Major depressive disorder (MDD) involves changes in lipid metabolism, but previous findings are contradictory. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is considered to be a regulator of lipid metabolism. To date, the function of MANF has been studied in many brain disorders, but not in MDD. Therefore, to better understand the role of lipids in MDD, this study was conducted to examine lipid levels in the serum of MDD patients and to investigate the potential function of MANF in MDD. First, the data on total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) in serum from 354 MDD patients and 360 healthy controls (HCs) were collected and analyzed. The results showed that there were significantly lower concentrations of TC and LDL-C in MDD patients compared with HCs, and TC levels were positively correlated with LDL-C levels. Bioinformatics analysis indicated that MANF/EWSR1/ANXA6 pathway might serve as the connecting bridge through which hypolipidemia played a functional role in MDD. Second, to verify this hypothesis, serum samples were collected from 143 MDD patients, and 67 HCs to measure the levels of MANF, EWSR1, and ANXA6 using ELISA kits. The results showed that compared to HCs, MDD patients had a significantly lower level of MANF and higher levels of ANXA6 and EWSR1, and these molecules were significantly correlated with both TC level and Hamilton Depression Rating Scales (HDRS) score. In addition, a discriminative model consisting of MANF, EWSR1, and ANXA6 was identified. This model was capable of distinguishing MDD subjects from HCs, yielded an area under curve of 0.9994 in the training set and 0.9569 in the testing set. Taken together, our results suggested that MANF/EWSR1/ANXA6 pathway might act as the bridge between hypolipidemia and MDD, and these molecules held promise as potential biomarkers for MDD. Major depressive disorder (MDD) is a major mental disorder with high mortality and disability , 2. The Nowadays, diagnosis in psychiatry is still based on symptomatology and lacks any molecular foundation, which limits the development of diagnostic measures. Psychiatric diseases are increasingly being studied by using peripheral tissues such as blood from patients –14, and These findings emphasize the importance of serum lipids in MDD and MANF in regulating lipid metabolism. However, the concentration and role of MANF in the serum of MDD patients have received considerably less attention. Further investigations of MANF in MDD can help us better understand the role of lipids in the onset and development of depression, as the results may provide a valuable network among serum lipids, MANF and MDD. Therefore, the present study was conducted to assess the changes of serum lipid levels in MDD patients and investigate the potential role of lipid metabolism regulator MANF in depression. Furthermore, the correlative relationships of MANF and its related molecules EWSR1 (Ewing sarcoma breakpoint region 1) and ANXA6 (Annexin A6) with clinical characteristics and serum lipid levels were also investigated.The study received approval from the local ethics committee of Chongqing Medical University. Written informed consent was obtained from each participant before blood sampling. In total, 714 subjects ) were included between November 2021 and June 2022 to assess the serum lipids level, and another 210 subjects (sample set 2: 143 MDD patients and 67 HCs) were enrolled to study the underlying role of serum MANF in depression. The diagnosis of MDD was confirmed by two trained psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders-version IV criteria, and the International Statistical Classification of Diseases and Related Health Problems criteria, 10th revision. All MDD patients enrolled from the Department of Psychiatry, First Affiliated Hospital of Chongqing Medical University. Considering that antipsychotic medication may affect the levels of serum lipids, MDD patients were divided into drug-naive MDD (DN-MDD) group and drug-treatment MDD (DT-MDD) group. The DN subgroup was defined as first-episode MDD and never received any antidepressant treatment. And the DT subgroup was defined as having only received antidepressant treatment before. HCs were recruited from the Medical Examination Center, First Affiliated Hospital of Chongqing Medical University. Subjects receiving fatty acid supplements, statins, interferon, or cortisone in the previous three months were excluded from all groups. Meanwhile, serum samples used for measuring the MANF, EWSR1, and ANXA6 levels were collected from subjects in sample set 2.Based on the basic clinical documentation, the depression severity in MDD patients was rated by the 17-item Hamilton Depression Rating Scale (HDRS). Clinical data were extracted from the patient discharge letters. We also collected patient’s information on marital status (single/married/divorced/widowed), levels of education (low/middle/high), work status (unemployed/others not working/working/in training/retired/housewife/-man), drinking status (never/moderate/heavy), and smoking status (never/moderate/heavy). Meanwhile, detailed information on antidepressant use was collected in the DT-MDD subgroup.The Ingenuity Knowledge Base’s ingenuity pathway analysis (IPA) is an accurate online biomedical analysis tool that helps researchers to predict existing interaction networks between molecules and understand their properties . Here, bBlood for lipid analyses was sampled at 7:00 AM after overnight fasting. The samples were transported to the Center for Clinical Molecular Medical detection, First Affiliated Hospital of Chongqing Medical University within one hour. Levels of TC, HDL-C, and LDL-C, as well as TG, were determined on the COBAS C8000 Modular Analyzer  according to routine laboratory methods.g for 15 min, aliquoted, and stored at −80 °C until use. Total MANF, EWSR1, and ANXA6 were measured using commercially available high-sensitivity ELISA kits from MEIMIAN  by two blind experimenters. All ELISA kits were used following the manufacturer’s instructions. The detection limit was 0.25 ng/ml for the MANF assay, 10 pg/ml for the EWSR1 assay, and 1 ng/ml for the ANXA6 assay. In addition, sample collection and storage time did not differ among groups.Peripheral whole-blood samples were collected from each participant by venous puncture. Consistent with our previous study , serum wt-tests , Mann–Whitney U test , Kruskal–Wallis test , or ANOVA followed by post hoc comparison with the Bonferroni test , where appropriate. The variances between MDD and HCs were calculated using Levene’s test; if the variances were not similar between the two groups, the adjusted p-value was used. Comparisons of categorical variables were performed using Chi-square test. Pearson correlation coefficient was used to characterize the correlation between two variables. To identify the potential biomarkers for MDD, the logistic-regression analysis was used here. To assess the diagnostic performance of the panel consisting of these potential biomarkers, the receiver operating characteristic (ROC) curve analysis was used to quantify its ability in discriminating MDD patients from HCs in both training and testing sets. The value of the area under the ROC curve (AUC) was the evaluation index of diagnostic performance: ‘excellent’ (AUC = 0.90–1.00), ‘good’ (AUC = 0.80–0.89), ‘fair’ (AUC = 0.70–0.79), ‘poor’ (AUC = 0.60–0.69), or ‘fail’/no discriminatory capacity (AUC = 0.50–0.59) [p values of < 0.05 for group comparisons and correlation analyses.Statistical analyses were performed using Statistical Package for the Social Sciences, version 20 . The results were presented as mean ± standard error of the mean (SEM). Comparisons of demographic and clinical data for continuous variables between groups were conducted using independent sample 50–0.59) . Signifip = 0.579) and age (p = 0.360) between HCs and MDD groups. Meanwhile, sample set 2 included 67 HCs and 143 MDD patients (75 DN-MDD and 68 DT-MDD). No significant difference in sex (p = 0.129) and age (p = 0.762) distributions were found between groups. The value of HDRS was significantly higher in MDD group than in HCs group (p = 8.17E−15). In addition, in sample set 1 and 2  and LDL-C (p = 2.20E−7), yet HDL-C (p = 0.201) and TG (p = 0.137) levels were similar between MDD patients and HCs. And LDL-C concentration in serum was found to be significantly positively correlated with TC level . Furthermore, TC and LDL-C concentrations were higher in the DT-MDD group relative to the DN-MDD group, especially in the 18–35 age subgroup .The concentrations of serum TC, LDL-C, HDL-C, and TG from subjects in sample set 1 were presented in Fig. To investigate the role of lipid regulator MANF in depression, the IPA database was used to explore the potential network of associations among cholesterol, MANF, and MDD. Analysis of the obtained molecular network showed that cholesterol might be involved in the pathogenesis of MDD via regulating the MANF/EWSR1/ANXA6 pathway Fig. .Fig. 2Pop = 2.66E−25) and higher levels of EWSR1 (p = 8.06E−22) and ANXA6 (p = 7.51E−28). Correlation analysis showed that MANF level  was significantly negatively correlated with HDRS score, and both EWSR1 level  and ANXA6 level  were significantly positively correlated with HDRS score , while lower levels of EWSR1 (p = 4.33E−09) and ANXA6 (p = 2.85E−17) , EWSR1  and ANXA6  were significantly correlated with TC level =1/(1 + exp(5.263*MANF − 0.028*EWSR1 − 0.368*ANXA6 + 17.506)). Then, we used ROC curve analysis to assess its diagnostic performance. The results showed that this discriminative model yielded an AUC of 0.9994 in the training set  was used here to independently assess the diagnostic generalizability of the identified potential biomarkers. This method has been successfully conducted in our previous studies , 51. In There were few reports about the relation of MANF/EWSR1/ANXA6 pathway to hypolipidemia-associated MDD. Our findings would provide a new direction for further investigating this relationship. Besides, MANF, EWSR1, and ANXA6 were found to be significantly altered in the serum of MDD patients and correlated with serum lipid and the severity of depression symptoms. These molecules might serve as additional state or trait biomarkers in depression. Our results also indicated a possible role of these molecules in the pathogenesis or progression of MDD, which could broaden our knowledge on the possible involvement of lipid metabolism in MDD. However, it is still unclear if hypolipidemia is the cause of depression or it occurs as a secondary effect in MDD patients. Thus, longitudinal studies involving serum lipid examination and MANF/EWSR1/ANXA6 pathway assessment are needed to further explain the possible role of hypolipidemia in the pathogenesis of MDD.Several limitations of this study should be mentioned here. First, the present study about serum lipid examination was retrospective, open, and uncontrolled design, although a large number of MDD patients were enrolled. Second, the effects of other influencing factors in this study, such as dietary habits or psychiatric comorbidity cannot be excluded –54. ThirOur study first measured the MANF/EWSR1/ANXA6 pathway in the serum of MDD patients. In conclusion, we found that compared to HCs, MDD patients had significantly lower levels of TC, LDL-C, and MANF, and higher levels of EWSR1 and ANXA6. Meanwhile, MANF, EWSR1, and ANXA6 were significantly correlated to TC concentrations and depression severity, and they could be potential biomarkers for diagnosing MDD. These results suggested that MANF/EWSR1/ANXA6 pathway might act as the bridge between hypolipidemia and depression. Our findings would be helpful for the future development of objective diagnostic methods for MDD and provide novel insights into exploring the pathogenesis of depression.Supplemental Information


