Estimation of the postmortem interval in advanced postmortem stages is a challenging task. Although there are several approaches available for addressing postmortem changes of a (human) body or its environment , most are restricted to specific timeframes and/or individual and environmental conditions. It is well known, for instance, that buried bodies decompose in a remarkably different manner than on the ground surface. However, data on how established methods for PMI estimation perform under these conditions are scarce. It is important to understand whether and how postmortem changes are affected under burial conditions, if corrective factors could be conceived, or if methods have to be excluded for respective cases. We present the first multi-methodological assessment of human postmortem decomposition carried out on buried body donors in Europe, at the Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA) in the Netherlands. We used a multidisciplinary approach to investigate postmortem changes of morphology, skeletal muscle protein decomposition, presence of insects and other necrophilous animals as well as microbial communities  from August to November 2018 associated with two complete body exhumations and eight partial exhumations. Our results clearly display the current possibilities and limitations of methods for PMI estimation in buried remains and provide a baseline for future research and application. Decomposition of a cadaver involves the processes of autolysis, putrefaction and decay . These pThe study of (human) decomposition is a very longstanding task in forensic science. In the context of PMI estimation, a main source of data comes from cross-sectional case observations of decomposing human bodies; however, such observations are limited in their contributions to theory building , mostly Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA) comprises a 500 m2 burial ground dedicated to study decomposition processes of buried human remains [Indeed, the establishment of anthropological research facilities has commenced a new era for decomposition studies. Researchers can seize the opportunity to conduct semi-controlled, longitudinal research studies using replicate human remains with known PMI . Practit remains .In the present multidisciplinary study, the decomposition process of two body donors, buried at the ARISTA facility, was monitored over a period of 15 weeks. At predefined time points, the bodies were exhumed, or partially exhumed to assess morphology , insect activity, microbial communities and skeletal muscle protein degradation. This study aimed to investigate the applicability and challenges of using a multidisciplinary approach to PMI estimation for advanced decomposition stages in buried human bodies.th) air temperature reached 28.5°C. The next three days were hot with a maximum temperature of 34.6°C on August 7th. Mean temperatures in all four months were constantly 1–2°C above Amsterdam average, while precipitation was significantly below average throughout the experiment  showed environmental temperature peaks at around 14:30 and fluctuations of approximately 11.5°C. The highest grave temperature peaked at 23:30 with fluctuations of 1.5°C. The body was warmest at 06:30 and only varied by about 0.5°C.Prior to the first exhumation, the probes for measuring the temperatures of the graves had been placed too deep, as the body temperature of both donors was almost always above the temperatures in the graves. Consequently, at the second burial (after the first exhumation) the probes were placed at middle depth of the body, as opposed to the bottom of the grave. Thereafter, a double-buffer effect was observed. Environmental temperature fluctuations led to temperature changes with a reduced amplitude and a phase shift in the grave, which again led to smaller and delayed body temperature oscillations . Data foth), no visible changes and no skin discoloration were observed except for livor mortis, consistent with a very early stage of decay. TBS and TDS assigned were respectively 3 and 6 for donor A and donor B corresponding to 67.3 (TBS) and 14.8 (TDS) estimated ADD. These scores are commonly assigned to bodies at very early stage of decomposition, although 17/16 (in donor A/donor B) days already passed since death corresponding to 68–64 ADD spent by the bodies in coolers at 4°C.On the day of the burial  and 122/121 days after death. Mummification was observed at each body part  with bone exposure of (way) less than half of the area being scored, and with no disarticulation. Based on the appearance of these late postmortem changes, high TBS and TDS values were assigned to donor A and B, with 24 and 12 respectively, corresponding to 916.2 and 204.2 ADD. Also for this second exhumation ADD predicted by both methods did not correspond to the actual ADDs of 2049/2027.On November 17Hence, all ADD predictions using morphology assessments, 28 and 105 days after the burial did not correspond to the actual values. TBS and TDS largely underestimated the ADD of the burial interval. However, the prediction by TBS was more accurate than TDS . No disaPartial exhumations and muscle biopsy sampling worked well in the early stages of the experiment. Until day 28 (including the first sample from the left thigh) each biopsy provided a sufficient amount of material, macroscopically easily identifiable as muscle tissue in both donors. Overall, it took about 15 to 20 minutes to complete sampling, specimen storage in the on-site freezer and re-burial. From day 40 onwards, the obtained biopsy material was increasingly difficult to identify as muscle tissue. Additionally, the thighs slipped alongside the femora, making it impossible to target a specific location/muscle. Nevertheless, a sufficient amount of material was collected until day 105, stored and analyzed according to the protocol.All investigated proteins depicted native bands at the day of burial and underwent qualitative changes during the time course of the experiment. Tropomyosin was present as a characteristic double band (at 38 and 36 kDa) until day 10 in both donors. While bands were lacking in all subsequent donor A samples, the pattern remained detectable on day 18 in donor B. Additionally, a single band was detected on day 28 in the tissue sample collected from the right thigh. Interestingly, no bands were found in the sample collected on the same day from the left thigh, as well as in all subsequent samples.The native vinculin band (117 kDa) was present until day 3 in both donors and did not appear in any samples with larger PMI. Meta-vinculin (135 kDa) was detected on day 0 in donor A, but in no other sample. A degradation product at 84 kDa was present from day 0  until day 10. A faint band with intensity below 1% of the native band was visible on day 18 in donor B, but no later than that in any of the donors. One sample  depicted a degradation product at approximately 75 kDa. From day 3 to day 10 both donors depicted another vinculin degradation product at approximately 63 kDa. While still detectable on day 18 in donor B, in donor A this band was lacking at this time point. The 63 kDa fragment was absent in all subsequent samples.The native α-actinin band (100 kDa) was present until day 5 in donor A and until day 10 in donor B. A degradation product of 80 kDa appeared on day 3 in both donors and disappeared again on day 18 (A) and day 28 (B). A second degradation product of approximately 65 kDa was detected during the same timeframe in donor B, however only on day 5 in donor A, together with a <1% faint band on day 10.The native GAPDH (40 kDa) vanished after day 10 in both donors. The native eEF1A2 band (50 kDa) disappeared after day 3 in donor A and after day 1 in donor B . OriginaCalliphora, Lucilia and Pollenia caught in the trap, together with 1–2 flesh flies (Sarcophagidae) and house flies (Muscidae) monitored on plants and fences on the site. Only a single female of the species Lucilia sericata was found on donor B during two afternoon hours  [During exhumations, insect activity on the bodies was very low. Donor A showed no infestation during both evaluation days, while Donor B revealed small spots of fly larvae (Day 28 and Day 105) and a few pupae (Day 105) which were distributed in quite different body regions (e.g. right foot and left elbow). The overall number of specimens was below 50, all larvae and pupae belonging to the scuttle fly horidae) , 21.Enchytraeus. Sequence comparisons with the published barcoding regions of E. moebii, E. albellus, E. crypticus, and E. albidus, as well as further so far unidentified Enchytraeus species from the collection of the Goethe-University Frankfurt, as well assigned the specimens to the genus Enchytraeus. A Neighbor Joining analysis with the sequence of Lumbricus terrestris was used as outgroup.Additionally, on the left arm of donor B, annelids, initially addressed as Enchytraeids, were collected. Sequence analysis of these specimens produced no exact match in NCBI and in the BOLD databases but has been assigned to the genus The original sample total intended for sequencing was 48, which included 24 per donor body representing different body sites and three time points . Of the 48 total, 16 samples did not amplify (often because of low DNA concentrations) after extraction and cleanup procedures, leaving a total of 32 samples that were sequenced. Sequencing of the V4 region of the 16S rRNA gene resulted in 2,453,935 total reads, averaging 61,348  reads per sample. Based on rarefaction curves, using observed sequence variants and Shannon diversity, a rarefaction of 4,000 reads per sample was chosen for additional analyses . After fThe overall postmortem microbial communities among body sites for the two donors combined was represented by eight phyla, nine families and 27 genera with relative abundances of >0.3%, >3% and >1% among all samples, respectively  Tables. Peptoniphilus was 20% of rectum communities), with the most dominate genera within a body site generally ranging from 5–16%  without accounting for differences between donors. The top bacteria genera used in the predictive model (determined by mean decrease accuracy) were mostly bacteria absent on the August 4th sampling but in higher abundances on later dates  when predicting body site. However, random forest modeling was able to distinguish among sample dates timepoint  with a 5.6% error rate  for bodies in earth graves. Research for this specific purpose is difficult because observations and collections are only possible with disturbance of the body and its environment. With the exception of muscle tissue sampling, which was possible with minor interference, the other assessments required complete body exhumations. Depending on the research question, a subtle balance between least possible disturbance and maximum outcome had to be defined. Similar compromises have to be considered, not only for buried bodies, but when several methods are to be investigated at once in a collaborative manner. Inclusion and exclusion criteria, physical properties of the site/graves  and the bodies (clothing), sampling/exhumation intervals and resources are among important aspects to work out. Despite the resulting limitations and the small sample size we did, however, collect valuable data important for assessing the potential and limitations for advanced-stage PMI estimation methods for buried human remains.Since Megyesi’s study in 2005 , decompoIn the assessment of TBS and TDS, no disagreement between the two examiners occurred. Both assessments are practical, simple and user-friendly to efficiently evaluate human decay. According to previous reports , 22–26 tMummification, as observed in both donors, is a process of preservation and is characterized by the desiccation of remnant tissues. Especially porous and permeable soil, as