Underground shopping districts (USDs) are susceptible to severe indoor air pollution, which can adversely impact human health. We measured 24 volatile organic compounds (VOCs) in 13 USDs throughout South Korea from July to October 2017, and the human risk of inhaling hazardous substances was evaluated. The sum of the concentrations of the 24 VOCs was much higher inside the USDs than in the open air. Based on factor analysis, six indoor air pollution sources were identified. Despite the expectation of a partial outdoor effect, the impacts of the indoor emissions were significant, resulting in an indoor/outdoor (I/O) ratio of 5.9 and indicating elevated indoor air pollution. However, the effects of indoor emissions decreased, and the contributions of the pollution sources reduced when the USD entrances were open and the stores were closed. Although benzene, formaldehyde, and acetaldehyde exhibited lower concentrations compared to previous studies, they still posed health risks in both indoor and outdoor settings. Particularly, while the indoor excess cancer risk (ECR) of formaldehyde was ~10 times higher than its outdoor ECR, benzene had a low I/O ratio (1.1) and a similar ECR value. Therefore, indoor VOC concentrations could be reduced by managing inputs of open air into USDs. Public-use facilities are defined as facilities that are utilized by unspecified groups of people [Hazardous materials may cause severe indoor air pollution in underground shopping districts (USDs). This is because USDs have many different types of stores due to the characteristics of the facilities, and because ventilation facilities are often inadequate. Various types of goods, furniture , construction materials , and household electrical appliances  emit a variety of volatile organic compounds (VOCs). These compounds are present in indoor air and can adversely affect indoor air quality ,2,3. To f people . In SoutBenzene, formaldehyde, and acetaldehyde are classified as carcinogenic substances belonging to Groups A, B1, and B2, respectively, in the United States Environmental Protection Agency (EPA) Integrated Risk Information System (IRIS); they have been identified as substances that pose potential risks to the human body . FormaldAs in other countries, USDs for pedestrian traffic have been constructed in Korea to facilitate the passage of citizens while constructing underground stations and large buildings. USDs enable efficient use of space in large-scale urban centers and are also equipped with commercial facilities. However, in Korea, where south and north are divided, USDs were also used for evacuation purposes during the war. Previous studies on formaldehyde and VOCs have focused on measuring the concentrations of hazardous materials in indoor air produced by indoor pollution sources, while others have focused on outdoor pollution sources, indoor air quality in living spaces, risk assessments of exposure to hazardous materials, and pollution source estimation ,19,20,212. The number of shops in each USD (excluding empty rooms) ranged from 16 to 319 and included clothing stores (56%), followed by shops selling shoes or bags (8%), electronics stores selling mobile phones (6%), food stores such as restaurants or cafes (3%), cosmetics shops (2%), and nail shops (1%). Other unclassified stores such as convenience stores, institutions, and flower shops accounted for 24% of the stores. We divided the USDs into “open,” “semi-open,” and “closed” types, depending on the type of the entrance to the outdoors. The open type had no door at the entrance of the USD, and air could be exchanged freely through the entrance. The semi-open type had one door at the entrance that partially blocked the air flow. The closed type had a double door or an air curtain to separate the indoor and outdoor air. We also divided USDs into “open,” “mixed,” and “closed,” depending on the store entrance opening up to the passageway. For closed-type facilities, stores were separated from the passageway, whereas for open-type facilities, stores were connected directly to a passageway and had no entrance. In mixed-type facilities, stores with and without entrances coexisted.We investigated 13 USDs in 7 regions of South Korea . Measure2, and we assumed that remodeling did not have a significant impact, as it was completed over 6 months prior to the measurement period. An air conditioner was operated in four of the seven stores, and cleaning was carried out 1–2 times per day (vacuum and wet cleaning). Because USDs were dominated by open-type stores, the VOC characteristics of each store type were difficult to determine, even if measurements were made at each store. Therefore, for separate stores  clothing stores; (2) fashion accessory (leather goods) stores; (3) electronics stores; (4) restaurants; (5) cafes; (6) cosmetics shops; (7) nail shops) that could exhibit distinct VOC characteristics from the USD, indoor VOC concentrations were measured for 7 days at one indoor location. Although all seven store types were located adjacent to the road at ground level, the entrances were kept closed at all times. The areas of these stores ranged from 24.5 to 94.3 m2, three for areas between 10,000 and 20,000 m2, and four for areas >20,000 m2. We sampled outdoor air at one point within 10 m distance the entrance of each USD. The sampling period spanned from July to October 2017, and sampling was performed on three consecutive days for each USD.We determined the number of sampling points according to the standard methods for indoor air quality specified by the National Institute of Environmental Research, Korea . We sampAlthough we measured 58 VOCs, we analyzed 24 VOCs. We excluded 34 VOCs for which the concentrations were sometimes below the detection limits. The 24 VOCs analyzed in this study were aromatics , aliphatics , terpenes (α-pinene and d-limonene), oxygenated VOCs , and total volatile organic compounds (TVOC). Of these, benzene, toluene, ethylbenzene, xylenes, styrene, n-heptane, n-octane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, nonanal, n-butanol, α-pinene, and d-limonene were measured twice in 30 min intervals at a 100 mL/min flow rate using an MP-∑30  pump fitted with a sorbent tube  filled with 200 mg of Tenax-TA. In the sorbent tube, a tube conditioner  was used for the desorption of high-purity nitrogen gas (99.999%) at a 100 mL/min flow rate at 320 °C for 180 min. After sample collection, the sorbent tube was closed with a ¼ inch thread-type stopper filled with polytetrafluoroethylene (PTFE) ferrules, sealed with Parafilm, and was refrigerated until analysis. To account for any contamination that may have occurred during transport, field blanks were pre-treated, transferred, stored, and analyzed under the same conditions as the samples and were used for correcting the measured values. 2) of the calibration curve exceeded 0.99, and the method detection limits (MDL) ranged from 0.90 ng (n-heptane) to 5.14 ng . TVOC concentrations were calculated by applying the response factor (RF) of toluene to the sum of the total peak area eluted from n-hexane to n-hexadecane [The VOCs collected using Tenax-TA were analyzed using thermal desorption‒gas chromatography mass spectrometry (TD‒GCMS) with a GC-2000 Plus . The analytical conditions are listed in xadecane .Formaldehyde, acetone, acetaldehyde, butyraldehyde, benzaldehyde, and propionaldehyde were collected twice for 30 min at a flow rate of 500 mL/min using an MP-∑100  pump and cartridge  filled with silica gel coated with 2,4-dinitrophenylhydrazine (DNPH). By installing an ozone scrubber  filled with high-purity potassium iodide (KI) at the cartridge intake, the interference of ozone (which reacts with DNPH derivatives) was reduced. After sample collection, the cartridge was closed with the stopper previously enclosed with the cartridge, sealed with Parafilm, stored in an aluminum zipper bag to prevent contact with light, and refrigerated at 4 °C until analysis. Using cartridges with the same lot number as those used for sample collection, field blanks were transferred, stored, pre-treated, and analyzed with the sample cartridges and were used for correction after analysis. TM C18 , and acetonitrile and water at a ratio of 7:3 (v/v) were added at a rate of 1.0 mL/min. The column oven temperature was set to 30 °C, and detection was conducted at a UV wavelength of 360 nm. For the standard solutions, a TO11/IP-6A aldehyde/ketone-DNPH Mix 15 μg/mL solution  was diluted with acetonitrile and analyzed in five steps . The R2 of the analytical calibration curve exceeded 0.99, and the MDL at different analytical conditions ranged from 0.0004 μg/mL  to 0.0017 μg/mL . Note that the MDL of carbonyl compounds is given in μg/mL, whereas that of other VOCs is given in ng; this is because it depends on measurement and analysis methods, which differ by compound.For each cartridge, 5 mL of 2,4-DNPH derivatized aldehyde was extracted using acetonitrile into a volumetric flask with a vacuum manifold , i.e., solid-phase extraction. The extracted solutions were characterized using high-performance liquid chromatography . The column used for analysis was an AllureTo estimate the VOC sources, SPSS for Windows 20.0  was used to conduct factor analysis with varimax rotation extraction using descriptive statistical analysis, correlation analysis, and principal component analysis of the VOCs. Furthermore, we examined the variations in group means with the USD environmental factors such as the region and entrance, store shape, and the number of stores, to estimate the contribution of each VOC source. For benzene, formaldehyde, and acetaldehyde , the excess cancer risk (ECR) was assessed using the following equations :EC = (CA3) for public-use facilities regulated by the Indoor Air Quality Control Act of the Ministry of Environment; however, two of the 30 locations had formaldehyde concentrations that exceeded the threshold. Although the TVOC concentrations were below the threshold (500 μg/m3) in 11 of the 13 USDs, two USDs had concentrations above the threshold. There were stores nearby selling fashion accessories, clothing, and other general goods in two USDs where the concentration of formaldehyde and TVOC exceeded the thresholds. In addition, chemical scent such as adhesives were detected during measurement. It was measured in the passage of the USDs, but since 70–80% of the stores are the open type, it was estimated that the effect of the stores had affected the passage.The VOC concentrations measured at 30 locations in 13 USDs throughout South Korea are presented in 3, respectively, which are higher than those obtained in the present study. During that time, indoor air quality did not have a significant impact on citizens; thus, the TVOC concentration greatly exceeded the threshold. However, the amount of time spent indoors has since increased, and the effects and importance of indoor air quality have consequently increased; therefore, the relevant regulations have been strengthened. The standards for outdoor and indoor air quality have also become more stringent. However, only the benzene concentration of VOCs is regulated as hazardous substances in outdoor air, while other VOCs are regulated as ozone.In