Rapid diagnostic tests (RDTs) are a cornerstone of HIV diagnosis and rely on good quality processing and interpretation, particularly in the era of test and treat. The Deki Reader  is a portable device designed specifically for analysing RDTs and was selected for evaluation in South Africa in the context of HIV RDT analysis.This study consisted of a laboratory evaluation and two-part field evaluation of the Deki Reader v100, covering two RDT testing algorithms, and an evaluation of the continuous quality monitoring through the Fionet™ web portal. Based on user feedback from the field evaluation, the device underwent hardware and software redesign, and the Deki Reader v200 was evaluated in the laboratory. Ethics approval for this evaluation was obtained from the University of the Witwatersrand Human Research Ethics Committee: M150160.The intra- and inter-device laboratory precision of the Deki Reader v100 were 98.3 and 99.2% respectively, and 99.3 and 100% for the Deki Reader v200. The laboratory concordances compared to standard-of-care reporting were 99.5 and 98.0% for the two respective models, while sensitivity and specificity were 99.5 and 99.4% for the Deki Reader V100 and 100 and 93.1% for the Deki Reader V200 respectively. Screening and confirmatory concordances in the field were 99.3 and 96.5% under algorithm 1 and 99.7 and 100% under algorithm 2. Sensitivity and specificity for the field evaluation were 99.8 and 97.7%. Overall robustness of the device was acceptable and continuous quality monitoring through Fionet™ was feasible.The Deki Reader provides an option for improved and reliable quality assessment for rapid diagnosis of HIV using RDTs to enhance the quality of healthcare at the point-of-care. However, the introduction of new RDTs and modification of current algorithms necessitates ongoing and agile RDT reader adjustments, which will require cost modelling to ensure sustainability of devices implemented into national HIV programs. Rapid diagnostic tests (RDTs), with their simple strip-based technology, have been a cornerstone of disease diagnosis for decades. In many resource-limited settings, HIV diagnosis is now achieved using only RDTs, allowing for rapid scale-up of HIV testing services (HTS) , in an eIn addition to robust manufacturing, RDTs rely on good quality processing and interpretation. A recent systematic review  showed tThe WHO recommends that all HIV testing sites participate in external quality assessment programmes, involving regular observation of RDT performance by trainers or supervisors, and evaluation of well-characterised HIV specimens , 15. DriThere are a number of automated RDT analysers available, some linked to specific RDT brands and some with the potential to adapt to different RDTs. Applications created for smartphones are also available and are increasingly used by self-testers. One such device is the Deki Reader , a portable device designed specifically for analysing RDTs and utilising smartphone technology Fig. a. The agThis evaluation consisted of four parts, summarised in Fig. A target product profile (TPP) for automated RDT analysis devices was also designed. While this was not definitive, the Deki Reader was compared to this TPP to determine needs met within the testing environment. A critical future element for inclusion in the TPP is costing, which was beyond the scope of this evaluation.Ethics approval for this evaluation (laboratory and field) was obtained from the University of the Witwatersrand  Human Research Ethics Committee . All specimens used were considered to be residual specimens as no specimens were obtained specifically for the evaluation and results from the Deki reader were not used for patient management.n = 774 specimens) were the First Response® HIV 1–2-0 , the Determine™ HIV-1/2 Ag/Ab Combo  or the Uni-Gold™ HIV . RDTs used for the Deki Reader v200 evaluation (n = 158 specimens) were the First Response® HIV 1–2-0 , the HIV 1/2/0 TriLine ) and the OneStep Anti-HIV 1&2 . Data was adjudicated by Fio as part of the software learning process and this data is presented.All laboratory evaluations were performed in the Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa. Different RDTs were used in the two evaluations, based on changing national algorithms. RDTs used for the Deki Reader v100 evaluation  CD4 Flow Cytometry Laboratory  and the NHLS Pathology Laboratory . RDTs were prepared according to manufacturer instructions and evaluated (read) by a laboratory professional. The visual (user or standard of care (SOC)) result was manually captured onto the Deki Reader and uploaded to Fionet™. The RDT was then inserted into the Deki Reader for automated analysis; this result was automatically uploaded to Fionet™. The two analyses were compared to assess the concordance between visual and automated interpretation, using the visual (SOC) result as the gold standard.n = 3) and each RDT was read three times per each of three Deki Reader v100 instruments. The Deki Reader v200 was tested using ten different specimens that were tested on each RDT (n = 3), with each RDT being read five times on one instrument and once across five different Deki Reader v200 instruments. The latter protocol introduced more stringent precision analysis to assist the developer for improved software optimisation as a lesson learnt during the v100 evaluation.Further to this, precision analyses were performed using both HIV positive and HIV negative specimens. The Deki Reader v100 was tested using five different specimens per RDT , between 5 May 2017 and 9 April 2018. Six key performance indicators were selected for this evaluation: (1) overall robustness of the Deki Reader, (2) efficient record upload speed, (3) connectivity needs and reliability, (4) quality data capturing concordance between visual and automated result interpretation, and (5) end-user (HCW) acceptance of the Deki Reader. HCWs and programme staff involved in the field evaluation attended a two-day training workshop, which included an introduction to the Deki Reader v100 hardware and software, and hands-on testing using the Deki Reader v100. Project managers received further training in the Fionet™ web portal software, learning how to remotely monitor and interact with the HCWs; filter data, reports and queries; and to manage quality related issues, including troubleshooting during RDT processing.All patients attending the clinics for HIV testing services (HTS) were informed of the study and, whether or not they elected to enrol, were managed according the South Africa National HIV Guidelines  and the phase 1: 5 May to 5 October 2017) assessed the suitability of the Deki Reader to withstand field conditions and the connectivity needs for reliable data transfer (indicators 1 to 3), as well as HCW acceptance (indicator 4). Three HTS clinics  participated in this evaluation (n = 20 Deki Reader v100 devices) and 23 HCWs were trained. Themba Lethu Clinic  is a fixed HTS clinic and had eight devices installed. The other participants were mobile HTS clinics servicing two different regions within the Johannesburg Health District and were managed by community teams; each were allocated six devices. The RDT testing workflow followed the algorithm stipulated in the HIV testing services policy (6), which was HIV screening using the Advanced Quality™ Rapid HIV (1&2) Test  and HIV positivity confirmed using the ABON™ HIV 1/2/0 Tri-line Human Immunodeficiency Virus Rapid Test Device (Abon Biopharm (Hangzhou) Co., Ltd., Hangzhou, China). The uploaded images were used for device ‘training’ and automated reporting was not available for this phase. Information regarding the record upload speed (acceptability criteria > 90% in < 10 min), and robustness of the instrument in the field (system failures) was captured from the Fionet™ portal. All data was stored on the Fio Corporation server with restricted access.The first part of the evaluation (phase 2: 8 October 2017 to 9 April 2018) assessed the quality of the data recorded , including error rates and invalid results obtained, the concordance between the visual and automated RDT assessments, and the HCW acceptance of the Deki Reader v100 (indicators 4 to 6). Eight sites, with a total of 23 Deki Reader v100 devices, participated in this evaluation: Diepsloot Clinic, Eyethu Clinic, Hillbrow Clinic, OR Thambo Clinic, Themba Lethu Clinic, Windsor Clinic, Yeoville Clinic and the Dreams Team Mobile HTS clinic. In total, 52 HCWs had training to participate in this evaluation.The second part of the field evaluation  was used for patient management.There was a change to the HTS algorithm mid-evaluation when the Gauteng Department of Health RDT supplier was changed as per tender RT41–2017 . The HTSEnd-user surveys were conducted both pre- and post-implementation to determine the HCWs’ perceptions of the usability and performance of the Deki Reader. To eliminate biases, researchers issued the structured questionnaires to managers who administered them to all the end-users. The end-users were asked to rank the Deki Readers based on the perceived usefulness and ease-of-use during testing using a Likert-scale   using the Deki Reader. This data can be monitored centrally on a continuous basis. The capabilities of the portal were examined to determine the extent of continuous quality monitoring  available to authorised individuals (e.g. region managers). Both the devices and the web portal are accessed through a registered user name and password based on access authorisation determined at study commencement, ensuring that data security is maintained. Only authorised personnel  had access to anonymised results.Data collected through the Fionet™ portal were downloaded as Microsoft® Excel  files and included study number, date of processing, HCW login, RDT used, visual interpretation and automated interpretation (both including HIV-1 and HIV-2 if included on the RDT). The visual RDT interpretation (SOC) was considered the reference for all analyses. All data was adjudicated by a Fio expert, in order to improve the analysis software. Performance evaluation analyses of the Deki Reader result outputs compared to visual interpretation (considered the reference standard) included concordance, sensitivity and specificity, and inter- and intra-device result reproducibility (only determined in the laboratory setting). Concordance included invalid specimens, but excluded errors and specimens that were not processed, while sensitivity and specificity only included specimens with a valid HIV positive or negative result. Acceptability for reproducibility, concordance, sensitivity and specificity was set at ≥98%. Statistical analyses were performed using Microsoft® Excel 2016, Stata version 12-SE  and MedCalc . The HCW user experience feedback  was captured in Microsoft® Excel 2010 tables and positive or negative experiences summarised using a heat map approach.n = 12) were not uploaded (connectivity) and there were two errors recorded. If HCW entry error (n = 1), which caused a false positive result when the Deki Reader was compared to SOC, is taken into account, the intra- and inter-device precision improves to 99.2%. The intra- and inter-device precisions were 99.3 and 99.2% respectively for the Deki Reader v200. The Deki Reader v200 intra-device sensitivity was 100% (95% CI: 95.2–100) with specificity of 98.7% (95% CI: 92.8–100), and inter-device sensitivity was 98.5% (95% CI: 92.1–100) with 100% specificity (95% CI: 95.1–100). The overall intra-device (n = 150) sensitivity and specificity were 100% (95% CI: 95.2–100) and 98.7% (95% CI: 92.8–100) respectively, while inter-device (n = 142) sensitivity and specificity were 98.5% (95% CI: 92.1–100) and 100% (95% CI: 95.1–100). If HCW entry error in the Deki Reader v200 evaluation was addressed, similarly to the Deki Reader v100 evaluation, the intra-device precision improved to 100%. Discordant results  were linked to the presence of artefact in the region of the RDT HIV-2 test line, which affected the overall HIV status (Table n = 33 concordant and n = 6 discordant with SOC), but did not affect the overall HIV status.The laboratory evaluations of both the Deki Reader v100 and the Deki Reader v200 showed that automated RDT analysis was comparable with SOC . Overall precision (Table n = 1) and Uni-Gold™ HIV RDT (n = 1)) were caused by target capture lines with a width < 30% of the cassette window; the standard Deki Reader configuration is that a line is not present if its width is < 50% of the window. The two false positive results (both Alere Determine™ HIV 1/2 Ag/Ab Combo RDT) were the direct result of air bubbles in the membrane at the time of analysis (15 min), which were misinterpreted as test lines. All errors and misclassifications were related to the individual RDT used, rather than to the performance of the Deki Reader v100.The concordance analysis of laboratory specimens Table  was perfn = 6). When this data was adjudicated (increased flexibility in the location of the target line within the RDT), the One Step Anti-HIV (1&2) concordance improved to 98.6% and overall concordance improved to 98.0% (acceptable). The HIV 1/2/0 Tri-Line concordance was 96.8%, with five false positive classifications by the Deki Reader v200; this may indicate that refinement of the new software is required (device learning). HCW entry errors  were observed when HIV-1 and HIV-2 results were entered in reverse  of Deki Reader v100 devices showed no decrease in functionality, However, 10% (n = 2) devices, both placed with mobile units, failed to function optimally towards the end of the evaluation.Nineteen (82.3%) trained HCW participated in the first field evaluation, with 3159 RDT results successfully uploaded to Fionet™. On average 527 results were uploaded per month, but with a wide range , and the majority were uploaded during July 2017. The average number of records uploaded in <10 min was 95.8% , and all records were uploaded within a maximum of seven days. The quality data transmission  was consistently 100%. No battery failures were experienced over the course of the study and 90% (Thirty-six (69.2%) of HCW trained participated in the second field trial , although thirteen HCW processed less than ten specimens. Four HCW processed more than 400 specimens, ten processed between 100 and 300 specimens, and a further nine HCW processed between 50 and 100 specimens. A total of 4460 patients were enrolled in this field evaluation, 2463 (55.2%) under Algorithm 1 and 1997 (44.8%) under Algorithm 2. After data adjudication, concordances were 99.3 and 96.5% (screening), and 99.7 and 100% (confirmatory) for Algorithms 1 and 2 respectively (Table n = 32) of invalid  Advanced Quality Anti-HIV (1&2) RDTs in Algorithm 1. Furthermore, 15 false positive screening RDTs were recorded, of which twelve were true negative results and three should have been repeated: in one case the RDT strip had shifted  and to blood being added to the buffer well , which was only observed with the Advanced Quality Anti-HIV (1&2) RDT. Both of these are HCW errors, although the former was reported by HCW to be caused by the Deki Reader v100 RDT drawer not detecting closure in multiple instances.There were a number 1.4%, %, n = 32HCW surveys were completed post-training and post-field evaluation, with 19 HCW (100%) participating in the first field evaluation feedback and 34 HCW 65.4%) participating in the second field evaluation feedback  were responsible 90.0 and 89.5% of these errors, which comprised 30% of time expired errors. Once the drawer was replaced both HCW were not linked to similar error issues. This type of incident can be addressed timeously to ensure continued quality at all testing sites, through assessment of HCW, devices and clinic procedures. Weekly encounters at clinics are tracked, as are error rates across all sites. Lastly, the actual RDTs can be monitored for lot errors; on the recommendation of the field trial managers, lot numbers can now be recorded on the devices.www.fionet.com) through a local mobile data service provider; (3) a robust, electricity-independent system with rechargeable batteries; and (4) the ability to log multiple patients concurrently, yet ensure that the correct RDT is analysed for each patient.A TPP was designed based on the existing TPP for viral load monitoring in resource limited settings  and on kThe HTS programme in South Africa has been expanding since 2000  and RDT This study included a number of evaluations. Sensitivity and specificity between the visual and automated RDT analysis showed variability, while concordance was consistently acceptable. The laboratory evaluations of both the Deki Reader v100 and the updated Deki Reader v200, showed intra- and inter-device precision of > 99%. Similarly, concordance was consistently above 95%  and, excluding HCW errors and artefact, close to 99%. Concordance in the field was also high, 99.3 and 99.7% for screening and concordance respectively for Algorithm 1, and 96.5% (screening) and 100% (concordance) for Algorithm 2. The decreased screening concordance under Algorithm 2 was primarily caused by high levels of artefact present in the region of the HIV-2 target line on the HIV 1/2/0 Tri-Line RDT, which skewed the automated analysis. Options to overcome this are being investigated further by Fio Corporation, and the addition of lot number recording to the software was recommended to determine whether specific RDT batches were affected. The artefact could also be linked to imperfect processing, which would indicate a need for HCW retraining; review of RDTs centrally (as is not currently possible) would allow this to be detected rapidly. The processing error rate (9.5%) was slightly higher than that reported by Kalinga and colleagues (2018) for malaria RDTs , but wasIn the field, the overall robustness, quality data capturing and connectivity, and record upload speed were good. The Deki Reader was able to capture and transmit reliable data for monitoring and evaluation purposes (100%), and to store patient results until data upload if no connectivity was available. Neither of these was previously widely possible when using RDTs for HIV diagnosis, with conventional data records being paper-based, although certain mobile phone-based applications have been suggested . The relWhile the HCW were initially positive regarding the Deki Reader v100 improving their workflow, the end-user acceptance of the Deki Reader showed more ambivalence or dislike of the device in the post-evaluation surveys. The primary reasons for this were the system freezes, which have been addressed and will be re-examined during the Deki Reader v200 field evaluation, and that the Deki Reader will not allow a strip to be analysed prior to completion of the incubation period. While the extended analysis time does “irritate the patient as they don’t want to wait”, ensuring proper adherence to protocols does ensure the provision of high quality HTS services. A recent study showed that many HCW read the results too early and misdiagnose patients , in addiIt is proposed that the Deki Reader may also enhance HTS tester performance through the on-board systematic guidance and quality control checks. The use of both the paper-based records and the Deki Reader v100 in the field evaluation did increase the HCW time burden, which may have influenced their opinions, and ideally paper-based registers will be discontinued if automated, connected RDT analysers are implemented. However, the registers were useful in the data analysis, as both field and laboratory staff reported that if the incorrect visual result was entered onto the Deki Reader (both versions), there was no option to correct it. When comparing the paper-based results to the RDT images and entered visual results, it was noted that the majority of discordant specimens (Deki Reader compared to SOC) were not truly discordant. Based on this, the software is being modified to allow result correction  prior to automated analysis reporting. The HCW will also have the option to dispute the automated result, such as in the case of artefact, although this is still under development.The Deki Reader v100 met the majority of criteria suggested for the TPP and the Deki Reader v200 will be similarly assessed in the field. The robustness of the device should be evaluated further in more stringent environments, including increased humidity and more extreme temperatures, and full costing analyses must be performed. Although not part of the TPP, it should be mentioned that the value of a fixed, rather than mobile, device is that the distance between the device camera and RDT, remains constant irrespective of the RDT. The closed chamber further standardises the light environment when an image is captured, irrespective of the surrounding environmental light conditions. The presence of artefact is being addressed, as this was the most worrying TPP criterion not met, albeit only with one RDT brand. However, ongoing software optimisation and HCW training, including repeating RDTs when artefact is visible, may address this issue.It was noted that a potential limitation of the technology is that the introduction of any new RDT requires software modification, which requires time and validation prior to implementation. In addition, the RDT reader developer will require access to the new RDT to be used in the field and implementation partners need to be aware of time delays for technology optimisation. However, devices not locked to specific RDTs are important when using the device in different settings, with varying algorithms, RDT types and HTS protocols being required dependant on country needs. The ability of the Deki Reader to accept new software  and the flexibility of the software, as shown during the algorithm change mid field evaluation, enables the device to adapt to the needs of the settings and allows the RDT market to remain competitive.Lastly, with the increased availability of HIV self-testing, which is performed by the untrained layman, and the need for linkage to care for persons with a positive result , 42, theContemporary technologies have the potential to enhance the provision of healthcare services, particularly at POC. The Deki Reader provides a means for continuous quality control and reliable quality assessment in rapid diagnosis of HIV using RDTs. This includes effective and efficient patient care and data capturing, as well as high connectivity capabilities, and the device has the potential to be deployed to rural areas of South Africa to optimise HTS coverage. The technology can also be deployed for HIV RDT monitoring in settings where the Deki Reader v100 already has a large footprint in the diagnosis of febrile malaria. However, ongoing and agile measures should be in place to mitigate against all end-user challenges to ensure continuous and consistent acceptability of the device throughout the HIV value chain.


