialist , who frequently recommended the clients for a specific genetic test. The physician seems to be an important reference even for managing the genetic result; indeed, clients who started their “journey” thanks to the physicians usually decided to continue to trust the physicians after the test for all other communication and for handling the results.Even if the majority of the participants decided to trust their physicians, a good percentage of clients preferred to receive their result privately and be guided by the services offered in a direct relationship with the genetic company (around 36% maintained direct contact with GenomaLab), thus demonstrating a certain desire for freedom in handling the result and its implications.Traditionally, genetic tests were the responsibility of health professionals such as doctors, nurses, and genetic counselors . The marAnother interesting aspect emerging from our results was that most clients were used to undergoing regular medical check-ups, followed a Mediterranean diet , had a mean BMI in the normal range, and practiced moderate or vigorous physical activity, thus depicting a population aware and careful of their health status. Furthermore, the analysis of risky behaviors, such as smoking or damaging food intake, described a population not particularly exposed to the risks assessed in the questionnaire. It thus seems that Italian clients are usually people who tend to behave guided by the need for prevention, and who tend to believe they can have an impact on their health . It would be important to investigate how personality characteristics might affect the attitudes in the uptake of genetic risk information .Our population of consumers often complained about sleep disorders and reported current or past experiences with psychological suffering. Despite 62.7% of participants reporting a family history of disease, only a small percentage, 24%, recognize that health problems could be correlated with their family history and thus have a “genetic” origin.A promising result is that more than half of consumers claim to be motivated to change their behaviors  after testing. Such intentions seem not to depend upon the level of knowledge or the source of information . These consumers are also convinced that genetic risk analysis is paramount to adopting behaviors that can improve the state of their health. Nevertheless, a recent review showed how behavioral and lifestyle changes depend on people's perception of risk, severity, controllability of the disease, and the availability of treatments .On the other hand, a worrying piece of data shows that a percentage of our population demonstrate a poor sense of agency and self-efficacy in the management of this kind of information: they declared “I do not care about genetic risk/screening information” or “I am not confident with these issues” (35%). This is indicative of a lack of genetic literacy and understanding of how genes work and interact with non-genetic factors, including the environment and lifestyle experiences, in determining disease onset or other clinical conditions . MoreoveDTC-GTs for health purposes are now mainstream, and probably will become increasingly common; for this reason, it is paramount to investigate the profiles of genetic companies' clients. Indeed, by knowing their current motivation, beliefs, literacy, and concerns, it will be possible to optimize consumers' experience with GT results and to empower them to make more informed choices. However, several population characteristics  may depend upon national education policies and health care systems; therefore, results about GT information impact may vary, also noticeably, in different countries.This kind of survey contributes to building a set of easily accessible information about DTC-GT users in different EU nations; also, they allow to take steps for empowering consumers to make more informed choices about their behavior and resource expenditures for health.These measures could include providing:Education focused on gaps in public genetic literacy and knowledge;Fine-grained tailored communications about specific test characteristics and their advantages/disadvantages to different profiles of DTC-GT clients;More detailed information about data protection in order to overcome the more common privacy concerns.We recognize some limitations for the current investigation. The sample of 152 Italian GT consumers is quite small, so the results could not be generalized outside of this specific context, and further research is needed to amplify the significance of what has been observed. However, the population of GT clients recruited in this study underwent a further investigation of their psychological profile  and two The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Institutional Review Board of the University of Milan, the principal coordination center of the survey, and by the Center for Research Ethics and Bioethics, University of Uppsala, coordinator of the Mind the Risk project. The patients/participants provided their written informed consent to participate in this study.SO collected data and wrote the main part of the manuscript. FS collected data and provided revisions. GM and LV provided the analysis and wrote the results section. IC and AG provided revisions and comments. GP supervised the work and contributed to discussion and conclusions section. All authors contributed to the article and approved the submitted version.FS was employed by the company GenomaLab. The remaining 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.


Acer ginnala, as a deciduous shrub/small tree, is a foundation species in many terrestrial ecosystems of Northern China. Owing to its increased use as an economic resource, this species has been in the vulnerability. Therefore, the elucidations of the genetic differentiation and influence of environmental factors on A. ginnala are very critical for its management and future utilization strategies. In this study, high genetic diversity and differentiation occurred in A. ginnala, which might be resulted from its pollination mechanism and species characteristics. Compared with the species level, relatively low genetic diversity was detected at the population level that might be the cause for its vulnerability. There was no significant relationship between genetic and geographical distances, while a significant correlation existed between genetic and environmental distances. Among nineteen climate variables, Annual Mean Temperature (bio1), Mean Diurnal Range (bio2), Isothermality (bio3), Temperature Seasonality (bio4), Precipitation of Wettest Month (bio13), Precipitation Seasonality (bio15), and Precipitation of Warmest Quarter (bio18) could explain the substantial levels of genetic variation (> 40%) in this species. The A. ginnala populations were isolated into multi-subpopulations by the heterogeneous climate conditions, which subsequently promoted the genetic divergence. Climatic heterogeneity played an important role in the pattern of genetic differentiation and population distribution of A. ginnala across a relatively wide range in Northern China. These would provide some clues for the conservation and management of this vulnerable species.Genetic diversity and differentiation are revealed particularly through spatio-temporal environmental heterogeneity.  