present in the ARISTA facility, can easily absorb most putrefactive fluids causing a rapid dehydration of soft tissues and leading to mummification rather than decomposition. Previous work showed the weaknesses of scoring scales and regression models developed to predict ADD when mummification and/or irregular decomposition occurs . Vass reSo far, the study of decomposition using ADD has been largely investigated in bodies exposed on surfaces , 23–25 aThe postmortem degradation of biomolecules has lately become of forensic interest. Among those molecules are DNA (e.g. ), RNA  Decomposition patterns within the first 10 days post burial are familiar to reference data. Notably, the recorded changes were in close temporal resemblance to data, gathered from non-burial experiments in animal models and humans. (ii) Novel degradation patterns, especially the complete loss of protein bands, were detected between 10 and 28 days post burial. (iii) After 28 days, there were no protein profiles detectable using the current standard protocols.In detail, (i) the presence of tropomyosin and GAPDH as native bands until 10 days post burial confirms results from previous studies in animal models ,51,52 anTo our knowledge, (ii) many of the detected postmortem protein band alterations have not yet been described in forensic context, or at least could not reliably be assigned to a specific PMI or ADD. These alterations include the total loss of the native bands of tropomyosin (after 18/28 days), α-actinin (10/18 days) and GAPDH (18 days), some of which have previously been used as loading controls due to their ubiquitous expression in mammalian body cells , 54. Bas(iii) 28 days appears to be the maximum limit for (sensible) application of the analysis of muscle protein degradation for PMI estimation in this study/setting. Buried conditions provide a well-protected environment for autolytic processes. In other conditions, such as natural surface environment, this limit does not necessarily have to be reached. Once the skin barrier ruptures, e.g. by insect activity/feeding, tissues can be severely damaged and protein analysis can be complicated, or impossible . This meLucilia sericata appeared at the scene and did not oviposit after detailed examination. The low interest in the bodies may have been due to their low temperatures. Just arriving from the cooler, the body temperatures were still < 10°C, even in the body placed on the sunny surface for two hours.In forensic entomology, most cases are concerned with blow flies as they are keen to colonize fresh dead bodies and are very abundant in urban and natural habits. However, concealed and buried remains are difficult or impossible for them to access. Shallow graves are often characterized by a distinguished fauna , which vMegaselia scalaris, the most common and distributed phorid fly, feed on a very broad spectrum of decaying organic materials, and consequently are also common on human bodies. M. scalaris is usually most frequent in the first wave of insect arrivals but will oviposit at any stage, when given the opportunity [While blow flies are unable to gain access to a buried or hidden corpse, Phoridae can still enter the smallest of openings. The adult flies burrow through the soil and oviposit on the corpse . The larortunity . Few larM. scalaris is associated with a minimum PMI of about 3 weeks for donor B (based on the finding of pupae during the second exhumation on Nov. 17th). This would be an underestimate of about 10 weeks, but compared to morphology and protein analysis, which were not informative after several weeks postmortem, this could be a helpful estimation depending on the case. When focusing on the first exhumation (day 28), the entomological findings were close to the real timeline, as the feeding larvae of M. scalaris indicated a minimum PMI of about 2 weeks, much closer to the real burial time. However, the present study highlights 3 important challenges of forensic entomology: i) it can (obviously) just be applied when insects colonize the body: while donor B could be analyzed with entomological methods, for donor A there was no entomological evidence despite the fact that the body was buried only a short distances away from donor B. Whether random factors or internal properties of the body were responsible for the lack of insect activity remains unclear. ii) forensic entomology always provides a minimum PMI, which can be identical to the time since death; but when it comes to PMI > 6 weeks this method is limited and much more variable, especially under special circumstances like burials. iii) Many insect species  are still not understood regarding their development and their biology, and there is a need for acquiring reference data.Following the survey and summary by Disney , temperaEnchtytraeus, species are extremely difficult to discriminate, and identification by DNA barcoding and database analysis via NCBI or BOLD is only possible for a few taxa, often from a single specimen. In addition, sequences of several unclassified specimens have been uploaded so that overall DNA barcoding is not yet a reliable tool for the identification of enchytraeids at the species level.In addition to entomological specimens several dozen Enchytraeids were collected from donor B. Enchytraeids, also known as potworms, are a widely distributed group of small- to medium-sized earthworm-like animals (Annelida: Clitellata), but we were not able to determine the exact species using the barcode region. Within the genus 2 and have a significant effect on processes such as decomposition, especially in soils where earthworms are absent. It has been shown that they accelerate the rate of decomposition of dead plant material by feeding on it directly and also by influencing microorganisms [So far, information on the effect of carcass decomposition on enchytraeids and vice versa is very limited. Some species commonly occur in large numbers and populations up to several hundred thousand per mrganisms , but somrganisms .Similar to other studies examining the microbiome associated with buried cadavers or their surrounding soil –62, we wTissierella) [Sphingobacterium) [Brevundimonas) [Bacillus). Our findings were similar to other work on microbial indicators of PMI estimates showing that while microbes can be used as predictors, the taxonomic groups and level which best predicts PMI are dependent on the surrounding environment [Though the microbiomes of the two donors displayed differing trends in microbial diversity, we were able to classify sample date  with a high degree of accuracy (only a 5.6% error rate), and identified a number of genera that were important to the classification model. The top ten genus level indicators used in the classification model included a variety of both aerobic and anaerobic bacteria. While some of the genera include taxa which are predominantly associated with human digestive systems (e.g. ierella) , others cterium) , as oppodimonas) , or are ironment ,69. Thouironment ,65,70,71Both soil composition and environmental conditions play a substantial role in determining the structure and abundance of microbes within the soil community –74, and PMI estimation in buried bodies is a challenging task and available approaches largely suffer from limited reference data. Distinct postmortem changes were observed with every used method in this study. Morphology assessments worked well, using existing reference tables, however, temporal coherences are inadmissible. Correction factors or specific calculation models are conceivable, but require additional research. Muscle protein degradation proceeded in similar patterns compared to data from un-buried corpses and animal models. Even temporal coherences appear likely, which could be explained by a similar degree of protection from exterior influences in buried and un-buried bodies. However, this approach turned out to be clearly restricted to early and intermediate postmortem stages (until approximately 28 days). Ecological markers, such as microbes, insects and other necrophile taxa can provide crucial clues for the estimation of the (minimum) PMI and are yet among the most important traits in forensic practice, especially entomology. However, reference data for buried bodies is scarce. The validity of this study is limited by a small sample size, the particular geographic site, soil composition and burial depth. Nevertheless, opportunities to study human decomposition in different geographic locations and under varying circumstances are limited, and offer exclusive chances to gain scientific knowledge. ARISTA is a unique research facility, providing the infrastructure and technical know-how for future endeavors.All experiments were performed in accordance with international and institutional ethics guidelines, in accordance with Dutch legislation and the regulations of the medical ethical committee of the Amsterdam UMC at the location Academic Medical Center.The Dutch Burial and Cremation Act  describes donation to science as one of the three possible final destinations of human remains . The procedure of body donation to the Department of Medical Biology of Amsterdam University Medical Center (Amsterdam UMC)- location AMC and the subsequent use of these bodies for scientific research is not subject to medical ethics review.The Dutch Act on Human Subject Research does not cover research with bodies, donated in the context of the Burial and Cremation Act. Moreover, Dutch legislation does not have other regulations in place that require review in case of concrete research protocols with donated bodies.The Department of Health Law of the AMC approved the procedure for body donation. The Medical Ethics Committee of the AMC, provided a waiver for individual studies that make use of donated bodies. The Department of Medical Biology, being responsible for the body donation program, reviewed the protocols, methodology, academic merits, eventual privacy and ethical issues, as well as environmental issues.Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA). The two males died little over two weeks before the initial burial  and were stored in body bags in 4°C cooling units. Two corpses were obtained through the body donation program of the Department of Medical Biology, Section Clinical Anatomy and Embryology, of the Amsterdam UMC at the location Academic Medical Center in The Netherlands and were buried at the th 2018 two earth graves with a mean length of 2 m, a width of 1 m and a depth of 60 cm were dug into the sandy soil at a distance of 2 m to each other , with the right thigh bent at right angle to the body axis. A concrete brick was placed under the right knee to prevent sinking of the leg and/or rotation of the body during the burial and to hinder soil from settling. Starting with the sand from the deepest layers, the graves were carefully re-filled, avoiding the formation of artificial soil cavities (e.g. under the limbs and head). Ultimately, the top layer was replaced on the surface of the grave resulting in a slight elevation (bump) of approximately 10 cm. Indicator flags were placed to mark the position of the hip and the knee. At predefined time points, the bodies were exhumed for subsequent measurements (see below).M. vastus lateralis) were collected for protein degradation analysis  was removed with a scoop until thigh skin was visible. Sand residue on the skin was removed using wet cloths. Two centimeters proximal to the previous opening, a small incision was made and a muscle biopsy sample was collected .M. vastus lateralis) for the following biopsies.On day 28 and 105 complete body exhumations were performed. Initially, microbial swabs were collected from the surfaces of the graves. Then, soil was removed, again differentiating three layers to retain the vertical structure. Once the upper 25–30 cm of soil had been cleared and the position and composition of the body was determined, additional sand was removed from above and beside the body using small shovels, trowels and brushes. Again, microbial swabs were collected from the skin (thigh) and soil next to the body (i.e. approx. 