K) and Twitch Potentiated (TPK), central and peripheral parameters respectively. For elbow extensor muscles, the protocol used sustained submaximal contraction at 10, 20, 30, 40, and 50% of MVC. The neuromuscular fatigue in upper limbs was identified by Twitch Potentiated (TPE) and multiple Twitch Superimposed (TSE) parameters. Using the relationship between MVC (%) and evoked force, the proposed protocol used several TSE to provide slope, y-intercept and R2. It is proposed that slope, R2, and y-intercept change may indicate peripheral fatigue and the identified relationship between y-intercept and R2 may indicate central fatigue or both peripheral and central fatigue. The results were compared using the non-parametric analyzes of Friedmann and Wilcoxon and their possible correlations were verified by the Spearmann test (significance level set at p < 0.05). After PerFat a decrease in TPE  was found but not in any TSE, indicating only peripheral fatigue in upper limbs. After CenFat a decrease in TPE  and TPK  were found but not in TSK, indicating peripheral fatigue in upper and lower limbs but not central fatigue. A non-significant increase of 15.3% after CenFat and a statistical reduction  after PerFat were found by slope. Despite R2 showing differences after both exercises (p < 0.05), it showed a recovery behavior after CenFat (p: 0.016). Although PerFat provided only peripheral fatigue, CenFat did not provide central fatigue. Considering the procedural limitations of CenFat, parameters resulting from the proposed protocol are sensitive to neuromuscular alteration, however, further studies are required.Neuromuscular fatigue evaluation is widely performed on different muscles through the conventional protocol using maximum voluntary contraction (MVC) with electrical stimuli in the analyzed muscle. In an attempt to use this protocol on elbow extensor musculature, previous studies and pilot studies showed co-contraction effects from antagonist musculature during muscular stimulations. The aim of this study was to propose a new neuromuscular fatigue protocol evaluation on elbow extensor musculature. Twenty participants preformed exercises to induce central (CenFat) and peripheral fatigue (PerFat). Neuromuscular fatigue was evaluated on knee extensor muscles by a conventional protocol that provides Twitch Superimposed (TS The conventional neuromuscular fatigue evaluation proposed by Despite this method’s effectiveness as a procedure for musculature such as the quadriceps femoris , studiesA possible frailty in triceps brachii in tasks involving maximum contractions was suggested, being susceptible to present synergistic muscle inhibitory effects (co-contraction phenomenon) such as the biceps brachii  and the Regarding neuromuscular fatigue analysis for elbow extensor musculature , stimulaNeuromuscular fatigue evaluation through muscle stimulation during a maintained submaximal contraction (30% of MVC) has already been evidenced in the lower limbs . MoreoveIt is important to note that The linear method (linear regression) made by several TS, resulting from stimulation at different submaximal contraction rates, may be a valid approach to compare the neuromuscular status (differentiate and/or quantify the type of fatigue), providing parameters capable of identifying central and/or peripheral fatigue. It is possible that this mathematical strategy is sensitive, since a linear relationship between the evoked force and the submaximal contraction rate has already been verified .Analysis that employs linear regression presupposes that the line extremities  are directly regulated by parameters already discussed in the literature, such as pulses applied with the relaxed musculature (TP) (representing one extremity of the line) and the use of 100% TS of MVC (representing another extremity of the line) .R2).In the abstinence of detailed studies with triceps brachii, we consider that for the biceps brachii, both curvilinear and linear methods are valid and similar to extrapolate the parameters . In thisR2 than in the situation where the participant would be exposed only to peripheral pathway stress. In the latter case, exposure only to central pathway stress compromises both slope and R2, considering that the y-intercept remains the same as a function of the TP value that remains unchanged  participated in the study with a mean age, body mass and height of 25.1 ± 6.8 years, 74.9 ± 11.4 kg, and 174.8 ± 9.7 cm respectively. The participants were: five females  and 15 males . After divulgation of the research and contact of interested participants, individual contact was made with each participant to explain the risks, procedures used, and study objectives. The participants were asked not to train or use any ergogenic, energy, or alcoholic resources in the 48 h prior to the evaluations. Participants were only included in the study after signing the consent form. All experiments were previously approved by the Ethics Committee (Process number: 97168618.4.0000.5659) and conducted in accordance with the Declaration of Helsinki.After three familiarization sessions, the volunteers made two visits to the laboratory to realize the principal focus of the present study: validation of the proposed evaluation protocol for elbow extensor musculature. In the two visits the participants made the same experimental design that included: pre-evaluation, fatigue induction by an exercise, and post-evaluation. However, each of these visits was covered with a different exercise  with randomly established sessions. Both environments had a controlled temperature of 71.6°F.Neuromuscular evaluation in lower limbs, due to the fact of having proven scientific validity , aimed tE) and knee (MVCK) was determined; (2) The progressive electrostimulation test (PET) were performed for the elbow extensor and knee flexor musculature to provide the lowest stimulation intensity that promotes the highest rate of evoked force (best stimulation rate). This was performed in the moment before the fatigue induction exercise (PREFATIGUE); (3) The application of the valid neuromuscular fatigue evaluation protocol for knee extension musculature (FATIGUE) and during the recovery (POST5MIN and POST10MIN respectively) in both muscular regions  The maximal voluntary isometric contraction of elbow . Elbow and knee extension were performed against different load cells  and data acquisition was made with a Labview 2015 environment  with 1000 Hz acquisition frequency.The optimized intensity of the electrostimulation was determined individually for each participant (both for upper and lower limbs) using the PET (in the first step). Electrical stimulation was performed through an electrostimulator prototype developed for this purpose with a 200 V peak-to-peak capacity  (®). For lower limbs stimulation electrodes were positioned at the upper limit (cathode) and lower limit (anode) of the vastus lateralis and for upper limbs stimulation were positioned on the triceps long head (cathode) and distal tendon (anode). It should be noted that the electrical pulses applied in the musculature were “doublets.” Doublets guarantee force evocation during muscle stimulation  were used in the triceps brachii   and two mulation  and are A specific chair for neuromuscular evaluation was used for force measurement, and consequently neuromuscular evaluation, so that the participant remained with the trunk supported by the chair with the arm relaxed and parallel to the trunk, while the elbow was maintained in 90° flexion. The load cell attached to a sliding block had an adjustable strap that was attached to the participant’s wrist (dominant arm). To perform submaximal contractions, the load cell was adapted into a routine where the participant had access to its simultaneous strength by visual access to a force target through a monitor. The second load cell, for knee extension force evaluation, was attached to the chair and fastened to the participant’s ankle (dominant foot) while their leg was kept relaxed with 90° of knee flexion, without visual force information.The order of evaluation was the upper limbs and lower limbs respectively to ensure the quality of neuromuscular responses to the proposed protocol. It should be noted that in less than a minute after the end of the exercises, the entire neuromuscular evaluation protocol (including upper and lower limbs) had already been performed, preserving the neuromuscular responses with the best quality possible .E). At the end of the submaximal task another stimulation was applied on relaxed muscle and the difference between the evoked force and weight value of the relaxed musculature was considered as TPE.The elbow extension protocol involved an evaluation where the participant maintained sequentially five submaximal intensities of MVC  for 5 s each. During each submaximal contraction, stimulations were applied and the evoked force during the stimulation was determined by the difference between the submaximal contraction force and the submaximal contraction rate maintained (TSR2).Considering that the most common evaluation model uses stimulation in maximal contractions and that this model does not work well with the elbow extensor musculature due to the fact of stimulation superimposed on maximal contractions not exhibiting evoked force impairment , the useR2 ; peripheral fatigue may show a reduction in y-intercept, slope, and R2  and; central fatigue may show maintenance of y-intercept with reduction of slope and R2 .This hypothesis is based on the basic principles of neuromuscular analysis established by K) and a stimulation with relaxed muscle 3 s after MVC (TPK). This procedure was able to identify the peak force and, through the TPK and TSK, it was possible to calculate the voluntary activation (VAK (%) = [1 - (TSK/TPK)] ∗ 100) (The procedure to identify neuromuscular fatigue for knee extension musculature was composed of a 5 s MVC with a stimulation on the third second (TS] ∗ 100) .To induce peripheral fatigue in the elbow extensor musculature, a specific exercise for the region was chosen, while in view of generating only central fatigue, an exercise involving only lower limbs was preferred. Once the central fatigue is installed via the lower limbs, it is hypothesized that the upper limbs will only be able to change their parameters related to the central pathway .E load  in the halter exercise. Each series was composed of 10 repetitions with a 20 s interval between each series   consisted of the halter elevation exercise (made behind the head), which was performed in the neuromuscular fatigue evaluation chair. The halter starts from an initial position with 180° shoulder flexion and 35° elbow flexion, ending the movement with 180° elbow extension, giving a 135° movement degree. As in the original exercise the volunteers only performed the concentric movement . As usedh series . When dirotocol) .