Geographical topology can play a key role in determining genetic divergence of species6. Climate conditions, in particular the most recent climatic oscillations, can also impact the evolution of the local organisms7. Under the changing conditions, if species unable to respond the environmental change, it will be at increased risk of extinction8. Indeed, species survival ultimately depends on its genetic diversity and variation9. Thus, investigating the genetic diversity and extent of genetic differentiation within/between populations, while estimating the impact of environmental factors and understanding the processes that maintain these variations not only are very useful toward gaining deeper insights into evolutionary history, but also are critical for the prudent formulation of conservation, management and utilization strategies13.Genetic diversity and variation are manifested through phenotypic, chromosomal, and proteomic variations, which are revealed particularly through spatio-temporal environmental heterogeneityAcer ginnala (Aaceraceae) is a deciduous shrub/small tree that has monoecious and anemophilous pollination with an outcrossing breeding system16. It is a foundation species in many terrestrial ecosystems, distributing in Korea, Japan, Russia, Siberia, and China15. In China, this species primarily distributed in the northern regions, including Inner Mongolia, Hebei, Henan, Shanxi, and Anhui provinces, growing on cloudy slopes, gullies and valleys. A. ginnala has important economic value as the abundant gallic acid within leaves21 that can use as a functional material or novel herbal medicine with extracted galloyl derivatives22. However, the increased developments as an economic resource have accelerated the degradation of A. ginnala populations so that this species has been listed as a vulnerable plant in a recent nationwide biodiversity report23.A. ginnala were firstly examined along an altitude gradient at Qiliyu in Shanxi, China25. In Qiliyu, high genetic diversity of A. ginnala was found. Significant differentiations in phenotype and genetics were observed in populations25. The level of genetic variation of the studied populations was observed to increase along an elevation gradient26, indicating the influence of climatic heterogeneity.To provide theoretical basis for formulating effective and reasonable protection strategies, in our previous studies, the genetic diversity and differentiation of A. ginnala, a small number of individuals from a small-scale district were characterized; thus, the results provided very limited information on the genetic diversity and variations of this species. The extent and pattern of genetic differentiation of A. ginnala at relatively wide geographic scales remained unknown. Meanwhile, in northern regions of China, there is a complex geographical topology with more heterogeneous climate. How these conditions have affected the pattern of genetic variation across the present range of this species is also unclear.Although these studies revealed the level of genetic diversity in A. ginnala. According to this hypothesis, significant genetic variation among populations/groups would provide an evidence and a significant relationship would be present between genetic differentiation and climatic-geographical factors.Being a long-lived species, the geographical history events and climatic oscillations would be predicted to leave a signature on the level of genetic diversity and variation of 29. Meanwhile, the sequence-related amplified polymorphism (SRAP) technology has been recognized as a useful molecular marker system for population genetics research31. It targets coding sequences and results in the identification of a number of codominant markers. SRAP is more reproducible than RAPD (Random Amplified Polymorphic DNA) and less complicated than AFLP (Amplified Fragment Length Polymorphism)32. Ferriol et al.31 reported that the information obtained from SRAP markers was more concordant with the morphological variations and the evolutionary history of the morphotypes than that found with AFLP markers.In the last few years, microsatellites (SSR) have become one of the most popular codominant molecular markers used with applications in many different fields. High polymorphism and the relative ease of scoring represent the two major features that make microsatellites of large interest for many populations genetic studiesA. ginnala across a wide range in China using two types of molecular markers (SSR and SRAP), with a specific focus on genetic differentiation in the population, in order to understand whether geographical and climatic factors affected the genetic variation and genetic structure. In particular, the main objectives were to: estimate the genetic variability of A. ginnala populations and geographical groups; analyze the genetic structures and relationships of A. ginnala populations; verify the potential influences of spatial and environmental factors on population differentiation patterns. The combined analysis of molecular markers and eco-geographical data would provide beneficial knowledge for the utilization and conservation of this wild plant germplasm, for further revealing the evolutionary history of forest community in northern regions of China  being 98.99%. A total of 170 bands were amplified with the SRAP marker, and 100% of the bands were polymorphic. Based on the SSR markers, the highest genetic diversity was present in the PQG population, followed by XTS and BYS , which was higher than the mean values at the population level  (Table FIS) was 0.4980 (by SRAP data) and 0.4972 (by SSR data) respectively.For the SSR markers, a few loci were found to significantly deviate from HWE, three in the BDG population, four in the JMLC and XTS populations, five in the MLG and LJS populations, and one in the TTZ and WCLC populations. These loci were removed in the further analyses. There were 177 polymorphic loci in 179 putative genetic SSR loci, with the percentage of polymorphic bands (YS Table . AccordiYS Table . At the 8) Table . In addiK) was 3 based on the ΔK, where all of the populations were divided into three groups , indicating that 16% of the total variance occurred within the populations. With SRAP markers, AMOVA  revealed that 88% (ΦST SRAP = 0.88) of the total genetic variation was found among the populations (P = 0.01), whereas the remaining 12% of the total variation occurred within the populations  of the total variation, while there was ~ 60% variation within the groups . Meanwhile, the gene exchange among groups was also low. Based on SRAP data, the migration occurred from Group I to II and the reverse was 0.1085 and 0.1124 respectively. Group II to III and the reverse was 0.1034, 0.1124, respectively. Group I to III and the reverse was 0.1109, 0.1101, respectively. For SSR data, the migration occurred from Group I to II and the reverse was 0.1158 and 0.1036, respectively. Group II to III and the reverse was 0.1132, 0.1136, respectively. Group I to III and the reverse was 0.1119, 0.1161, respectively , bio2 , bio3 , bio4 , bio13 (Precipitation of Wettest Month), bio15 , and bio18 (Precipitation of Warmest Quarter). Among these seven bioclimatic variables, the first four  were associated with the temperature dimension, while the last three  were associated with the precipitation dimension. The PCA  showed that the explanatory percentage of the first two PCs  of the temperature dimension (45%) was estimated to be higher than that of the precipitation dimension (38%).SRAP = − 0.1532, P = 0.284; ρSSR = − 0.0133, P = 0.474), or between genetic and geographic distances . A significant relationship between genetic and environmental distances was found . While, the partial Mantel test did not detect a significant correlation between genetic and geographic distance when conditioning on the environmental effect . Nevertheless, there was a significant association between genetic and environmental distances, when the geographic distance was considered as a covariate in the partial Mantel test . In terms of the joint effects of geographic and environmental distances in MMRR (Multiple Matrix Regression with Randomization) analysis, the geographic distance did not have a significant impact, while the environmental distance did impact the genetic distance significantly . These results revealed that the genetic variation of the populations was linearly correlated with climatic differentiation, but not geographical differentiation.To assess whether geographic or environmental differences drove genetic divergence between populations, isolation-by-distance (IBD) and isolation-by-environment (IBE) tests were conducted using the Mantel test. The Spearman correlation showed no significant association between geographic and environmental distances (ρP < 0.0001), and that bio2 and bio3 had the highest explanatory proportions for predicting the genetic variation of the population. Moreover, seven environmental variables could be separated into two categories: temperature and precipitation. Three bioclimatic variables  of the temperature dimension had significant F statistics  could be explained by climatic variables in dbRDA   was observed between populations, whereas ~ 10% of the differentiation existed within populations. High genetic variation values  in this study.There are reports that some plants have suffered from a general decline in pollinator insects, a phenomenon referred to a pollination crisisA. ginnala