5 cm beside the thigh). Below the shoulder and hip regions, channels were dug to bring straps into position and lift the bodies out of the graves onto plastic tarps. The clothing was removed, and excess sand was brushed and wiped off again to facilitate photographic documentation and further investigation (see below). When completed (after approximately one hour), the bodies were carefully lowered into the graves and re-buried with soil as described above. After the first exhumation, the bodies were placed on the other (= right) side, with the left thigh in the position described above. This was necessary to provide additional sampling sites for muscle tissue (® Excel® (Ver. 14.5.1) was used for documentation and calculations.Pre-burial and during the complete body exhumations a total body score (TBS) according to Megyesi et al.  and a toMuscle sampling and protein analysis. Tissue samples from M. vastus lateralis were collected by biopsy from the right  and left thigh . After a punctual incision with a scalpel through the skin, the iliotibial tract and the muscle fascia, a 5 mm diameter biopsy needle was inserted to a depth of approximately 6 cm and muscle tissue was extracted. Approximately 100 μg of tissue material was transferred into a vial tube containing 1 ml of extraction buffer (RIPA buffer (SIGMA) and a protease inhibitor cocktail (ROCHE)). Tubes were stored at -20°C until further processing. After samplings, the openings were sealed with cyanoacrylate glue (Loctite® super glue). The initial sample was collected at one-third the distance from hip to knee along the lateral line of the thigh. Each subsequent sampling was taken 1–2 cm distal from the previous site. All samples were taken from the middle third of the M. vastus lateralis.All samples were homogenized in a two-step process. Initially, the tissue material was disrupted using an Ultra Turrax (IKA Werke GmbH & CO. KG). The dispersed samples were treated with high frequency sonication (Hielscher Ultrasonics GmbH) to break up cellular and sub-cellular structures and centrifuged at 1000 × g for 10 minutes. The supernatant was collected and stored at -20°C for further analysis. BCA assay was used to determine total protein concentration, and the samples were diluted to the same level prior to further analysis.®PVDF, Carl Roth GmbH) and stored at -20°C. Membranes were blocked in blocking buffer , and subsequently incubated with primary- and secondary antibodies. Between each incubation step, the membranes were rinsed and washed (3 × 10 min) in washing buffer . Primary antisera against the following proteins were used: tropomyosin, vinculin, α-actinin, GAPDH and eEF1A2. HRP-conjugated polyclonal goat anti-mouse, or goat anti-rabbit immunoglobulins were used as secondary antiserum. Staining was visualized by adding chemiluminescence substrate  and documented with a digital gel analysis system . Protein band intensities were measured using ImageJ software . Alterations, such as the disappearance of a native band or appearance of additional bands, were considered degradation events. Signals < 1% the intensity of the native bands were considered background and thus no band.SDS-PAGE were run on 10% polyacrylamide resolving gels and 5% polyacrylamide stacking gels, according to standard protocols . A total® Flycatchers  flytrap was placed in the facility on the day of burial to obtain a baseline characterization of insect activity. In addition, one of the two bodies (donor B) was kept on the surface for additional 2 hours, freely accessible to insects. Activities of insects were recorded prior to the burial.A modified original Red Top complete body exhumations) and the soil itself, as well as the bodies were visually checked for insects, and other biological activity. If present, specimens were transferred directly into 70% ethanol for killing and storage. Colonization on the surface, e.g. by blow flies, was prevented manually by permanent monitoring. After an initial morphological sorting and classification, genomic DNA from two specimens, which could morphologically be identified as enchytraeids was extracted from the whole individual using a slightly modified phenol–chloroform extraction [5′- GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGT GACCAAAAAATCA-3′) [® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The protocol included a total reaction volume of 20 μl consisting 3 μl Big Dye, 2.5 μl 5× sequencing buffer, 5 pmol primer and 1 μl PCR product. Protocol for sequencing reaction was 28 cycles of 96°C for 10 s, 50°C for 5 s, and 55°C for 4 min. Sequencing products were purified using gel-filtrated columns  and run on an ABI3130 genetic analyzer (Applied Biosystems). Sequence data for forward and reverse DNA strands were edited and aligned manually using the software CodonCode aligner (Version 5.1.5). Sequences were blasted to NCBI reference sequences and afterwards implemented in an NJ analysis and compared with the sequences of specimens of different Enchytraeidae using MEGA (Version 7.0.26).On day 28 and 105, specimens of insects and other necrophilous taxa were manually taken during body exhumation. Soil was removed (see above traction . SubsequATCA-3′) . AmplifiATCA-3′) . PCR proForensic grade DNA free cotton swabs  were used to sample microbial communities from different body sites and soil. The swabs were firmly scrubbed against the respective target areas for approximately 10 seconds. On the day of the burial, as well as the two complete body exhumations (day 28 and day 105), the following body sites were swabbed: eyes, ears, nose, mouth, thigh skin and rectum. The entire accessible surfaces  were swabbed while twisting the cotton tip to maximize the contact area. Avoiding contact to any other surfaces or aerosols the tips were inserted into test tubes, containing 50 μl of 100% ethanol, snapped off and stored in the on-site –20°C cooling units until further processing.®) was used for DNA isolation, with the addition of lysozyme  during the lysis step. Once isolated, DNA was quantified fluorometrically using a Qubit 2.0  and a dsDNA High Sensitivity Assay Kit (Invitrogen). All DNA preparations were then stored at -20°C until further processing. Library preparation and sequencing (2 x 250 bp paired-end reads) were performed using an Illumina MiSeq platform, following previously described methods [5′- GTGCCAGCMGCCGCGGTAA -3′, 5′- GGACTACHVGGGTWTCTAAT -3′) as previously described [For DNA isolation and downstream analyses, swabs were processed using previously published protocols . Briefly methods . To profescribed –82. DemuUsing default settings in QIIME 2 (v 2020.2) raw sequencing reads were quality filtered , while Dhttps://github.com/BenbowLab/ARISTAproject).Sequencing data were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under the accession code PRJNA643564. Differences in multivariate dispersions between/among groups  were tested using PERmutational Multivariate Analysis Of Variance (PERMANOVA) and weighted UniFrac distance implemented using vegan package v 2.5–4 . DiffereS1 Raw images(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 FigFamilies shown were represented by at least 3% among samples.(TIF)Click here for additional data file.S3 FigPhyla shown were represented by at least 0.3% among samples.(TIF)Click here for additional data file.S4 FigFamilies shown were represented by at least 0.3% among samples.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S1 TableMean values are represented by shades of color, with darker indicating more relative abundance among the taxa within a body site or soil.(XLSX)Click here for additional data file.S2 TableSamples from donors were pooled for the averages. Blue shaded cells represent positive change, while pink represent negative change.(XLSX)Click here for additional data file.S3 TableTaxa are arranged from greatest to lowest relative abundance.(XLSX)Click here for additional data file.S4 TableTaxa are arranged from greatest to lowest relative abundance.(XLSX)Click here for additional data file.S5 TableTaxa are arranged from greatest to lowest relative abundance.(XLSX)Click here for additional data file.S6 TableMean values are represented by shades of color, with darker indicating more relative abundance among the taxa within a body site or soil.(XLSX)Click here for additional data file.S7 TableMean values are represented by shades of color, with darker indicating more relative abundance among the taxa within a body site or soil.(XLSX)Click here for additional data file.S8 TableMean values are represented by shades of color, with darker blue and darker red indicating more or less relative abundance among the taxa within a body site, respectively. The mean percentage change between the first and last sampling dates is given, with taxa bolded that showed >/ = 5% absolute change (for those body sites with multiple sample dates).(XLSX)Click here for additional data file.S9 TableMean values are represented by shades of color, with darker blue and darker red indicating more or less relative abundance among the taxa within a body site, respectively. The mean percentage change between the first and last sampling dates is given, with taxa bolded that showed >/ = 5% absolute change (for those body sites with multiple sample dates).(XLSX)Click here for additional data file.S10 TableMean values are represented by shades of color, with darker blue and darker red indicating more or less relative abundance among the taxa within a body site, respectively. The mean percentage change between the first and last sampling dates is given, with taxa bolded that showed >/ = 5% absolute change (for those body sites with multiple sample dates).(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.S12 TableTaxa were chosen based on the effect their removal from the random forest model had on overall model accuracy (Mean Decrease Accuracy).(XLSX)Click here for additional data file.