®). After the warming up stage (20–30 repetitions with subjectively low load), the participant was asked to establish a load for which they could perform a maximum of 10 repetitions. The load and the number of repetitions to perform this task were applied to The central fatigue induction exercise (CenFat) was the leg extension exercise  in a maxThe choice of central fatigue inducing-exercise was made considering that central fatigue is more quickly established when there is greater muscle involvement in the effort , while t∗Power , it was possible to identify that 19 participants were necessary for the present study to obtain a significant statistical power . Data were processed using JASP® (Version 0.12.2). Due to the lack of normality and sphericity, the data were treated in a non-parametric manner. In order to investigate the difference between situations, moments, and multiple pulses, Friedmann’s variance ranking analysis and a Wilcoxon classification test were used. The Wilcoxon classification test was also used for the differences between the two exercises’ parameter results. A p < 0.05 significance level was considered for all results. Effect size is given by the matched Rank-Biserial Correlation.Using software GThe reliability of the data (comparison between different days) was also tested with intraclass correlation coefficient (ICC), typical error (TE), coefficient of variation (CV%), standard error of the measure (SEM), and minimal difference needed to be considered real (MD) . The TE K between PREFATIGUE and the two last measurements . After CenFat, statistical differences were found for TPK between the PREFATIGUE, POSTFATIGUE and both recovery moments . CenFat also shows statistical differences for MVCK between the PREFATIGUE, POSTFATIGUE and both recovery moments   .E and several TSE between PREFATIGUE, POSTFATIGUE, and both recovery parameters (p < 0.05). For TPE, effect sizes of the significant differences ranged between 0.853 and 0.453 after CenFat and between 1 and 0.965 after PerFat. Statistical differences were also observed for TSE between POST5MIN and POST10MIN (p < 0.05)  .K values in the POSTFATIGUE, POST5MIN, POST10MIN were lower after CenFat than PerFat . MVCK showed significant differences on POSTFATIGUE, POST5MIN, and POST10MIN   .E than CenFat on: POSTFATIGUE for TSE on 10, 20, 30, 40, and 50% of the MVCE ; POST5MIN for TSE on 10, 20, and 30% of the MVCE  and; POST10MIN for TSE on 10, 20, 30, 40, and 50% of the MVCE . PerFat also showed lower TPE than CenFat on POSTFATIGUE, POST5MIN, and POST10MIN  (In upper limbs in the PerFat showed lower TS < 0.01) .FATIGUE was lower than PREFATIGUE  and higher than POST10MIN . After PerFat, y-intercept on PREFATIGUE was higher than POSTFATIGUE, POST5MIN, and POST10MIN . After PerFat, slope on PREFATIGUE was higher than POSTFATIGUE and POST5MIN . After PerFat R2 on PREFATIGUE was higher than POSTFATIGUE, POST5MIN, and POST10MIN   .FATIGUE   (Slope was lower after PerFat than CenFat on POST < 0.05) . R2 was  < 0.05) .E at 10% (0.84) and 20% of MVC (0.82), TPE (0.83) and TPK (0.82). Moderate indices for TSE were also found at 30% (0.74), Slope (0.50), R2 (0.62), MVCK (0.59), TSK (0.72), and AVK (0.67). All reproducibility data are shown in The reliability tests indicated good ICC indexes for TSThe objective of the present study was to test a specific protocol for analyses of neuromuscular fatigue in elbow extensor muscles. Our main data show that the proposed protocol was able to verify changes in neuromuscular parameters using stimulation in submaximal contractions in the elbow extensor muscles.E) and the respective TSE presented was able to find differences between the two efforts (K) change (from 62.9 ± 50.6 N on PREFATIGUE to 48.5 ± 48.1 N on POSTFATIGUE).The linear relationship between maintained submaximal force  did not result in peripheral fatigue for lower limbs. However, a substantial decrease of these parameters was observed after CenFat, confirming peripheral fatigue manifestation (from 262.3 ± 58.1 N on PREFATIGUE to 206.7 ± 44.9 N on POSTFATIGUE). Finally, it is important to point out that one of the exercises used (CenFat) was a limitation of the study because it did not cause central stress by changing TSK . These results suggest that both exercises provided some disturbance of the extensor elbow muscles , which contained support loops so that the participant could hold with their hands during the leg extension. However, further investigations should be made regarding this parameter’s representativeness for the neuromuscular protocol involving stimulation in the elbow extensor musculature.It is possible that the TPE reduction in all submaximal contraction intensities (5MIN and POST10MIN) was different after each exercise. It was evidenced that, after PerFat, most of the TSE  were similar, exhibiting a plateau behavior without signs of recovery, unlike the results after the CenFat, which showed signs of recovery . Thus, the linear mathematical method used by As previously reported, in addition to identifying changes in neuromuscular parameters, the evaluation protocol suggested by the present study also showed linearity between the TSin vitro) methods. In view of the possible underestimation of the evoked force, we used stimulation intensity that most evoked force (during the PET) and not necessarily the maximum intensity or near to the pain threshold of the subjects (The inherent “co-contraction phenomenon” (simultaneous contraction of the agonist and antagonist musculature) of the brachial musculature during stimulation is treated as an abnormality, and several authors describe this situation as limiting for neuromuscular analysis . Howeversubjects .Upon these considerations, the linear method used assumes the state of co-contraction as a natural state of the elbow extensor musculature. The linear method utilized by the present study shows the possible recovery tendency after the CenFat by the y-intercept parameter, whereas after the PerFat this parameter does not show recovery signs .R2 values remained high, indicating low dispersion of the points for line formation.It is important to note the contrary response of slope after both exercises . While PE and TSE at 10% of MVCE); increase by the extremity comprising “lowest” stimulation values (TSE at 40 and 50% of MVCE); and the set of both alterations mentioned.The slope reduction after the PerFat may contrast three situations: reduction by the extremity that includes the “highest” stimulation values (TPK did not show a significant change after PerFat (from 55.5 ± 38.7 N to 62.6 ± 50.6 N), a state that, theoretically, would not significantly change a possible TSE at 100% of the MVCE in the elbow extensor muscles (if we extend the linearity for higher contractions) (E sudden reduction after PerFat (to 2.8 ± 1.0 N from 1.2 ± 0.6 N) should be considered. This reduction is greater when compared after CenFat  . FurtherE at 40 and 50% of MVCE) accompanied by a reduction less accentuated by TPE  is an effective method to identify peripheral fatigue. However, its ability to discriminate the origin of fatigue is limited, since the CenFat did not promote significant changes in central pathway.The products of the linear relationship between maintained submaximal force and TSFinally, linear regression appears to be a good method to investigate neuromuscular fatigue, although other non-linear methods should also be tested before this protocol has its validity tested in the practical environment.The study was designed by MP and MSN; data were collected and analyzed by MSN and TBA; critical procedure support was provided by MP; data interpretation and manuscript preparation were undertaken by MSN, TBA and MP. All authors approved the final version of the paper. 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. The study was possible due to the São Paulo Research Foundation (FAPESP) incentive and funding support . Special thanks to all researchers in the aquatic activity laboratory for systematic support throughout the study progression.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of School of Physical Education and Sport of Ribeirão Preto (Process number: 97168618.4.0000.5659) and conducted in accordance with the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.MP and MN designed the study. MN and TA collected and analyzed the data. MP support the procedure. All authors approved data interpretation and manuscript preparation and the final version of 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.