have a large wing and can be dispersed by the wind, the potential for exchange might be more sensitive to geographic barriers than that of other wind-pollinated and wind-dispersed species. In the A. ginnala distribution areas, the discontinuous distribution of mountains, such as Taihang Mountains and Wuling Mountains, provide a complex landform that likely blocks gene exchange among populations50. These factors presumably isolated populations; thus, promoting population differentiation by limiting the potential for gene exchange. Interestingly, the Mantel test and Spearman correlation revealed no significant relationship between genetic and geographical distances. For A. ginnala, gene flow might also be obstructed by the pollination pattern. Being an insect pollinated species, pollen-mediated gene flow was generally limited, as the range of most pollinators might be less than 20 km36, and they tend to visit neighboring plants51. Thus, at a relative larger-scale area, A. ginnala populations might be geographically and/or ecologically too isolated to be connected via pollen exchange. Besides, the considerable genetic differences between populations might also be attributed to the absence of generative reproduction53. During the field observations, we found that some populations ruled out generative reproduction, due to the absence of flowering individuals, or their inability to produce fruit.The seeds of 54, which might favor higher genetic variation within Group I and III. Meanwhile, observed genetic differences between three groups were relatively large and statistically significant. Climate heterogeneity and/or discontinuous mountains in Northern China could segment large populations into multiple-small fragmented populations, further block the gene exchange among groups  might have played an important role in shaping the genetic divergence of populations, corresponding to the environmental heterogeneity that occurred for A. ginnala. As revealed by dbRDA analysis, temperature and precipitation are frequently found to play prominent roles as a driver for genetic variation in various plant species61. Furthermore, in our study, the temperature seems to be more overwhelming and dominate to affect the genetic differentiation of A. ginnala than the precipitation variables. Within the studied populations, each was located in areas under different environment conditions. The maximum of Mean diurnal range (bio2) was 116 in the MLG population, and the minimum was 78 in the FZL population. For Isothermality (bio3), it was from 270 in the BYS population to 236 in the BDG population. Meanwhile monsoon rainfall may occur due to the orientation of different mountains, such as Qinling and Taihang Mountains, in the geographical distribution of A. ginnala66. Bio15 had the highest adjusted R2 value between three major precipitation ranges  for the pre-experiments (primer screening). These abided by the following principles: the primer length was controlled between 18 and 25 bp, the GC content was from 40 to 60%, and the annealing temperature was from 50 to 60 °C. Hairpin structures, dimers, hat structures, and mismatches were avoided as much as possible. The primers were synthesized by Shanghai Sangon Biological Engineering Technology . Finally, ten pairs of polymorphic SSR primers with distinct bands and high stability were selected to amplify 310 individuals of the A. ginnala populations , 94 °C denaturation (30 s), 55  °C annealing (45 s), 72 °C extension (65 s), 36 cycles, and a final extension for 9 min at 72 °C.SSR markers were obtained from primers previously developed for 30, we also referred to the combination mode of forward and reverse primers in the closely related species of the genus Acer designed by Liu71. After primer screening, ten pairs of primers with clear and stable bands were also selected , and 10.5 μL double-distilled water. The reaction procedure included initial denaturation at 94 °C (5 min), followed by 36 cycles at 94 °C (40 s), annealing at Tm temperature under different primers (25 s), extension at 72 °C (65 s), and final extension at 72 °C (10 min).While for SRAP markers, according to the primer design principles developed by Li and Quiros’sThe PCR amplified products were separated by polyacrylamide gel electrophoresis (12%), with an electrophoresis time of ~ 2.5 h at 200 V, and the fragments were visualized by silver staining.PPB), Shannon's Information index (I) and expected heterozygosity (He) were calculated using GENALEX72. Additionally, departures from Hardy–Weinberg equilibrium (HWE) were tested also using GENALEX72 for SSR data. To elucidate the extent of genetic variation between and within populations or groups, the hierarchical analysis of molecular variance (AMOVA) was performed in GENALEX72. The groups were assigned according to the STRUCTRE results, DAPC results and geographical distribution of the studied populations. The gene flow was calculated by using the formula: Nm = (1 − FST) /4FST to estimate the migration rates among populations. To further investigate gene flow among the three groups, BAYESASS software73 was used. The burn‐in period of the model was set at 1 × 106, while MCMC iterations were set at 1 × 107. Several instances of the model were run with different starting seeds. Results were similar among runs, and only the values from the first run were reported. The inbreeding coefficients (FIS) of this species was also estimated using BAYESASS73.For both two molecular data, the genetic diversity parameters, such as the percentage of polymorphic bands  was estimated by 15 independent runs using two-million steps Markov chain Monte Carlo (MCMC) replicates after one-million-steps burn-in for each run to evaluate consistency. The optimum K value was determined using the Delta K method (ΔK statistic) in STRUCTURE HARVESTER75. These 15 runs were aligned and summarized using CLUMPP.STRUCTURE analysis76. The genetic data was initially transformed according to Principal Component Analysis (PCA). These components explained most of the genetic variation based on PCA was then used to perform linear Discriminant Analysis (DA), which provided variables to describe the genetic groups that minimized the genetic variance within populations, while maximized the variation between populations.To further confirm the population genetic structure, a discriminant analysis of principal components (DAPC) was conducted using the R package ‘adegenet’A. ginnala variation, we extracted the environmental factors with DIVA-GIS software77 and determined the key environmental factors by the “vif” function in the R package ‘usdm’78. Variables with a high variance inflation factor (VIF). were removed to reduce multicollinearity. The VIF values of all the remaining variables were < 10, where finally seven bioclimatic variables were retained for further analyses.To investigate the influence of the environment on A. ginnala, the Mantel test of Spearman correlation was performed among genetic, geographic, and environmental (climatic) distances using R package ‘vegan’80. Pairwise FST was calculated between populations based on Nei’s81 method, then FST/(1 − FST) was used to estimate the genetic distance metric. Geographic distances were estimated using Euclidean distances, according to three dimensional factors . Environmental distances with seven bioclimatic variables were also calculated using Euclidean distances. A partial Mantel test between genetic and environmental distances controlled for the geographic distance was also performed. Further, multiple matrix regression with randomization (MMRR)82 was performed using R package ‘PopGenReport’83 to test whether genetic distances responded to variations in geographic and/or environmental distances. The joint effects of both geographic and environmental distances on the genetic distances were also examined. Regression coefficients of the Mantel test (ρ) and MMRR (β), and their significance, were estimated based on 9,999 random permutations. Additionally, partial distance-based redundancy analyses  were performed to explain the effects of climatic variables on the genetic distribution of populations using R package ‘vegan’80. The generalize linear regression model (GLRM) was employed to test the effects of climatic variables on populations.To test how the geographic distance and environmental differences affect the genetic composition of Supplementary Information.