Pinus wallichiana, Abies spectabilis and Picea spinulosa, were collected to look for the local causes of EMF community composition and diversity patterns. The EMF colonization rate, diversity and taxonomic community structure were determined by morphotyping and sanger sequencing of the fungal ITS gene from the root tip samples. Soil exploration types were identified based on the morphologies of the ectomycorrhizas, coupled with soil properties analysis and plant diversity survey. Contrasting patterns of EMF community and functional diversity were found across the studied three forests types dominated by different coniferous tree species. In terms of associations between soil and EMF properties, the total phosphorus (TP) and nitrate (NO3−) contents in soil negatively correlated with the colonization rate and the Shannon diversity index of EMF in contrast to the positive relationship between TP and EMF richness. The soil total nitrogen (TN), ammonium (NH4+) and plant diversity together caused 57.6% of the total variations in the EMF taxonomic community structure at the three investigated forest systems. Whereas based on the soil exploration types alone, NH4+ and TN explained 74.2% of variance in the EMF community structures. Overall, the findings of this study leverage our understanding of EMF dynamics and local influencing factors in coniferous forests dominated by different tree species within the subalpine climatic zone.The symbiotic relationship between ectomycorrhizal fungi (EMF) and the roots of host plants is significantly important in regulating the health and stability of ecosystems, especially of those such as the climate warming affected subalpine forest ecosystems. Therefore, from the coniferous forest systems located in the Southern Qinghai-Tibetan Plateau, root tips from three forest tree species:  The impacts of climate change on forest processes are driven by disturbances in biogeochemical cycling and decline in soil biodiversity . The convia enzymatic decomposition of the organic matter in soils  from host plants is critical for EMF who in return, acquire nutrients for host plants by directly emanating their hyphae to scavenge inorganic nutrients, or through indirect provisioning of organic nutrients in soils . The EMFin soils . Seemingin soils . Such chin soils . Whereasin soils . Furtherin soils  and, incin soils . Contrasin soils . These iIncreasing local-scale studies suggested soil properties as the dominant factors shaping EMF communities across various ecosystems . In EuroThe divergent EMF taxa differ largely in nutrient uptake strategies, the differences are partially represented in the features of their emanating mycelium, which are relevant to the function of nutrient exploitation and transport . GeneralThe Qinghai–Tibet Plateau is a unique location comprising of different forest systems vulnerable to climate change at regional scale ,b. ThereThe EMF colonization rate, diversity and community structure would differ across three different coniferous forests dominated by different tree species;Considering the interactions between above-below ground biotic communities and soil properties, any changes in plant diversity and soil properties will be reflected by variations in EMF community diversity;Under varying environmental conditions and plant diversity, the EMF communities will have to adapt and alter nutrients foraging strategy, thereby indicating significant changes in EMF functional traits.Pinus wallichiana, Abies spectabilis and Picea spinulosa were selected. The three forest sites were approximately 300 m distance apart. Five forest plots (20 m × 20 m) with a 20 m distance grid, at each of the three forest sites  were systematically chosen for soil sampling purposes.The study site is in a mountainous region on the Southern Qinghai–Tibet Plateau . The location is near the Keelung Port, and at an elevation level of 3,000 m a.s.l (meters above sea level). Indian ocean Monsoons impact the weather of the study area, with mean annual precipitation (MAP) and mean annual temperature (MAT) of 635 mm and 3.9°C, respectively; This region experiences long sunlight hours throughout the year . The main vegetation at the site is natural secondary subalpine coniferous forest on a dark brown soil . From this subalpine coniferous forest zone, three separate coniferous forest sites dominated separately by In each forest plot, soil adhering to fine roots at 0–10 cm depth was collected by shaking and then sieved through > 2 mm ethanol sterilized mesh. These root samplings were done along four different directions and at a distance of ~ 0.5 m from the same tree trunk. The fine root samples were collected from five randomly selected but similar sized dominant trees (~ 30 cm diameter at breast height). Care was taken to trace the fine roots belonged to the same tree. The four fine root and soil samples from each tree were bulked to form one composite sample per tree. Subsequently, the fresh soil and root samples were stored at −20°C and 4°C for soil properties analyses and mycorrhizal morphotyping, respectively.4+) and nitrate (NO3−) contents were, respectively, determined by the indophenol blue colorimetric technique and phenol-disulfonic acid colorimetric method (−1 potassium chloride (KCl) solution. We used the molybdenum blue method after extraction to colorimetrically quantify available phosphorus (AP) from the soil in a 0.5 M sodium bicarbonate (NaHCO3) solution at pH 8.5 (2SO4–HClO4).Soil pH value was determined by a conductivity meter  in a 1:2.5 soil: water solution. Soil organic carbon (SOC) and total nitrogen (TN) concentrations were measured with a CN-element analyzer , according to their morphological characteristics such as the shape, color, bifurcation, and the texture of the surface of the mycorrhizas . The numFor each EMF morphotype, ~ 20 root tips were carefully selected and stored at −20°C until DNA extraction and molecular identification.−1), 2.5 μl of 10 × Taq buffer, 3 μl MgCl2 (23 mM), 0.5 μl of 10 mM dNTP mix and 14.375 μl ddH2O. The PCR thermal profile included following steps: initial denaturation at 95°C for 1 min, followed by annealing at 55°C for 30 s and extension for 72°C for 1 min, and a final extension at 72°C for 5 min. The amplified PCR products were purified using Axy Prep DNA Gel Recovery Kit as described by the manufacturers.The total genomic DNA of EMF morphotypes was extracted from the root tips using modified cetyl-trimethylammonium bromide (CTAB) method as described by The purified PCR products were then used for Sanger sequencing and the sequences were assembled using Staden Package 4.10.Prior to the statistical analyses, Shapiro–Wilk and Levene test were used to test the normality and homogeneity of the data. Where not normal, the data were log transformed or non-parametric tests were used for the statistical analysis. Analysis of Variance (ANOVA) was conducted to determine the significant differences in the means of plant diversity indices and soil properties and their influence on the EMF colonization. The EMF alpha-diversity was estimated by richness (Chao1) and diversity (Shannon index). The Pearson’s correlation analyses were used to determine the associations between EMF alpha-diversity and environmental variables .Redundancy analysis (RDA) were conducted to investigate the impacts of plant diversity and soil properties on EMF communities structure , and Anap > 0.05; P. wallichiana and A. spectabilis in comparison to the P. spinulosa dominated forest site  remained unchanged (3−) and total phosphorus (TP) had lower contents in the P. wallichiana forest soils than the contents in A. spectabilis and P. spinulosa dominated forest soils . The highest values for these soil contents were found in forest site where P. spinulosa were abundant  contents were significantly higher (p = 0.02) and twice the amount in the A. spectabilis forests (108.32 mg kg−1), than the P. wallichiana and P. spinulosa forest sites. Except for nitrate N (NO3−) contents, the amounts of all other soil parameters were significantly higher in the A. spectabilis dominated forests than the other two forests and showed a trend of A. spectabilis > P. spinulosa > P. wallichiana. The soil pH and available phosphorus (AP) did not significantly differ among three forest soils , total nitrogen (TN), soil nitrate , while the highest rate was observed in the P. wallichiana forests (95.1%). The EMF richness (Chao1 index) ranged from 3 to 10 and was significantly lower in the P. spinulosa (3.6) than in the P. wallichiana (9.4) and A. spectabilis forests . The EMF diversity (Shannon index) of P. spinulosa forest ranged between 0.259 to 0.543, and was significantly lower than P. wallichiana (0.789) and A. spectabilis (0.749) forests  except for NO3− and EMF richness, which had no significant negative correlationship  and Sebacina cystidiata (24.75%) were highest in P. wallichiana forest, followed by Russula xerampelina (15.23%), Tomentella sp. (8.29%), while S. cystidiata (25.04%), R. xerampelina (27.67%) and Tomentella stuposa (22.41%) were dominant in A. spectabilis forest. The main dominant species of the P. spinulosa forest were Lactarius dombangensis (51.93%) and Pseudotomentella sp.  indicated soil TN, NH forests  and S3.  forests .Helotiales sp., S. sibiricus and Mortierella alpina (p < 0.05). The soil TN and SOC negatively correlated with decreased relative abundance of Helotiales sp., S. americanus, S. sibiricus, C. geophilum and M. alpine (p < 0.05), but had significant positive relationship with the relative abundance of T. stuposa (p < 0.05). Likewise, NO3− and TP negatively correlated with the relative abundance of Helotiales sp., S. americanus, S. sibiricus, Tomentella sp. and M. alpina, but had positive relations with relative abundance of L. dombangensis. The NH4+ also had significant positive relation with the relative abundance of T. stuposa, C. delectabile, Neobulgaria sp., Thelephoraceae sp. and R. xerampelina (p < 0.05). While, plant diversity was significantly negatively related with the relative abundance of L. dombangensis  were identified in total.P. wallichiana forests (relative abundance: 28.90%) than in the A. spectabilis (67.76%) and P. spinulosa abundant forest sites  forest ecosystems , nearly 75% of phosphorus (P) and 80% of nitrogen (N) acquisition seems to be facilitated by mycorrhizal fungi . Compareosystems  and N acosystems . Albeit P. spinulosa and higher soil nutrient contents. The nitrate and phosphorous contents in soil were the primary indicators of EMF variability in our study as seen by RDA analysis. This is in line with previous studies showing soil environmental variables  having significant influence on EMF diversity at local scale  depicted wide host range  by directly altering the EMF community composition through changes in the quantity of photosynthetic C supplied to the symbiotic partner , and ii. In otheociation . SimilarPinaceae . In othest range . Accordist range , which m4+) and total nitrogen (TN) were the key regulators in our study, and this is in line with the results of previous studies displaying close relationship between soil NH4+ and EMF community composition in forest soil systems  through their hyphal network (A. spectabilis (relative abundance: 67.76%) and P. spinulosa (94.19%) forests, than P. wallichiana (28.90%) forests where soil TN contents were comparatively low. No long-distance exploration types were detected in A. spectabilis and P. spinulosa forests, compared to the relative abundance of 31.27% in P. wallichiana forests. This is in line with the recent study (Tomentella sp.) and long-distance  EMF exploration types.EMF can mineralize organic forms of N and P by releasing hydrolytic and oxidative enzymes, while also can effectively forage nutrients from inorganic sources . The EMF community and functional diversity differed across the three forest sites. The soil total phosphorus (TP) and nitrate (NO3−) contents were key predictors of EMF colonization and EMF Shannon diversity index. While soil TP controlled EMF richness. Additionally, the EMF taxonomic community structures were explained by soil total nitrogen (TN), ammonium (NH4+) and plant diversity, while NH4+ and TN in soils were the local causes for the differences observed in exploration types of EMF communities in this study. The findings of this work advocate the complex interactions between above and below ground biotic communities especially the EMF and soil properties. However, the inferences were only related to soil chemical characteristics influencing root tip scale EMF colonization rate and diversity. Therefore, further work on understanding the effects of other abiotic and biotic variables such as the soil moisture, plant trait and plant productivity on EMF fungal trophic guilds is needed. Regardless, the results contribute to comprehensive understanding of EMF community diversity and characteristic and local causes determining their regulation in subalpine coniferous forests dominated by different coniferous tree species.This study explored how ectomycorrhizal community and functional diversity respond to varying soil chemical properties and plant diversity across three different coniferous forests (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/NY: investigation, conceptualization, visualization, formal analysis, writing—original draft, and writing—review and editing. JH: investigation, visualization, formal analysis, and writing—original draft. JZ, YZ, and XL: investigation and methodology. PB and DL: writing—review and editing. HR: resources, funding acquisition, validation, and project administration. WX and LM: conceptualization, funding acquisition, visualization, supervision, and writing—review and editing. All authors contributed to the article and approved the submitted version.The study was support by the following programs: Strategic Priority Research Program of the Chinese Academy of Sciences (no. XDB31000000), National Key Research and Development Program of China (no. 2021YFD2200403), and Natural Science Foundation of China .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.