the previous studies of hazardous materials in shopping malls and department stores , formaldehyde concentrations were similar to those obtained in this study ,23. The In open air, the concentrations of benzene, toluene, ethylbenzene, xylenes, and styrene measured in Seoul in 1996  were not2/C3). In this study, the C2/C3 was approximately 2.65, which is similar to the results of Santarsiero and Fuselli [The indoor/outdoor (I/O) ratios of temperature, formaldehyde, and VOCs (except relative humidity) all exceeded 1. Compared to open air, indoor temperatures had relatively small deviations, and the relative humidity was lower; thus, suitable temperature and humidity conditions were maintained by air conditioning equipment in the USDs. Except for benzene and propionaldehyde, the I/O ratios of the VOCs exceeded 2, indicating that the effects of indoor pollution sources were more significant than those of open air sources. Benzene and propionaldehyde were highly correlated with open air, which suggested that they originated from open air emissions. Benzene, xylenes, ethylbenzene, and toluene were reported to be derived from automobile exhaust fumes ,35. In t Fuselli  and suggBased on the I/O ratios of VOCs in the USDs, the VOC contents were dominated by the types of stores in the USDs, the products sold at these stores, and the construction materials used in the USDs. Accordingly, the formaldehyde and VOC concentrations were measured in seven representative stores that reflected the types of stores in USDs. Formaldehyde concentrations were the highest in clothing stores, followed by nail shops, cafes, and electronics stores . TVOC co3) in the aisles of USDs [Formaldehyde concentrations were high in furniture shops that contained lumber products . In the  of USDs  determinFactor analysis was conducted using principal component analysis of the 24 types of indoor VOCs analyzed in this study. Six groups had eigenvalues > 1 and accounted for 85.6% of the cumulative variation . Prior tFactor 1 had high loadings of acetone, butyraldehyde, n-pentadecane, benzaldehyde, toluene, d-limonene, n-butanol, formaldehyde, styrene, and acetaldehyde and accounted for 28% of the total variation. Because materials with high I/O ratios, such as acetone, formaldehyde and acetaldehyde (oxygenated VOCs), styrene (aromatics), and d-limonene (terpenes), were included , factor Factor 2 had high loadings of n-tetradecane, benzene, n-undecane, n-dodecane, and n-tridecane and accounted for 15% of the total variation. Benzene is emitted by almost all processes involving VOCs, including volatilization of gasoline and automobile emissions ,41. DecaFactor 3 contained high loadings of aliphatics  and accounted for 13% of the total variation. Nonane, heptane, and octane are utilized in floor and wall coverings and are emitted from the volatilization of gasoline and diesel, which is similar to factor 2 . AccordiFactor 4 had high loadings of α-pinene, nonanal, and propionaldehyde and accounted for 11% of the total variation. Although pinene has the potential for external factors, such as biogenic or landfill emissions , the I/OFactor 5 had a high n-decane loading and accounted for 11% of the total variation. N-decane was also included in factor 2 and is emitted by solvent use, volatilization of gasoline, and surface coatings ,45,46. Tm,p,o-xylenes and accounted for 8% of the total variation. Although the I/O ratio exceeded 1, the correlation with outdoor VOCs was also high, likely due to the high emission contributions from fossil fuels [Factor 6 had high loadings of ethylbenzene and il fuels ,41. Howeil fuels .Increases in the number of stores resulted in increases in the total concentrations of the 24 VOCs; however, these increases were not due to constant changes in pollution source contributions. By increasing the number of stores, the store types diversified, and consequently, the pollutants generated by these stores also diversified , B1 , B2 , C (possible human carcinogen), D (not classifiable), and E (evidence of non-carcinogenicity) . Accordi−6 and 10−4 based on the maximum concentration near the pollutant source  [−6 indoors; they were present at levels at which the carcinogenic risk could not be regarded as negligible. In particular, the 95th percentile concentration of formaldehyde was approximately equivalent to the threshold stated in the Indoor Air Quality Control Act. However, the ECR indicates a close level that requires immediate action. In order to manage the formaldehyde concentration, it is necessary to prepare a management plan for periodic ventilation and operation of air conditioners in the USDs. Weng et al. [−3, which was higher than the value obtained in the present study.The EPA sets goals for ECR of 10ctively) . In thisg et al.  found thThe indoor and outdoor ECRs of benzene and acetaldehyde were not as high as those of formaldehyde; however, these pollutants need to be managed through continuous monitoring. In the USDs, benzene did not have a significant pollution source indoors and showed a high correlation with the open air. Hence, controlling benzene concentrations in the atmosphere is expected to be sufficient to reduce its risk indoors.In this study, VOC sources were estimated using indoor and outdoor concentrations of 24 VOCs for the purpose of managing indoor air quality in 13 USDs in South Korea. In general, formaldehyde and TVOC concentrations were below their respective thresholds and the concentrations reported in previous studies. This suggests that the indoor air quality in the studied USDs is generally well managed. Although the majority of VOCs had high I/O ratios due to the effect of indoor pollution sources, benzene and propionaldehyde were significantly affected by artificial impacts of open air sources such as automobiles and coal power generation circumjacent to the USDs.Based on factor analysis, the sources of indoor VOCs in USDs were of six types, including (1) emissions within the store, (2) indoor fuel use, floor and wall coverings, (3) outdoor fuel use, floor and wall coverings, (4) fossil fuel combustion and cleaning products, (5) solvent use and surface coatings, and (6) vehicle emissions and painting/printing products. The contributions of store emissions and indoor fuel  were high . Because the effect of indoor emission was significant, the effect of open air was insufficient to significantly impact indoor air quality.Highly carcinogenic benzene, formaldehyde, and acetaldehyde appeared to be present at harmful levels both inside and outside of USDs. The ECR of the 95th percentile concentration of benzene, formaldehyde, and acetaldehyde indicates that these substances should be under continuous observation. Benzene had a high correlation with the open air; therefore, its indoor risk would be reduced if the benzene concentrations in the open air were controlled.This study was based on data collected in the aisles of USDs and identified the VOC concentrations in these facilities. However, measurements were not conducted inside the stores, which limits the interpretations of the data. In future studies, additional measurements in stores selling various products should be conducted in USDs to identify the effects in different types of stores.


Objectives: To explore the prevalence of post-traumatic stress disorder (PTSD) symptoms and the factors influencing mental health symptoms in students aged 8–18 in Wuhan, China at 6 months after the COVID-19 pandemic was controlled.Methods: Questionnaires were distributed to students aged 8–18 in Wuhan through an online platform from September to October 2020, and 15,993 valid surveys were returned, resulting in a response rate of 75.4%. The data related to symptoms of PTSD, anxiety, depression, stress and psychological inflexibility levels, as well as demographic information about the population. Hierarchical multiple regression analyses were performed to examine the predictive effects.Results: In total, 11.5% of the students met the criteria for clinically concerning PTSD symptoms. Psychological inflexibility was associated with PTSD symptoms, depression, anxiety, and stress symptoms  in children and adolescents.Conclusion: This study investigated the impacts of COVID-19 on the mental health status among students aged 8–18 in Wuhan. Even at 6 months after the outbreak was brought under control, some students were still affected. Psychological inflexibility was correlated with psychological symptoms in students. Therefore, methods to reduce psychological inflexibility may help improve the mental health states of students as part of psychological interventions. The recent novel coronavirus disease (COVID-19) outbreak is characterized by rapid transmission and wide infection range, presenting great difficulty for effective control. As of 5:08 pm. CEST, July 12th, 2021, COVID-19 had spread to 223 countries, with 186,638,285 people infected and 4,035,037 related death . This puThe deadly nature of COVID-19 and its very high infection rate can induce post-traumatic stress disorder (PTSD) in people . PTSD isMany factors affect the level of mental health, including gender, age , educatiMost recent studies have focused on the mental health status of adults with COVID-19, with little study of children and adolescents. Therefore, the goals of this cross-sectional study were to understand the incidence of PTSD symptoms in students aged 8–18 in Wuhan 6 months after COVID-19 was controlled, and to explore the relation between psychological inflexibility and students’ mental health.We surveyed students aged 8–18 (fourth grade of primary school through third grade of high school) in Wuhan from 16th September to 9th October 2020, approximately half a year after COVID-19 was brought under control in Wuhan. Questionnaires were distributed through an online platform. Before the test began, students and their parents were told the purpose of the measurements and read the informed consent to understand that they could refuse to answer or quit answering at any time. A total of 21,218 questionnaires were collected. An indicator question in the questionnaire, which was “Please select the last option for this question,” was included. Participants who consistently chose one option and those who answered the indicator incorrectly were considered as not being serious and, therefore, were excluded. Data on 15,993 students remained for analysis (75.4%). The average age of the participants was 12.26 (SD = 2.14), and the detailed demographic information is shown in The Children’s Revised Impact of Event Scale  was considered statistically significant and a value of VIF < 5 was considered free of multicollinearity.SPSS 26.0 software was used for data analysis. Means and standard deviations (SD) were calculated to describe the scale scores. Cronbach’s α coefficient was used to represent internal consistency of CRIES-13, DASS-C21, and AFQ-Y8. Pearson correlation coefficients were calculated to describe the correlations between variables. Using the two category variables of gender and only child or not, the relationships between psychological inflexibility and psychological symptoms were explored using a hierarchical multiple regression analysis. Model 1 included