Vultur gryphus) to investigate whether contemporary low genetic variability is the result of recent human expansion and persecution, and compared this genetic history to that of California condors .We then explored historic demographies for both species via coalescent simulations. We found that Andean condors have lost at least 17% of their genetic variation in the early 20th century. Unlike California condors, however, low mtDNA diversity in the Andean condor was mostly ancient, before European arrival. However, we found that both condor species shared similar demographies in that population bottlenecks were recent and co‐occurred with the introduction of livestock to the Americas and the global collapse of marine mammals. Given the combined information on genetic and demographic processes, we suggest that the protection of key habitats should be targeted for conserving extant genetic diversity and facilitate the natural recolonization of lost territories, while nuclear genomic data should be used to inform translocation plans.While genetic diversity of threatened species is a major concern of conservation biologists, historic patterns of genetic variation are often unknown. A powerful approach to assess patterns and processes of genetic erosion is via ancient DNA techniques. Herein, we analyzed mtDNA from historical samples (1800s to present) of Andean Condors ( Andean condors have lost at least 17% genetic variation by the early 20th century. Demographic bottlenecks are associated with global anthropogenic processes, however, unlike California condors, low mtDNA diversity in Andean condors is mostly ancient. Conservation measures require to include both environmental and genetic management. Photo credit: Gonzalo Ignazi.  The latter has been an object of intense genetic studies due to its near extinction in the 1980s and its subsequent breeding program in the 1990s  by incorporating 10 μl 1 M of dithiothreitol (DTT) and 3 μg of RNA carrier to the digestion buffer. Contemporary DNA was extracted from molted feathers collected from roosting sites in central Argentina , nucleotide diversity (π), and the average number of nucleotide differences (k) in DnaSP 6.0  to take into account the possible influence of the early European settlers and modern civilization  without the historical‐recent bottleneck  between the ancient and historical‐recent bottlenecks  and comparing their 95% confidence intervals  of Andean condors between 50 and 1,000 for the northern region and between 500 and 5,000 for the south. These priors were based on the estimates of regional population censuses of <2,570 individuals from Peru to Venezuela, >3,000 between Bolivia, Chile, and Argentina, and an estimated total number of 6,700 mature individuals in the whole continent , including five samples previously amplified by Hendrickson et al.  that matp < .001, exact‐p = .01) and southern Andes , but not between central and south . The spatial sorting of the haplogroups between central‐southern  and northern Andes  showed that HIII was exclusively found in the north; HIV, HV, and HVI were only present in the south, while HII was predominant in the south (93.75%) and HI was evenly distributed across regions , indicating a history of evolutionary divergence between north and south . However, our hierarchical AMOVA showed no statistical differences across time, while regions exhibited marked differences within time periods, with most of the genetic variation explained within regions  in the historic samples and five haplotypes (HI‐V) in contemporary populations, resulting in the loss of one haplotype (HVI) in the 20th century, or a 17% of decline Figure . All lins Figure . Mantel h Figure . Nucleot3.2Pr = 0.568, CI: 0.562–0.573) and southern Andean condors  with 95% confidence intervals not overlapping with alternative scenarios. Northern Andean condors showed lower posterior probabilities , although with no confidence interval overlapping with other scenarios (details in Table t1), and an ancient population size of 32,200–47,400 (Nef2) between 2,576 and 23,730 years ago (te) and a lower population size reaching 853–14,300 (Nef4), 52,500–227,500 years ago (t2). Historical population size in northern Andean condors was 170–503 (Nef1), with an historical bottleneck around 1662–1781 (t1), and an ancient population size of 6,760–15,900 (Nef2). Historical population size of southern Andean condors was 855–2,320 (Nef1) after a bottleneck around 1844–1963 (t1), harboring an ancient population size between 39,700–47,100 individuals, 1,792–20,020 year ago, prior a lower population size between 22,300–33,200 individuals, 329,000–452,900 years ago  and comparison of posterior probabilities between scenarios showed consistent results, supporting scenario 4 in California and Andean condors from central‐south of South America, whereas scenario 2 was the most supported in northern Andean condors. Principal component analysis revealed that the observed data fitted well within the simulated prior combinations of the supported scenarios and comparatively similar or better than in the alternative models Figure . Posteri44.1Our study of contemporary and historical mitochondrial data in conjunction with a continental‐level sampling of the Andean condor allowed us to characterize the spatiotemporal genetic composition for this species of special conservation concern. We showed that Andean condors have lost at least 17% of genetic variation by the early 20th century. As expected, genetic erosion was associated with the loss of a mitochondrial lineage in the peripheral distribution during the contraction of its southern geographic margin in the Atlantic coast. However, we found no evidence of lost haplotypes in the northern distribution where populations have been driven to near‐extinction , the red kite (Milvus milvus), and the Spanish Imperial eagle  exhibit one of the lowest mtDNA diversity levels with ≤ 10 haplotypes reported for the Control region  and Andean condors (310 bp from 12S and Control region). However, if this pattern is indeed species‐specific, factors other than life‐history traits should be invoked to explain such disparity in the mutation rates between condor species. For instance, it is possible that species have experienced disparate evolutionary histories during phylogenetic radiation such as contrasting levels of ancestral genetic variation, sex‐biased asymmetries, different strengths of genomic sweeps, introgression events, or mito‐nuclear conflicts  nature of mtDNA of Andean condors, we suggest that when possible, rather than translocating individuals from disparate regions, current conservation measures should focus on enhancing gene flow to assist natural augmentation and recolonization. Ideally, future management measures should rely on high‐resolution nuclear genomic data capable of identifying possible cryptic evolutionary units of the species in order to inform which lineages should be used to restock specific geographic locations.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Julian Padro: Conceptualization ; Data curation (lead); Formal analysis (lead); Funding acquisition ; Investigation (lead); Methodology (lead); Project administration ; Resources ; Visualization (lead); Writing‐original draft (lead). Sergio Lambertucci: Conceptualization (supporting); Funding acquisition ; Investigation ; Project administration ; Resources (lead); Supervision ; Writing‐review & editing . Paula Perrig: Investigation (supporting); Methodology (supporting); Writing‐review & editing (supporting). Jonathan Pauli: Conceptualization ; Funding acquisition ; Project administration ; Resources (lead); Supervision (lead); Writing‐review & editing (lead).SupinfoClick here for additional data file.