The AHN3 point cloud was obtained with Light Detection And Ranging (LiDAR) technology and contains for each point the x, y, z coordinates and additional characteristics . Moreover, the point cloud has been pre-processed by ‘Rijkswaterstraat’ (the executive agency of the Dutch Ministry of Infrastructure and Water Management), comes with a Digital Terrain Model (DTM) and a Digital Surface Model (DSM), and is delivered with a pre-classification of each point into one of six classes . However, no detailed information on vegetation structure is available from the AHN3 point cloud. We processed the AHN3 point cloud (∼16 TB uncompressed data volume) into 10 m resolution raster layers of ecosystem structure at a national extent, using a novel high-throughput workflow called ‘Laserfarm’ and a cluster of virtual machines with fast central processing units, high memory nodes and associated big data storage for managing the large amount of files. The raster layers (available as GeoTIFF files) capture 25 LiDAR metrics of vegetation structure, including ecosystem height , ecosystem cover , and ecosystem structural complexity . The raster layers make use of the Dutch projected coordinate system (EPSG:28992 Amersfoort / RD New), are each ∼1 GB in size, and can be readily used by ecologists in a geographic information system (GIS) or analytical open-source software such as R and Python. Even though the class ‘1: Unclassified’ mainly includes vegetation points, other objects such as cars, fences, and boats can also be present in this class, introducing potential biases in the derived data products. We therefore validated the raster layers of ecosystem structure using >180,000 hand-labelled LiDAR points in 100 randomly selected sample plots (10 m × 10 m each) across the Netherlands. Besides vegetation, objects such as boats, fences, and cars were identified in the sampled plots. However, the misclassification rate of vegetation points (i.e. non-vegetation points that were assumed to be vegetation) was low (∼0.05) and the accuracy of the 25 LiDAR metrics derived from the AHN3 point cloud was high (∼90%). To minimize existing inaccuracies in this country-wide data product , we provide an additional mask that captures water bodies, buildings and roads generated from the Dutch cadaster dataset. This newly generated country-wide ecosystem structure data product provides new opportunities for ecology and biodiversity science, e.g. for mapping the 3D vegetation structure of a variety of ecosystems or for modelling biodiversity, species distributions, abundance and ecological niches of animals and their habitats.The third Dutch national airborne laser scanning flight campaign  conducted between 2014 and 2019 during the leaf-off season (October–April) across the whole Netherlands provides a free and open-access, country-wide dataset with ∼700 billion points and a point density of ∼10(–20) points/m Specifications TableValue of the Data•Ecosystem structure data are important for understanding, modelling and predicting biodiversity because the species richness, composition, distribution and abundance of organisms and their habitat preferences (e.g. nest sites and shelter), food provisioning and foraging are tightly linked to the horizontal and vertical heterogeneity of vegetation.•The physical structure of ecosystems also influences microclimates at the land–air interface via respiration, heat and energy exchange. This affects species behavior, growth, reproduction, and survival. Predictions of species and ecosystem responses to global change thus require high resolution data of ecosystem structure to account for temperature buffering near the ground and microrefugia within landscapes.•2 concentrations.Measurements of the vertical structure of forests and other ecosystems are also critical for accurately assessing biomass and carbon storage, and how land use changes, ecosystem restoration or variations in climate may impact atmospheric CO•Spatially contiguous, high resolution raster layers of ecosystem structure will thus be beneficial for a range of users, including field biologists, ecologists, conservationists, ecological modelers, geoinformaticians, land managers and environmental system analysts. Researchers from other domains (e.g. hydrology and climatology) might also take advantage of such data.•Ecosystem structure data can be used as predictors in statistical models  to correlate species observations with environmental layers. Such predictive habitat distribution models aim to quantify and map the determinants of species’ ecological niches and their ability to cope with climate or land use change.1This dataset was generated during the development of the Laserfarm workflow, a high-throughput pipeline for generating geospatial data products of ecosystem structure from airborne laser scanning point clouds. The aim was to create a high-resolution (10 m) geospatial dataset of 25 LiDAR metrics (i.e. raster layers in GeoTIFF format) from multi-terabyte LiDAR point clouds at a national extent (i.e. across the Netherlands), capturing various aspects of ecosystem structure, including vegetation height, cover and structural complexity of vegetation. This provides a standardized set of Essential Biodiversity Variables (EBVs) derived from LiDAR for the EBV ‘Ecosystem Vertical Profile’ 22.1https://pypi.org/project/laserfarm/), using the feature extraction module from the 'Laserchicken' software (https://laserchicken.readthedocs.io/en/latest/#features). For each of the 25 calculated LiDAR metrics, the GeoTIFF file name, the Laserchicken feature name, the formula, a general description and its ecological relevance is provided .The 100 randomly selected 10 m × 10 m plots were widely spread across the whole Netherlands . Most pln = 25 LiDAR metrics), ranging from 0.87–1. The number of plots with differences in LiDAR metric values and the degree of difference varied among LiDAR metrics .Besides the 25 LiDAR metrics, we additionally calculated the point density for each 10 m x 10 m grid cell to quantify the variability in available AHN3 point densities across the Netherlands. This represents the spatial distribution of the point cloud density and can be used for additional analyses, e.g. to test how LiDAR metrics of ecosystem structure vary with point densities. The AHN3 point density was calculated for each 10 m x 10 m grid cell using the ‘point_density’ feature from the 'Laserchicken' software  using the same spatial resolution (i.e. 10 meter) and projected coordinate system (EPSG:28992 Amersfoort / RD New) as the other GeoTIFF files. We aggregated the water, building and road polygons from the TOP10NL shapefiles into one type, and rasterized them using a binary classification . The mask allows users to minimize errors in the generated country-wide ecosystem structure data because not all points in the ASPRS point class ‘1: Unclassified’ are vegetation points  from the Dutch cadaster data which allows the user to minimize inaccuracies related to misclassifications, e.g. ships on water surface, chimneys on roofs, or cars on roads which might incorrectly be considered as vegetation. A mask of water surfaces and human infrastructures (buildings and roads) was created from the shapefiles of the 2018 Dutch cadaster data  and a script for automatic download (https://github.com/eEcoLiDAR/downloadAHN). Data were downloaded to the GRID storage infrastructure (http://doc.grid.surfsara.nl/en/latest/Pages/Advanced/grid_storage.html) from SURF, the trans-national IT infrastructure for the Dutch academic community (https://www.surf.nl/en/ict-facilities).The whole LiDAR point cloud dataset from AHN3 is large and contains ∼700 billion points and ∼16 TB uncompressed data volume, available in 1,367 point cloud files (LAZ format). We downloaded all 1,367 files from the AHN3 repository using the PDOK webservices (3.2https://userinfo.surfsara.nl/systems/hpc-cloud). Each of the 11 VMs had 2 cores , 32 GB or 64 GB RAM, and 256 GB local HDD. We used the Laserfarm workflow (version 0.1.5) available from PyPI (https://pypi.org/project/laserfarm/), GitHub (https://github.com/eEcoLiDAR/Laserfarm) and Zenodo (https://zenodo.org/record/5636773) to process the multi-terabytes of AHN3 point clouds into GeoTIFF files of LiDAR metrics capturing ecosystems structure. The Laserfarm workflow is implemented in Python and makes use of open-source tools such as ‘Laserchicken’ https://pdal.io/), the Geospatial Data Abstraction Library , the Dask library https://pypi.org/). All steps of the AHN3 processing using the Laserfarm workflow are available in Jupyter Notebooks (https://github.com/eEcoLiDAR/AHN/tree/main/AHN3) and involved the following processing steps:For processing the files, we set-up a cluster of 11 virtual machines (VMs) using the HPC Cloud from SURF (As a first step (‘Re-tiling’), we split the 1,367 LAZ files into a grid with 1 km × 1 km size (512 × 512 cells across the Netherlands), making use of the Dutch projected coordinate system (EPSG:28992 Amersfoort / RD New). The LAZ files were retrieved from the storage and then split using functionality from the PDAL library In the second step , we calculated the normalized height for each individual point as the height relative to the lowest point within a 1 m × 1 m cell. This pipeline employs the ‘Normalize’ module of the 'Laserchicken' software In the third step (‘Feature extraction’), we calculated the LiDAR metrics using the ‘Features’ and 'Compute Neighbors' modules of the 'Laserchicken' software In the fourth step (‘Rasterization’), we merged and exported the PLY tiles for each metric as a single-band GeoTIFF file in the Dutch projected coordinate system (EPSG:28992 Amersfoort / RD New).3.3https://www.rdocumentation.org/packages/raster/versions/3.5-15/topics/sampleRandom). We then segmented the point cloud of each plot from the raw AHN3 point clouds using the ‘lasclip’ tool from the Lastools software (https://rapidlasso.com/lastools/), using the polygons of the sampled plots. We then hand-labelled all segmented point clouds  into the classes of vegetation, ground, and others . This was done with the ArcGIS Pro interactive editing tool for LAS classification (see https://pro.arcgis.com/en/pro-app/latest/help/data/las-dataset/interactive-las-class-code-editing.htm). Each of the 25 LiDAR metrics was then calculated