demographic factors. In Model 2, the psychological inflexibility total score was added to the regression equation. Durbin-Watson (DW) was used to calculate the independence of the residuals of the dependent variables, and variance inflation factor (VIF) was used to measure the multicollinearity of the independent variables. A value of The average CRIES-13 score was 15.91 ± 11.14, with scores of 5.5 (SD = 4.51) for intrusion, 4.34 (SD = 4.53) for avoidance, and 6.06 (SD = 4.76) for hyperarousal. A total of 840 (11.5%) CRIES-13 scores were equal or higher than 30 points, the standard of clinical concern. The average scores of AFQ-Y8 and depression, anxiety, and stress dimensions in DASS-C21 were 34.42 ± 7.01, 9.65 ± 4.84, 9.59 ± 4.54, and 10.74 ± 5.39, respectively.p-values less than 0.01).Correlation analysis results are presented in A hierarchical multiple regression analysis of the CRIES-13 data showed that DW = 1.93, indicating that residual values of dependent variables were independent and lacked autocorrelation. PTSD symptoms measured by the CRIES-13 total score were regressed on demographic variables, with a 9% co-explanation variation for demographic factors. Gender, only child or not, age, perceived family atmosphere, perceived family economic level, personal physical health status and knowing people who were infected, were all found to be significantly associated with PTSD symptoms Model 1, . After aThe DW values of the regression models of depression, anxiety, and stress were 1.98, 1.97, and 2.01, respectively, indicating that the residuals of the dependent variables were independent of each other and lacked autocorrelation. This is the first study to explore the possible effects of COVID-19 on the mental health of students in Wuhan. The results showed that approximately half a year after COVID-19 was controlled, 11.5% of students aged 8–18 in Wuhan had clinically concerning PTSD symptoms caused by the epidemic. This result differs somewhat from previous research. Results of a meta-analysis of 39 studies showed that the prevalence of PTSD in children and adolescents 1, 2, 3, and 4 months after earthquake and flood disasters was 19.2, 30.0, 24.4, and 20.4%, respectively . Three yThis study revealed a significant correlation between PTSD symptoms caused by COVID-19 and adolescent depression, anxiety and stress levels, which is consistent with previous research. Early studies showed that depression is a factor that predicts PTSD symptoms . SubsequIn addition to psychological processes, such as psychological inflexibility, some demographic factors may also contribute to psychological symptoms. Age, perceived family atmosphere and family economic level may be predictors of four types of mental health disorders in adolescents. It is difficult to improve these factors through clinical interventions, but they allow us to identify at-risk children and may help involve adolescents’ parents into the process of psychological intervention, thereby comprehensively promoting the mental health of adolescents. As the first city to report COVID-19, the mental health status of Wuhan’s residents is of great significance for follow-up studies on psychological effects related to the epidemic. Future research may further explore the causal relationship between psychological inflexibility and psychological symptoms, as well as the significance of this relationship for clinical intervention.This study had the following limitations: First, 8–18 years old were studied, and the results may differ among smaller age groups. Future research should explore the mental health characteristics of students at different stages of education. Second, this study only collected the cross-sectional data of a large sample. Future studies should collect longitudinal data to better understand the development and interactions of mental health problems in adolescents after traumatic events, as well as the role of psychological inflexibility in this process. In addition, this study used a self-report instrument, which could make the results less clinically accurate. A more objective approach to assessing the mental health of participants may be used in the future. Finally, all the subjects in this study were from Wuhan; consequently, the measured results may not be representative of all populations. Future research should focus on the characteristics, developmental trajectory and influencing factors of PTSD and mental health in adolescents on a wider geographic scale.This study found that 6 months after the control of COVID-19, the prevalence of PTSD symptoms among adolescents in Wuhan was 11.5%. PTSD symptoms caused by the epidemic were significantly related to the adolescents’ current mental health states. Psychological inflexibility was associated with mental health disorder symptoms. This is the first study to investigate the prevalence of PTSD symptoms, and their relationship with psychological inflexibility, in children and adolescents. The results of this study have important implications for future research and planning for emerging diseases. They will help public health and educational systems better address the impacts of COVID-19, or similar disasters, on adolescent mental health.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Research Ethics Committee, Institute of Psychology, CAS. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.YC and ZZ made the main contributions to this study including the concept, and writing and revising the manuscript. FL and SL provided data analysis strategies for this study. JC provided suggestions on revisions and confirmed the final version to be published. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ichneumonidae of Germany is provided. The list represents an updated version of an earlier checklist published in 2001. The present list includes several records of species that are new for the German fauna and species that were discovered since the last checklist was published. The present checklist was compiled as part of the DNA barcoding projects at the Zoologische Staatssammlung München.A revised checklist of the Ichneumonidae from Germany, with 48 species recorded for the first time. Compared to the checklist published 20 years ago, the number of ichneumonid species recorded from Germany has increased by 312 species.The checklist includes 3,644 species of  Ichneumonidae is the largest family of insects in Germany, with over 3,600 species already recorded for the country and an estimated number of 4,000 species in total  and Bavaria (Barcoding Fauna Bavarica). A new checklist was deemed necessary because of the relatively large number of species that were added since the last checklist was published 20 years ago by Klaus The list was compiled as part of DNA barcoding projects at the Zoologische Staatssammlung München (ZSM) that aims at assembling DNA barcode libraries for Germany (German Barcode of Life Project, Ichneumonidae provided by The present checklist is an updated version of the checklist of German Cryptinaesensu lato into the three subfamilies Ateleutinae, Cryptinae and Phygadeuontinae (Neorhacodinae (Theroniini is recognised as a tribe of the subfamily Pimplinae, Pseudorhyssa is listed under Pimplinae and Hemiphanes under Cryptinae.The classification follows uontinae  and recoacodinae . FurtherIchneumonidae for Germany (Suppl. material Ichneumonidae, led to changes to the internal classification of the family (e.g. The present checklist comprises 3,644 species of ily e.g. . Table 2Latreille, 18025CE9081F-703E-5E54-86F7-9FF5968262D4Ichneumonidae, see Suppl. material For a complete checklist of German Ichneumonidae for Germany was presented by Horstmann as part of the "Verzeichnis der Hautflügler Deutschlands" (The first checklist of chlands" . An essechlands" .Sphecidae, 32 specialists were responsible for 247 species, whereas for the 3,332 species of Ichneumonidae, only one specialist was available (Ichneumonidae, in particular, species in genera of the subfamilies Banchinae, Campopleginae, Phygadeuontinae, Ctenopelmatinae and Ichneumoninae, are poorly defined and their identification remains most challenging.Klaus Horstmann initially hesitated with compiling the checklist because of the daunting task and because, in his opinion, a scientifically satisfactory checklist could not be achieved . The maivailable . Due to Using the comprehensive keys in With these limitations in mind, Horstmann followed a set of rules when preparing the checklist. First and foremost, only original records were considered for compiling the checklist. Faunistic records were excluded if they were suspected to be based on misidentifications, if they were based on varieties of a particular species or if their collecting locality was unclear. For example, species that were recorded from Germany before WWII without giving the exact locality might be extralimital and were, therefore, not included. Species were excluded if the original description was published before 1945 and the species never treated in a taxonomic revision or mentioned in the taxonomic literature after that year. Finally, species were excluded if their records were based on males from genera where males are virtually impossible to identify or genera where males had not been associated with conspecific females. Only in a few cases were these species included on a case by case basis . SpeciesThe present checklist basically follows the same rationale. Species that were added to the current checklist are based on records of reliably identified specimens in the taxonomic and faunistic literature. In a few cases, species not included in Horstmann's checklist were added, based on a recent critical evaluation of the species or the literature source.Ichneumonidae (The catalogue of umonidae  lists 4,umonidae . It is eumonidae . AlthougIchneumonidae are still undescribed. For Germany, this percentage is probably lower, but, assuming that 15-20% or even up to 30% of the German species are undescribed, the estimated number of species occurring in Germany ranges from 4,000 to 4,500 species. Most of these are species in genera with large numbers of undescribed species, including Campoplex Gravenhorst, Diadegma Förster, Lissonota Gravenhorst, Olesicampe Holmgren, Phygadeuon Gravenhorst and Stenomacrus Förster ( Förster .Ichneumonidae by 312 species or about nine percent. Closing the gap between the currently-known species and a complete inventory of German species will require substantial efforts in terms of collecting, specimen processing and taxonomic research, but the application of integrative taxonomic approaches that combine independent character systems, including morphology, DNA sequence data and host information, hold promise of expediting the discovery of new species of Ichneumonidae in Germany and the clarification of their taxonomy and biology.Twenty years after the last checklist was published, the present checklist raises the number of Germany species of E6862676-2000-55F7-9E7D-ED2B91BE36E310.3897/BDJ.9.e64267.suppl1Supplementary material 1IchneumonidaeChecklist of German Data typeSpecies checklistFile: oo_526684.xlsxhttps://binary.pensoft.net/file/526684Riedel, M., Humala, A., Schnee, H., Schwarz, M., Schmidt, S.