The data obtained broadened the view on the microbial communities that were associated with the various products examined, revealing the presence of previously underreported subdominant species. Moreover, the composition of these communities could be linked to the specificities of individual products, in particular pH, salt content, and geographical origin. In contrast, no clear links were found between the volatile organic compound profiles of the different products and the country of origin, distinct processing conditions, or microbial communities. Future application of the HTS method offers the potential to further unravel complex microbial communities in fermented meats, as well as to assess the impact of different processing conditions on microbial consortia.Insight into the microbial species diversity of fermented meats is not only paramount to gain control over quality development, but also to better understand the link with processing technology and geographical origin. To study the composition of the microbial communities, the use of culture-independent methods is increasingly popular but often still suffers from drawbacks, such as a limited taxonomic resolution. This study aimed to apply a previously developed high-throughput amplicon sequencing (HTS) method targeting the 16S rRNA and  The geographical origin of fermented foods plays an important role in conferring cultural and gastronomic meanings as well as in influencing certain product characteristics ,2,3. NatStaphylococcus xylosus and Staphylococcus equorum, whereas in Northern Europe there is typically a much higher prevalence of Staphylococcus carnosus [Latilactobacillus sakei and Pediococcus pentosaceus and in the case of CNS is limited to S. xylosus and/or S. carnosus [S. equorum, Staphylococcus saprophyticus, and S. xylosus being the most prevalent species [Such distinct differences in processing are known to influence the growth and composition of the predominant LAB and CNS communities . For inscarnosus ,13,14,15carnosus ,17,18. Icarnosus ,20. Depe species .Culture-dependent as well as culture-independent identification methods have been used to study the bacterial communities of fermented meats ,22,23. TStaphylococcus genus. It is, therefore, necessary to apply other phylogenetic marker genes, such as the rpoB or tuf gene, when analyzing diverse staphylococcal communities [Recently, culture-independent high-throughput sequencing (HTS) technologies have been applied to analyze microbial communities in fermented foods, including fermented meats, in greater detail ,27,28. Fmunities ,31,32.tuf genes [The objective of this study was to assess if a previously developed amplicon-based HTS method targeting the 16S rRNA and uf genes  can be sA total of 15 fermented pork products was obtained from supermarkets in and around Brussels, Belgium. The commercial products originated from Belgium, Germany, France, Italy, and Spain. Three fermented meat products were selected per country of origin for dedicated HTS analysis. Products originating from Belgium, France, and Germany came from at least two different producers per country and products originating from Spain and Italy came from at least three different producers per country. For each product, the label was analyzed to monitor relevant information, i.e., salt concentration and country of origin, and triplicate measurements of pH and the bacterial loads of presumable LAB, determined using de Man–Rogosa–Sharpe (MRS) agar, and CNS, determined using mannitol–salt–agar (MSA), were obtained as described previously .5-fingerprinting method [Most fermented meat products were typical representatives of their geographical region of production with respect to their bacterial loads, acidity, and salt content, and were selected based on the data from an earlier study . For allg method . These dg for 10 min to remove coarse impurities. This recovery diluent consisted of a sterile solution of 0.85% (m/v) NaCl  and 0.1% (m/v) bacteriological peptone . Thereafter, the resulting suspensions were filtered through a 20-μm average pore-size 50-mL Steriflip filter  to remove remaining contaminants. Cell pellets were then obtained by centrifuging at 4000× g at 4 °C for 20 min.From each fermented meat product, two samples were taken, representing biological duplicates, to analyze the bacterial composition. Bacterial DNA was extracted by first centrifuging a homogenized mixture of sample and recovery diluent at 900× Thereafter, DNA extraction was carried out, using a combination of enzymatic, chemical, and mechanical cell lysis. This was followed by phenol/chloroform/isoamyl alcohol extraction and column purification of the DNA, as described in , except tuf gene with sufficient discriminatory power to distinguish different CNS species was amplified using primer set Tuf387/765 , and allows distinguishing staphylococcal communities to species level [S. carnosus, Staphylococcus epidermidis, S. equorum, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, S. saprophyticus, Staphylococcus sciuri, Staphylococcus succinus, and S. xylosus, was used to check the performance of the PCR assays and subsequent sequencing.To target the overall bacterial communities, the V4 region of the 16S rRNA gene was amplified using the primer set F515/R806 , furtherf387/765 . This pres level . The PCRtuf gene sequences as described above. In the case of the tuf gene, ASVs were only classified to species level if a 100% match with a sequence in the custom database was found. If taxonomy could not be assigned using the custom database, ASVs with more than 1000 reads were aligned to the non-redundant nucleotide database nt of the National Center for Biotechnological Information , using blastn as an alignment tool [Processing of the V4 amplicons was similar to the method described in Zhang et al. , wherebyent tool . ASVs wem/z range of 35 to 400 after 2 min. The raw data were deconvoluted by TOF-DS software (Markes). Afterward, peaks were identified based on the NIST library  and the use of commercially available standards [Non-targeted VOC profiling was conducted in triplicate by headspace/solid-phase microextraction coupled to gas chromatography and time-of-flight mass spectrometry (HS/SPME-GC-TOF-MS), using a Trace 1300 gas chromatograph  equipped with a Stabilwax-MS column  and coupled to a BenchTOF-HD mass spectrometer operating with electron impact ionization at 70 eV . For analysis, approximately 10 g of the fermented meat product sample was frozen using liquid nitrogen  and grounded into a fine powder with a coffee grinder . Subsequently, 1.5 g of grounded powder was incubated in a 10-mL screw-top headspace vial at 60 °C for 10 min, after which the sample was exposed to an SPME fiber  at 60 °C for 20 min. To each sample, 10 µL of a 10 ppm toluene-D8 