for each plot using the Laserfarm workflow, once using all points from the ASPRS point class ‘1: Unclassified’ and once using only the vegetation points from the hand-labelled point clouds (as ground truth). The values of each LiDAR metric  for each plot were then extracted using the extract function in R (https://www.rdocumentation.org/packages/raster/versions/3.5-15/topics/extract).We randomly located 100 plots (each of 10 m × 10 m size) across the Netherlands using the sampleRandom function in R (n = 100). This allowed us to quantify how accurately the 25 LiDAR metrics capture vegetation structure, and to what extend their values might be affected by non-vegetation points that remain in the class ‘unclassified’ of the AHN3 pre-classification.The misclassification rate was calculated for each plot as the number of points which were incorrectly classified as vegetation divided by the total number of points in the ASPRS class ‘1: Unclassified’. Accuracy of the 25 LiDAR metrics was assessed by taking the number of plots in which the LiDAR metric calculation did not differ between hand-labelled and originally classified points (i.e. difference = 0) and dividing it by the total number of plots (3.4https://github.com/eEcoLiDAR/AHN/tree/main/AHN-mask).The mask layer was generated using the water, buildings and road polygons from the TOP10NL cadaster data (see above ‘Data source location’). We aggregated the polygons and exported them as a raster layer with a binary classification  and 10 m resolution, using a Jupyter Notebook (https://www.elsevier.com/authors/policies-and-guidelines). The work does not include chemicals, procedures or equipment that have any unusual hazards inherent in their use, nor does it involve the use of animal or human subjects. No studies on patients or volunteers have been performed.This work meets the requirements for ethical publishing (W. Daniel Kissling: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing. Yifang Shi: Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – review & editing. Zsófia Koma: Investigation, Methodology, Writing – review & editing. Christiaan Meijer: Software, Validation, Methodology. Ou Ku: Software, Validation, Methodology. Francesco Nattino: Software, Validation, Formal analysis, Methodology. Arie C. Seijmonsbergen: Funding acquisition, Writing – review & editing. Meiert W. Grootes: Conceptualization, Methodology, Resources, Software, Supervision, Validation, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Alphavirus and Flavivirus, and Costa Rica is endemic to several of them. The aim of this study was to describe and analyze the community structure of such vectors in Costa Rica.Mosquitoes are vectors of various arboviruses belonging to the generaSampling was performed in two different coastal locations of Costa Rica with evidence of arboviral activity during rainy and dry seasons. Encephalitis vector surveillance traps, CDC female gravid traps and ovitraps were used. Detection of several arboviruses by Pan-Alpha and Pan-Flavi PCR was attempted. Blood meals were also identified. The Normalized Difference Vegetation Index (NDVI) was estimated for each area during the rainy and dry seasons. The Chao2 values for abundance and Shannon index for species diversity were also estimated.Culex quinquefasciatus was the most caught species. The differences in NDVI were higher between seasons and between regions, yielding lower Chao-Sørensen similarity index values. Venezuelan equine encephalitis virus, West Nile virus and Madariaga virus were not detected at all, and dengue virus and Zika virus were detected in two separate Cx. quinquefasciatus specimens. The primary blood-meal sources were chickens (60%) and humans (27.5%). Both sampled areas were found to have different seasonal dynamics and population turnover, as reflected in the Chao2 species richness estimation values and Shannon diversity index. A total of 1802 adult mosquitoes belonging to 55 species were captured, among which  Seasonal patterns in mosquito community dynamics in coastal areas of Costa Rica have strong differences despite a geographical proximity. The NDVI influences mosquito diversity at the regional scale more than at the local scale. However, year-long continuous sampling is required to better understand local dynamics.The online version contains supplementary material available at 10.1186/s13071-022-05579-y. Alphavirus  and genus Flavivirus  and dengue virus [DENV]) [Mosquitoes are vectors of several arboviruses belonging to the genus  [DENV]) . In some [DENV]) –4. Vecto [DENV]) . Moreove [DENV]) –7. These [DENV]) .Vertebrate host diversity and species richness have also been established as significant predictors for vector-borne disease transmission risk , 9. DepeResearch on the effect of vector diversity on disease prevalence is scarce. One study has found that vector richness can increase the prevalence of malaria , but empDeinocerites pseudes and from Aedes taeniorhynchus collected with CDC light traps [Haemagogus spegazzinii was identified as the possible vector [. More recent mosquito research in Costa Rica has focused chiefly on the primary vectors of dengue (DENV), ZIKV and chikungunya (CHIKV) viruses, namely Aedes aegypti and Aedes albopictus [Costa Rica is among those countries in the world with the highest number of mosquito species per area unit . Consequht traps . During e vector . More ree vector . More rebopictus –23, due In contrast with the neighboring country of Panama and other countries of Latin America, mosquito vector communities in arbovirus endemic areas of Costa Rica are mostly unknown –26. TherThe study was performed in two coastal locations with evidence of arboviral activity in Costa Rica: Cuajiniquil (CU) and Talamanca (TA) . CU is a2 at a 300 l/min flux using the CO2 tank adapter (BIoquip Products Inc.), and GT were baited with a hay infusion. EVS traps and GTs were set between 18:00 h and 6:00 h for one night per season at each sampling location; the OVs were left at each study site for 2 weeks. The EVS trap was the only trap placed at all four sampling settings ; GTs and OVs were not set at DO. A manual collection of larvae in natural breeding sites was also performed in each locality to enable a better description of the species composition. All visible larval habitats were sampled, including artificial containers, phytotelmata, ponds and puddles. Depending on the water volume, each container was tested using a turkey baster or a D-Frame Water Net (Bioquip Products Inc.). Larvae were sorted in a larval tray (Bioquip Products Inc.), and all visualized individuals were deposited in 70% ethanol.Mosquitos were caught in CU from May to December 2017 (rainy season) and from January to April 2018 (dry season); in TA, trapping was carried out from May to July 2018 (rainy season) and from August to October 2018 (dry season). Three different mosquito trapping methods were used per sampling location: four encephalitis vector surveillance (EVS) traps , three CDC female gravid traps (GTs) , and three ovitraps (OVs) . EVS tra Key for the Mosquitoes of Costa Rica [The adult mosquitoes collected were freeze-killed and transported to the Vectors Research Laboratory (LIVe) at the University of Costa Rica to be identified at the species level. Larvae (fixed in 70% ethanol) were later mounted on glass slides using a polyvinyl alcohol mounting medium (Bioquip Products Inc.). All specimens were identified using thesta Rica . Adults  Alphavirus and Flavivirus molecularly. For Flavivirus, a semi-nested PCR was used to amplify a 220-bp fragment of the NSP4 gene using primers cFD2 (5ʹ-GTGTCCCAGCCGGCGGTGTCATCAGC-3ʹ) and MAMD (5ʹ-CATGATGGGRAARAGRGARRAG-3ʹ) for the first reaction, and primers cFD2 and FS778 (5ʹ-AARGGHAGYMCDGCHATHTGGT-3ʹ) For the second reaction [Flavivirus.Pan-PCRs were used to detect captured species of reaction . PCR amp Alphavirus, a nested PCR protocol was applied to amplify a 210-bp product using the primers PanAlphaOutF (5’-TTTAAGTTTGGTGCGATGATGAAGTC-3ʹ) and PanAlphaOutR (5ʹ-GCATCTATGATATTGACTTCCATGTT-3ʹ) for the first reaction and PanAlphaInF (5ʹ-GGTGCGATGATGAAGTCTGGGATGT-3ʹ) and PanAlphaInR (5ʹ-CTATGATATTGACTTCCATGTTCATCCA-3ʹ) for the second reaction [Forreaction . Cyclingreaction . The PCRAll amplicons were visualized by electrophoresis in 1.5% agarose gel  stained with GelRed™  and using Borate-EDTA 0.5× (Thermo Fisher Scientific) as a medium buffer. Amplicons were compared with a 100-bp molecular ruler (Thermo Fisher Scientific) with its corresponding positive control size. Positive amplicons were purified using ExoSap IT™ (Thermo Fisher Scientific) and sequenced (sense and antisense) elsewhere .Ae. aegypti (Rockefeller strain) blood-fed on Mus musculus (BALB/c) was used as a positive reaction control. The PCR reaction mix, amplicon visualization and sequencing steps were performed as described above.Only blood-fed females collected with GTs were used for blood-meal detection. Blood meals were scored according to the BF1-3 classification, and any individual with evidence of blood-feeding within the last 72 h was included in the analysis . A 244-bViral and blood-meal consensus sequences were generated and aligned using the MEGA X™ software. Each consensus sequence was identified according to its closest percent similarity with reference sequences using the BLAST® tool. We considered a percentage of similarity > 98% for virus sequences as a correct identification . For verE = e ∧ H '/S) was also estimated using the Shannon diversity index [For diversity analyses, EstimateS™ software 9.1 version was used . Accordity index . The Chaty index . In addity index .Culex) on edges of fields and forest [The Normalized Difference Vegetation Index (NDVI) and the Normalized Difference Water Index (NDWI) for a local and regional scale was calculated to correlate seasonal fluctuations (absolute difference) with the Chao-Sørensen similarity index. The NDVI and NDWI values were calculated in QGIS with Landsat-8 satellite imagery obtained from LandsatLook™ . Each NDVI and NDWI was calculated for each county during each season. The selection criteria for the image were: (i) it had to be taken during each sampling season; (ii) there had to be a maximum of 25% cloud coverage; and (iii) the buffer area for the sampling point in each image (radius: 2 km) must