Chrysosporum, Cuspidothrix, Cylindrospermopsis, Raphidiopsis, and Sphaerospermopsis, using genetic markers such as rbcLX, rpoB, rpoC1, and cpcBA, and determined the amplification conditions for each pair of primers. The primer performances were verified on single or mixed nostocalean cyanobacterial isolates. The five primers allowed selective identification of all the target genera. In field samples collected during summer, when cyanobacteria flourished in the Nakdong River, the respective PCR product was observed in all samples where the target genus was detected by microscopic analysis. Besides, weak bands corresponding to Sphaerospermopsis and Raphidiopsis were observed in some samples in which these genera were not detected by microscopy, suggesting that the cell densities were below the detection limit of the microscopic method used. Thus, the genus-specific primers developed in this study enable molecular monitoring to supplement the current microscopy-based monitoring.The geographical range of invasive cyanobacteria with high toxigenic potential is widening because of eutrophication and global warming, thus, monitoring their appearance is necessary for safe water quality control. Most invasive cyanobacteria are nostocalean species, and their accurate identification by classical morphological methods may be problematic. In this study, we developed polymerase chain reaction (PCR) primers to selectively identify five invasive cyanobacterial genera, namely,  Anthropogenic freshwater eutrophication causes harmful cyanobacterial blooms in streams and lakes worldwide. With the air and water temperatures increasing because of global warming and climate changes causing sudden intense rainfalls or increased water shortage, the intensity and duration of harmful cyanobacterial blooms are expected to increase. Moreover, these environmental changes extend the geographical range of invasive cyanobacteria from their original to new habitats ,2,3,4. NCylindrospermopsis spp., Sphaerospermopsis spp., Cuspidothrix spp., and Chrysosporum spp., which have been reported to invade new habitats in various environments [Members of the genera belonging to the order Nostocales of cyanobacteria have the advantages of producing dormant cells and fixing nitrogen. Some members of this order are ronments ,6,7,8. TrbcL), nitrogen fixation (nif) functions, RNA polymerase beta subunit (rpoB), gyrase beta subunit (gyrB), and polyphosphate kinase (ppk) have been investigated as systematic genome markers [rbcLX, and rpoB genes were used to compare the results obtained by morphological classification and phylogenetic analysis of Nostocales [Aphanizomenon spp. using the 16S rRNA, rbcLX, and cpcBA genes [Anabaena depending on their habitat were revealed using the 16S rRNA, rbcLX, and rpoC1 genes [rbcLX, and hetR genes [Sphaerospermopsis, Cuspidothrix, and Chrysosporum, were separated from the original genus Aphanizomenon based on the polyphasic approach, including ultrastructural and molecular characterization along with the conventional morphological classification [Cylindrospermopsis and Raphidiopsis, is still under debate [Molecular phylogenetic systematic research has been conducted utilizing various genome sequences of cyanobacteria. In particular, genes encoding 16S/23S rRNA, the internal transcribed spacer (ITS), ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit  primers developed in this study are expected to allow accurate early detection of potentially toxic invasive cyanobacteria and, consequently, enable their effective management.In this study, as the first step in the molecular biological monitoring of invasive cyanobacteria of the order Cylindrospermopsis, Cuspidothrix, Chrysosporum, Sphaerospermopsis, and Raphidiopsis. To obtain the target gene region of the target cyanobacteria, Cylindrospermopsis sp., Cuspidothrix sp., Sphaerospermopsis spp., and Raphidiopsis sp. were isolated from the Nakdong River in Korea and maintained in monocultures [Chrysosporum spp. were not confirmed to appear in the Nakdong River, and therefore, Chrysosporum ovalisporum CS-1034 was obtained from the Australian National Algae Culture Collection. As a positive control, Cuspidothrix issatschenkoi NIVA CYA-711 was obtained from the Norwegian Culture Collection of Algae. Raphidiopsis mediterranea CS-1037 and Cylindrospermopsis raciborskii CS-1101 were obtained from the Australian National Algae Culture Collection  was used for genomic DNA extraction from cultured cyanobacteria and field samples according to the protocol provided with the kit. To increase the DNA extraction efficiency, the filtered sample stored at −80 °C was incubated at room temperature for 5 min, followed by the addition of lysis buffer. This mixture was sonicated twice for bursts of 10 s each, at a constant amplitude of 20% of 130watt using VCX 130PB . The concentration and purity of the DNA were measured using an Infinite M200 PRO microplate reader . The extracted genomic DNA was stored at −20 °C.Aphanizomenon, Cuspidothrix, Cylindrospermopsis, Sphaerospermopsis, and Raphidiopsis species were collected from the GenBank database registered in the US NCBI  and all sequence information registered at the time of this study was used; 147 sequences of 16S rRNA, 140 sequences of rbcLX, 133 sequences of cpcBA, 134 sequences of rpoC1, and 65 sequences of rpoB. The DNA sequence similarity and genetic distances of target genes within nostocalean cyanobacteria were analyzed. Gene segments with the same base sequences were used for common sequences in the same species to eliminate sequence variations such as a single nucleotide polymorphism (SNP) within strains. DNA sequence similarity and genetic distance analysis was conducted after cutting off both ends of the aligned sequences, and the Kimura two-parameter model [To develop genus-specific primers, the sequences of housekeeping genetic markers from er model  was usedMicrocystis aeruginosa was used as the outgroup.Phylogenetic analyses of target gene regions of the nostocalean genera were conducted using MEGA v.6  and Clushttp://bioinfo.ut.ee/primer3/ accessed on 20 February 2019). For the selection of genus-specific primers, amplification test by touchdown PCR was performed with annealing temperatures incrementally decreasing by −0.2 °C for each cycle from 62 °C to 50 °C. The characteristics and potential hairpin formation of the search-specific primers were verified using Oligo Calculator version 3.27. Optimization of the annealing temperatures for the selected primers was carried out by performing gradient PCR with annealing temperatures in the range of 50 °C to 62 °C. For amplification of the target gene region, a 20-μL reaction mixture was made by mixing 1 ng of genomic DNA, 10 pmol of each PCR primer, and 10 μL of AccuPower® Taq PCR premix . PCR was performed at 95 °C for 5min, followed by 30 cycles at 95 °C for 30 s, 55–59 °C  for 30 s, and 1 min at 72 °C, and finally 10 min at 72 °C.Based on the analysis of the collected sequences described in To verify the developed primers, one to three strains were cultured for each of the seven nostocalean genera used in this study, including the five-target invasive nostocalean genera a. GenomiTo confirm the developed PCR primers’ applicability to field samples, surface water was collected 500 m upstream of the Gumi weir on 1 June 2020. Seven cultured cyanobacterial strains were added at different combinations to the collected water, resulting in eight samples c, and PCCylindrospermopsis, Cylindrospermopsis-specific PCR primer pair (cyl2-cyl4) developed by Wilson et al. [To compare the specificity of the PCR primer pair developed in this study to genus n et al.  was usedFive target cyanobacterial strains were used as a positive control a, and strbcLX, rpoB, rpoC1, and cpcBA genes were used to identify the generic specificity of the target genes through phylogenetic classification.In this study, to design PCR primers that could selectively distinguish the typical invasive nostocalean cyanobacterial genera, the 16S rRNA, Sphaerospermopsis, Cuspidothrix, and Chrysosporum of the target cyanobacteria formed independent clades, and the genera Raphidiopsis and Cylindrospermopsis formed one clade with separate branches  formed an independent clade. C. issatschenkoi also formed one clade with Dolichospermum spp., split into two branches based on the phylogenetic analysis using the rbcLX sequences , and Sphaerospermopsis, Cuspidothrix, Chrysosporum, and Raphidiopsis showed a similarity of 94.6% with other cyanobacteria  showed a similarity of 83.4% with the other nostocalean cyanobacteria, which was lower than the total average sequence similarity of 88.3 ± 5.5% (n = 28), and the cpcBA gene was 74.6% similar to those of other compared taxa, which was close to the total average sequence similarity of 74.4 ± 10.2% (n = 45)  and 25 bp (reverse) in Sphaerospermopsis spp. was selected by a comparative analysis of multiple sequences from the nostocalean cyanobacteria listed in the NCBI database  . For thedatabase . ConsequrpoB sequences, C. issatschenkoi was 78.2% similar to the other nostocalean cyanobacteria, which was slightly higher than the total average sequence similarity of 78.0 ± 8.4% (n = 15). Based on the analysis of the cpcBA, rbcLX, and rpoC1 gene sequences, C. issatschenkoi had a similarity of 77.5%, 86.8%, and 76.0%, respectively, to the other nostocalean cyanobacteria, which were slightly higher or lower than the total average sequence similarity of 74.4 ± 10.2% (n = 45), 88.3 ± 5.5% (n = 28), and 75.6 ± 6.7% (n = 55)  and 6 bp (reverse) from those in the other genera. Thus, Cuspidothrix genus-specific primers, Cus-rpoB-F/R, were designed using this segment  . As C. i segment .rpoC1 and cpcBA genes of R. mediterranea to those of the other compared taxa were 66.4% and 71.7%, respectively, which was lower than the total averages . However, these genes in R. mediterranea showed 96.4% and 98.1% sequence similarity, respectively, with those of C. raciborskii, which was in the same clade by phylogenetic analysis  between R. mediterranea and C. raciborskii, found a characteristic sequence with differences of 1 bp (forward) and 3 bp (reverse) between two genera. Based on these results, Raphidiopsis genus-specific primers, Raphi-rpoC1-F/R, were designed  showed that the values  were lower than the total average values   and 8 bp (reverse) between C. ovalisporum and other species, and Chrysosporum genus-specific primers, Chry-cpcBA-F/R, were designed  . Based odesigned .DNA from morphologically and genetically similar members of the seven nostocalean cyanobacterial genera a, includCuspidothrix genus-specific primers Cus-rpoB-F/R were used with DNA from 11 strains of C. issatschenkoi (NRERC-650–652 and 654–661), while the Sphaerospermopsis genus-specific primers Sph-rbcLX-F/R were used with DNA from 7 strains of S. aphanizomenoides (NRERC-600–603 and 605–607) and 2 strains of Sphaerospermopsis reniformis  (NRERC-604 and 608). The results confirmed the amplification of PCR products of the correct sizes for all isolates  developed by Wilson et al. [Cylindrospermopsis and Raphidiopsis, suggesting that the two genera could not be differentiated  and one strain of R. curvata (NRERC-701), which were isolated from the Nakdong River, was only positive for the respective targets when using the primers Raphi-rpoC1-F/R and Cyl-rpoC1-F/R; however, PCR products were observed for the members of both genera when using the cyl2-cyl4 primers  and organic materials, that could affect the reaction of the five developed PCR primer pairs, cultures of the seven nostocalean cyanobacterial genera were added to the surface water collected from the Nakdong River (spike), and the reaction status of the primers was verified. Based on the microscopic analysis of the phytoplankton in the surface water collected at the Gumi weir section on 1 June 2020, the cell density of the total phytoplankton was 26,344 cells·mL strains c, PCR pr strains b. Based Aulacoseira spp. and Skeletonema spp.) and cryptophytes (Cryptomonas spp.) were dominant in two (Nos. 10 and 13) and one sample (No. 8), respectively. Cyanobacteria dominated the remaining ten samples. Microcystis spp., Dolichospermum spp., and Merismopedia spp. were dominant in seven , one (No. 3), and two (Nos. 1 and 5) samples, respectively. Among the cyanobacteria targeted by the PCR primers developed in this study, Cuspidothrix spp., Cylindrospermopsis spp., and Sphaerospermopsis spp. were observed in eight, two, and six samples, respectively, while Chrysosporum spp. and Raphidiopsis spp. were not observed in any of the 13 samples . Raphidiopsis spp. were not observed by microscopic analysis, but a weak band was detected by gene analysis (Nos. 2 and 7)  .rbcLX sequence contains rbcL and rbcX, the genes encoding two subunits of the RuBisCO (ribulose-bisphosphate carboxylase), separated by an intergenic spacer (IGS). rbcLX was used to bypass the limitations of rbcL, which had been conventionally widely utilized and was first used to establish the phylogenetic relationship between the genera Dolichospermum (formerly Anabaena) and Aphanizomenon by Gugger [rpoB and rpoC1 genes encode the beta and gamma subunits of RNA polymerase, respectively, and were used in previous studies for lineage differentiation, including a study on the evolutionary association of chloroplasts of