solution (Sigma-Aldrich) was added as an internal standard. The VOCs adsorbed to the SPME fiber were thermally desorbed at 250 °C with a split rate of 50 mL/min. The GC oven temperature program consisted of an initial step at 40 °C for 1.5 min, followed by a continuous increase to 225 °C at 10 °C/min, and finally, the temperature was kept constant at 225 °C for 15 min. Helium gas  was used as the carrier gas at a flow rate of 1 mL/min. The TOF-MS was scanned in the tandards ,41. For RStudio software  was used for all statistical analyses and tests .Assessment of inter-sample diversity (beta-diversity) was achieved by performing a permutational multivariate analysis of variance (PERMANOVA) based on Bray–Curtis dissimilarity scores to ascertain differences in bacterial composition between the fermented meat products investigated. Subsequently, a series of pairwise PERMANOVA comparisons and a similarity percentage analysis (SIMPER) were performed to assess the differences in bacterial communities between fermented meat products produced in the countries mentioned above. The vegan (version 2.5-4)  and RVAiHeatmaps of the non-targeted VOC compound profiling were calculated and clustered using the packages ComplexHeatmap (version 2.0.0)  and circThe characteristics with respect to the pH, salt content, and bacterial loads of each sample of the commercial fermented meat products examined are provided in Lactobacillus was present in all samples, no matter the geographical origin of the product. In contrast, Pediococcus was only found in samples from Belgian and German fermented meat products and Pseudomonas was exclusive to one sample of a Belgian product. Samples from Spanish fermented meat products showcased the greatest microbial diversity, with genera such as Bacillus, Brochothrix, Carnobacterium, Corynebacterium, Leuconostoc, and Tetragenococcus being present. Staphylococcus was only found sporadically via this approach in Belgian, German, and Spanish fermented meat products, whereas it was present in all French and Italian ones.In addition, the bacterial communities present in the various fermented meat products were assessed through amplicon-based HTS, based on 83,642 and 71,971 raw V4 and Tuf387/765 sequence reads on average per sample, respectively. The V4 sequence reads provided a first overview of the bacterial genera present in the different fermented meat products . It unvetuf gene sequencing gave a more detailed view of the CNS communities present in the different fermented meat products  could be ascribed to these genera.The Tuf387/765 sequence reads obtained by partial products . All speStaphylococcus species using partial tuf gene sequencing, differences were found between the CNS communities of the fermented meat products from different countries  impact on the composition of the microbial communities. Pairwise PERMANOVA tests showed that microbial communities from Spanish fermented meat products were significantly different from those found in fermented meat products from all other countries. German fermented meat products had different (p < 0.05) microbial profiles than French, Italian, and Spanish variants. The microbial communities of Belgian fermented meat products were not significantly different from their German or French counterparts, whereas French fermented meat products had comparable microbial profiles to the Italian ones. SIMPER highlighted that differences in microbial communities were primarily due to the presence of S. equorum in Spanish fermented meat products, S. carnosus in Belgian and German ones, and S. xylosus in French and Italian fermented meat products , 16% were aromatic hydrocarbons , 10% were esters , 9% were alcohols , and 9% were ketones . Minor VOCs consisted of aldehydes , alkanes , carboxylic acids , furans , sulphuric compounds , and nitrogen compounds  . DespiteDue to variations in ingredients and processing technologies, the geographical origin of fermented foods can have a profound impact on their technological properties and microbial diversity ,4. This The differences in technological properties, namely salt content and pH, of fermented meat products in the present study are in line with the findings of an earlier study, in which the same fermented meat products were investigated, albeit at another moment in time, thus representing other batches . GeneralLactobacillus was omnipresent in fermented meat products across all producing countries examined. Although the genus Lactobacillus has recently been reclassified into 25 genera, of which Latilactobacillus and Lactiplantibacillus contain the most relevant species for meat fermentation [Lactobacillus is reported here. The presence of Lactobacillaceae is not surprising as they are frequently encountered as one of the most important genera in fermented sausages, with multiple species such as Latilactobacillus sakei (formerly known as Lactobacillus sakei), showing great adaptation to the meat matrix [Pediococcus in some Belgian and German fermented meat products might be a consequence of its addition as a starter culture, as pediococci, such as P. pentosaceus, are sometimes added in Northern Europe and Northern America to hasten acidification under higher fermentation temperatures [Bacillus, Brochothrix, Carnobacterium, Corynebacterium, and Tetragenococcus. These species have nonetheless all been encountered previously to some degree in the natural microbiota of fermented meat products [With respect to the microbial diversity, application of amplicon-based HTS methods allowed studying the microbial communities with greater resolution compared to previous culture-dependent studies ,25. Sequentation , the SILt matrix ,25,49. Teratures ,50. Spanproducts ,53,54,55Staphylococcus was not encountered across all Belgian and German fermented meat product samples when targeting the V4 region, did not necessarily infer its absence, as differences in biomass and 16S rRNA copy numbers may cause less abundant genera to be under the detection limit [Staphylococcus, the MSA and MRS counts were comparable, with less than 1.0 log (cfu/g) of difference. Furthermore, high-throughput amplicon sequencing of a part of the tuf gene revealed diversified CNS communities in all fermented meat products examined. The CNS communities in French and Italian fermented meat products demonstrated a high relative abundance of S. xylosus, which was almost always accompanied by S. equorum. Other CNS species, such as S. carnosus and S. saprophyticus, were encountered intermittently and mostly in low relative abundances. This was in line with previous studies, where S. xylosus and S. equorum were frequently found to characterize Southern-European fermented meat products because they preferred relatively high pH and low fermentation temperatures [S. equorum, but