not have any cloud coverage. Because of the reduced number of available images, a time series reflecting changes in the NDVI and NDWI was impossible. Therefore, we only could make a comparison between seasons. Any value between - 1 and 0 was filtered to extract any data coming from the ocean or due to heavy cloud cover . For thed forest . Each 2-Culex quinquefasciatus (n = 514) was the most frequent adult mosquito species captured in both sampling localities . For example, no Anopheles albimanus (n = 0) was caught during the dry season, and the number of Cx. quinquefasciatus captured decreased from 149 to five individuals between the rainy and dry seasons. De. pseudes (n = 75) was the only species for which the number of captures was not reduced during the dry season . The OV traps were relatively unsuccessful in capturing mosquitoes during the dry season due to water evaporation and lack of rain, with only a few individuals of Ae. aegypti caught in one trap. Human-made habitats, such as rice plantations, were widely used by An. albimanus, Culex coronator and Culex (Melanoconion) theobaldi and, interestingly, this was the only habitat where An. albimanus larvae were found.The number of individual mosquitoes captured in the EVS traps in CU was much higher during the rainy season than during the dry season (Cx. quinquefasciatus (n = 360) and Cx. coronator (n = 96). Medical important species belonging to the genus Mansonia (n = 66) were also captured in EVS traps in TA, as well as other species in lesser numbers  were obtained from the GTs. The only medically important species captured in the OVs was Ae. aegypti. Although the Melanoconion subgenus is of medical importance and Culex (Melanoconion) psathaurus was captured, its vector capacity has not been tested.The most captured species in the EVS traps in TA were The species accumulation curve in both study sites shows that we did not attain the total sampling of all species Fig. . AlthougJohnbelkinia leucopus and De. pseudes, respectively. The most abundant species sampled were Cx. coronator, which was found in eight different habitat types, followed by Limatus durhammii, which was found in four habitat types. Other medically important species, such as Ae. aegypti, Culex nigripalpus, Mansonia dyari, Haemagogus iridicolor and Haemagogus lucifer, were also collected. In TA, Cx. coronator and Ha. iridicolor were collected from tree holes. Other mosquitoes, such as Culex (Microculex) spp., which only use phytotelmas as breeding sites [R = − 0.1) and NDWI (R = − 0.2) (Table R = − 0.76)  with 97.25% identity in BLAST. RNA of DENV type 3 was identified in a blood-fed Cx. quinquefasciatus collected in a GT located in a PE setting from TA during the rainy season. The sequence showed 96.7% similarity with a 2015 Colombian isolate (Accession number: MH544650.1).RNA of ZIKV was detected in a pool of Culex (n = 255), with Cx. quinquefasciatus (n = 126) and Cx. corniger (n = 92) being the most frequent species. Of these, 90 were gravid females, and 181 were blood-fed in the last 72 h. An attempt to obtain sequence data from blood in both gravid and blood-fed mosquitoes resulted in 65 identified blood meals. Overall, chicken was the main blood meal detected in engorged mosquitoes (39/65), most of which were collected in FE settings, followed by human blood (18/65). Of note, chicken blood was dominant in mosquitoes from the PE and PN settings, but it was not detected in those from the FO. Other blood-meal sources are detailed in Fig. The blood meals identified in mosquitoes were mainly from domestic animals and humans. Most of the captured mosquitoes belonged to the genus Our results show that mosquito populations in CU and TA form complex communities that can change between seasons. The highest species richness estimations were obtained in TA in the rainy season. During this period, more habitats may be available for larval development due to rainfall, not only in artificial containers but also in phytotelma. Although the sampling effort in this study was limited, most of the species present in each area were captured based on the rarefaction curves Fig.  and ChaoCx. quinquefasciatus, which accounted for 47.6% (360/756) of the total captured mosquitoes in our samples  was higher in CU, although the species estimation was higher in TA, which suggests that CU populations are more homogeneous or contain fewer dominant species. The lower diversity values (high regularity) in TA may be due to the dominance of les Fig. . Interes tropics . Low div tropics , and funnditions . FurtherAn. albimanus and De. pseudes. In this context, Cx. quinquefasciatus is a proven vector for several zoonotic arboviruses, including WNV and SLEV [Changes in species composition (Chao-Sørensen similarity index) was different among the sampling locations; nonetheless, their degree of variation between seasons differed for each county. Values for TA were nested within seasons, showing small changes in their species composition. In contrast, CU had a more dissimilar population between seasons, with a high turnover in the presence of medically important species, such as and SLEV . This vaand SLEV .De. pseudes). It has been proposed that the Shannon evenness index is a strong predictor of disease risk in multiple host communities [The evenness in CU indicates that some species are dominant in this community year-long (e.g. munities . Historimunities . TherefoR = − 0.20) when compared with Chao-Sørensen values of the same sampling points between seasons. In contrast, the correlation of regional mean NDVI  shows a stronger negative correlation (R = − 0.76) with the absolute difference in the NDVI values. The regional Chao-Sørensen index can represent a more representative change in the overall community since it considers more subsets of the total population, giving a more robust correlation when compared with the regional NDVI. However, the NDVI has been broadly used to predict population changes in different environments [R = − 0.61) was weaker than the correlation with the NDVI. Nonetheless, the NDWI has also been proven to predict mosquito abundance in swimming pools and with longer mosquito seasons [The variation in seasonal NDVI, which is associated with the forest phenology, was higher for both regional and local values  in CU, where the forest is deciduous during the dry season, giving a higher variation in NDVI , 52. In ronments . Arbovirronments , 55. In ronments –58. Rega seasons , 60.Haemagogus spp. and Sabethes spp., which can be vectors of YFV and Mayaro. Similarly, in CU, rice fields were only present during the rainy season [An. albimanus [An. albimanus are rice fields [An. albimanus population to drop off almost completely. Currently, there is no active transmission of Plasmodium spp. in CU, although it is present in other areas of Costa Rica due to human movement and anthropogenic landscape changes, including illegal gold mining [Plasmodium transmission in CU during the wet seasons. Moreover, the severe dry season can also influence arboviral incidence in the region, considering that some mosquito populations (e.g. Anopheles quadrimaculatus) can increase drastically after severe drought [These variations in the NDVI and NDWI were also reflected in the larval abundance between seasons in CU, where several of the OVs placed were completely dry upon later evaluation during the dry season  was also detected, which is of interest given that neutralizing antibodies for WNV and SLEV have been detected before in this species [Cx. quinquefaciatus and De. pseudes, are feeding on putative enzootic hosts and dead-end hosts, which is necessary for viral transmission to horse and human populations. The implications of reptile blood in terms of transmission are probably less important than those of avian blood, although some species can serve as amplifiers for WNV [Most engorged females belonged to the le hosts . Since o species . Althoug for WNV .The frequent detection of chicken blood in the mosquitoes collected at both sites can have repercussions on the epidemiological cycle. Chickens are refractory to WNV  infectioCx quinquefasciatus that contained human blood, it is likely that the viral RNA was from a viremic human since Cx. quinquefasciatus is not epidemiologically relevant as a DENV vector [The detection of DENV and ZIKV RNA was not unexpected in these areas since both viruses are prevalent in Costa Rica. DENV type 3 has not been detected in humans in Costa Rica since 2016 . AlthougV vector .Cx. quinquefasciatus has a significant role in DENV or ZIKV transmission to humans. Overall, viral detection was unexpectedly low. Arboviruses usually circulate at a low prevalence between vectors [Cx. quinquefasciatus: 514/1360).Regarding ZIKV, this species has only been proven to be an efficient vector in a few vector competence studies, but the consensus is that it is not a primary ZIKV vector . We do n vectors . AlthougDespite their geographical closeness, CU and TA districts have different seasonal dynamics and population turnover. These factors can be important in further prediction and ecological modeling for arboviruses in Costa Rica and other neotropical countries that share tropical rain and dry forests. In addition, the NDVI can have more influence on mosquito diversity on a regional scale than on a local scale. However, year-long continuous sampling is required to understand local dynamics better. This can further relate to how anthropogenic pressure  can affect the mosquito vectors present in the area. Since mosquito feeding preferences are strongly guided by host availability, these changes in land use and resource availability can modify the community of putative vectors in an epidemiological context.Additional file 1: Table S1. Mosquitoes captured with EVS traps in Cuajiniquil. DO: Domiciliary, PE: Peridomiciliary, PN: Animal Pen, FO: Forest.  Table S2. Mosquitoes captured with EVS traps in Talamanca. DO: Domiciliary, PE: Peridomiciliary, PN: Animal Pen, FO: Forest. Table S3. Mosquito larvae captured with manual collection.  Table S4. Regional Chao- Sørensen Index. Table S5. Chao- Sørensen Similarity Index.  Table S6. Chao- Sørensen Index and NDVI .  Table S7. Chao- Sørensen Index and NDWI.  Table S8. Mosquito larvae captured with ovitraps.  Table S9. Mosquitoes captured with GT traps.