Ultra-processed foods have known negative implications for health; however, their effect on skeletal development has never been explored. Here, we show that young rats fed ultra-processed food rich in fat and sugar suffer from growth retardation due to lesions in their tibial growth plates. The bone mineral density decreases significantly, and the structural parameters of the bone deteriorate, presenting a sieve-like appearance in the cortices and poor trabecular parameters in long bones and vertebrae. This results in inferior mechanical performance of the entire bone with a high fracture risk. RNA sequence analysis of the growth plates demonstrated an imbalance in extracellular matrix formation and degradation and impairment of proliferation, differentiation and mineralization processes. Our findings highlight, for the first time, the severe impact of consuming ultra-processed foods on the growing skeleton. This pathology extends far beyond that explained by the known metabolic effects, highlighting bone as a new target for studies of modern diets. Many signaling pathways control the patterning, growth and maturation of skeletal structures from early development to adulthood.2 the latter of which is responsible for long bone development. EO is initiated in the fetus and continues postnatally in the growth plates (GP) until growth cessation in adolescence.3 The rate of longitudinal bone growth is determined by the rate of chondrocyte proliferation, the size of the hypertrophic chondrocytes, and cartilage degradation and replacement by bone. Chondrocytes in different zones of the GP are distinguished by their differentiation stage, morphology and matrix production. In the proliferative zone (PZ), chondrocytes are arranged in longitudinal columns, where they rapidly proliferate and deposit typical cartilage extracellular matrix (ECM) components, especially collagen type 2 (Col2) and aggrecan (Acan). Next, chondrocytes exit the cell cycle and become prehypertrophic by expressing the type X collagen (Col10) and Indian hedgehog (Ihh) genes. Prehypertrophic chondrocytes increase in size to become hypertrophic cells.4 In the hypertrophic zone (HZ), cells become enlarged, swollen and vacuolated and are characterized by secretion of Col10, alkaline phosphatase, and angiogenic factors.5 Fully differentiated hypertrophic chondrocytes at the chondro-osseous junction undergo apoptosis or transdifferentiation, and cartilaginous ECM is removed by osteoclasts, allowing the penetration of blood vessels and bone tissue formation by osteoblasts.6 The EO process is tightly orchestrated by various signaling molecules and transcription factors, including transforming growth factor β/bone morphogenetic protein (BMP), fibroblast growth factor (FGF), Wnt, hedgehog, and the transcription factor Sox9.2The vertebrate bony skeleton is formed through two processes, intramembranous ossification or endochondral ossification (EO),7 Proper intake of macronutrients, minerals and vitamins is therefore of great importance in achieving optimal bone structure and quality.8Bone elongation and hence whole-body longitudinal growth are strictly regulated by genetic and environmental elements. To maximize growth and peak bone mass, modifiable environmental factors, particularly the balance of nutrition, should be optimized before the onset of puberty and maintained throughout this period of rapid growth.10 Over the past 30 years, children’s ultra-processed food (UPF) intake has increased markedly, with 50% of the children in the US consuming these foods.11 Only in the US does UPF comprise 57.9% of energy intake, of which 89.7% is derived from added sugars. This reflects children’s excessive consumption of food and drink that are high in fat and refined sugars but do not provide appropriate levels of the proteins, vitamins and minerals required for growth.12 The health outcomes resulting from this type of nutrition have been described in many studies and include obesity, metabolic syndrome and diabetes.14 However, the critical effect of such a diet on postnatal skeletal growth from birth until puberty has not been assessed.12Food supplies in recent decades have been dominated by heavily processed, ready-to-eat products. Essentially, 75% of all world food sales are of processed foods.15 the effect of excessive consumption of UPF on bone development and growth has not been evaluated at the cellular level, nor has its effect on bone quality been studied. Here, we used young rats as our experimental model for postnatal development and examined the effect of an unbalanced ultra-processed diet (UPD) during postnatal growth on skeletal development and quality.While previous studies have shown that malnutrition or malabsorption result in longitudinal growth retardation and short stature,16To study the link between UPF consumption and postnatal skeletal development, we conducted a 6-week-long in vivo trial in young female rats (3 weeks to 9 weeks of age). This time frame extends from weaning to puberty and therefore represents the growth period before sexual maturation and GP closure in humans.12 The experimental design included two groups: the control group, which was fed a standard rat diet, and the UPF + CSD group, which was fed a diet comprised of a typical UPF meal (see methods for preparation) and a caloric soft drink. All rats had ad libitum access to food and liquids.The diet chosen here exemplifies the Western UPD with unbalanced levels of micro- and macronutrients.During the experiment, the body weight and total body length of all rats were measured. In addition, the femur and lumbar vertebra lengths were assessed by µCT after 3 and 6 weeks of the experiment. Weight gain was reduced in the UPF + CSD group Fig. , and totTo assess the effect of the UPD on bone quality, femora and lumbar vertebrae were scanned by µCT to examine their trabecular and cortical bone properties.The trabecular bone parameters of the UPF + CSD group were inferior to those of the controls. The bone volume fraction (BV/TV) of the femora decreased significantly, from 35.54% to 19.17% and from 23.06% to 16.27% at 6 and 9 weeks of age, respectively. The mean trabecular number (Tb. N) and mean trabecular thickness (Tb. Th) in the femora were significantly lower in the UPF + CSD group, while trabecular separation (Tb. Sp), which represents the mean distance between trabeculae, was significantly higher than that in the control group at both time points  and BMD, which was 30% lower for rats consuming UPF + CSD Fig. . Ct. Po 17 A significant and dramatic decrease was demonstrated in bone mechanical parameters such as stiffness, yield load, maximum load and fracture load  revealed an uneven widening of the GP in rats in the UPF + CSD group. This widening was characterized by a mass of avascular nonmineralized cartilage lesions that extended from the epiphyseal GP into the metaphysis of the tibia , -10 Fig. . IntriguFst), noggin (Nog), Gdf10 and inhibin beta A subunit (Inhba),4 suggesting the attenuation of BMP signaling  was upregulated in the UPF group in tandem with a significant increase in the expression of the downstream genes Gdf10, Acan, Col2 and Col10  and phosphate-regulating endopeptidase homolog X-linked (Phex), encoding proteins that enhance mineralization,23 and upregulation of the gene encoding matrix gla protein (MGP), a mineralization inhibitor.24 Interestingly, while mineralization was downregulated, vascularization genes  were not differentially expressed , indicating that bone resorption was taking place , and the second group was fed a high-sucrose diet that consisted of the control diet + drinking solution with 10% sucrose (sucrose). The results showed normal trabecular and cortical bone parameters and normal GP organization in the Corn and Sucrose groups  and P (1.21 mg·g−1) levels and ratio in the UPD (Ca/P group). The Ca/P group exhibited growth retardation in terms of body weight, body length and femoral length that similar to the UPF group  in the UPD . Despite the lower consumption of calories, Ca and P in the 30%/70% group Fig. , the gro11 However, to the best of our knowledge, the impact of these food choices on skeletal development has not been studied.26Extensive consumption of processed food and soft drinks is endemic in the modern era, with many known metabolic implications.We showed that consumption of a diet consisting exclusively of UPF in growing rats leads to severe disruption of skeletal development, which is characterized by major alterations of the GP. We observed significant deterioration of the quality of trabecular and cortical bone, widely distributed porosities in the compact bone, and a dramatic decrease in the BMD, all of which were correlated with retardation of longitudinal growth and bone fragility.16 To shed light on the underlying mechanism of the disorder of the GP, we performed mRNA-seq of control and UPD GP samples. The mRNA profile of the UPF group exhibited an impaired EO process.The most striking result was the observation of aberrant cartilage occupying the GP of rats fed a UPD. Histological and SEM analyses of these bones revealed a disordered GP characterized by a noncalcified, nonvascularized cartilage lesion that extended from the epiphyseal GP into the metaphysis. This rare and severe phenotype differs greatly from other abnormal GP phenotypes, such as rickets and protein-energy malnutrition.Fst, Nog, Gdf10 and Inhba) without modifications of BMP or its receptors. BMP plays an essential role at every stage of endochondral bone development.4 It can induce bone and cartilage formation and stimulate chondrocyte proliferation and differentiation.28The UPD caused a significant increase in the synthesis of ECM components, while there was no difference in the expression of matrix-degrading enzymes, leading to an imbalance in the ECM formation–degradation process. This might have led to the formation of cartilaginous plaques in the GP of the UPF group. BMP signaling was weakened due to high expression of BMP inhibitors , suggesting the intensification of the early chondrocyte differentiation process.Another fundamental factor for EO is Sox9 elevation, the levels of Col10, a hypertrophic matrix component, were upregulated in the UPF group. He et al.32 confirmed the requirement of Sox9 for hypertrophic chondrocyte development. They demonstrated that Sox9 and Jun bind and coactivate a Col10a1 enhancer. Furthermore, Dy et al.33 claimed that Sox9 is necessary for GP chondrocyte hypertrophy. They showed that Sox9 transactivates Col10a1 along with Mef2c to