exhibited a greater CNS species diversity. The latter was largely due to the presence of S. xylosus, S. saprophyticus, S. sciuri, S. gallinarum, S. carnosus, and S. vitulinus, which have also previously been found in spontaneously fermented Southern-European fermented meat products [The fact that the genus on limit ,57. Indeeratures ,54,58,59products ,61,62,63S. carnosus in Belgian and German fermented meat products does indicate starter culture use, as this species is not common in the natural microbiota of spontaneously fermented meat products but routinely used as a starter culture in Northern Europe [S. carnosus was still found, the relative abundance of S. xylosus increased and S. saprophyticus and S. vitulinus also emerged, all of which have been encountered in previous studies in spontaneously fermented meat products of similar acidity [When compared to French and Italian fermented meat products, the occurrence of less common genera and species and the overall lower bacterial loads in Spanish products suggest differences on the level of starter culture application, either because the process is based on spontaneous fermentation or because of poor adaptation of the starter culture bacteria involved to the processing conditions ,16,64. In Europe ,54,65,66 acidity ,67,68. A acidity ,64.Compared to the culture-dependent methodology applied previously on the same commercial fermented meat products , amplicoSeveral VOCs were detected in the fermented meat products examined, which could either be related to microbial metabolism , the addition of certain spices and herbs , or the processing applied  ,71,72,73tuf genes allows for an improved exploration of the species diversity of the microbial communities in fermented meats, in particular the staphylococcal communities. Amplicon-based HTS broadened the view on the microbial communities to also encompass several subdominant CNS species that previously may have been underreported. Furthermore, it permitted to emphasize the influence of the processing conditions on the bacterial diversity, indicative of pH and starter culture impact. In the present study, only bacterial communities were targeted using partial 16S rRNA and tuf gene sequencing. In the future it might be valuable to target other marker genes as well, to obtain a higher resolution image of the microbial communities, including an overview of yeast species diversity. Future applications of the aforementioned amplicon-based HTS methods offer great potential to further unravel complex microbial communities in fermented meat products and other fermented foods as well as to assess the impact of different processing conditions on the entirety of the microbial consortia present.The present study demonstrated that an amplicon-based HTS method targeting regions of the 16S rRNA and


Litsea cubeba L. essential oil (LCEO) is a natural essential oil with considerable antimicrobial activity, and it can gradually replace some chemical additives in the food industry. However, the genetic evidences of stress response of bacteria under sub-lethal treatment with LCEO is limited. To this end, transcriptomic analysis of Staphylococcus aureus 29213 under a low concentration of LCEO was performed. Bacterial RNA samples were extracted from 1/4 MIC (0.07 μL/mL) of LCEO-treated and non-treated S. aureus 29213. The transcriptional results were obtained by RNA sequencing (RNA-Seq). After treated with LCEO of S. aureus 29213, 300, and 242 genes were significantly up and down-regulated. Up-regulated genes were mainly related to cell membrane  stress stimulon including genes related to two-component regulatory system (VraS), histidine metabolism (hisABCD etc.) and L-lysine biosynthesis . Significant differences were also founded between LCEO-treated and non-treated groups in peptidoglycan biosynthesis related pathways. Down-regulated genes were related to nitrogen metabolism (NarGHIJ etc.), carotenoid biosynthesis  and pyruvate metabolism  of S. aureus 29213 in an LCEO-existing environment compared to the control. At the same time, we confirmed that LCEO can significantly affect the staphyloxanthin level of S. aureus 29213 for the first time, which is closely related to the redox state of S. aureus 29213. These evidences expanded the knowledge of stress response of S. aureus 29213 strain under sub-lethal concentration of LCEO. Litsea cubeba L. (L. cubeba) is a plant of the Lauraceae family and distributed in many countries in East Asia, and it is mainly distributed in the Southern and western parts of China . The main component of L. cubeba in the fruit leaf, flower and twig oils is citral, 1,8-cineole, while in the stem oil there are limonene, citronellal, and citronellol  is a serious bacterial pathogen that causes infections  and food poisoning in many animals and humans (S. aureus was stronger than that of any other months (S. aureus (MRSA) caused the leakage of intracellular macromolecules in cell  and the chemical compositions of LCEO was shown in S. aureus ATCC 29213 strain has the ability to form biofilm and to generate staphyloxanthin as well as it was often used in antimicrobial research  then inoculated into tryptic soy broth (TSB) at 37°C for 36 h with 250 RPM shake, then 10 μL cultured solution was incubated into TSB at 37°C, 12 h with 250 RPM shake for viability recover. One microliter cultured solution was taken by inoculation hard loop to inoculate on a plate with tryptone soy agar (TSA) at 37°C for 24 h. A single colony was incubated in a tube with 3.5 mL of TSB at 37°C with shaking at 250 rpm for 8 h before subsequent trials.One milliliter S. aureus 29213 strain was determined by the serial-dilution culture method.Minimum Inhibitory Concentration values of LCEO on S. aureus 29213 inoculum (approximately 104 CFU/mL), with a final concentration of LCEO solution of 71.4, 35.7, 17.8, 8.9, 4.5, 2.2, 1.1, 0.56, 0.28, 0.014, 0.070, and 0.035 μL/mL. After incubation at 37°C for 24 h, MICs were measured by visual inspection of the turbidity of broth in tubes  contained 1.75 mL LCEO solution and 1.75 mL of diluted in tubes . That isS. aureus 29213 strain culture (500 μL) which mentioned above and then incubated at 37 °C with 250 rpm shaking. Cell samples were taken for LCEO stimulation as the optical density value (OD600) at 0.8 (1 × 106 CFU/mL) and subsequently, 3 samples of O_S29 group  were treated with 1/4 MIC (0.07 μL/mL) of LCEO for 15 min. The control group  was without incubated with LCEO and were also kept in the same conditions for 15 min.A conical flask (150 mL) with TSB (45 mL) was inoculated with fresh Total RNA was extracted from 6 bacterial samples  separately by a Total RNA Isolation System (Promega) according to the manufacturer’s protocol. The quality of the RNA samples was examined using the Agilent 2100 Bioanalyzer. Library construction and Illumina sequencing was performed by Novogene China. An RNA-seq analysis was performed according to the protocol recommended by the manufacturer (Illumina Inc.) .S. aureus Newman (NC_009641). Differentially expressed genes (DEGs) were identified by the edgeR package based on Genes with two criterions: |log2(fold change)| > 0.58499 (|fold change| >1.50003) and p < 0.05For data analysis, RNA-Seq reads were mapped to the reference genome of To ensure the accuracy of RNA-seq data, 8 DEGs were selected for quantitative real-time PCR (RT-PCR) verification and they were based on two standards: (a) Functional classification. The 8 DEGs were selected from different GO terms and KEGG pathways which were significantly enriched. (b) The value of fold change. 