Paspalum belongs to the family Poaceae and has several species that are native to Brazil. The Paspalum Germplasm Bank (GB) of the Brazilian Agricultural Research Corporation comprises approximately 450 accessions from 50 species. Among these accessions, Paspalum atratum (BGP 308) has economic potential for forage purposes. However, the endophytic and rhizospheric microbial communities within this accession and their ability to promote plant growth remain unknown. The present study aimed to isolate the endophytic and rhizospheric bacteria associated with P. atratum and to assess their potential for plant growth improvement, so-called plant growth-promoting bacteria (PGPB). For the in vitro tests, the ability of nitrogen-fixing bacteria (NFB), phosphate solubilization (PS) and indoleacetic acid (IAA) production were evaluated. A total of 116 endophytic and rhizosphere bacteria were obtained from the isolation. In the in vitro tests, 43 (37.00%) of these isolates showed positive NFB, PS, and IAA results. These isolates were identified by 16S rDNA sequencing. The phosphate solubilization index (PSI) ranged from 2 to 3.61, all 43 strains performed biological nitrogen fixation and the IAA production ranged from 12.85 to 431.41 μg ml−1. Eight of these 43 isolates were evaluated in vivo in a greenhouse using P. atratum caryopsis. The pots were filled with soil prepared with three different phosphate sources and one control without phosphate. After growth, the plants were submitted to morphological, bromatological and chemical determination. Data were analyzed using analysis of variance (ANOVA) and principal component analysis (PCA). In the in vivo test, treatments 105 (Pseudomonas sp.) and 458 (Pseudomonas sp.) were the most significant for the crystalline phosphate source, 109 (Bacillus sp.) for the sedimentary phosphate source and, as for the soluble phosphate source most treatments that received bacterial isolates had higher phosphorus content in the dry matter than the uninoculated soluble phosphate control. The 105FCR , 109FSE (sedimentary phosphate + Bacillus sp.), and 110 FSE (sedimentary phosphate + Enterobacter sp.) treatments showed the best results for plant growth promotion. This work made it possible to determine the bacterial community associated with P. atratum (BGP308) and to obtain new potential plant growth-promoting strains.The genus  Urochloa currently constitutes the majority of Brazilian pastures used for animal feed. The main problem in regard to Urochloa spp. is exposure to ecological imbalances owing to its low genetic variability , Brazil exported approximately 8.50 million tons of beef in 2020 and 4.38 million tons in the first half of 2021, significantly contributing to Brazil’s economy  and the crystalline rocks of Cajati/SP (5.0% P2O5)  .Paspalum accessions from the germplasm bank of Embrapa Pecuaria Sudeste have been evaluated for different uses, such as forage , nitrogen-fixing bacteria (NFB) and indoleacetic acid (IAA) production. In the in vivo stage, P. atratum plants were characterized in a morphological, nutritional and mineral manner (emphasizing phosphate solubilization), when inoculated or not with plant growth-promoting bacterial strains.The work was divided into steps P. atratum BGP 308  were collected in August 2016 (dry season) and January 2017 (rainy season). Access BGP 308 belongs to the Paspalum germplasm bank, located at Brazilian Agricultural Research CorporationSamples  from an adult plant of −1) to prevent fungal growth.The endophytic bacterial community was isolated according to −1). Bacterial cultures were preserved in tryptone soya broth (TSB) supplemented with glycerol (1:1) at −80°C until further study.The isolation of rhizospheric bacteria was performed according to 2, and 1.25 mM dNTP) and 3 pmol of each primer was performed for the selected isolates. The reaction conditions consisted of an initial 95°C step for 3 min, followed by 31 cycles of 95°C for 30 s., 60°C for 30 s., 72°C for 1 min. and a final extension of 10 min at 72°C in a BioRad T100 thermocycler. The amplicons were examined by 0.7% agarose gel electrophoresis and purified by using a QIAquick (Qiagen) kit. The sequencing reactions were performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and sequenced using an ABI 3730 DNA Analyzer. Sequences were analyzed by Sequencing Analysis 5.3.1 software using the Base Caller KB, and the low-quality sequences were visualized and edited using BioEdit software  sequence was used as an outgroup.The phylogenetic tree was obtained by multiple alignments of software  using thsoftware . HalobacP. atratum were qualitatively/quantitatively screened in triplicate for their ability to solubilize inorganic calcium phosphate. Strains were incubated in nutrient agar supplemented with Ca3(PO4)2 for 96 h at 28°C  assay was carried out by growing the strains in a semisolid nitrogen-free medium twice for 72 h/28°C . For theP. atratum were germinated and inoculated with eight different strains that presented PSI ≥ 2.0. The experiment was conducted in a greenhouse at the Brazilian Agricultural Research Corporation (see footnote 1), São Carlos, SP, Brazil. A randomized block design with three replications was used in a factorial scheme of 8 × 3 + 1 with pots containing 0.5 l and 4.5 l of soil.Caryopsis of 2O5 soluble in neutral ammonium citrate + water), (2) sedimentary phosphate—Arad phosphate rock concentrate , and (3) crystalline phosphate—Cajati phosphate rock concentrate  with a dose of 200 mg kg−1 P or 458 mg kg−1 P2O5.The soil was prepared with three different sources of phosphate plus the control without phosphate, totaling 36 treatments and 108 pots. The three sources of P used were (1) soluble phosphate—triple granulated superphosphate (46.0% P2SO4 (60% K2O) until K reached 3% of the cation exchange capacity (CEC).The chemical characteristics of the soil were determined according to 9 CFU/ml and used in this step. Thus, seedlings were transplanted into 500 ml pots containing limed soil without correction of nutrients. Five more inoculations were performed: in 500 ml pots, they received two more inoculations (15 and 30 days after transplant), and in 4.5 kg pots, they received three more inoculations . Finally, the 6 ml volume of the standardized bacterial suspension was inoculated into the soil close to each plant’s root. The control was inoculated with phosphate buffered saline (PBS) solution without the bacterial isolate.The caryopses of the spikelets were removed and subjected to the disinfection process in a closed desiccator using the protocol described by The seedlings remained in the 500 ml pots for 43 days and were transplanted into the 4.5 kg pots for the four treatments. At 50 days after transplanting, the aerial parts of the plants were cut 15 cm from ground level and measured.The aerial part samples were placed in an oven with forced circulation at 60°C for 72 h. After drying the samples and determining the dry leaf weight, the material was crushed in a Wiley mill with 1 mm sieves. The collected material was stored in a plastic bottle.in vitro digestibility), FDN , Ll (leaf length), PHe (plant height), Fe (iron), FDA (acid detergent fiber), MS (dry matter), EE (ether extract) and Cu (copper). Dry matter, crude protein, neutral detergent fiber, acid detergent fiber, lignin, and in vitro digestibility were determined using a near-infrared spectrometer , EN (nitrogen extract), ECa , CP (crude protein), MM , LIG (lignin), Ca , Mg (magnesium), P (phosphorus), K (potassium), S (sulfur), Mn (manganese), Zn (zinc), N (nitrogen), SPAD (SPAD index), AFW , ADW , LW (leaf width), NL (number of leaves), NT (number of tillers), ANT (presence of anthocyanin), LA (leaf area), EZn (zinc extract), DIV  were analyzed. Data were analyzed using analysis of variance (ANOVA) and principal component analysis (PCA) with SASsoftware .−1 cfu gm−1 (leaf) to 43 × 10−3 cfu gm−1 (root), whereas in the dry season isolation, the bacterial population ranged from 05 × 10−1 cfu gm−1 (leaf) to 35 × 10−2 cfu gm−1 , and in the dry season, it was 5.6 (water) and 5.0 (CaCl2).In the rainy season isolation, the bacterial population ranged from 02 × 10P. atratum BGP 308, where 43 (37.00%) were obtained from rhizospheric soils, 42 (36.20%) from roots, and 31 (26.70%) from leaves. A total of 74 (63.70%) strains were isolated in the rainy season and 42 (36.20%) in the dry season.A total of 116 bacterial isolates were collected from Enterobacter (46.50%), Pseudomonas (32.50%), and Pantoea (13.90%) were the most abundant  for the groups formed of samples from each genus and its correspondent reference sequences for the genus. Moreover, nodes forming a group with all the samples from genera Enterobacter, Pantoea and Pseudomonas and another group with all the samples from genera Bacillus, Microbacterium and Micrococcus also showed significant support (>90%), showing the relation among genera identified in this study  showed positive NFB, SF, and IAA results. From these, the strains that belonged to the genera abundant . On the is study .Enterobacter genus, showed the best results, with PSI values of 3.61, 3.58, and 3.56, respectively. All 43 strains in −1. The endophytic leaf strain 170 (Pantoea sp.) showed the highest IAA production (431.41 μg ml−1), followed by 18 Enterobacter spp.; endophytes from roots presented 105.05–263.74 μg ml−1 of IAA production.The phosphate solubilization index (PSI) ranged from 2 to 3.61 . StrainsMicrobacterium, Micrococcus, Pantoea, Bacillus, and Enterobacter were endophytes, while the Pseudomonas strains were either endophytic or rhizospheric. Among the 43 strains selected for the in vitro tests, six were isolated from the rhizosphere, 27 from the root, and 10 from the leaf (All strains belonging to the leaf .in vivo test were Isolates 25 (Pseudomonas sp.) and 458 (Pseudomonas sp.), and the endophytic bacteria were Isolates 103 (Enterobacter sp.), 105 (Pseudomonas sp.), 109 (Bacillus sp.), 110 (Enterobacter sp.), 161 (Pseudomonas sp.), and 170 (Pantoea sp.).In the plant growth promotion assay, the selected strains belonged to the rhizosphere, root and leaf. The PSI ranged from 2 to 3.61, and all were positive for NFB and IAA. The rhizospheric bacteria selected for the p-value = 0.0023.The triple interaction [Source of Phosphorus (3) vs. Isolate (9) vs. Cut (3)] for Phosphorus extract (P) was significant with –1, considering treatments and cuts (Pseudomonas sp.) and 458 (Pseudomonas sp.) had a higher phosphorus content than the control crystalline phosphate in the first cut and showed a drop in the second third cut. The treatment containing Strain 110 (Enterobacter sp.) presented a high level of phosphorus in the second cut compared to the control. Within this treatment, P extract increased in the second cut, and decreased it in the first and third cut. Despite the numerical variations, in the phosphorus content, there was no statistical difference (p > 0.05). In treatments with sedimentary phosphate, the mean phosphorus content in dry matter ranged from 0.89 to 11.82 kg ha–1, considering treatments and cuts. The treatment that received the bacterial isolate 109 (Bacillus sp.) had a higher phosphorus content