Ixodes ricinus, one of the most common tick species in Europe, has been poorly investigated for WNV to date. As such, to evaluate the vector competence, laboratory reared Ixodes ricinus nymphs were in vitro fed with WNV lineage 1 infectious blood, allowed to molt, and the resulting females artificially fed to study the virus transmission. Furthermore, we studied the kinetics of WNV replication in ticks after infecting nymphs using an automatic injector. Active replication of WNV was detected in injected nymphs from day 7 post-infection until 28 dpi. In the nymphs infected by artificial feeding, the transstadial transmission of WNV was confirmed molecularly in 46.7% of males, while virus transmission during in vitro feeding of I. ricinus females originating from infected nymphs was not registered. The long persistence of WNV in I. ricinus ticks did not correlate with the transmission of the virus and it is unlikely that I. ricinus represents a competent vector. However, there is a potential reservoir role that this tick species can play, with hosts potentially acquiring the viral agent after ingesting the infected ticks.West Nile virus (WNV) is a mosquito-borne agent that has also been isolated from several tick species. Vector competence of  Flavivirus genus alongside more than 70 viruses, the majority being transmitted by mosquitoes and ticks. WNV is maintained in an enzootic transmission cycle between ornithophilic mosquitoes (the most important representative of which is Culex pipiens) and avian species that act as amplifying hosts [West Nile virus (WNV) is a zoonotic arbovirus with a recognised worldwide dispersal causing neurological diseases in animals and humans. Since its first isolation from the blood of a febrile woman in the West Nile district of Uganda in 1937, WNV has extensively propagated, and is at present endemic in all continents apart from Antarctica [ng hosts .Amblyomma tigrinum, A. ovale and A. tonelliae larvae successfully acquired the virus after feeding on viremic chicks. Furthermore, unfed nymphs of A. ovale and A. tonelliae maintained WNV transstadially, being positive for the virus after plaque assay. In the same study, infection of A. tigritum nymphs after feeding on WNV-infected chicks was also successful while the virus persisted in the adult stage at a low rate (3.7%) [Ixodes scapularis, Dermacentor andersoni and D. variabilis after the feeding of larvae and nymphs on infected mice and hamsters [Ixodes pacificus is another ixodid tick species for which maintenance of WNV from larval stage to nymph was indicated by RT-PCR and plaque assay after infecting larvae by feeding on viremic song sparrows [WNV has also been isolated from several species of ixodid (hard) and argasid (soft) ticks from endemic areas of Asia, Europe and Africa ,4,5,6,7,e (3.7%) . WNV trahamsters . Ixodes sparrows . None ofHyalomma marginatum ticks, experimentally infected with WNV by feeding on viremic rabbits and confirmed positive by RT-PCR, showed potential to transmit the virus to a naïve host after maintaining the agent through the molting process [Ornithodorus moubata was also able to successfully transmit WNV to naïve hosts (mice and day-old chicks) under experimental conditions [In contrast,  process . Infectenditions ,13.Ixodes ricinus for WNV, one previous study experimentally infected the nymphal stage of this species by placing ticks to feed on infected BALB/c mice [In order to determine the vector competence of B/c mice . The sucIxodes ricinus has the most widespread geographic distribution in Europe [I. ricinus are Borrelia burgdorferi sensu lato (Lyme disease agent) and the Western European subtype of tick-borne encephalitis virus [Flavivirus genus closely related to WNV [Ixodes ricinus immature stages frequently parasitize birds [n Europe . This tin Europe ,16,17. Tis virus ,19, a med to WNV . Ixodes ze birds , the natThe WNV outbreaks in recent years have been described in countries from Southern and South-eastern Europe but also registered an expansion to countries from Central Europe . The cliI. ricinus ticks are abundant [I. ricinus represented the arguments for undertaking a vector competence study using laboratory reared I. ricinus ticks and WNV lineage 1. Therefore, the aims of this study were to explore (i) the kinetics of WNV lineage 1 replication in Ixodes ricinus nymphs (ii) the transstadial transmission of WNV from nymphs to adults and (iii) the potential transmission of WNV by Ixodes ricinus females during blood feeding.The spread of WNV virus towards regions in which abundant  corroborI. ricinus nymphs were collected at different days post-infection  and tested for their virus load by RT-qPCR and by inoculating tick homogenates on Vero cell monolayers for confirmation of infectious viral particles. Out of 98 nymphs injected with WNV, 79 (80.6%) died within the first seven days post-infection, while from the control group, five out of 12 (41.7%) nymphs died within the same interval. The 19 surviving ticks of the WNV injected group were collected as mentioned above  and WNV RNA load was relatively quantified by RT-qPCR. All tick homogenates and Vero supernatants collected at day 7 after inoculation were confirmed positive for WNV RNA  (p = 0.0249), followed by a continuous increase in RNA load until 28 dpi (3.89 log10TCID50/mL), indicating that WNV was injected percoxally, replicated during the incubation period in the I. ricinus nymphs   each having 30 nymphs and one female to stimulate attachment to the membrane. In total, the feeding lasted for 9 days, after which 48.3% (58/120) of nymphs from the experimental group fed until repletion , while in the control group, 43.3% (13/30) nymphs were engorged. Two engorged nymphs from the experimental group were collected right after repletion, being confirmed as positive for WNV RNA after RT-qPCR from both tick homogenates and Vero supernatants. In total, 56 engorged nymphs fed with infectious blood were incubated. The molting of nymphs into adult stage started 31 days from the completion of feeding, resulting in a molting rate of 94.6% . After processing each individual male tick, 14 samples  tested positive for WNV RNA in both types of samples (RNA from ticks and from Vero supernatants) . RelativCID50/mL . In addiCID50/mL .Ixodes ricinus females that were fed with WNV infectious blood meal at nymphal stage were placed in individual FUs in order to investigate the virus transmission during artificial feeding. In total, 23 FUs were used for the ticks that were fed on infectious blood meal as nymphs, while the control group was added in one FU . Blood meals were collected at 24 h intervals starting from the second day of feeding for four consecutive days. During the collection of the blood samples, FUs were visualised under a stereomicroscope and the attachment of females to the membrane was noted. At day 1, eight females from the experimental group were feeding while, for the next days, eight, 11 and 13 females were observed to be feeding at days 2, 3 and 4, respectively . The results showed two positive Vero supernatants of two female ticks partially fed, collected at day 3 and 4 of feeding and evaluated as dead at the time of collection, with a titre of 0.29 and 0.02 log10TCID50/mL, respectively.ectively . After tIn total, nine females from the experimental group and three from the control group fed until repletion and they were incubated to lay eggs , Table 1All samples from the negative control groups were tested negative by RT-qPCR for WNV RNA in either tick homogenates, blood meals or Vero supernatants.Ixodes ricinus ticks for West Nile virus lineage 1. As such, ticks were successfully infected with WNV lineage 1 by percoxal injection and an in vitro feeding method was applied to also infect ticks and further on, to observe the virus transmission from infected ticks to the blood meal during feeding. The main findings of the study refer to (i) active replication of WNV in injected I. ricinus nymphs during 28 days of incubation, (ii) successful infection of nymphs with WNV by artificial feeding and (iii) transstadial persistence of the virus from the immature nymphal stage to the adult stage.The experimental work in this study was done to acquire new insights regarding the vector competence of I. ricinus ticks infected parenterally with WNV support the active replication of the virus, this being observed in ticks incubated for 28 days post infection. The inoculation of pathogens by microinjection requires special attention as a high proportion of ticks can die due to the injection injuries [Dermacentor reticulatus, but the decrease in RNA copies quantified by RT-qPCR was observed during the first 2 dpi [Our data showed that injuries . After ist 2 dpi .I. ricinus nymphs were successfully infected and maintained the virus transstadially, with 46.7% of the tested males being positive. The presence of infectious virus in these samples could also be confirmed through the inoculation of Vero cells, which lead to increased titres in some samples and allowed viral detection from samples in which the direct detection had failed. These results contradict the findings of a previous study published by Lawrie et al. (2004), during which WNV was not able to replicate in I. ricinus tick species [By in vitro feeding with blood spiked with WNV lineage 1,  species . The autI. ricinus nymphs using an in vitro feeding method while, in previous reports, infection of ticks was done by allowing them to feed on WNV-infected hosts. In these previous studies, several tick species acquired the virus from infected hosts: larvae and nymphs of Amblyomma ovale, A. tigrinum, and A. tonelliae after feeding on chicks [Ixodes scapularis larvae, larvae and nymphs of Dermacentor andersoni and D. variabilis from infected mice and hamsters [Ixodes pacificus larvae infected from viremic song sparrows [Hyalomma marginatum from rabbits [Ornithodorus moubata and I. ricinus nymphs after feeding on mice [10TCID50/mL in the artificial feeding system) than in the other study (viremic mice with 2–3 log10 PFU/mL).Furthermore, the results of our study are in concordance with previous reports that indicated the transstadial persistence of WNV in hard ticks without any proof of viral transmission to the host during feeding ,9,10. A n chicks , Ixodes  rabbits , second  on mice . We demo on mice ,29,30,31I. ricinus ticks are not competent to transmit the virus while feeding on a host. In contrast to these results, one report showed that Hyalomma marginatum nymphs and females were able in fact to transmit the virus to uninfected hosts during feeding, concluding that this species could be involved in the natural transmission cycle of WNV [H. marginatum were infected by feeding on viremic rabbits. The infected ticks were allowed to molt into the next stages and, after feeding on naïve rabbits, WNV antibodies were detected by indirect immunofluorescence assay in the blood of the infested hosts. The putative vector competence of H. marginatum for WNV could be an outcome of the existent strong association between immature stages of this species and passerine birds, an association that is missing between birds and I. ricinus immature ticks. Larvae and nymphs of H. marginatum preferentially feed on birds  rather than rodents [H. marginatum ticks in the transmission cycle. These results place H. marginatum next to soft tick Ornithodorus moubata, for which the potential viral transmission to uninfected mice was also indicated in brain samples from one mouse infested with 20 infected soft ticks, with the brain samples testing positive for WNV RNA by RT-PCR but negative by immunofluorescence assay [When surveying for the tick-to-blood meal transmission by feeding of females, we did not detect WNV in the blood, leading to the assumption that e of WNV . Immatur rodents ,33, and ce assay .I. ricinus engorged nymphs that molted into adult males, which subsequently tested positive for WNV, totalized 53 days. This long period during which WNV can persist in ticks corroborated with the life cycle characteristics and longevity of these arthropods could indicate their potential role as reservoir hosts for the virus. Immature stages of I. ricinus (larvae and nymphs) frequently feed on resident and migratory birds, especially on ground-feeding birds [In order to be further transmitted by ticks during feeding, a viral agent must reach the salivary glands (SG) by overcoming, in addition to the innate immune system, several barriers  . When itng birds , and canng birds , represeng birds ,38,39.I. ricinus females for WNV and, apart from two ticks, collected at days 3 and 4 of feeding, all samples tested negative for the virus. This low rate of transstadial persistence of WNV in adult females compared to males might be a result of the tick immune system activation during blood feeding. It could also indicate the inability of WNV to reach and infect the salivary glands prior the feeding of ticks. In contrast, it is known that tick-borne encephalitis virus and Powassan virus are able to reach salivary glands of ticks before the onset of feeding and, as a result, are successfully transmitted to the hosts at very early stages of feeding [After carrying out artificial feeding to study transmission of WNV lineage 1 by ticks, we tested partially engorged  feeding ,41.Ixodes ricinus nymphs can successfully be infected with West Nile virus lineage 1 by in vitro feeding and the virus persists transstadially