4 DEGs were up-regulated and the other 4 DEGs down-regulated and with |fold change| > 1.8. All experiments were performed in triplicate with at least three independent experiments.TM Real-Time System (Bio-Rad). The thermal cycling conditions were as follows. After activation of the polymerase and a DNA denaturation step at 95°C, 40 amplification cycles were performed with a denaturation step at 95°C followed by an annealing and extension step at 55°C. The cDNA values were normalized with the value of 16s rRNA, which was constant in different conditions (data not shown).Quantitative real-time PCR assay was carried out as described previously with some modifications . The cDNS. aureus 29213 strain cultures were diluted 1:100 in tubes with 3.5 mL TSB at 250 RPM shaken and 37°C for 24 h with different lever of LCEO: 1/2 (0.14 μL/mL), 1/4 (0.07 μL/mL), and 1/8 (0.035 μL/mL) MIC. The control samples (without LCEO) were incubated in the same culture conditions. All the culture in tubes were centrifugated and then washed with distilled water for two times. Each tube was added into 2 mL methanol for the ultrasonic extraction of staphyloxanthin under 600 watt at 55°C for 20 min, and the supernatant with staphyloxanthin was obtained by centrifugation. The value of OD465 was measured as the biosynthesis level of staphyloxanthin. Three independent experiments were run in triplicate.Staphyloxanthin production was measured as described previously with some modifications . Brieflypost hoc analysis using Tukey test. Differences were considered significant at P < 0.05.Statistical analysis was performed using SPSS . Differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by S. aureus Newman (NC_009641)  and control (S29) cDNA libraries, respectively. These clean reads were mapped to the reference genome of _009641) . RNA-seqCRA002405 that is publicly accessible at https://bigd.big.ac.cn/gsa.The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive  in BIG Data Center (Nucleic Acids Res 2019), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession numbers p < 0.05, 542 DEGs of LCEO-treated group with 300 and 242 genes were up and down-regulated, respectively, compared to the control (Identified by adjusted |log2(fold change)| >0.58499 (|fold change| >1.50003) and  control . In the S. aureus 29213 which was treated by 1/4 MIC LCEO. As shown in The 542 DEGs were processed by Gene Ontology (GO) enrichment analysis for understanding the stress response at genetic level of Ten GO terms were enriched in the biological process, including amino acid transport, anion transport, organic anion transport, organic acid transport, carboxylic acid transport, amino acid transmembrane transport, anion transmembrane transport, organic acid transmembrane transport, membrane fusion, and cell adhesion.Ten GO terms were enriched in the cellular component, including extracellular space, viral envelope, viral membrane, extracellular region, cell wall, extracellular region part, integral component of membrane, virion, intrinsic component of membrane, and immunological synapse.In the category of molecular function, Ten GO terms  were significantly enriched.The 542 DEGs were mapped to the KEGG database, and then analyzed by KEGG pathway enrichment analysis to understand the involved pathways . As showS. aureus. In this experiment, 1/4 mic of LCEO also triggered cell wall (membranes) stress to S. aureus 29213.Study has shown that the leakage of intracellular macromolecules was caused by the destructive effect of 0.5 mg/mL (1 MIC) LCEO on the cell membrane of MRSA . ActuallS. aureus  annotated organism-specific genes in these steps were also down-regulated. These results suggested that the histidine biosynthesis of S. aureus 29213 was increased under the stimulation of 1/4 MIC of LCEO (Histidine protein kinases (HPKs) are a large family of signal-transduction enzymes. The typical HPK is a transmembrane receptor and it can autophosphorylate the conserved histidine residue of itself . Bacteri. aureus . Here, i of LCEO .S. aureus 29213 has increased under the stress of LCEO.Like alanine and glutamate, L-lysine is not only used to synthesize proteins but also a component of peptidoglycan of cell wall in gram-positive bacteria. Meso-2,6-diaminopimelate can be utilized in the biosynthesis process of peptidoglycan and L-lysine . ResultsvraS) encoding the sensor histidine kinase VraS in two-component system was up-regulated in LCEO treated S. aureus 29213  and its key enzyme (glucose-6-phosphate dehydrogenase) activity of methicillin-resistant S. aureus were inhibited by LCEO treatment (S. aureus 29213 was treated with LCEO for a short time (only 15 min). Although there was no significantly enriched, present data showed that almost all the screened genes involved in fructose and mannose metabolism, starch and sucrose metabolism, glycolysis/gluconeogenesis and citrate (TCA) cycle were down-regulated  and resource independence (like anaerobic metabolism under indirect nitrate supplyment) . Denitrisistance . TherefoFrom this part, we assumed that LCEO inhibited the metabolism of carbohydrate and nitrogen, so that in order to survive, the bacteria has to increase activity of fatty acid degradation.S. aureus and it distinguishes S. aureus from other Staphylococci even gram-positive cocci. Staphyloxanthin is biosynthesized by a series of steps and is the only C30 golden carotenoid of membrane-embedded. Staphyloxanthin is an important antioxidant as well as an anti-host neutrophil-based killing substance. Also, the biosynthesis of staphyloxanthin is under the oxygen-sensing and redox-signaling regulation of the two-component system  [EC:2.3.1.12] and dihydrolipoamide dehydrogenase: subunit E3[EC:1.8.1.4] were down-regulated compared with the control  in BIG Data Center (Nucleic Acids Res 2019), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession number WZ and HS designed this work. All authors reviewed and approved the final manuscript.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.