eubacteria and cyanobacteria [rpoB was first used to supplement the limitations of the 16S rRNA in a study by Dalhllöf [rpoC1 was used, along with the 16S rRNA, hetR, and nifH genes, in a study of Thomazeau on the phylogeny of cyanobacteria in sub-Saharan Africa [C. raciborskii [cpcBA includes sequences of the cpcB and cpcA genes, encoding for phycocyanin subunits, and an IGS. cpcBA was first used as a complementary gene for addressing the limitations of the 16S rRNA gene in a phylogeographical study of the genus Synechococcus [cpcBA was also used as a geographical marker of the genus Cylindrospermopsis in a study by Dyble [The housekeeping genetic markers used in this study are all genes widely utilized in various phylogenetic studies of cyanobacteria to compensate for the limitation of 16S rRNA. The y Gugger . The rpobacteria . rpoB waDalhllöf , and rpon Africa , which siborskii ,33. cpcBhococcus . cpcBA wby Dyble . PhylogeCylindrospermopsis spp. and Raphidiopsis spp., which have a similar trichome shape, were distinguished by forming heterocytes. If conical heterocytes were formed at the end of the trichome, the organism was classified to the genus Cylindrospermopsis; if no heterocytes were formed, the organism was classified to the genus Raphidiopsis [Raphidiopsis and Cylindrospermopsis form a monophyletic lineage and are not distinguished [Raphidiopsis based on a phylogenetic study that used the 16S rRNA gene, 16S–23S ITS, and cpcBA sequences [C. raciborskii as Raphidiopsis raciborskii [rpoC1, and cpcBA genes  and sesame (Sesamum indicum) [Cylindrospermopsis and Raphidiopsis spp. in the 1st and 4th to 5th position from the 3′ terminus of the reverse primer can cause a mismatch with a probability of 22.5%, 31.7% and 32.9%, respectively. As a result, it was possible to distinguish these two genera in the actual PCR amplification. This result signifies that a high overall similarity of genes does not prevent the primer design and the taxonomic distinction between the two genera. Furthermore, this primer design method can increase primer specificity and be applied in other cyanobacterial species with high genetic similarity.In the conventional morphological classification system, idiopsis . Howevernguished . Hence, equences . Consequiborskii ,37,38,39BA genes . Howeverindicum) . AccordiS. aphanizomenoides from Aphanizomenon spp. or Dolichospermopsis spp. if an akinete adjacent to a heterocyte is not found, while Raphidiopsis spp. are morphologically similar to Cuspidothrix spp. and Cylindrospermopsis spp., particularly when the trichomes are young. Therefore, for a trichome at its early stage of proliferation, morphological characteristics that serve as identification keys might not have appeared, and thus there is a possibility that it was falsely identified as Aphanizomenon spp. or Cuspidothrix spp., which appeared at relatively high densities. Moreover, we cannot exclude that S. aphanizomenoides and Raphidiopsis spp. appeared at a low density and could not have been detected by our protocol of microscopic analysis but were detected by PCR. In the filamentous cyanobacteria, a few or dozens of cells form one filament, and if the concentration is less than one filament per milliliter, no microscopic detection is registered if 1 mL of water is analyzed, and also more time-consuming methods such as the concentration of samples are needed to detect by microscopy. However, molecular methods such as PCR amplification allow the detection of even a single cell, thus enabling identification at a very low cell density. In this study, PCR products were detected in samples in which target cyanobacteria were not detected by microscopic analysis of 1 mL, whereas DNA was extracted after filtering a minimum of 50 mL of river water. Furthermore, the shape of special cells, such as heterocytes, akinetes, and apical cells, is used for morphological identification of most nostocalean cyanobacteria. However, if a cyanobacterium is present at low concentration, it is difficult to observe a specific cell shape and to identify the organism accurately. Therefore, the use of the PCR primers developed in this study is expected to allow the accurate identification of the target cyanobacteria present at a low concentration.It is assumed that the discrepancy between the microscopic results and molecular monitoring results in the field samples is due to the difference in the two methods’ detection limits. It is challenging to distinguish Cuspidothrix and Cylindrospermopsis. For the genus Sphaerospermopsis, after isolating S. aphanizomenoides and S. reniformis and applying the primers developed in this study, the PCR product was amplified from DNA of both species, suggesting primer specificity, not for S. aphanizomenoides but the genus Sphaerospermopsis. In the case of Raphidiopsis spp., the PCR product was generated not only for R. mediterranea, isolated in Australia, but also for R. curvata, isolated from the Nakdong River, indicating that the primer selectively targeted the genus Raphidiopsis. Therefore, the primers developed in this study were determined to be genus-specific primers that can distinguish the target genera. We plan to additionally confirm their genus-specific properties after securing genetic information and cultured strains for other species in the target genera.Although this study was initially aimed to develop species-specific primers, due to the limited sequence information in the NCBI database (number of sequences and differences among the regions sequenced) and a shortage of positive control strains, primers were developed for representative species in each target genus. Although the developed primers distinguished the target genera, the reaction status was not investigated for different species in the genera C. issatschenkoi and C. raciborskii are toxic species that have been reported to produce anatoxin-a and cylindrospermopsin [Raphidiopsis brookii has been reported to secrete saxitoxin [R. curvata has been reported to secrete cylindrospermopsin [R. mediterranea secretes anatoxin [Chrysosporum spp. do not secrete saxitoxin, they carry the sxtA gene, suggesting their potential toxigenicity [S. aphanizomenoides has also been reported to have a potential toxin-producing capability [Among the target species used in this study, ermopsin ,19,36,41axitoxin , and R. ermopsin . Furtheranatoxin . Althouggenicity ,46, and genicity ,48. S. apability ,50. MoreChrysosporum, Cuspidothrix, Cylindrospermopsis, Raphidiopsis, and Sphaerospermopsis. Five pairs of PCR primers based on housekeeping genetic markers could accurately detect the target cyanobacteria in the culture and field samples. These results are expected to enable the early detection of potentially toxic invasive cyanobacteria and their effective management. Moreover, these primers can distinguish two genera which have high overall genetic similarity. These results show the applicability of molecular monitoring for morphologically and genetically similar cyanobacterial genera using highly specific primer pairs. The developed primers could distinguish different genera; however, species-level identification could not be achieved because the necessary genetic information for each species is not currently available and because of the lack of corroborative positive control possessed. If these challenges are addressed in the future, primers that can more accurately distinguish different species could be developed.In recent years, the habitats of invasive toxic nostocalean cyanobacteria are continuously expanding, so it is necessary to monitor their appearance in waters where they have never been found. In this study, we developed PCR primers that specifically identify and detect five common invasive nostocalean genera,


Background: During the COVID-19 pandemic, lockdown strategies have been widely used to contain SARS-CoV-2 virus spread. Children and adolescents are especially vulnerable to suffering psychological effects as result of such measures. In Spain, children were enforced to a strict home lockdown for 42 days during the first wave. Here, we studied the effects of lockdown in children and adolescents through an online questionnaire.Methods: A cross-sectional study was conducted in Spain using an open online survey from July (after the lockdown resulting from the first pandemic wave) to November 2020 (second wave). We included families with children under 16 years-old living in Spain. Parents answered a survey regarding the lockdown effects on their children and were instructed to invite their children from 7 to 16 years-old (mandatory scholar age in Spain) to respond a specific set of questions. Answers were collected through an application programming interface system, and data analysis was performed using R.Results: We included 1,957 families who completed the questionnaires, covering a total of 3,347 children. The specific children's questionnaire was completed by 167 kids (7–11 years-old), and 100 adolescents (12–16 years-old). Children, in general, showed high resilience and capability to adapt to new situations. Sleeping problems were reported in more than half of the children (54%) and adolescents (59%), and these were strongly associated with less time doing sports and spending more than 5 h per day using electronic devices. Parents perceived their children to gain weight (41%), be more irritable and anxious (63%) and sadder (46%). Parents and children differed significantly when evaluating children's sleeping disturbances.Conclusions: Enforced lockdown measures and isolation can have a negative impact on children and adolescent's mental health and well-being. In future waves of the current pandemic, or in the light of potential epidemics of new emerging infections, lockdown measures targeting children, and adolescents should be reconsidered taking into account their infectiousness potential and their age-specific needs, especially to facilitate physical activity and to limit time spent on electronic devices. Nearly 80–90% of school-age youth could not physically attend school in more than 160 countries during the first wave of the coronavirus disease 2019 (COVID-19) pandemic , 4. Herevia social media and the landing page of the Kids Corona Project. The questionnaire . Mann–Whitney test is applied to numerical answers; the reported p-value results from the corresponding one-sided test.The analysis was performed using R language . The sigThe study was conducted in accordance with the protocol and the Declaration of Helsinki , and following the relevant legal requirements . The study protocol, informed consent forms and data collection tools were approved by the Ethics Committee for clinical research of the Hospital Sant Joan de Déu, Barcelona, Spain (C.I. PIC-123-20), prior to study initiation.A total of 2,054 families answered the survey, of which 1,957 met the study inclusion criteria. The included surveys covered a total of 3,347 children: 1,463 aged 0–6 years, 1,284 aged 7–11 years, and 600 adolescents aged 12–15 years. All parents answered a questionnaire, one for each daughter or son, independently. Additionally, 13% of the older children (167 answers) and 17% of the adolescents (100 answers) completed the questionnaire specifically addressed to their respective age group. In 57% of the households there was at least 1 adult working in a highly COVID-19 exposed environment . The main participants sociodemographic characteristics are presented in At the time of survey completion, 6% of the households reported at least one family member having been affected or currently infected by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , and 12% reported at least one suspected case. The majority (84%) of families knew someone infected and 20% knew someone close who had died of COVID-19. Among families with either a suspected or microbiologically confirmed infection, 47% considered a 5–15 year-old child to have been the index case, 22% a 0–4 year-old, 12% a >16 year-old, and 19% reported a simultaneous infection among more than one household member (of varying ages). Median time to subsequent secondary cases was 6.5 days (interquartile range [IQR]: 3–10). When a case was confirmed or suspected at home, measures adopted varied widely between families: 42% followed a strict lockdown of the family, 21% a strict lockdown of the affected person, 16% used a facemask at home, 19% ensured an exclusive use of the sleeping room and bathroom, 19% restricted the use of common spaces, and 39% did not follow any of the aforementioned measures. Regarding parents' opinion about children's role in the pandemic, 42% perceived them to be at high risk of getting infected by SARS-CoV-2, 50% thought strict lockdown measures for children were necessary, 60% considered that children should have had permission to go outside in a controlled way since the first day, and 65% found re-opening of schools was appropriate (98% of them suggested to do it with safety measures in place). During strict lockdown, 88% of children never set foot on the street, while during relaxed lockdown the majority (54%) spent time outdoors at least 3–6 days a week for <1 h/day. Masks were worn by 95% of older children and adolescents and by almost half of the children below 6 years of age, even if this was not mandatory in Spain.The overall rate of good coping with confinement  was generally very