in this species. Even with this persistence, ticks did not transmit WNV during feeding and it is unlikely that I. ricinus represents a competent vector for the virus. However, there is the potential reservoir role that this tick species can have, as it is able to maintain the viral agent for long periods of time compared to the natural vectors represented by mosquitoes.This study demonstrated that laboratory reared The procedures for the feeding of laboratory tick colonies on rabbits and gerbils were approved by the State Office for Agriculture, Food Safety and Fishing of Mecklenburg-Western Pomerania (LALLF) (reference number: 7221.3-2-008/16). The experimental infection of ticks was done in accordance with the 3R principles of animal experimentation: replace, reduce and refine for scientific purposes.Ixodes ricinus ticks  were obtained from Institute of Parasitology and Tropical Veterinary Medicine, Freie Universität, Berlin, Germany. Ticks were further reared in special designated tick-rearing facilities within the Institute of Infectology, Friedrich Loeffler Institut, in order to obtain the first new generation of I. ricinus nymphs that were used in all experiments. Briefly, adult stages of I. ricinus were fed on New Zealand white rabbits , by ear bag infestation, while larval and nymphal stages were fed on gerbils . During tick infestation, gerbils were held in cages with steel grid at the bottom and the cages placed inside a water pool. Between feeding and infection experiments, ticks were kept in an incubator at 22 °C with an atmosphere of 85–90% relative humidity (rH) and 16 h light/8 h dark cycle.Adult laboratory reared I. ricinus ticks. In order to obtain the viral stocks, the virus was passaged on Vero cells (Collection of Cell Lines in Veterinary Medicine (CCLV), Friedrich-Loeffler-Institut, Insel Riems-Greifswald, Germany) in modified minimum essential medium (MEM) with 2% fetal calf serum (FCS). The virus was harvested at day 7 post infection (7 dpi) and the titre was determined on Vero cells by using the endpoint dilution assay and the Spearman–Kärber algorithm [10 50% tissue culture infective dose (TCID50) per mL while a viral stock with a titre of 6.25 log10 TCID50/mL was used for the infection of ticks by in vitro feeding. The virus was stored at −70 °C in aliquots of 1 mL.The WNV lineage 1  was used for the experimental infection of lgorithm . The titI. ricinus nymphs were infected by percoxal route with 69 nL of approximately 3.59 log10TCID50. In total, 98 Ixodes ricinus nymphs were injected into the haemocel through the coxal plate of the fourth pair of legs (Video S1). The injection of ticks was performed using a digital microinjector system  and 5 µL fine-pulled capillaries in a glovebox  by placing ticks on a double sided sticky tape with the ventral side up and exposing the coxal plate with fine forceps (Video S1). In addition, 12 nymphs were injected with the identical quantity of clear MEM as controls. After injection, ticks were kept in an incubator at 22 °C, 90% rH and 16 h light/8 h dark cycle for 28 days. During the incubation period, injected ticks were sampled at different timepoints , with four ticks per timepoint being analyzed . After sampling, ticks were stored at −70 °C until further processing.In order to study the viral replication kinetics of WNV, unfed I. ricinus nymphs while membranes with a thickness of 80–100 µm were used to feed adult females. The membranes were glued, using Elastosil E41  to acrylic glass tubes  of 5 cm length, 2.6 cm inner diameter and 3 cm outer diameter. The membranes were then checked for leakage and surface sterilised by placing them in a 6-well plate  with 70% ethanol for 30 min. On the membranes used for feeding I. ricinus females, a square piece of mosquito netting material was glued for adding tactile stimuli. Bovine hair extract was obtained by soaking chopped hair of approximately 1 cm length in a beaker with 10 mL of methanol  and left at room temperature for at least 4 days to allow alcohol evaporation [The artificial feeding system used in the current work was built based on the protocols published by Kröber and Guerin  and KrulI. ricinus nymphs was done to evaluate the transstadial transmission of WNV while in vitro feeding of molted female ticks was performed to study the potential virus transmission from ticks to the blood during feeding. For the infection of nymphs with WNV, four feeding units (hereafter FUs) each containing 30 nymphs and one I. ricinus female were used, while one FU with 30 nymphs and one female served as negative control. Adult female ticks were added to each FU as stimuli for nymphs to attach to the membrane . The artificial feeding system was kept in an incubator  under controlled conditions . Both nymphs and females were fed with sterile heparinized bovine blood  supplemented with 4 g/L glucose  by adding 3.03 mL blood meal for each FU.10TCID50/mL and 30 µL of ATP 0.1 M  as a phagostimulant, the viral titre of the blood meal being approx. 5.95 log10TCID50/mL. The spike of the blood meal with WNV was done after 24 h from the debut of tick feeding, to allow nymphs to attach and have an active feeding process. The infectious blood meal was maintained during four consecutive days, being changed twice a day at 8 and 16 h intervals. The infectious blood meal was titrated before and after feeding on Vero cells for confirmation of the viral titre.For infection of nymphs with WNV, each FU belonging to the experimental group was placed on blood meal containing 2.5 mL bovine heparinized blood, 500 µL of the virus stock with a titre of 6.25 logI. ricinus females, blood meal samples were collected at 24 h intervals (100 µL for inoculation of Vero cells and 100 µL for detection of WNV RNA).The composition of uninfected blood meal consisted of 3 mL bovine heparinized blood and 30 µL ATP 0.1 M. During artificial feeding with non-infectious blood meal, changing of blood was done once every 24 h. Feeding of female ticks that molted from the nymphs of the experimental group was done by placing one individual tick per FU together with a laboratory-reared uninfected male. During the entire feeding process of 2 atmosphere for seven days and prior formalin inactivation of the 96-well-plate, 100 µL of the cell supernatants were collected from all samples, regardless of the cytopathic effect (CPE) and placed in TRIZOL reagent for RNA extraction.In order to analyse tick samples for the presence of WNV, each tick was homogenized in a 2 mL tube containing 200 µL of MEM and one 5 mm sterile steel bead. Homogenization of ticks was done using Tissue Lyser II  twice for 1 min, at 30 Hz. A volume of 100 µL of the tick homogenates was transferred to a 1.5 mL tube containing TRIZOL  reagent for total RNA extraction and stored at −70 °C until further processing. The remaining 100 µL of the homogenate were added on Vero cells layer seeded in a 96-well-plate for the isolation of infectious virus. The cells were kept in MEM with 2% FCS and antimicrobials . Vero cells were incubated at 37 °C and 5% COI. ricinus females of the experimental group, 200 µL of blood meal was collected daily during in vitro feeding, following a similar protocol as for the tick homogenates with the exception that only 50 µL of blood meal were added on Vero cells monolayer and incubated until day 7. For the isolation of total RNA, 100 µL of blood meal and Vero cell supernatants were placed in TRIZOL LS  and TRIZOL reagent, respectively.To study the vector competence of Female ticks were allowed to feed until repletion and to lay eggs for the evaluation of the transovarial persistence of WNV. Egg batches and the females that laid eggs were processed similarly as tick samples but underwent only RT-qPCR analysis for the identification of WNV RNA.® VET kit  and the King Fisher® Flex Purification system , following the manufacturer’s instructions. Total RNA was eluted in 100 µL of elution buffer then stored at −70 °C until further use.Total RNA extraction of all samples was done by initial phase separation using TRIZOL reagent, then the obtained aqueous phase was further processed for an automatic RNA extraction by using NucleoMagTM Universal Probes One Step Kit  and CFX-96 Real-Time system  as previously described [Amplification of RNA extracted from tick samples and blood meal was done by RT-qPCR targeting the non-structural NS2A region of WNV genome, using iTaqescribed . The samt-test was done using GraphPad Prism version 8.1.0 for Windows  for the comparison of viral titres between WNV injected nymph groups collected at different timepoints post-infection, with differences being considered significant at p-values lower than 0.05.Graphical presentations were built using R version 3.6.2 in R Studio ,47. One-


Vitis vinifera L. cv. Cabernet Sauvignon) to different applied water amounts based on the replacement of fractions of crop evapotranspiration (ETc) during two growing seasons with contrasting precipitation patterns. The experiment consisted of three irrigation treatments based on the weekly replacement of 25, 50, and 100% of ETc. Grapevine stem water potential decreased during the growing season reaching its lowest value  at harvest in the more stressed vines (25 and 50% ETc). Leaf gas exchange variables were measured during the two seasons and 100% ETc had the highest rates of photosynthesis and stomatal conductance and better instantaneous water use efficiency, also resulting in higher leaf chlorophyll and carotenoid content. Mineral nutrient content for nitrogen and potassium increased linearly with the increase in applied water. At harvest, no differences were observed in the number of clusters per vine; however, the 25% ETc had the lowest berry size and yield per vine with no difference in sugar content of berry. Conversely, sugar allocation to reserve organs was highly affected by applied water leading to different shoot to root biomass partitioning, where shoot:root ratio, leaf non-structural carbohydrates, and photosynthetic pigments increased with greater applied water. Likewise sucrose:N ratio and root non-structural carbohydrates decreased with the lower applied water. Altogether, carbon allocation between the source and sink organs likely controlled the response of grapevines to water deficits in a hot climate, and replacing 50% ETc was sufficient to sustain the grapevine performance given the enhancement of sugar transport, which could slow down the detrimental effect of water deficits on yield.Majority of viticulture regions are located in mid-latitudes characterized by weather variability and stressful environments relying on irrigation for mitigating environmental stress during the growing season and to ensure a profitable yield. The aim of this study was to characterize the response of grapevine ( Vitis vinifera L.) is the most economically important fruit crop with more than 7.4 million cultivated hectares worldwide in 2016  demand in the immediacy of the root zone without using the storage capacity of the soil  are responsible for providing energy and carbon for grapevine growth, being stored as reserves in grapevine perennial organs. The role of stored NSC in the early season is crucial until bloom when leaf photosynthesis becomes the primary source of carbon . The capreserves . Water areserves  and for reserves . Indeed,reserves . Howeverreserves .c on grapevine physiology, as well as to assess their effect on carbon partitioning among the source and sink organs during two growing seasons.The objective of this study was to characterize the primary metabolism response of grapevines to different applied water amounts based on the replacement of fractions of ETc replacement treatments: (i) 25% ETc, (ii) 50% ETc, and (iii) 100% ETc, with six replicates each consisting of five experimental units, three of which were used for