than the sedimentary phosphate control in the first cut (p > 0.05). Within this treatment, the phosphorus content was higher in the first cut, followed by the second and third cut (p ≤ 0.05). The treatment that received strain 103 (Enterobacter sp.) also showed an increase in phosphorus content in the first cut. In the other treatments there were no (p > 0.05) significant changes in the phosphorus content in the cuts.The P extract showed that in treatments with crystalline phosphate, the mean phosphorus content in dry matter ranged from 0.90 to 3.82 kg haand cuts . The tre–1, considering treatments and cuts. Regardless of treatment, the phosphorus content was higher in the first cut (p ≤ 0.05). In this cut, the lowest phosphorus content in the dry matter was in the treatment that received the strain 103 (Enterobacter sp.) and the highest was in the treatment that received the strain 161 (Pseudomonas sp.) with 11.65 and 24.00 kg ha–1, respectively. The treatment that received the bacterial isolate 110 (Enterobacter sp.) showed a significant difference (p ≤ 0.05) between the three cuts, for the phosphorus content, with 21.66, 7.73 and 3.98 kg ha–1, respectively. The phosphorus content, in cut1, for the control of uninoculated soluble phosphate was 15.21 kg ha–1. In cuts 2 and 3, the maximum phosphorus content reached was 7.73 kg ha–1, regardless of the treatments.In treatments with soluble phosphate, the mean phosphorus content in dry matter ranged from 3.14 to 24.00 kg haThe results of the principal component analysis (PCA) showed that Component 1 (PRIN1 = 65.31%) and Component 2 (PRIN2 = 8.52%) explained 73.83% of the variance observed. A total of 22 variables were significant (correlation > 50%) in the discrimination of treatments  among thWhen evaluating the phosphate source used in the experiment, the analyzed variables divided the treatments into two groups , directePrincipal Component 2 showed strong associations with aerial fresh weight (AFW), aerial dry weight (ADW), leaf width (LW), number of leaves (NL), number of tillers (NT), anthocyanin (ANT), area leaf (LA), phosphorus extract (EP), calcium extract (ECa), calcium (Ca), magnesium (Mg), phosphorus (P), sulfur (S), manganese (Mn), nitrogen (EN) and mineral matter (MM) . These vPseudomonas sp.), 109FSE (sedimentary phosphate + Bacillus sp.), and 110FSE (sedimentary phosphate + Enterobacter sp.) treatments.In In the production of pastures for animal nutrition, there is great concern regarding the cultivation of plants. For cultivation, plants obtain most of their nutrients from some industrialized source. In 2018, Brazil consumed 852.4 thousand tons of triple superphosphate  and 2,437.7 thousand tons of ammonium phosphate . For nitrogen, phosphorus, and potassium (NPK) fertilizer, Brazil consumed 15507.1 thousand tons and imported 13338.7 thousand tons, the largest importer of NPK on a worldwide scale . MicroorPaspalum accessions are scarce. Enterobacter strains from Paspalum vaginatum and evaluated their influence on promoting salt tolerance in the plant. P. arundinaceum in Mina Santa Cruz, Bolivar., Colombia, aiming to obtain plant growth-promoting strains with potential for phytoremediation of mercury-contaminated soil. Paspalum and evaluated the effect of inoculation on P. regnellii, P. atratum, and P. malacophyllum. The genotypes were also collected from the Paspalum germplasm bank at the Brazilian Agricultural Research Corporation; however, the genotype of P. atratum studied was cv. Pojuca BGP 098. For studies involving phosphate solubilization in Paspalum with potential for forage, this work is the first to examine phosphate rock in the nutrition of P. atratum plants, intermediated by cultivable endophytic and rhizospheric microorganisms.Studies involving the endophytic and rhizospheric microbiota of 6 and 6.8 ± 1.20 × 103 for rhizospheric and endophytic bacteria in maize plants.Based on the present study results, the bacterial population was higher in the rhizosphere and the root. The rhizosphere exhibits different physical and chemical characteristics than nonrhizospheric soil, as plants can produce root exudates, which provide bacterial nutrition and make this environment favorable for bacterial colonization . In the Among the 116 strains isolated, 43 showed positive results for NFB, SF, and IAA. The 43 strains belonged to Proteobacteria, Firmicutes, and Actinobacteria. Genetic diversity studies have reported that these phyla are both endophytic and rhizospheric  and genein vitro tests, the main strains that showed the potential to promote plant growth belonged to Bacillus, Enterobacter, Microbacterium, Micrococcus, Pantoea, and Pseudomonas . The mineral descriptor was potassium (K), and as for the results of the bromatological analysis, the crude protein (CP) content was the most significant. These descriptors showed lower values in treatments that received the soluble phosphate source. For Group 2, the most significant morphological descriptors were aerial fresh weight (AFW) and aerial dry weight (ADW), the mineral was calcium extract (ECa), and the bromatological descriptor was nitrogen extract (EN). Phosphorus was the 11th most significant descriptor in Principal Component 1 and the 12th most significant in Principal Component 2; thus, it had equal importance for both groups.In the in vitro tests showed that the PSI ranged from 2 to 3.61, and the phosphate-solubilizing bacteria belonged to Bacillus, Enterobacter, Microbacterium, Micrococcus, Pantoea, and Pseudomonas. These genera are already described in the literature as phosphate-solubilizing bacteria. Pseudomonas sp. with 5.3 and 8.3 of PSI and Pantoea sp. with PSI = 6.0. Enterobacter, presenting 2.2–5.8 of PSI. Similarly, the strain with the highest PSI also belonged to the Enterobacter genus in the present study.The in vivo test in a greenhouse, the treatments with crystalline phosphate plus Isolates 105 (Pseudomonas sp.) and 458 (Pseudomonas sp.) showed available phosphorus in the initial period of plant development. However, the treatment that received the 110 (Enterobacter sp.) strain showed higher phosphorus content in the second cut, suggesting that the 110 strain needs a more extended period to make the phosphorus available to the plant. In the literature, there is a search for an alternative source of phosphorus for plant nutrition, with Arad rock phosphate being one of those rocks with potential for this purpose. Trifolium repens) and ryegrass (Lolium multiflorum) pastures. Megathyrsus maximus than in Urochloa brizantha, noting that the success in fertilization was dependent on the cultivated species and soil acidity. These studies sought to explore the gradual capacity of natural rocks to release phosphorus. However, they only used Arad’s natural phosphate in crops without studying the endophytic and rhizospheric microbiota of the host plant. In the present study, the primary method was the optimization of the phosphorus contained in the phosphate rock through selected microorganisms isolated from soil and plant tissue of P. atratum. Another characteristic observed in P. atratum was the acidic soil; both in the rainy and dry seasons, the soil pH of the soil was not higher than 5.6. As mentioned by Regarding the Enterobacter sp.) and 458FCR (Pseudomonas sp.) treatments were very close. The 105FCR (Pseudomonas sp.) treatment stood out, being the treatment with crystalline phosphate that came closest to the treatments that received soluble phosphate. The literature shows that the main phosphate solubilizers are Arthrobacter, Azospirillum, Azotobacter, Bacillus, Beijerinckia, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Mesorhizobium, Microbacterium, Pseudomonas, Rhizobium, Rhodococcus, and Serratia  treatment was the closest to the treatments with soluble phosphate and presented the highest phosphorus content in the first cut. One hypothesis to explain the higher content of phosphorus in the first cut is that there was great solubilization of sedimentary phosphate during the initial periods of the plant, thus depleting almost all available phosphorus sources in the first cut and, consequently, reducing these in dry matter in the second and third cut. This fact shows the importance of topdressing after grazing.In treatments with sedimentary phosphate, the 109FSE (Enterobacter sp.). Therefore, future studies should explore the possibility of using less soluble phosphorus by inoculating phosphate-solubilizing bacteria, seeking to optimize the use of soluble phosphorus in agriculture. When looking for bacteria with the potential for phosphate solubilization in peas, Bacillus sp. and Pseudomonas sp. They used soluble phosphate as a control and tricalcium phosphate (Ca3(PO4)2) insoluble in the treatments. As a result, it was observed that the strains increased the phosphorus content in the plant compared to the insoluble control. Nevertheless, no treatment equaled or surpassed the phosphorus content present in the dry matter of the plants treated with soluble phosphate. Similar to the results presented by The results observed in treatments with soluble phosphate that included the bacterial isolates also suggest that the source of phosphorus was depleted during the initial stages of plant development. Of the eight treatments that received the strains, seven showed higher phosphorus content than the soluble phosphate control dry matter. The exception was treatment 103FSO , a nitrogen-fixing bacteria, found by Bacillus and Burkholderia. As in these studies, no strains of Azotobacter were obtained in the present study. However, the growth of other microorganisms in the NFB medium was justified because the medium was not highly selective; therefore, strains with the potential to use malic acid as a carbon source and with a pH of 6.8 can grow  and nitrogen (N) content correlated with the morphological descriptor index SPAD in the plant. The CP descriptor was calculated by multiplying the nitrogen content by 6.25 . The SPAPseudomonas sp.), 103FSE (Enterobacter sp.), 25SF (Pseudomonas sp.), CAFCR, and 109SF (Bacillus sp.) were the ones with the highest SPAD index. On the other hand, the 103FCR (Enterobacter sp.), 25FCR (Pseudomonas sp.), 109SF (Bacillus sp.), 161SF (Pseudomonas sp.), and 105 FSE (Pseudomonas sp.) treatments presented the highest levels of CP and N.Among the most significant descriptors to assess the variation between treatments and plant growth promotion, crude protein content was significantly crucial in Group 1 treatments. The 161SF , Stargrass (Cynodon), and marandu grass (U. brizantha cv. Marandu), respectively.P. atratum) and by U. brizantha, U. decumbens, U. humidicola, and U. Ruziziensis, which ranged from 6.4 to 7.5% CP in dry matter.Crude protein contents lower than 7% in the dry matter limit animal nutrition . All treIn the present study, none of the treatments received nitrogen fertilization. Nevertheless, many of them had similar or superior CP results compared to other studies that evaluated cultivars already on the market. Even the phosphate-free control (CSF), which did not receive any phosphorus source or bacterial inoculum, showed CP results superior to those found by Pseudomonas sp.), 103 (Enterobacter sp.), 105 (Pseudomonas sp.), 109 (Bacillus sp. and 161 (Pseudomonas sp.). The 103FSO (Enterobacter sp.) treatment stood out due to high levels of CP and N in the dry matter. The Isolate 103 (Enterobacter sp.) also showed good levels of CP and N in treatments with a sedimentary and crystalline phosphate source, making it a good candidate for biological nitrogen fixation investigation. Potassium was another significantly important mineral descriptor observed in Group 1 treatments. In the K+ format, potassium regulates the osmotic potential and activates enzymes involved in respiration and photosynthesis in the plant (Pseudomonas sp.), 103FSE (Enterobacter sp.), 170FSE (Pantoea sp.), 25FCR (Pseudomonas sp.), and 161FCR (Pseudomonas sp.), followed by the control treatments CASF, CAFCR, and CAFSE. The microbiological modification technique enables the direct application of rocks in agriculture. Citric and oxalic acids produced by microorganisms release potassium from biotite, a common mineral in the silicate class , 109FSE (Bacillus sp.), 110FSE (Enterobacter sp.), 103SF (Enterobacter sp.), 458SF (Pseudomonas sp.), and 458FCR (Pseudomonas sp.) showed growth similar to those treated with a soluble phosphate source. Therefore, they can be selected for future plant growth-promotion experiments.The plants inoculated with the bacterial Isolates 105FCR , Pseudomonas (32.50%), and Pantoea (13.90%), and Bacillus, Microbacterium, and Micrococcus strains represented 6.90%.As for the capacity of the plant growth-promoting bacterial strains, 43 (37.00%) strains showed positive NFB, SF, and IAA results and belonged to Pseudomonas spp.) to 3.61 (Enterobacter spp.) and the IAA production ranged from 12.85 (Pseudomonas spp.) to 431.41 (Pantoea spp.) μg ml−1.The phosphate solubilization index (PSI) ranged from 2  and 458 (Pseudomonas sp.) were the most significant for the crystalline phosphate source, 109 (Bacillus sp.) for the sedimentary phosphate source and, as for the soluble phosphate source most treatments that received bacterial isolates had higher phosphorus content in the dry matter than the uninoculated soluble phosphate control.In the For the morphological, mineral and bromatological variables, the principal component analysis showed that Principal Component 1 had significant associations with the SPAD index (SPAD), potassium (K), zinc (Zn), crude protein (CP), lignin (LIG) and nitrogen (N). While, principal Component 2 showed strong associations with the other 16 descriptors.Pseudomonas sp.), 109FSE (sedimentary phosphate + Bacillus sp.), and 110 FSE (sedimentary phosphate + Enterobacter sp.) treatments showed the best results in the plant growth promotion assay.These diverse cultivable bacterial genera have the potential to promote plant growth, and the 105FCR  can be found at: All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by grants from the São Paulo Research Foundation, FAPESP (Proc. No. 2020/11315-6).ND, WJ, AB, BV, and AF were employed by Embrapa Pecuária Sudeste.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Complete recovery after adequately treated neuroborreliosis is common, but studies report that some patients experience persistent symptoms like self-reported cognitive problems and fatigue. Persisting symptoms are often termed post-Lyme disease syndrome, of which etiology is not clearly understood. The aim of this study was to investigate cognitive function, possible structural changes in brain regions and level of fatigue. We have not found previous studies on neuroborreliosis that use standardized neuropsychological tests and MRI with advanced image processing to investigate if there are subtle regional changes in cortical thickness and brain volumes after treatment.We examined 68 patients treated for neuroborreliosis 6 months earlier and 66 healthy controls, with a comprehensive neuropsychological test protocol, quantitative structural MRI analysis of the brain and Fatigue Severity Scale.p = 0.001), and more patients (25.4%) than controls (5%) had severe fatigue (p = 0.002), but neither mean score nor proportion of patients with severe fatigue differed from findings in the general Norwegian population.We found no differences between the groups in either cognitive function, cortical thickness or brain volumes. The patients had higher score on Fatigue Severity Scale 3.8 vs. 2.9 (The prognosis regarding cognitive function, brain MRI findings and fatigue after adequately treated neuroborreliosis is favorable. Although neuroborreliosis is effectively treated with antibiotics, previous studies have found that 10–50% of the patients report persistent symptoms like fatigue, malaise, pain and subjective cognitive problems . PLDS isThe aim for this study was two-fold: firstly, we aimed to assess cognitive function with neuropsychological tests and measure self-reported fatigue in patients treated for neuroborreliosis six months previously as compared to a healthy age- and gender matched control group. Secondly, we aimed to assess cortical thickness and brain volumes in patients and controls using quantitative MRI.The study is part of a randomized, double-blinded non-inferiority trial comparing 2 and 6 weeks of doxycycline treatment in patients with acute neuroborreliosis . Betweenrd edition [n = 58) were asked to grade subjective memory/concentration problems 6 months after treatment as no problems, mild without influence on daily living, or serious with influence on daily living.Patients and controls underwent testing of general abilities using the subtests Block design, Matrixes, Vocabulary, Information, Digit-span, Arithmetic, Symbol search and Coding from Wechsler Adult Intelligence Scale 4th ed. to estimate full-scale IQ . To asse edition . We defiFatigue severity scale (FSS) was used to identify level of fatigue . FSS hasThe whole brain scan for this study was a sagittal 3D T1 weighted MPRAGE (magnetization-prepared rapid gradient-echo) sequence. Subjects were included at two locations with different 3 T scanners, one with a Siemens Skyra and one with General Electric Signa where 55 patients and 58 control subjects were scanned on the Skyra and 11 patients and 5 controls were scanned on the Signa. Head coil with 64 and 32 channels, respectively, was used. The study protocol was set up to be as similar as possible with the following parameters (Skyra/Signa): Slice thickness 1.1/1.0 mm, field of view read 256 mm and phase 96.9%/256 mm, repetition time 2300 ms, echo time 2.98 ms/minimum and inversion time 900/943 ms.https://surfer.nmr.mgh.harvard.edu/. Details about the method are described elsewhere [For the image analysis, we used FreeSurfer software package version 7.1.1 available at lsewhere –34, brielsewhere  resultinIBM SPSS statistics for Windows version 28 was used for statistical analysis . IndepenWe used Spearman rho to investigate correlation between FSS and cortical thickness, volumes and neuropsychological tests, and Chi-Square test to investigate proportions. To compensate for multiple comparisons, False Discovery Rate (FDR) was applied in the analysis of differences in mean neuropsychological tests scores, in ROI based cortical thickness and in brain volumes . FDR wasSeven patients were unable to perform one or more of the neuropsychological tests, and five patients missed one or more subtests at random. For the control group, one participant was unable to perform two neuropsychological tests, while four participants missed one or more subtests at random. Missing data were handled by pairwise deletion in the analysis.For the FreeSurfer analysis, one patient and five controls were excluded from the analysis due to errors in segmentation.p = 0.011). There were no differences in proportions in any other subtests. The patients had a higher level of fatigue. Out of 63 patients, 16 (25.4%) reported severe fatigue ≥ 5, vs. 3 (5.0%) in the control group (p = 0.002). Out of 58 patients being asked, 41 (70.7%) reported no subjective memory/concentration problems, while 17 (29.3%) reported mild problems without influence of daily living. No patients reported serious problems with influence of daily living. Fifty-four of these patients also completed CVLT-II. Out of 38 patients reporting no subjective memory/concentration problems, 10 patients (26.3%) had very low score on CVLT-II trial 1, while 28 patients (73.7%) had normal scores. Out of 16 patients reporting mild problems, 3 (18.8%) had very low score on trial 1, while 13 (81.3%) were in the normal range. No association was found between subjective memory/ concentration problems and very low score on CVLT-II trial 1 . Patients who were on sick-leave related to neuroborreliosis (n = 11), did not have lower scores on neuropsychological tests compared with patients not on sick-leave, but a higher level of fatigue .Demographic and clinical data for patients and controls are presented in Table We found no differences in cortical thickness in frontal, parietal, temporal or occipital lobe Table . Whole br = 0.329, p = 0.009), color word inhibition  and color word inhibition switching . P-values in analysis of correlations are unadjusted.There was no correlation between FSS and cortical thickness in brain lobes and brain volumes. In the patient group, we found a weak, but significant correlation between FSS and color word naming  . A possiWe did not find any differences in cortical thickness or volumes between patients and controls. Research in patients with ME have shown these patients tend to score lower in certain cognitive domains, mainly reduced processing speed . ImagingThe strengths of our study are a large cohort of patients with well-characterized neuroborreliosis and an age and gender matched control group. We included a comprehensive neuropsychological protocol covering several cognitive domains, including full-scale IQ to ensure premorbid cognitive function in the patient group was comparable with the control group. Seven patients missed one or more subtests due to fatigue, while only one of the control persons missed one or more subtests for the same reason. We cannot rule out missing data may have influenced the results on some of the neuropsychological tests. None of the participants missed Color Word subtests due to fatigue. As these subtests were the only ones that weakly correlated with fatigue, we interpret the influence of missing data to be modest. Considering no other studies have investigated morphological alterations in patients with neuroborreliosis using the same method, our research questions regarding MRI findings of cortical thickness, volume and subcortical volume were wide and explorative. We chose to merge some regions of interest and volumes instead of focusing on very numerous specific regions, and minor changes in specific structures might have been wiped-out.Cognitive function, cortical thickness, and brain volumes do not seem to be affected in patients with adequately treated neuroborreliosis. The patients report a higher level of fatigue compared to the control group, but not compared to the general Norwegian population. Overall, our results indicate a favorable outcome in a majority of patients with adequately treated neuroborreliosis.