increase hypertrophy and maintain a functional GP.33 We showed that while Sox9 and Col10 were upregulated in the UPF group, Jun and Mef2c, though highly expressed, were not differentially expressed. This might imply that there are more candidates responsible for the elevated Col X levels, such as FoxA factors. Indeed, FoxA2 and A3 were upregulated in the UPF group by 2.8- and 3.2-fold, respectively. FoxA2 and A3 regulate chondrocyte hypertrophy through direct induction of Col10.27 Accordingly, Ionescu et al.28 showed that chondrocyte hypertrophy is delayed in Col II–Cre, FoxA2flox/flox, and FoxA3−/− mice, suggesting that these factors are responsible for the elevation of Col10a1 in our model.In addition to Dmp1 and Phex and upregulation of MGP. Dmp1 and Phex belong to a group of proteins that enhance mineralization,23 whereas MGP is a mineralization inhibitor,24 suggesting the inhibition of the bone mineralization process. Ye et al.34 showed that Dmp1−/− mice have shorter bones and increased tibia width and cortical porosity. They further reported that Dmp1−/− mice have a disorganized and irregular GP with an expanded HZ that is poorly calcified.34 Phex−/− mice exhibited a very similar phenotype, including a widened and irregular HZ with hypomineralization and increases in cartilage remnants from the GP in both trabecular and cortical bone.35 These results correspond to the phenotype exhibited in the UPF group in our experiment.Our results also demonstrated downregulation of the crucial ossification genes Dmp1 or Phex leads to overexpression of FGF23 by osteocytes and, as a result, to hypophosphatemic rickets.37 This contradicts our findings of a decrease in serum FGF23 and hyperphosphatemia. These differences may result from the fact that knockout mice are characterized by complete deficiency in Dmp1 and Phex, whereas our UPF group exhibited a decrease in Dmp1 and Phex mRNA levels. Moreover, our experimental group also exhibited hypocalcemia and thus the elevation of serum PTH, leading to a decline in OPG levels. OPG is known as a receptor antagonist for RANKL that prevents it from binding to and activating RANK, thereby inhibiting osteoclast differentiation.38 In our experiment, OPG downregulation led to osteoclast differentiation and bone resorption, as detected by TRAP staining in the cortex. Interestingly, there was no difference in osteoclast activity at the chondro-osseous junction, highlighting the differences between osteoclasts in the GP and cortex. Correspondingly, we revealed increased porosity in the UPF group femoral cortex and similar TRAP mRNA levels in the UPF GP. In summary, the consumption of UPF stunted growth and disrupted bone quality and EO. The GP of the UPF group was occupied by a massive cartilaginous plaque that was assembled from a large number of cells and ECM. Transcriptome analysis revealed that this aberrant cartilage resulted from an imbalance in ECM formation and degradation, disrupted proliferation and differentiation mechanisms, and reduced mineralization.Inactivation of either Next, we wanted to understand what components of the UPD cause growth retardation and the bone phenotype. We demonstrated neither the soft drink nor the macronutrients was the cause, but the Ca and P deficiency and imbalance caused damage that was similar to but less severe than that caused by the UPD. This suggests that Ca/P deficiency leads to the aberrant phenotypes and that there are other cofactors responsible for these detrimental phenotypes in UPD.39 oil or sugars may not be as detrimental to human health without additional or excess processing. Overprocessing itself is not always classified as a damaging process since well-balanced healthy products such as infant formulas are also included in this food group. However, the combination of sugar and oil overload with micronutrient deficiency and harmful processing procedures  may be the driving force behind impaired bone development. Behavioral changes in the rats consuming the UPF 41 made the observation that “people do not choose nutrients, they select combinations of foods…researchers should be more creative and…bolder in assessing the health implications of common combinations of foods.” This is the research approach used here by studying the effect of a “whole diet” rather than the effect of individual nutrients, which in this case was the UPD or so-called “new malnutrition“.42 There is general agreement that the consumption of processed foods and sugar-sweetened beverages is associated with increased rates of obesity,43 diabetes44 and cardiovascular disease.46 We demonstrate a new target for the harmful effect of such a diet and suggest it as a direct risk factor for bone health.Finally, we established a UPD model in young rats that simulates modern Western eating habits. Providing rats with a whole diet that is equivalent to that eaten by humans is a novel scientific approach. The common nutritional methodology involves adding or removing one component at a time from the diet to study its effect separately, as in pharmaceutical or genetic studies. However, nutrition is different; a recent paper in NatureAll experiments were conducted on female Sprague-Dawley (SD) rats after weaning (3 weeks of age). We chose young female rats before sexual maturation as our study model because female subjects are more prone to suffer from bone diseases. Thus, improving bone quality in young females is a preventative approach.ad libitum access to food and drink. After 4 days of acclimation, the rats were randomly divided into experimental groups. All procedures were approved by the Hebrew University Animal Care Committee #AG-11-13225-2, AG-13-13952-2. Throughout the experiments, body weight, body length from the tip of the nose to the end of the tail, food and fluid consumption were measured twice a week, and daily food intake in kilocalories was calculated for each rat per day. At two time points, after 3 and 6 weeks of the experiment , animals were anesthetized with isoflurane, blood samples were collected, and they were sacrificed. Their internal organs and bones  were harvested. The femora and 3rd–5th lumbar vertebrae were manually cleaned of soft tissue and stored at −20 °C until micro-computed tomography (µCT) scanning and mechanical testing. Tibiae were fixed immediately after sacrifice  or frozen in liquid nitrogen for RNA sequencing analysis. For the full experimental setup of this study, see Supplementary Fig. The rats were purchased from Harlan Laboratories  and housed under standard environmental conditions with a 12 h light:12 h dark cycle and n = 8) received a standard diet based on the composition for growing rats recommended by Harlan Laboratories (Control), and the other (n = 8) was offered a diet based on UPF, which was rich in fat and sucrose and included, a typical caloric soft drink (UPF + CSD). The UPD included a roll, hamburger, tomatoes, lettuce, ketchup (without onion or pickles) and French fries. The whole meal was homogenized, shaped into patties and frozen at −20 °C  were divided into four groups that received (i) the standard diet for growing rats  and (ii) a diet based on UPF, which was rich in fat and sucrose and included a soft drink containing 10% sucrose . In the third and fourth groups, the main ingredients of the fast food diet—fats and sugar—were isolated, and the animals received (iii) a high-fat diet with the addition of corn oil  and (iv) a high-sucrose diet consisting of the regular control diet + drinking solution with 10% sucrose  ; (ii) a diet based on UPF without the caloric soft drink ; and (iii) a custom diet with an altered Ca:P ratio, 62 mg Ca per 100 g and 121 mg P per 100 g of diet  (n = 8). This ratio corresponded to the Ca:P ratio in the UPF group, while the other micronutrients and macronutrients were similar to those in the control diet. The diet was prepared by Envigo   and phosphorus (P) in the UPD causes the lesion phenotype in UPF-consuming rats, 24 rats were divided into three groups receiving (i) the standard diet ; (ii) the UPD ; and (iii) the Control diet 30% of the week and the UPD the rest of the week . Group (iii) mimicked UPF eating patterns  the standard diet  for the UPD. Calories from each macronutrient were obtained by multiplying grams of protein, fats and carbohydrates by the factors 4-9-4, respectively.47 Supplementary Fig. The macro- and micronutrient contents of the control and UPD diets were analyzed by Aminolab Laboratories , which provides analytical services certified by the FDA and Israeli Ministry of Health , and hormonal profile analysis was performed at American Medical Laboratories .50Femora and lumbar vertebrae were scanned in a SkyScan 1174 X-ray computed microtomograph scanning device. Images were obtained with an X-ray tube voltage of 50 kV and current of 800 μA. The bones were scanned using a 0.25-mm aluminum filter with a 4 500 ms exposure time and at high spatial resolution (13.8 μm). For each specimen, a series of 900 projection images were obtained with a rotation step of 0.4° by averaging 2 frames for a total 360° rotation. For analysis of the diaphyseal cortical region, 200 slices were chosen. Global grayscale threshold levels for the cortical region between 87 and 255 were selected. For the trabecular analysis of the secondary ossification zone, 150 slices were selected, and the adaptive grayscale threshold levels of 85 and 255 were used.49 The following bone biomechanical parameters were derived from load-displacement curves: bone stiffness, yield load, force to fracture, maximal force and total energy to fracture.52The mechanical properties of the femora from each group were determined by three-point bending tests using a custom-made micromechanical testing device.53 In situ hybridization was performed to detect Col2 and Col10 expression.54 The widths of the whole GP and the PZ, HZ and lesion zone (LZ) were measured at 10 different points along the GP and averaged with measurements from 6 other plate samples in each group. The percentages of PZ and HZ/LZ from the whole GP were calculated. For the measurements, H&E staining was used. Stained tissue sections were viewed under an Eclipse E400 Nikon light microscope at various magnifications using light filters. Pictures were taken with an Olympus DP71 camera controlled by Cell A software (Olympus).The effect of the different diets on the GP of growing rats was examined by histological techniques (to determine the structure and cell types). Histological staining included hematoxylin and eosin (H&E), safranin O, Masson’s trichrome staining, and