data collection and the two on distal ends were treated as border plants. Plants were irrigated weekly with two drip emitters per vine.The experiment was conducted during two consecutive seasons (2018–2019 to 2019–2020) in Oakville, CA , with row orientation NW–SE. The vineyard was planted in 2011 with Cabernet Sauvignon (clone FPS08) on 110R rootstock at a spacing of 2.4 × 2.0 m (row × vine). The grapevines were trained to a bilateral cordon on a vertical shoot positioned trellis with a cordon 96 cm high above the vineyard floor and pruned to one-bud spurs. All other cultural practices, including vineyard fertilization, were standard for the area and conducted before treatment application. The experiment was designed as a randomized block with a one-way arrangement of the following fractions of ETc. Vineyard ETc was calculated using the following equation: ETc = ETo × Kc, where ETo is reference evapotranspiration and Kc is the crop coefficient. During the experiment, evaluation of vineyard Kc over the course of the growing season was calculated using the shade cast beneath grapevines grown with 100% ETo application from April 1 and the VSP-specific equation developed by The irrigation treatments were applied by varying water application rate based on calculated ETc treatment plots. Thus, by reducing the emitter output from 8 to 2 L/h and 4 L/h per emitter, respectively, the 25 and 50% ETc plots necessarily received water at a fraction of 100% ETc plots. The irrigation treatments started in April of each year. Harvest commenced when the berry total soluble solids reached ca. 24°Brix on average in all treatments on 25 September 2019 (114 DAF) and 8 September 2020 (115 DAF), respectively.The irrigation treatments were imposed by varying the emitter output per vine in the drip line, and the irrigation pump was scheduled to run based on the application rate of the 100% ETo was obtained using a modified version of the Penman–Monteith equation  throughout both growing seasons every 2 weeks. A fully expanded leaf from each treatment-replicate, sun-exposed, and without sign of disease or damage from the northeast side of the canopy was selected and measured. Two hours prior to taking the measurements, foil-lined zip bags were placed on sun-exposed leaves and sealed before excising the petiole in order to suppress transpiration. SWP was then directly determined with a pressure chamber .2 window. For each date and experimental unit, three measurements were made ca. solar noon (11:30 to 13:30 h) on a healthy leaf under light-saturating conditions  and values were averaged. The cuvette was oriented perpendicularly to sunlight, which was always in saturating conditions . Measurements were taken at 40% relative humidity, a CO2 concentration of 390 μmol CO2 mol–1, and using a flow to the chamber of 300 ml min–1.Leaf gas exchange was measured with a CIRAS-3 portable infrared gas analyzer system  featuring a broad-leaf chamber with a 4.5-cmTo summarize the temporal information for plant water status and leaf gas exchange, the area under the curve for all the parameters was calculated by using natural cubic splines for both years individually and collectively. The resultant values were divided by the number of the days between the first and the last day of measurements in each year, and then normalized by the first measurement in the 2018-2019 season and 2019-2020 season to make the data comparable to each individual measurement.via automated combustion analysis (method B-2.20), while phosphorus (P), potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), zinc (Zn), manganese (Mn), boron (B), iron (Fe), and cuprum (Cu) were analyzed via nitric/perchloric acid digestion (method B-4.20) as described by In June of each growing season (ca. anthesis), 50 leaves per treatment-replicate were collected, leaf blades were removed, and petioles were dried at 70°C in a forced air oven. Then, mineral nutrient analysis was carried out using inductively coupled plasma-mass spectrometry by Dellavalle, Inc., Fresno, CA, United States, as reported elsewhere . Total na + b) and total carotenoids according to At mid-ripening of the second growing season, two leaves of each treatment-replicate were collected and 25 mg of tissue was used for determining total chlorophylls  and using the regressive relationship between leaf dry mass and leaf area. A subsample of oven-dried leaves (30 mg) from each treatment-replicate was collected for sugar and starch analysis. Then, leaf area to fruit ratio was calculated by dividing the leaf area by the yield per vine and reported as mSix weeks following the harvest of 2020 (15–19 October 2020), one grapevine per treatment-replicate was removed from the vineyard by using a mechanical spade. The grapevine was then portioned into trunk and cordon, roots, and stems (shoots). Then, each portion was weighed on a top-loading balance to obtain the fresh biomass of the portions. A subsample of shoots and fine roots was collected for organ-dried biomass estimation and sugar and starch analysis. Harvest index (ratio of yield to biomass) was calculated after oven drying the samples.Subsamples of leaves, shoots, and roots were oven-dried at 70°C to a constant weight. Dried tissues were ground with a tissue lyser . Thirty milligrams of the resultant powder was extracted in ethanol:water (75:25) solution. Briefly, 1.5 ml was added to each sample and extracted for 10 min at 90°C in a water bath. Then, they were centrifuged at 10,000 rpm for a minute, and the supernatant was collected for sugar determination. The procedure was then repeated for starch determination in which the resultant pellet was used.D-glucose, D-fructose, D-sucrose, and D-raffinose were injected to obtain the retention time for each compound, and detection was conducted by RID. Sugar standards were purchased from VWR International . Sugar concentration of each sample was determined by comparison of the peak area and retention time with standard sample curves.Total soluble sugars (SS) and individual sugars were determined in the shoot, leaf, and root ethanolic extracts and in the diluted berry must samples (1:10). Samples were filtered with PTFE membrane filters  and transferred into high-performance liquid chromatography (HPLC) vials and subjected to reversed-phase HPLC analysis. The equipment consisted of an Agilent 1100 system coupled to a diode array detector (DAD) and an Infinity Refractive Index Detector (RID) . The reversed-phase column was Luna Omega Sugar  with a guard column of 5 mm. The temperature of the column compartment was maintained at 40°C and the RID flow cell was kept at 35°C. The mobile phase consisted of isocratic elution with acetonitrile:water  at a flow rate of 1.0 ml/min with a run time of 22 min. Standard solutions of 10 mg/L of 2O was added to a total volume of 5 ml. Next, 500 μl of the above sample and 500 μl of starch assay reagent were mixed and incubated for 15 min at 60°C. Negative controls with the starch assay reagent blank, sample blank, and glucose assay reagent blank and positive controls with starch from wheat and corn were performed. Reaction started with the incubation of 500 μl of each sample and 1 ml of glucose assay reagent at 37°C and was stopped with the addition of 1 ml of 6 M sulfuric acid after 30 min. The reaction was followed with a Cary 100 Series UV-Vis Spectrophotometer  and starch content was expressed as percent of starch per tissue dried weight.Starch content of the roots, shoots, and leaves was conducted using the Starch Assay Kit SA-20  in accordance with the manufacturer’s instructions. Briefly, pellets of different tissues were dissolved in 1 ml DMSO and incubated for 5 min in a water bath at 100°C. Starch digestion commenced with the addition of 10 μl α-amylase and then incubated in boiling water for another 5 min. Then, the ddHF value was significant (p ≤ 0.05), Duncan’s new multiple range post hoc test was executed using “agricolae” 1.2-8 R package  for Windows. Seasonal integrals of SWP and gas exchange variables for each growing season and for both seasons were calculated by using the same software. All data were subjected to Shapiro–Wilk’s normality test. Data were normally distributed and subsequently submitted to an analysis of variance (ANOVA) to assess the statistical differences between the different irrigation amounts. For seasonal integrals, a two-way ANOVA was applied to assess the effect of the growing season (year) and irrigation amounts on SWP and gas exchange parameters. For all data, means ± standard errors (SE) were calculated, and when the  package . Percent package .o of 23 mm higher. On the other hand, the lower available water for grapevine growth resulted in smaller canopy development decreasing the ETc, which explained the lower irrigation amount of 2020 compared with 2019  for both seasons were affected by the interaction between irrigation amount and year. During the 2019 season, there was a significant increase of SWP with 100 and 50% ETc siSWP compared with 25% ETc siSWP  and gas exchange parameters  were shown. A higher grapevine water status (si2019–20SWP) was positively related to an increased growth of roots, shoots, and leaves. Similarly, leaf starch content was strongly correlated with si2019–20SWP, si2019–20gs, and si2019–20AN . On the other hand, a significant relationship between increased leaf starch content and yield per vine was observed . Moreover, the increases in yield per vine were related with enhancements in leaf, shoot, and root biomasses. Berry must SS were positively correlated with si2019–20WUE and negatively with si2019–20gs . Finally, increased vegetative growth of trunk, leaves, and shoots negatively affected root SS . Conversely, positive relationships between trunk, root, leaf, and shoot biomasses with leaf starch content were recorded .To analyze the carryover effect of irrigation amounts on grapevine growth and sugar metabolism, a correlation analysis was conducted . Thus, sc on grapevine physiology and metabolism, Pearson correlations between shoot:root ratio and petiole N content with the total biomass (BM) and primary metabolites were conducted  (c) never reached values lower than -1.5 MPa for SWP and/or 50 mmol m–2 s–1 for gs, which have been reported to impair grapevine performance and berry ripening  . Moreoveripening . As Kellclimates . Previouclimates . Thus, lmilation .c. AN and gs peaked around veraison and then declined in all the treatments similar to several studies conducted in a warm climate before  or active (upregulation in abscisic acid) signals  from roots to shoots (AN/gs), no effect due to growing conditions was observed in contrast to previous studies on vines subjected to mild water stress . Contrarily, grapevines subjected to 100% ETc had higher photoassimilation rates throughout the course of the study that led to higher SS and starch content and, consequently, to the improvement of BM and, therefore, higher harvest index. Therefore, the reduced growth rate of both sink and source organs in response to water deficits indicated that the availability of carbon is a major growth constraint. The yield per plant of 50% ETc was lower than 100% ETc, but not as low as 25% ETc. However, canopy BM was greatly reduced in both 50% ETc and 25% ETc compared with 100% ETc. Accordingly, Grapevines subjected to 25% ETc likely resulted from higher sugar content in the roots as our data supported. It was recently reported that increases in root elongation and hexose contents were due to the VvSWEET4 overexpression, a gene implied as a grapevine response to abiotic stress  available for new growth and exported to sinks, and a lower plant BM due to water restriction. Conversely, 100% ETc showed the highest photosynthetic performance and water status, which led to increased contents of soluble sugars and starch in the