positive. Parents scored their children an 8 [IQR: 6–9] both during strict and relaxed lockdown. Older children scored themselves a 7 [IQR: 5–10] and an 8 [IQR: 6–10], and adolescents scored themselves a 7 [IQR: 6–9] and an 8.5 [IQR: 7–10], during strict and relaxed lockdown, respectively . When asAccording to parents' perception, during strict lockdown 90% of older children and 81% of adolescents practiced less sports than usual , and 64%p = 0.000004 and p = 0.009). In older children, higher self-perception was observed for awakening in the middle of the night , sleeping more than usual , having a hard time falling asleep , and having nightmares . Similarly, increased self-perception in adolescents was observed for awakening in the middle of the night , sleeping more than usual , having a hard time falling asleep , and having nightmares , irritable (55%), and sadder (52%), with median intensities from 4 to 5 out of 10 during strict lockdown. In adolescents, these feelings were reported in 49, 46, and 41% individuals, respectively, with median intensities from 3 to 5 out of 10 during strict lockdown.Parents reported that at least one type of electronic device  was used more than usual during strict lockdown in 77% of younger children, 93% of older children, and 95% of adolescents. The most widely used device was television together with online audiovisual content, with 75% of younger children, 85% of older children, and 82% of adolescents using it more often than before the lockdown. Playing videogames also increased in 65% of older children and 62% of adolescents. In addition, adolescents also increased use of their smartphone (68%) and social media (61%). The use time of electronic devices increased with age, being 1–3 h/day the predominant option among all children and 3–5 h/day the predominant option among adolescents. Higher use of social media increased during lockdown in comparison with the usual utilization before lockdown, and was self-reported by 28% of older children and 52% of adolescents. These results are summarized in Parents scored satisfaction with remote schooling in a 0–10 scale, with a median of 6 [IQR: 4–9] for younger children, and with a median of 7 [IQR: 5–8] for older children. Children themselves scored it with a median of 7 [IQR: 5–8]. The most common score was 10 among the three groups. Both parents of adolescents and teenagers themselves scored schooling satisfaction with a median of 7 [IQR: 5–9], but in this group the mode score was 10 for parents and 8 for the students. When children were asked if they were satisfied with school activities, 26% of children and 35% of adolescents were not. Importantly, 81% of children reported to feel comfortable with schools re-opening compared to a 65% of adolescents, and 52% of the latter thought it was necessary.p = 0.047). In general, sleeping problems were less likely to happen among children who did sport during lockdown vs. the ones who did not (49.15 vs. 41.67%), especially regarding having trouble falling asleep and sleeping more than usual. Doing sport activities as usual seems to be mildly protective against sleeping problems when compared to spending less time than usual (p = 0.04). Spending more than 5 h per day looking at screens was a risk factor for some of the sleeping disturbances in teenagers, when compared to 1–3 h of daily use (p = 0.00087).In all age groups, we found higher number of reported sleeping problems (either by parents or by children) among those knowing someone who died of COVID-19 compared to those who did not. Older children who reported to have felt fear or anxiety about going out reported a higher incidence of sleeping disturbances than the ones who did not feel that way  and adolescents  who did sport as compared to those who did not. In addition, less physical activity than usual was also found to be associated with an increased feeling of sadness in adolescents, as compared to spending more time than usual doing sport (p = 0.01). The increased use of social networks above usual levels was found to be associated with increased anxiety levels in teenager reported answers (p = 0.0077). Among adolescents, being unsatisfied with academic training during lockdown was found to be associated with anxiety (p = 0.04), and fear of going out in the street was associated with increased perceived sadness (p = 0.00017).Regarding the reported feelings by children and adolescents, the score on irritability was significantly lower among kids  were also those who went most often outside their homes , 28. ThiA major strength of our study is the large number of families who answered the survey, largely superior to previous studies carried out in Spain and capturing information about 3,347 children. Another major strength is the collection of questionnaires directly from children and adolescents, which is not often included in similar studies. On the other hand, since our study is based on questionnaires answered by parents and their children after the lockdown periods studied, it could be affected by a recall bias and social desirability bias. However, the surveys performed to Spanish children and adolescents during the strict lockdown showed similar emotional and behavioral alterations , 24, 29.In summary, our study showed a negative impact of strict lockdown in the mental health and well-being of children and adolescents but also showed their high resilience and capability to adapt to new situations. Lockdown measures should consider their age-specific needs in future waves of the pandemic and in similar potential emergencies, facilitating physical activity and limiting time spent on electronic devices. Maintaining schools open or, at least, ensuring children can spend a minimum time outdoors, can help address these priorities.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study protocol, informed consent forms, and data collection tools were approved by the Ethics Committee for Clinical Research of the Hospital Sant Joan de Déu, Barcelona, Spain (C.I. PIC-123-20), prior to study initiation. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.QB, JG-G, SAj, BB, NB, RV, PM-M, and SAr: conceptualization and methodology. QB, AC-S, BN, and JG-A: survey preparation and software development. JG-A, JF, and AP-L: data collection, curation, data analysis, and statistics. SAj: writing-original draft. BB, NB, RV, PM-M, SAj, and JG-A: writing-review and editing. JG-G and QB: funding acquisition and project administration. All authors contributed to the article and approved the submitted version.www.mineco.gob.es) TEC2014-60337-R, DPI2017-89827-R, Networking Biomedical Research Center in the subject area of Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), initiatives of Instituto de Investigación Carlos III (ISCIII), and Share4Rare project (Grant Agreement 780262). B2SLab is certified as 2017 SGR 952. BB is a Beatriu de Pinós postdoctoral fellow granted by the Government of Catalonia's Secretariat for Universities and Research, and by Marie Sklodowska-Curie Actions COFUND Programme . NB was supported by an FPU predoctoral fellowship from the Spanish Ministry of Universities (FPU18/04260). JF acknowledges the support from the Serra Húnter program.This study was supported by Banco Santander and Stavros Niarchos Foundation, through KIDS corona platform. This study has been partially funded by Stavros Niarchos Foundation (SNF), Banco Santander and other private donors of Kidscorona. ISGlobal receives support from the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa 2019–2023 Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. This work was supported by the Spanish Ministry of Economy and Competitiveness (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plant breeding reduces the genetic diversity of plants and could influence the composition, structure, and diversity of the rhizosphere microbiome, selecting more homogeneous and specialized microbes. In this study, we used 16S rRNA sequencing to assess the bacterial community in the rhizosphere of different lines and modern cowpea cultivars, to investigate the effect of cowpea breeding on bacterial community assembly. Thus, two African lines (IT85F-2687 and IT82D-60) and two Brazilian cultivars (BRS-Guariba and BRS-Tumucumaque) of cowpea were assessed to verify if the generation advance and genetic breeding influence the bacterial community in the rhizosphere. No significant differences were found in the structure, richness, and diversity of bacterial community structure between the rhizosphere of the different cowpea genotypes, and only slight differences were found at the OTU level. The complexity of the co-occurrence network decreased from African lines to Brazilian cultivars. Regarding functional prediction, the core functions were significantly altered according to the genotypes. In general, African lines presented a more abundance of groups related to chemoheterotrophy, while the rhizosphere of the modern cultivars decreased functions related to cellulolysis. This study showed that the genetic breeding process affects the dynamics of the rhizosphere community, decreasing the complexity of interaction in one cultivar. As these cowpea genotypes are genetically related, it could suggest a new hypothesis of how genetic breeding of similar genotypes could influence the rhizosphere microbiome. Particularly, studies about plant-microorganism interactions in the rhizosphere have focused on bacterial communities that represent the most versatile and diverse groups of microbes found in soils2. Recently, Geisen et al.2 have estimated more than one million bacterial species in soils acting on some important biological processes, such as primary production and nutrient cycling.The interaction between plants and microorganisms in the rhizosphere regulates several biological processes important to plants. The rhizosphere is known as the narrow zone of soil, which is driven by root traits, being a specific zone where thousands of microbial species live in close association with plants3. On the other hand, different traits of the rhizosphere presented by distinct plants species can shape the pattern of the bacterial community. Indeed, different plant species4, the stage of development5, characteristics of root exudation6, and different cultivars8, can select specific bacterial groups and contribute to change diversity and richness of species. For instance, Araujo et al.5 assessed the influence of the stage of development in maize and cowpea on bacterial community in the rhizosphere and found that the structure and diversity of bacterial community varied significantly according to different plant species and, to a minor extent, to their development stage.In the rhizosphere, the bacterial community interacts with plant roots and stimulates plant development, inhibits pathogens, and solubilizes nutrients, so conferring positive effects on plants9. This process is usually done with the use of advancing generations and selection of lines with the obtention of modern cultivars10. Although being positive to humans being, plant breeding promotes a reduction in the genetic diversity of plants11 and it can, consequently, select more homogeneous and specialized microbes12. Therefore, plant breeding can potentially modify the composition, structure, and diversity of the bacterial community in the rhizosphere. Indeed, previous studies have reported that plant breeding and the advance of generation significantly impacted rhizosphere microbial communities and network assembly14, promoting a homogenization and specialization of the bacterial community in the rhizosphere as influenced by the decreased genetic diversity16.The influence of the rhizosphere on the response of the bacterial community in several plants species and conditions is well known, but it remains unclear how plant breeding shapes the assembly of the bacterial community in the rhizosphere. Plant breeding is a process involving the changes in plant traits to obtain superior characteristics, such as biotic and abiotic stress tolerance and grain yieldVigna unguiculata L. Walp.) has been submitted to intensive plant breeding, mainly to figure out modern cultivars with higher tolerance to biotic and abiotic factors and yield17. However, little attention has been paid to the effect of cowpea breeding on the bacterial community in the rhizosphere. The knowledge of the behavior of the bacterial community in the rhizosphere of cowpea is important since it could positively influence plant performance. Here, we hypothesized that the process of cowpea breeding would influence plants traits and consequently drive the pattern of the bacterial community. Thus, we used 16S rRNA sequencing to assess the bacterial community in the rhizosphere of different lines and modern cowpea cultivars, to investigate the effect of cowpea breeding on bacterial community assembly.As an important tropical plant species, cowpea (p = 0.0023). However, no significant differences were found in bacterial community structure between the rhizosphere of the different cowpea genotypes . The results also showed no differences in both richness and diversity of the bacterial community between each rhizosphere and the bulk soil  explained 58.9% of the total variation in bacterial operational taxonomic units (OTU) of which 41.8% and 17.1% are displayed on the first two