tartrate-resistant acidic phosphatase (TRAP).55 The polished surfaces were studied using reflected-light microscopy . Images were captured using a 12.1-megapixel resolution DP71 digital camera attached to the microscope.Transverse slices of ~1.2 mm were prepared from the middle of the diaphysis of the left femora from five rats. Each slice was marked to identify its axial position and proximal, medial and cranial aspects. The slices were ground and polished with successively finer abrasive paper (from 2 500 to 4 000 grit), followed by 3 and 1 cloth with diamond paste.Paraffin-embedded tibia bones were deparaffinized in xylene and rehydrated, carbon-coated and scanned. High-resolution images of the tibial GP surface were examined by SEM  at various working distances with an electron energy of 10 keV in high-vacuum mode using secondary electron detector mode.n = 8) and UPF (n = 8) groups were collected. The GP area was immediately isolated with a scalpel, avoiding contamination with adjacent unwanted tissue . The isolated GP was then pulverized manually in liquid nitrogen. Total RNA was extracted using TRI reagent  according to the manufacturer’s protocol. The structural integrity of the RNA is of great importance in preparing mRNA libraries. Therefore, we used the Agilent 2200 TapeStation system to assess RNA quality. The TapeStation instrument produces gel images and RNA integrity numbers (RIN) for each sample. The RIN is presented as a value between 1 and 10, where 10 represents the highest quality RNA.56 Twelve samples with high RIN values (>7) were selected for mRNA library preparation. Nine libraries (control n = 4 and UPF n = 5) were successfully prepared using Lexogen’s QuantSeq 3’ mRNA-Seq library prep kit. Libraries were quantified by a Thermo-Fisher Qubit and Agilent TapeStation and sequenced in an Illumina NextSeq 500 sequencer using the NextSeq 75 bp kit to produce single-end reads. The output was ~25 million reads per sample. The sequencing process was conducted by the Core Research Facility at The Faculty of Medicine - Ein Kerem, The Hebrew University of Jerusalem, Israel.After 3 weeks of the experiment, animals were sacrificed, and the right tibia bones from the control (Raw reads (fastq files) were inspected for quality with FastQC v0.11.4. Raw reads were trimmed for quality to remove the poly (A) tails and adapter sequences using the Trim Galore default settings.Single-end reads were mapped to the rat genome (Rattus_norvegicus. Rnor_6.0.94) using STAR v2.201. Mapping files were further processed for visualization by Samtools Utilities v 0.1.19.P value was lower than 0.05. Additionally, we used a fold change filtering value of |fold change | >1.Differential expression analysis: The uniquely mapped reads per gene were counted using HTSeq-count, and differential expression analysis was performed using the DESeq2 R package. Genes were considered significantly expressed if the adjusted 18GeneAnalytic was used to search for possible biological processes, canonical pathways and networks. This software is a gene set analysis tool for contextualization of expression patterns and functional signatures embedded in postgenomics big data domains, such as RNA sequences. To perform this analysis, we converted the rat gene IDs to those of the homologous human genes using the OMA web-based database interface for orthology prediction. Next, using Biomart, we converted the human IDs to NCBI IDs. We then uploaded the differentially expressed genes to the GeneAnalytics website.P ≤ 0.05. Groups with the same letter were not measurably different, and groups that were measurably different are indicated by different letters. Groups may have more than one letter to reflect the “overlap” between the sets of groups, and sometimes a set of groups is associated with only a single treatment level.All data are expressed as the mean ± SD (standard deviation). The significance of differences between groups was determined using JMP 12.0 Statistical Discovery Software (SAS Institute 2000) by one-way ANOVA followed by the Tukey–Kramer HSD test and t-test. Differences were considered significant at Supplemented material


Pinus edulis E.). Our results show that pinyon shoot growth after 6 years of these treatments was reduced more by drought than warming. The combined drought and warming treatment reduced the abundance and diversity of EMF more than either treatment alone. Individual ectomycorrhizal fungal taxa, including the drought tolerant Cenococcum geophilum, were present in all treatments but the combined drought and warming treatment. The combined drought and warming treatment also reduced the abundance of dark septate endophytes (DSE), but did not affect their diversity or species composition. The current year shoot growth of the trees correlated positively with ectomycorrhizal fungal diversity, highlighting the importance of diversity in mutualistic relationships to plant growth. Our results suggest that EMF may be more important than DSE to aboveground growth in P. edulis, but also more susceptible to the negative effects of combined climate stressors.Changing climates can cause shifts in temperature and precipitation, resulting in warming and drought in some regions. Although each of these factors has been shown to detrimentally affect forest ecosystems worldwide, information on the impacts of the combined effects of warming and drought is lacking. Forest trees rely on mutualistic root-associated fungi that contribute significantly to plant health and protection against climate stresses. We used a six-year, ecosystem-scale temperature and precipitation manipulation experiment targeted to simulate the climate in 2100 in the Southwestern United States to quantify the effects of drought, warming and combined drought and warming on the root colonization (abundance), species composition and diversity of ectomycorrhizal fungi (EMF), and dark septate fungal endophytes in a widespread woodland tree, pinyon pine ( Changes in climate, including the combined effects of increased drought and warming temperatures, are significantly affecting temperate forest ecosystems . These sMicrobial plant mutualists, such as root-associated fungi, significantly affect plant responses to climate change  and two plant species (Picea abies and Betula pendula). On the other hand, inoculation with DSE improved host plant responses to drought in the studies reviewed by The roots of many plant species, including some of those that host EMF, also are colonized by dark septate endophytes (DSE), ascomycete fungi grouped by the morphology of their highly melanized hyphae within host roots . Unlike Pinus edulis, a western United States tree species that occupies a large area of semi-arid landscape where it occurs with co-dominant members of the genus Juniperus. Warm temperatures combined with extreme drought resulted in significant P. edulis mortality across 12,000 km2 of the southwestern United States in 2002–2003 , drought (~50% reduction in precipitation), warming (temperature 5°C above ambient) and a combination of drought and warming treatments for 6 years at the Los Alamos Survival-Mortality (SUMO) experiment located in Los Alamos County, New Mexico . The SUMO site, established in summer 2012, consists of five treatments with 5–6 trees per treatment. These treatments are: control with trees experiencing ambient temperature and precipitation, heat with trees inside open-top chambers where temperature was maintained constantly at 4.8°C above ambient temperature, drought with trees located within a precipitation exclusion structure constructed of polyethylene troughs about 1.5 m above the soil surface covering ~50% of the ground area and directing ~45% of the precipitation off the site, a combined drought and heat treatment, and a chamber control treatment with open-top chambers kept at ambient temperature  and one-seed juniper [Juniperus. monosperma (Engelm.) Sarg.], with shrubby Gambel oak (Quercus gambelii Nutt.) and an occasional ponderosa pine (Pinus ponderosa C. Lawson) occurring in the vicinity. The climate is semi-arid, with a mean annual temperature of 10.4°C (1987–2017) and a mean annual precipitation of 358 mm (1987–2017) of which about 50% falls during the North American Monsoon season from July to September , heat (n = 4), drought (n = 6) and combined drought and heat treatments (n = 5). In addition to the effects on precipitation and air temperature, the treatments influence soil temperature measured continuously at the base of all target trees with thermocouples installed at 5, 10, 15 and 30 cm depths. The drought treatment alone had negligible effect (<0.1°C) on soil temperature while the warming treatment increased soil temperature on average by 3.6°C. In April 2016, the coverage of the precipitation exclusion structure was briefly increased to 90% by adding additional clear polymer troughs to increase the stress experienced by the trees. To prevent excessive heating of the soil surface and airspace below the troughs, thermal bubble insulation was installed underneath the polymer troughs, and portable blower fans  were placed throughout the drought and drought and heat treatments. To ensure the effectiveness of the cooling, soil temperature was additionally measured continuously  over a 0–30 cm depth at the base of each tree. Mean daily soil temperature under the structure was on average 1.4 ± 0.9°C higher than ambient conditions  at a depth of 0–30 cm, pooled from two locations per tree. Roots were collected right at the tree base and well within each treatment footprint, traced to the focal tree, carefully excavated using a trowel, and placed in a cooler prior to transport to Northern Arizona University where they were stored at −20°C until processing. Root colonization by EMF was measured on each sample by counting the number of living ectomycorrhizal root tips relative to non-colonized root tips based on differences in their morphology as described in c of EMF . Root tiTo assess DSE colonization, a sample of the remaining fine roots from each sample  was cleared for 20 min in boiling 10% KOH and then left an additional 12 h at room temperature in fresh 10% KOH followed by several rinses in tapwater. Around 10-1 cm segments of root were mounted on glass slides, and observed using a compound microscope at 400× magnification. The presence of melanized, septate hyphae and microsclerotia were used as indicators of DSE and quantified using the grid-line intersect method  using ~1Standard methods for DNA extraction, PCR, and Sanger sequencing for EMF root