leaves and greater yield. However, 100% ETc showed clear signs of being excessive as leaf biomass increased disproportionately to the point that it had a higher leaf area to fruit ratios. Our data revealed that in the 50% ETc treatment, the enhancement of sugar transport, mainly sucrose and raffinose, could slow down the detrimental effect of water deficit on yield. Finally, an important role of sugar and nitrogen was suggested due to their significant relationship with biomass partitioning.Our results provided evidence that differences in carbon assimilation and partitioning between the source and sink organs largely explained the response of grapevines to water deficits as it would be scheduled by irrigators and water purveyors in hot climates. Therefore, grapevines supplied with 25% ETThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SK conceptualized, designed, and acquired the funding. EK, JM-L, NT, and RY executed and curated the data. NT and RY wrote the first version of the manuscript. All the authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ischemic postconditioning (PC) is proved to efficiently protect diabetic patients with acute myocardial infarction from ischemia-reperfusion injury. We aimed to explore the protective roles of ischemic PC on diabetic retinopathy in tree shrews with diabetic cerebral ischemia. A diabetic tree shrew model was established through high-fat diet feeding combined with streptozotocin (STZ) injection, while cortical thrombotic cerebral ischemia was induced photochemically. Tree shrews were divided into the normal control group, sham operation group, diabetes mellitus group, diabetes mellitus+cerebral ischemia group, and diabetes mellitus+cerebral ischemia+PC group . H&E staining was used to examine the pathological changes in the retina, and immunohistochemistry was performed to determine the retinal expression of VEGF . The modeling resulted in 77% tree shrews with diabetes. Ischemic PC reduced the blood glucose levels in the tree shrews with diabetic cerebral ischemia. Tree shrews with diabetes had thinned retina with disordered structures, and these pathological changes were aggravated after cerebral ischemia. The retinopathy was alleviated after ischemic PC. Retina expression of VEGF was mainly distributed in the ganglion cell layer in tree shrews. Diabetes and cerebral ischemia increased retinal VEGF expression in a step-wise manner, while additional ischemic PC reduced retinal VEGF expression. Therefore, ischemic PC effectively alleviates retinopathy in tree shrews with diabetic cerebral ischemia, and this effect is associated with reduced retinal VEGF expression. Diabetic retinopathy (DR) is one of the serious microvascular complications of diabetes mellitus (DM), and it is also a disease with a high incidence of blindness –3. ApproThe pathogenesis of DR is very complex and multifactorial , 10. CurWith an extremely complexed structure, the retina contains a large number of highly differentiated neurons. Since the metabolism in the retina is profoundly active, the tolerance of retinal cells to ischemia and hypoxia is poor. Therefore, the mechanisms of retinal ischemia and anti-injury protection have always been the hot topics in the field of DR pathogenesis , 14, 15.Tupaia glis), which have similar biological characteristics with monkeys, to establish the models of type 2 DM and cerebral ischemia, and examined the changes in body weight and blood glucose levels. Based on the structural changes in the retina and the retinal expression of VEGF, the protective mechanisms of ischemic PC on the DR of tree shrews were revealed to be associated with reduced retinal VEGF expression. Our study provides additional experimental basis for using ischemic PC in clinical prevention and treatment of DR.Multiple studies using rodent models have confirmed the protective roles of PC in DM progression –23. MoreHealthy adult male tree shrews (body weight 130 ± 30 g) were provided by the National Primate Research Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences (contract no. SYXK-Dian-K2013-0001). The tree shrews were maintained under standard feeding conditions  in an animal room with artificial light cycles (light time 08: 00-20: 00), room temperature (25 ± 3)°C, and humidity 40%-60% in the animal center of Kunming Medical University, Kunming, Yunnan Province, China. The chow for tree shrews was from Chengdu Dashuo Experimental Animal Co., Ltd., China. The formula of high-fat diet was as follows: basic chow supplemented with 1% cholesterol, 0.1% sodium cholate, 10% lard, 5% egg yolk powder, and whole milk powder.According to the model to be established and the interventions to be taken, tree shrews were randomly assigned into 5 groups with initial 18 animals in each group, as follows: A, normal control group; B, sham operation group; C, diabetes mellitus group; D, diabetes mellitus+cerebral ischemia group; and E, diabetes mellitus+cerebral ischemia+ischemic conditioning group . Tree shThe citric acid and sodium citrate solution was made by mixing citric acid, sodium citrate solution , and distilled water at a ratio of 1 : 1 : 32 to achieve a concentration of 0.01 mol/L. The pH value was adjusted to 4.2, and each time, only the freshly generated solution was used for dissolving streptozotocin. Streptozotocin  was made as a 2% solution in the ice-cold citric acid and sodium citrate solution and stored in the dark. Rose Bengal sodium salt  was formulated to a concentration of 1.5% solution, and stored with protection from light exposure at 4°C for later use.After feeding a high-fat diet for 8 weeks, tree shrews were weighed on the day of modeling and deprival of food and water for 8 hours . Anesthe2 and a central wavelength (λ) of 560 nm can photochemically react with the Rose Bengal sodium salt and oxygen in the cerebral blood vessels to induce endothelium damage and thrombosis. During the experiment, the surface temperature of the skull was maintained at 36 ± 1°C, and the time was electrically controlled (15 min). After the operation, the aluminum piece was taken out, and the incision was disinfected and sutured layer by layer. Temperature was maintained until the animal was awakened, and they were placed in the cages for further observation.The diabetic tree shrews were fed a high-fat diet for 8 weeks and used for the thrombotic local cerebral ischemia model. The tree shrews were weighed and anesthetized through intraperitoneal injection of 1% thiopental solution at a dose of 200 mg/kg body weight. After being fixed in a prone position on the operating table, the animals were disinfected on the right top skin. An incision about 1 cm long was made between the right ear and the right eye, and the diaphragm was separated to expose the skull. At this time, the intracranial blood vessels were visible through the skull. A sterile aluminum piece was embedded in the incision. The center of the aluminum piece had a circular hole with a diameter of 0.5 cm. After the area around the aluminum sheet was covered, the Rose Bengal sodium salt was injected into the femoral vein. After 10 minutes of circulation, the SQ-III cerebral thrombosis device (patent number: ZL201420068737.2) was used to permeate light at the skull surface through the above circular window. The special light beam with a light intensity of 1.0 W/cmThe diabetic tree shrews were anesthetized again at 4 h after the photochemically induced thrombosis modeling . After bAfter anesthetization with intraperitoneal injection of 1% thiopental into the animal, the right eyeball was quickly removed. The eyeball with a central puncture in the cornea was immersed in 10% neutral formalin for fixation. After 12 hours, it was washed with distilled water, and the eyeball was cut along the limbus to remove the eye. After removing vitreous body, the eyecup containing retina was soaked in 10% neutral formalin and fixed again for 24 h. The slices were dewaxed, hydrated, and stained with hematoxylin, 1% hydrochloric acid ethanol, and 0.5% eosin dye for 1-3 min, respectively. After washing with distilled water, gradient alcohol dehydration, and clearance and mounting, the slices were observed under a light microscope for morphological changes of the retina.The prepared sections were subjected to antigen retrieval and blocking following the standard protocol. The sections were incubated with primary Abs against VEGF , at 4°C overnight. After washing with cold PBS, the sections were incubated with HRP-conjugated goat anti-rabbit secondary antibody  at room temperature for additional 30 min. The sections incubated with normal rabbit IgG were considered negative controls. An inverted fluorescence microscope  was used for visualization (magnification: 400x), and brown staining was considered a positive signal. Five individual fields per slide were selected for the evaluation of staining intensity.β-actin (Cat. No. HC201) was from TransGen Biotech, Inc. . The secondary horseradish peroxidase- (HRP-) conjugated anti-rabbit IgG  antibody was used. Proteins of interest were visualized using enhanced chemiluminescence kit . The band intensities were quantified by densitometry using ImageJ software .After washing with cold PBS, retinal tissues were ground and lysed in radioimmunoprecipitation assay lysis buffer  containing a protease and phosphatase inhibitor cocktail on ice for 30 minutes. After centrifugation at 13000 g for 20 minutes at 4°C, proteins in the supernatants were quantified and equal amounts of total proteins were loaded. Samples were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and incubated with the primary anti-VEGF D antibody  overnight. The antibody for the loading control P value < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 17.0 software . Quantitative data were described by the mean ± standard deviation, and differences between groups were compared using one-way analysis of variance (ANOVA). A In our animal models, tree shrews were fed a high-fat diet and treated with STZ . The purP < 0.05). There was no difference in animal body weight between the sham operation group and the normal control group, either before or after modeling , suggesting that cerebral ischemia can enhance the expression of VEGF in diabetic tree shrew, while in the DM+cerebral ischemia+ischemic PC group, the thickness of the retina layers was close to that of normal retina and the morphology of pigmented cells was normal. Notably, the expression of VEGF was also significantly decreased (P < 0.01), suggesting that ischemic PC can prevent retinal ischemia and hypoxia and delay the development of DR. Consistent with our observation, the increase of retinal VEGF expression was also noticed in rats with DM, and ischemic conditioning or pulses could significantly decrease VEGF expression [Compared with the DM group, the DM+cerebral ischemia group had significantly increased expression of VEGF in the retinal tissue of tree shrews  and inhibition of positive feedback regulation of VEGF expression by tissue hypoxia , 34. TheCurrent investigations suggest that although PCs require more stringent execution times, postconditioning and preconditioning show equal benefits on cardioprotective and neuroprotective functions . Since tIn summary, we established a tree shrew diabetic model through high-fat diet feeding combined with STZ injection in animals. The retinal expression of VEGF in tree shrews significantly increased with the aggravation of diabetes and cerebral ischemia, and ischemic postconditioning can effectively reduce the expression of VEGF in the retina of tree shrews with diabetic cerebral ischemia. Our preliminary exploring on establishing the tree shrew diabetic model and the protective roles of ischemic postconditioning supplements further experimental evidence for applying ischemic PC for DR therapy in clinics.