axes, respectively , followed by Proteobacteria (23%), Firmicutes (16%), Planctomycetes (10%), Acidobacteria (8.5%), and Chloroflexi (4.5%) , Polyangiaceae (2), Nitrososphaeraceae (2), and Gemmataceae (2) presented negative correlation with cowpea genotypes traits, while Nocardioidaceae (3) and Unclassified Chloroflexi (2) showed positive correlations.To analyze the correlation between individual bacterial groups (at OTU level) and specific plant performance traits we calculated all possible Spearman’s rank correlations to identify possible bioindicators that influence cowpea plant growth and health Fig. . The anaStreptomyces, Acidothermus, Nitrososphaeraceae, and Paenibacillus. In line IT82D-60, the top five key species were Bradyrhizobium, Acidobacteria - Subgroup 6, Blastocatellaceae, Syntrophobacteraceae, and Solirubrobacter. Regarding cultivar BRS-Guariba, the top five key species belonged to Isosphaeraceae, Azospirillum, Pirellula, TK10, and Alicyclobacillaceae; while the key species found in the rhizosphere of cultivar BRS-Tumucumaque were Rhizobiaceae, Betaproteobacteriales, Candidatus Udaeobacter, Conexibacter, and Blastocatellaceae.The complexity of connections in the bacterial community was assessed by the co-occurrence network analysis Fig. . Bulk soThe putative prediction of the potential functional profile of the soil bacterial community has shown a significant core of potential functions represented by chemoheterotrophy, aerobic chemoheterotrophy, cellulolysis, and nitrification  to Brazilian cultivars (BRS-Guariba and BRS-Tumucumaque), accessed through their genealogies, did not affect the richness, diversity, and overall structure of the bacterial community in the rhizosphere. This similarity among the different cowpea genotypes could be explained due to their genetic similarities  than the African lines (26.7%), revealing a higher similarity within the modern Brazilian cultivars.In this study, we assessed the bacterial community of two African lines and two modern Brazilian cowpea cultivars to address the hypothesis that the breeding process affects the microbiome assembly in the rhizosphere. First, the PCA analysis showed a different bacterial community structure between bulk soil and rhizosphere. This is expected since the rhizosphere is influenced by plant exudates and promotes differences in the microbial communities’ structure when compared with the bulk soil24. Firmicutes comprises some well-known plant growth-promoting bacteria, such as Bacillus and Paenibacillus, which are known to colonize the rhizosphere of cowpea5. Regarding Bacteroidetes and Gemmatimonadetes, these phyla present the ability to efficiently use carbohydrates released by the roots, being favored by root exudation25. Thus, our results showed a significant influence of rhizosphere on both bacterial phyla. Indeed, some studies with leguminous plants have found an increased abundance of these same phyla in the rhizosphere of soybean, common bean, and cowpea20.The composition of the bacterial community showed an enrichment of the phyla Proteobacteria, Firmicutes, Bacteroidetes, and Gemmatimonadetes in the rhizosphere of cowpea. Proteobacteria consists of a group of beneficial bacteria, mainly those involved in biological N fixation, which is commonly found in leguminous plants, such as cowpea5. Also, the order Gaiellales and the genera Ammoniphilus, Conexibacter, and Sphingomonas are aerobic bacteria that use several organic sources that can be released by the roots29 while Xanthobacteraceae and Frankiales are recognized as N-fixers in roots of leguminous plants30. Thus, these bacterial groups are positively influenced by the rhizosphere environment and present a beneficial whole to the plant host.In general, when considering the top 10 most abundant bacterial OTUs, which together accounted for 21.1% of the total sequences, we observed the dominance of some interesting bacterial groups in the rhizosphere. For instance, 10% of all the sequences were affiliated to the order Bacillales, which consists of a group of bacteria involved in the plant growth-promoting in the rhizospherePaenibacillus, Polyangiaceae, Nitrososphaeraceae, and Gemmataceae, do not contribute to increasing plant yield. Interestingly, Paenibacillus is a well-known plant growth-promoting bacteria that contributes to cowpea yield31, but in our study, this bacterium was negatively correlated with yield traits (seeds per pod and pod length). It may mean that this native Paenibacillus could differ from those improved Paenibacillus strains used in cowpea. Also, maybe the beneficial traits of Paenibacillus are not related to plant yield under the conditions of our experiment. It was previously reported that this genus induces drought tolerance and antagonizes pathogens32, which is not the case in this study. On the other hand, groups affiliated to Nocardioidaceae and Unclassified Chloroflexi were positively correlated with growth and productive traits. For instance, Nocardioidaceae is a family of nitrifier bacteria that acts on nitrification and contribute to increasing soil nitrate33. Thus, these bacteria can influence positively the growth and yield of cowpea.The analysis of correlation showed that specific bacterial groups presented a negative correlation with yield. This result indicates that some bacterial groups that colonize the cowpea rhizosphere, such as 35. Second, there was a variation between genotypes where both African lines presented similar numbers of nodes and edges, while Brazilian cultivars were different. The bacterial community in the rhizosphere of BRS-Guariba and BRS-Tumucumaque presented the highest and lowest complexities, respectively. This result clearly shows that plant breeding affects the complexity of the rhizosphere microbiome. Rossmann et al.14 showed that microbial networks of wheat landraces formed a more intricate network topology than wheat cultivars, revealing a loss of microbial interactions within the rhizosphere niche. We also observed this trend in the BRS-Tumucumaque, but for the BRS-Guariba the network complexity increased. The decrease in complexity of bacterial communities in the rhizosphere of the cultivar BRS-Tumucumaque may be related to your parentage with the cultivar BRS-Guariba , with loss of genetic variability (inbreeding) and due to the genetic constitution of each cultivar, which are the result of different crosses  and an annual mean temperature of 28 °C. The soil used in this study is classified as a Fluvisol, the type of soil where cowpea is commonly cultivated in Northeastern, Brazil, and it was collected at 0–20 cm depth. The soil samples were collected in an area where cowpea has been growing during the last ten years. Soil chemical properties were estimated according to Tedesco et al.44, being: pH – 6.1; organic C – 9.2 g kg-1; P – 4.9 mg kg-1; K – 32.5 mg kg-1; base saturation (V) – 58%.The experiment was conducted in a greenhouse at the Department of Soil Science, from the Federal University of Piauí, Brazil. The climate is tropical dry with a mean precipitation of 1,300 mm yr45. All genotypes are identified and deposited in the Germplasm Bank of Cowpea from ‘Empresa Brasileira de Pesquisa Agropecuária – EMBRAPA’, in Teresina (Embrapa Meio-Norte), Piauí, Brazil. The permission to use these genotypes was obtained from Embrapa Meio-Norte. Five seeds of each genotype (lines and modern cultivars) were sown per pot and ten days after germination, plants were thinned, leaving one individual plant per pot. Pots were irrigated daily with sterilized water to maintain soil moisture at 80% of field capacity. The temperature of the greenhouse was controlled at 30 °C. In this study, polyvinyl chloride pots  were filled with 5 kg of soil and the experiment was arranged in a completely randomized design with three replications. Two African lines (IT85F-2687 and IT82D-60) and two Brazilian cultivars (BRS-Guariba and BRS-Tumucumaque) of cowpea were selected following a generation advance and genetic breeding (Table Plants were collected 40 days after emergence (corresponding to the flowering period). Rhizospheric soil sampling was performed as follows: roots and adhering soil of each plant were placed onto a 1 mm mesh sieve and washed thoroughly with a gentle stream of tap water to remove the soil. All soil samples were stored at − 20 °C prior to DNA extraction and analysis.DNA was extracted from 0.5 g  of soil using the Power Lyzer Power Soil DNA Isolation Kit , according to the manufacturer’s instructions. The DNA extraction was performed in triplicate for each soil sample. The quality and concentration of the extracted DNA were determined by using NanoDrop 2000 spectrophotometer .46. Each 25 μl PCR reaction contained the following: 12.25 μL of nuclease-free water , 5.0 μL of buffer solution 5x (MgCl2 2Mm), 0.75 μL of solution of dNTP’s (10 mM), 0.75 μL of each primer (515 YF 40 μM e 806 R 10μM), 1.0 unit of Platinum Taq polymerase High Fidelity in a concentration of 0.5 μL , and 2.0 μL of template DNA. Moreover, a control reaction was performed by adding water instead of DNA. The conditions for PCR were as follows: 95 °C for 3 min to denature the DNA, with 35 cycles at 98°C for 20 s, 55 °C for 20 s, and 72 °C for 30 s, with a final extension of 3 min at 72°C to ensure complete elongation.The V4 region of the 16S rRNA gene was amplified with region-specific primers (515F/806R)After indexing, the PCR products were cleaned up using Agencourt AMPure XP – PCR purification beads , according to the manufacturer’s manual, and quantified using the dsDNA BR assay Kit  on a Qubit 2.0 fluorometer . Once quantified, equimolar concentrations of each library were pooled into a single tube. After quantification, the molarity of the pool was determined and diluted to 2 nM, denatured, and then diluted to a final concentration of 8.0 pM with a 20% PhiX  spike for loading into the Illumina MiSeq sequencing machine .47, using the consensus method to remove any remaining chimeric and low-quality sequences. After filtering, approximately 355,000 high-quality sequences were obtained, with an average of ~19,600 sequences per sample. Singletons and doubletons were removed, and the samples were rarefied to 8600 sequences, following the number of the lowest sample. The taxonomic affiliation was performed at 97% similarity using the Silva database v. 13248, and the generated matrix was further used for statistical analyses. The sequences were submitted to the NCBI Sequence Read Archive under the identification PRJNA751574.Sequence data were processed using QIIME 2 version 2019.10. Firstly, the sequences were demultiplexed and quality control was carried out using DADA249. We also used Past to calculate the richness of observed OTUs and Shannon’s diversity index, and the comparison was based on Tukey’s HSD test. A Venn diagram was constructed to verify the proportion of groups exclusive and shared between treatments using the webtool Venny 2.150. To determine the differential composition of bacterial groups among treatments, we used the software STAMP51. P-values were calculated based on two-sided Welch’s t-test, and correction using Benjamini-Hochberg FDR. In addition, to investigate the correlation between bacterial groups and cowpea plant performance properties we used Spearman’s rank correlation coefficient using R52. We then used network analysis to assess the complexity of interactions among bacterial taxa in each treatment. For this, non-random co-occurrence analysis was carried out using the Python module ‘SparCC’53. For this, a table of frequency of OTUs was used for analysis, and SparCC correlations were calculated, and only strong (>0.9 or <− 0.9) and highly significant (p < 0.01) correlations were selected. The nodes in the reconstructed network represent OTUs, whereas the edges represent significantly positive or negative correlations between nodes. The network graphs were based on a set of measurements, including the number of nodes, number of edges, modularity, number of communities, average node connectivity, average path length, diameter, and cumulative degree distribution. The network visualization and properties measurements were calculated with the interactive platform Gephi54. Finally, to further predict the relevant potential functions performed by the bacterial community, we performed a functional annotation using FAPROTAX53, a database that maps prokaryotic clades (e.g. genera or species) to established metabolic or other ecologically relevant functions, based on cultured strains. For this, a table of frequency of taxa at the genus level was used as input and converted into a putative functional table and the comparisons were done between bulk soil and the rhizosphere of the different cowpea genotypes.To compare the bacterial community structure between treatments we used Principal component analysis. First, the data matrix was initially analyzed using Detrended correspondence analysis (DCA), indicating a linear data distribution and the best-fit mathematical model was the PCA. To test whether the different niches  and cowpea genotypes harbored significantly different active bacterial communities we conducted a two-way permutational multivariate analysis of variance (PERMANOVA). PCA analysis was conducted using Canoco 4.5  and PERMANOVA using Past v.4Supplementary Information 1.Supplementary Information 2.


Ramadan fasting creates changes in lifestyle, causing biochemical alterations that affect glucose metabolism and insulin sensitivity. This study aims to assess the impact of Ramadan fasting on glycemic control and Fetuin-A, a glycoprotein that affects insulin resistance, in patients with type 2 diabetes mellitus (T2DM).This was a prospective study done among 37 patients with T2DM from Internal Medicine Polyclinic in a hospital in Jakarta, Indonesia. Anthropometric data as well as Hemoglobin A1c (HbA1c), Fasting Blood Glucose (FBG), and Fetuin-A levels of the subjects were measured in three time points: before, during, and after Ramadan fasting. A bivariate analysis was done to see the effect of Ramadan fasting on those parameters.Ramadan fasting reduced Fetuin-A levels  four weeks after the end of Ramadan compared to pre-Ramadan. After two weeks of Ramadan fasting, we found a significant reduction in body weight, BMI, FBG, and HbA1c levels which rebounded to baseline level after Ramadan.Ramadan fasting was associated with a significant decrease in Fetuin-A level post Ramadan. Ramadan fasting, Diabetes mellitus, Fetuin-A, Insulin resistance, Glycoprotein Home of the largest Muslim population in the world, Indonesia has reported a dramatic increase in the prevalence of DM which reached 10.9% in 2018 , 4.Ramadan fasting is the act of restraining oneself from eating, drinking, taking medications and/or smoking between dawn and sunset every year for one lunar month . These cIt is important to note that in addition to inconsistencies between studies regarding the impact of Ramadan fasting on the metabolic and clinical profile of DM patients, only limited data are available studying the impact of Ramadan fasting on molecular markers of insulin resistance, the main pathogenesis of T2DM. Fetuin-A is a glycoprotein produced primarily by hepatic cells and adipocytes  that hasIn summary, the impact of Ramadan fasting on the metabolic and clinical outcome of DM patients might be on the slightest, however, its impact on subclinical level has rarely been assessed. Our study aims to assess the impact of Ramadan fasting on Fetuin-A level, a glycoprotein associated with insulin resistance.2This prospective observational study was conducted among T2DM patients who fasted during Ramadan at the Metabolic Disorder, Cardiovascular and Aging Laboratory of The Indonesia Medical and Education Research Institute (IMERI), Faculty of Medicine Universitas Indonesia (FMUI). This study was part of The Diabetes and Ramadan Study Project 2019 held by the Division of Endocrinology and Metabolism, Internal Medicine Department, FMUI, Jakarta, Indonesia. Detailed methods of the Ramadan study projects have been published elsewhere . Ethics The inclusion criteria were type 2 DM patients aged 40–70 years who fasted for a minimum of 14 days and consented to participate in this study. Subjects with cardiovascular disease, kidney disease, severe liver disease, chronic gastrointestinal disease, autoimmune disease, and history of hospital admission due to severe hypo- or hyperglycemia one month prior to Ramadan were excluded from the study. Changes of fetuin-A level throughout Ramadan were the primary outcomes of this study. The sample size was calculated using the formula for the average of two independent populations, which yielded a minimum of 30 subjects.2.12 was considered as overweight, while BMI of ≥25 kg/m2 was considered as obese according to WHO Asia Pacific criteria and median [Interquartilerange (IQR)]. We analyzed the changes of the study parameters before (T0), during (T1), and after Ramadan fasting (T2) using paired t-test between T0 and T1, T1 and T2, as well as T0 and T2 for normal data distribution. Wilcoxon test was done for non-normal data distribution. P value of <0.05 was considered significant.3Thirty-seven subjects were included in this study. Most subjects were women (54.1%) with a median age of 48 (44–53) years old. The baseline characteristics of the subjects are available in After 14-days of fasting, we observed a reduction in Fetuin A level (from 5.35 (2.91–7.81) to 3.68 (2.33–5.89), The reduction of Fetuin A level after 14 days of fasting was in line with a parallel significant decrease of body weight (p < 0.001), BMI (p < 0.001), FBG (p = 0.013), and HbA1c (p = 0.004). However, four weeks after the end of Ramadan, in contrast to the further reduction we observed in the Fetuin A level, all of those metabolic and clinical parameters rebounded to their baseline levels .4This prospective observational study was performed among patients with T2DM who practiced Ramadan fasting consecutively for a minimum of 14 days. In this study, Fetuin-A levels were significantly lower in the 4 weeks after Ramadan fasting compared to baseline. In addition, we found a significant decrease in bodyweight, BMI, FBG, and HbA1c after 2 weeks of fasting, which rebounded 4 weeks after Ramadan fasting.As increase in Fetuin-A levels are associated with the increase of insulin resistance, the decrease levels of Fetuin-A during and after Ramadan fasting found in our study might show the beneficial effect of Ramadan fasting in the cellular levels. Lifestyle interventions, such as dietary changes and physical activity, have been shown to decrease Fetuin-A levels , 18. PreIn contrast to the rebounded metabolic and clinical parameters 4 weeks after the end of Ramadan fasting, interestingly, the serum levels of Fetuin-A still decreased, even statistically significant compared to baseline. This might be caused by a delayed effect of body weight decrease during fasting or continuous beneficial response of Ramadan fasting. Either way, it might suggest the presumably long-term effect of Ramadan fasting. A similar finding also observed by Blüher et al  in obesePrevious studies have shown a similar weight-decreasing effect of Ramadan fasting , 24, 25.Another mechanism thought to play a role in the weight loss during Ramadan fasting is the more efficient fat utilization observed during fasting  A study The improved glycemic control during Ramadan fasting in our study, as shown by FBG and HbA1c levels, supports findings from previous studies , 34, 35.In our study, in concordance with previous studies , 15, we This study is the first to observe the impact of Ramadan fasting on T2DM patients toward Fetuin-A level in association with other clinical and metabolic parameters. Observation and measurement of subjects before, during, and after Ramadan fasting could be considered as the strength of this study although with limited number of subjects. However, Fetuin-A level and other parameters were significantly decreased after Ramadhan fasting could be affected due to the naturalistic observation condition from this study. In addition, we treated the same attention to all participants and this can be applied in primary care setting in Indonesia to give safety recommendation for patients with T2DM who choose to fast during Ramadhan. Moreover, the length of Ramadhan is only 30 days annually, thus extended intermittent fasting might have a beneficial changes in T2DM patients due to the changes of feeding habits and calorie restriction during fasting.5In summary, Ramadan fasting is associated with a persistent reduction in Fetuin-A level despite a temporary improvement in other metabolic parameters. Future studies with more patients and multivariate analysis are needed in order to analyse the inflammatory markers and insulin resistance levels this would be needed to get a clearer picture of the effect of Ramadan fasting on Fetuin-A levels and its associations with those parameters.Dante Saksono Harbuwono and Dicky Levenus Tahapary: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Brama Ihsan Sazli: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Budiman Darmowidjojo and Sukamto Koesnoe: Conceived and designed the experiments; Analyzed and interpreted the data.Farid Kurniawan: Performed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.10.13039/501100006378Universitas Indonesia (NKB-4110/UN2.RST/HKP.05.00/2020).This work was supported by Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.