Fixed orthodontic appliances are common and effective tools to treat malocclusion. Adverse effects of these appliances, such as dental caries and periodontitis, may be associated with alteration of the microbiome. This study investigated the impact of these appliances on the dynamics of the oral microbiome. Seventy-one patients were selected. Supragingival plaque samples were collected before placement (T0) and six months after placement (T1). Saliva samples were collected at T0 and T1, and then when appliance removal (T2). Microbial DNA was analyzed by 16S rRNA meta-sequencing. The diversity analysis indicated dynamic changes in the structure of the oral microbiome. Taxonomic analysis at phylum level showed a significant increase in Bacteroidetes and Saccharibacteria  and decrease in Proteobacteria and Actinobacteria over time, in both plaque and saliva. Genus level analysis of relative abundance indicated a significant increase in anaerobic and facultative anaerobes in both plaque and saliva. Fixed orthodontic appliances induced measurable changes in the oral microbiome. This was characterized by an increase in relative abundance of obligate anaerobes, including periodontal pathogens. It can be concluded that this dysbiosis induced by fixed orthodontic appliances is likely to represent a transitional stage in the shift in microbiome from healthy to periodontitis. The mouth comprises complex structures of hard and soft tissue, such as teeth, tongue, gingiva, and palate; unique variation in oral microbiome structure is observed according to the different surface properties3.The mouth harbors more than 700 bacterial species, constituting one of the most diverse bacterial communities in the human body6. Therefore, it has been suggested that the risk of white-spot lesions, dental caries, and periodontal complication are due to the change in oral microbiome. The incidence and prevalence rates of white-spot lesions in patients undergoing orthodontic treatment are high, but these are incipient carious lesions that can be remineralized by application of fluoride7. Previous studies have suggested that an increase in Streptococcus mutans bacterial counts, generally regarded as a major risk factor for dental caries, is associated with placement of fixed orthodontic appliances11.Fixed orthodontic appliances are a common and effective tool used to treat malocclusion, but can be associated with secondary effects, such as a change of microbiome and subsequent infections. The complicated undercut shape of orthodontic appliances makes teeth cleaning more difficult and induces plaque accumulation as well as need for restorationsFusobacterium, Treponema, and Porphyromonas spp., have been detected in dental plaque around orthodontic appliances12. Furthermore, the frequency of Tannerella forsythia, Campylobacter rectus, and Prevotella nigrescens increased after placement of orthodontic appliances13. Severe clinical attachment loss during orthodontic treatment has also been reported14. These reports suggest that fixed orthodontic appliances may change the oral microbiome and have the potential to shift the bacterial ecosystem toward a pathogenic state. However, most previous reports have focused on only specific species and very few have focused on the microbiome and its dynamics, including unculturable bacteria, following orthodontic treatment.It is understood that anaerobic microorganisms in plaque play a key role in the initiation and acceleration of periodontal diseases. Periodontal pathogens, such as 15. However, there are few reports about the relationship between orthodontic treatment and the dynamics of oral microbiome. In previous report using NGS, periodontal pathogens were highest during orthodontic treatment, but the data was confined about plaque16.Conventional methods of bacteriological identification, such as cultivation, present limitations to the analysis of microbial community structure and diversity, because the human bacterial flora contains many unculturable species. Emergence of Next Generation Sequencing (NGS) technology has enabled analysis and comparison of bacterial composition, including unculturable bacteria, with unprecedented depth compared to previous methodologiesThe aim of this study was to assess the changes in oral microbiome dynamics caused by fixed orthodontic appliances using 16S rRNA gene meta-sequencing of supragingival plaque and saliva.In total, 13,506,556 reads were generated from 144 samples, with an average length of 300.4. After sequence trimming, 8,284,029 high quality reads remained, with an average length of 222.5 bp. In plaque samples, 44 samples were successfully analyzed both at T0 and T1. In saliva samples, 16, 23, and 17 samples were successfully analyzed at T0, T1 and T2 respectively. We detected 983 OTUs in total, with an average of 341 OTUs per sample. The number of OTUs increased according to time course both in plaque  and saliva samples  though the difference was not significant.Alpha diversity, a measure of microbial community evenness and richness in each sample, was calculated and compared between time points  was slightly more diverse than at T0  to T1 (7.53%), although the difference was not significant.Bacteria which decreased over time in supragingival plaque are listed in Table Prevotella, Porphyromonas, Capnocytophaga, Tannerella, Fusobacterium, Selenomonas, and Atopobium spp. significantly increased stepwise from T0 to T2. All the other bacteria that increased in saliva were facultative or obligate anaerobes.Increased bacterial abundance in saliva over time is shown in Table Neisseria, one of the core genera in saliva, decreased stepwise from T0 (9.16%) to T1 (7.58%) and T2 (3.62%) and Streptococcus decreased from T0 (15.97%) to T1 (12.86%) and T2 (11.73%), although these differences were not significant. All bacteria that decreased with time were aerobes or facultative anaerobes, consistent with the results in plaque.Bacteria in saliva that decreased over time are shown in Table S. mutans bacterial counts were seen with fixed orthodontic appliances18. Similarly, placement of orthodontic appliances influenced clinical parameters and colonization of periodontal pathogenic bacteria such as P. gingivalis, P. intermedia, P. nigrescens, and F. nucleatum21. In this study, the number of detected OTUs slightly increased with time both in plaque and saliva, although there was no significant difference, suggesting that bacterial structure may have become more diverse after application of orthodontic appliances. The relative segregation in PCoA demonstrated discrepancies between bacterial structures at different time points, especially in plaque samples , both anaerobes, significantly increased over time. Bacteroidetes are widely distributed in gut, oral cavity, and skin and include periodontal pathogenic bacteria, such as P. gingivalis and P. intermedia. Laboratory culture of Saccharibacteria has so far been impossible, here NGS analysis allowed us to detect this phylum. Saccharibacteria lives on the surface of its host bacterium. They affect oral microbial ecology by modulating the microbiome structure hierarchy and functionality through affecting the host’s physiology and the relative abundance of the host via direct killing22. Both in plaque and saliva samples, Saccharibacteria abundance significantly increased over time. The average age of the population sampled at T2 was different from that at T0 and T1, therefore we believe that this is likely also linked to the differences in microbiome, including Saccharibacteria abundance, observed at T2. Some reports suggest that Saccharibacteria increase with advancing age and have a role in initiating periodontitis24.Analysis of relative abundance at phylum level revealed more detail on bacterial distribution Fig. a–c such Prevotella, Capnocytophaga, Atopobium, Selenomonas, and Campylobacter significantly increased with time in both plaque and saliva  which is obligate anaerobe and regarded as a major pathogen of periodontitis with highly proteolytic activity became higher in abundance during orthodontic treatment in plaque samples although there was no significant difference36. Aggregatibacter actinomycetemcomitans, one of the pathogens of periodontitis, was reported to significantly increase three or six months after the placement of orthodontic appliances and remained higher level after removal38. These data suggest that the bacteria that significantly increased in relative abundance, in both plaque and saliva, were obligate and facultative anaerobes; notably most have been reported to be associated with periodontal diseases. No significant increase in the relative abundance of aerobes were detected after placement of appliances. These results strongly suggest that fixed orthodontic appliances alter the oral microbiome towards an anaerobic and periodontopathogenic state.The genera va Table a,c. The Actinobacillus, Actinomyces, Corynebacterium, Kingella, and Neisseria and the species Haemophilus parainfluenzae, Lautropia mirabillis, and Rothia dentocariosa significantly decreased over time in plaque samples. Regarding Actinomyces, some studies reported that they significantly increase in plaque by orthodontic treatment. Koopman et al. found the genus Actinomyces increased with time, while Actinomyces naeslundii mainly increased after removal of the appliances16. Tanner et al. reported Actinomyces to be associated with gingivitis caused by orthodontic treatment26. These diverse results indicated that the genus Actinomyces showed variable behaviors by multiple factors such as host age on genus level. Neisseria has been reported to became higher in abundance twelve weeks after and became lower over time to the removal, and to be associated with low gingivitis group of orthodontic patients26. Most of these organisms that significantly decreased over time in plaque are aerobes or facultative anaerobes and are not associated with periodontal diseases. Commensal oral bacteria, such as Neisseria and Haemophilus spp., have previously been shown to decrease with time.Abundance of organisms belonging to the genera Streptococcus also decreased over time both in plaque and saliva, although this difference was not statistically significant. Lucchese et al. reported in their systematic review study that the increase of S. mutans and Lactobacillus was influenced by the placement of orthodontic appliances39. We analyzed the relative abundance of bacteria in plaque and saliva by NGS, in contrast, their studies analyzed the whole volume of bacteria or bacterial DNA in plaque by colony counting and PCR. These methodological differences may account for the different conclusion.In our study, the genus 41. The species of Solobacterium moorei, Parvimonas micra, Selenomonas noxia, and P. melaninogentica which showed significant increase in supragingival plaque in our study . Verbal informed consent was obtained from all the patients following provision of a full explanation of the study. This study included subjects under the age of 18, in which case informed consent was obtained from the parent or legal guardian. All samples were anonymized following collection.All subjects participating in this study were Japanese and underwent orthodontic treatment from July 2016 to April 2018 at the Department of Orthodontics, Hiroshima University Hospital  immediately before placement of fixed orthodontic appliances, (T1) 6 months after the beginning of orthodontic treatment, and (T2) immediately after removal of the appliances . Supragingival plaque samples were collected from upper and lower anterior teeth at T0 and T1. The area of tooth is above gingival margin at T0 and between gingival margin and cervical side of the brackets at T1. The sampling area was isolated with sterile cotton rolls and saliva removed by gentle air drying; supragingival plaque samples were then collected with a sterilized explorer. Unstimulated saliva samples were collected into a clean paper cup at T0, T1, and T2. To ensure standardization, the subjects were instructed to avoid eating, drinking, and tooth brushing at least 2 h before the sample taking. All the samples were collected at the beginning of the visits and pooled approximately into a 1.5 mL microcentrifuge tube containing 0.5 mL of phosphate buffered saline (PBS) and stored immediately at − 80 °C until use49, with minor modifications.Microbial DNA was extracted from supragingival plaque and saliva samples using a MasterPure Complete DNA and RNA Purification Kit  according to the manufacturer’s instructions as previously performed by50. Amplification was performed on a Veriti Thermal Cycler . PCR products (approximately 450 bp) were visualized by agarose gel electrophoresis, purified using Agencourt AMPure XP magnetic beads , and DNA concentrations were measured using a NanoDrop ND1000 spectrophotometer and Invitrogen Qubit 4.0 Fluorometer (Thermo Fisher Scientific). Tag-indexed samples were diluted and pooled into a low DNA-binding tube in equal amounts from each sample. DNA concentration in the pooled samples was confirmed by qPCR, using KAPA SYBR FAST qPCR MasterMix (KAPA Biosystems). The library was applied to a MiSeq sequencing platform (Illumina). Nucleotide sequence data reported are available in the DNA Data Bank of Japan (DDBJ) Sequenced Read Archive under the accession number DRA010713.The V1-V2 hypervariable regions of 16S rRNA gene sequences were amplified by PCR using KAPA HiFi HotStart ReadyMix  and primers from the Nextera XT Index Kit https://greengenes.secondgenome.com). Additional statistical analysis such as Mann–Whitney U test and Kruskal–Wallis test were conducted using StatView 5.0 J . Mann–Whitney U test was used for comparisons between two groups and Kruskal–Wallis test was used in multiple comparisons.Sequence data were analyzed with CLC Genomics Workbench ver.9 . Sequences were assigned to the same operative taxonomic units (OTUs) when within 97% similarity, in reference to the Greengenes database (All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendment or comparable ethical standards.Supplementary Information