tips were used e.g., . BrieflyWe sequenced the fine roots described above to assess DSE community characteristics using the Illumina platform. We extracted DNA from 2.0 g wet mass samples (one per tree) using DNeasy Plant Extraction Kits . PCR was performed using primers and conditions described by DNA sequences of EMF root tips were aligned and trimmed in Bioedit  and idenFor the DSE data set, the forward and reverse reads of ITS sequences were stitched using FastqJoin  and qualCommunity composition of EMF and DSE was compared among treatments using separate Permutational MANOVAs (PERMANOVA) with the Bray-Curtis dissimilarity index in Primer 7 . The Shannon diversity (H′ log base e) was calculated for EMF and DSE using Primer 7 and compared among treatments using a one-way ANOVA in SPSS (IBM SPSS v. 20) followed by a Tukey’s test to locate treatment differences. Data on EMF colonization, DSE colonization, and shoot growth also were analyzed using one-way ANOVAs followed by Tukey’s tests. Hyphal exploration type was evaluated using a MANOVA in SPSS.F3,16 = 3.573, p = 0.038; F3,16 = 4.389, p = 0.02; Root colonization by EMF was, on average, 50% lower in trees that experienced both drought and warming than in trees that experienced ambient conditions , individual taxa were significantly affected by the combined drought and warming treatment  in the ambient, drought and warming treatments, but was ~20% lower in the combined drought and warming treatment  followed by Paraphoma (n = 6), while the most common OTUs identified at the ordinal level were found in the Pleosporales and Helotiales, with six OTUs each.The root DSE community consisted of 101 OTUs, with most of these (57%) occurring at less than 1% relative abundance in any treatment group. Thirty-two percent of the OTUs were identified to species, 31% to genus, 9% to family, 20% to order, and 8% to phylum (Ascomycota). The genus F3,16 = 0.397, p = 0.757; F3,16 = 1.35, p = 0.163). This similarity is illustrated by the relative abundance of the 10 most common OTUs which make up between 36 and 40% of the community in all four treatments . There was no association between shoot growth and DSE colonization  or DSE diversity .Mean shoot length during the growing season in which fungi were sampled was most strongly positively correlated with EMF diversity , moderate drought increased the importance of EMF to uptake of inorganic N, but this effect was EMF species specific, even differing among members of the same genus , but previous studies utilizing the same experiment provide clues. Over the years, the drought, heat and combined drought and heat treatments have affected the carbon fixation and water uptake as well as growth of the on light , 2018a. on light  and stemon light  in the con light , and redon light . These oon light  they genon light . For exaon light . In addiP. edulis; this difference also may contribute to the different sensitivities of the two groups to the combined stressors in our study.At our site, reductions in plant photosynthesis and growth were accompanied by reduction in leaf-area-specific plant hydraulic conductivity, but no change in the depth of main water sources used by the trees , or the Our experimental study of the effects of warming and drought on the fungal communities of an arid land conifer provides an important contrast to similar studies in cooler, wetter climates. Heating alone caused little change, but combined heat and drought had strong negative effects on root-associated fungi. Our results also indicate that EMF are more sensitive than DSE, with the former showing declines in both abundance and diversity. The differences among root symbionts could be due to differences in stress tolerance, host plant specificity, degree of dependence on plant hosts for carbon, or a combination of these factors. Our data on aboveground plant growth and EMF species diversity support the view that EMF are mutualists, and emphasizes the importance of community diversity rather than simple abundance to plant vitality. Less is known about the importance of DSE to plant performance in arid land trees or how DSE and EMF interact with one another and thereby affect their shared host. Obtaining this information is critical for understanding potential acclimation and adaptation of forest ecosystems to changing climate as well as for predicting bottle necks and tipping points that influence forest health.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CG contributed to data collection and analysis and led the writing of the manuscript. SS helped to construct and maintain the experiment, contributed to data collection, and helped to draft the manuscript. AP and DU contributed to data collection and revised the manuscript. CK initiated the collaboration and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Future increases in the intensity of hurricanes and El Niño periods predicted by climate change models have focused attention on their role in stimulating harmful algal blooms (HABs). A series of hurricanes that recently impacted Florida (USA) provided a unique opportunity to explore the relationships between hurricanes, El Niño and HABs in two Florida estuaries subject to repeated intense ecosystem disruptive HABs, the Indian River Lagoon and the St. Lucie Estuary. The roles that hurricanes and El Niño play in contributing to HAB events are examined in the context of key structural and functional features of each estuary and their watersheds, including morphology, water residence time and hydrology, such as the influence of Lake Okeechobee discharges into the St. Lucie Estuary. The most direct impact was the increase in rainfall associated with hurricanes and El Niño, resulting in enhanced nutrient loads which drive HABs in the Indian River Lagoon and Lake Okeechobee. Major HABs in Lake Okeechobee also present an indirect threat of freshwater HAB blooms in the St. Lucie Estuary via mandated discharges from the lake into the estuary during high rainfall periods. Conversely, during the absence of HABs in Lake Okeechobee, short water residence times produced by discharges into the St. Lucie Estuary can result in lower bloom intensities. There are also growing concerns that anticipated future changes in climatic conditions will increase threats for HABs3. Among these climatic threats are increases in the intensity and duration of hurricanes and El Niño periods6. High rainfall and winds associated with storms and elevated rain during El Niño periods in certain regions of the world can impact a range of processes relevant to phytoplankton dynamics and HAB development, including nutrient loads, physical disruption of ecosystems and ecosystem flushing rates10. These impacts can be exacerbated by human influences on nutrient loads and hydrology12.There is a consensus about the role that human activity plays in nutrient enrichment of aquatic environments as drivers of harmful algal blooms (HABs)14.A series of hurricanes that recently impacted the peninsula of Florida, and the long record of El Niño/La Niña cycles, provide an opportunity to explore the potential impacts on HABs. The effects of storm and El Niño-driven changes to the hydrology and nutrient status of coastal estuaries can be difficult to define without a basic understanding of the structure and function of both the core estuary and its watershed. For example, storm enhanced watershed runoff can increase external nutrient loads that fuel algal blooms, or contain high HAB biomass from freshwater ecosystems in the watershed, or conversely, in some ecosystems elevated flushing rates can limit the intensity of autochthonous HABs by reducing water residence times. In addition to bringing high rainfall to impacted areas which enhance nutrient loads, the winds associated with storms can cause physical damage (e.g. erosion) or disruption (e.g. sediment re-suspension) of aquatic ecosystems, contributing to internal nutrient loading and re-distribution of nutrient sources and sinks15. Consequently, other variables can take on greater importance in driving phytoplankton dynamics, often on longer and less predictable time intervals16. Two such factors are multi-year cyclical patterns (e.g. El Niño/La Niña cycles) and stochastic variability in rainfall intensity and wind associated with storm events. Both the St. Lucie Estuary and Indian River Lagoon have experienced significant hurricane activity, but each illustrates a different set of drivers and consequences associated with storm events as it relates to HABs. In the St. Lucie Estuary, hurricanes can indirectly impact HABs by increasing the potential for introduction of high algae biomass from freshwater systems in the watershed , El Niño periods and HABs in two sub-tropical ecosystems, the St. Lucie Estuary and the Indian River Lagoon Fig. . In subthed Fig. . In the 2 and is shallow throughout (i.e. mean depth 2–2.5 m)20. In addition to two natural inflows, Ten-mile Creek and Old South Fork, three man-made canals  were added to the system in the first half of the 20th Century to control hydrology in the region, including the regulation of water releases from Lake Okeechobee into the St. Lucie Canal (C-44) for flood control 22.In the St. Lucie Estuary, the study focused on two sampling sites in the inner regions of the St. Lucie Estuary, Site SL1 in the North Fork region and Site SL2 in the South Fork region . It is shallow (2.7 m mean depth), eutrophic and frequently subject to intense cyanobacteria blooms17. It has been impacted repeatedly by hurricanes, sometimes several in the same year. The lake has distinct zones that differ in their ecological structure and function, including a large comparatively deep (i.e. 4–5 m) central zone characterized by flocculent muddy sediments, a shallower (<3 m) perimeter zone characterized by firmer sediments, and a shallow northern perimeter zone subject to the largest external inflows from the water shed24. In terms of HAB events, blooms often begin in the shallow perimeter regions of the lake because of higher light availability and proximity to external nutrient inputs, but can spread throughout the lake24. In order to capture potential variability in conditions, data for two sites were included in these analyses, i.e. Sites LO1 and LO2 in the nearshore region of the lake, near the outflow to the St. Lucie Canal, which flows into the St. Lucie Estuary , is the largest lake in the southeast United States , relative to the size of the receiving basins , but their watersheds differ in terms of percent distribution of land-uses28. The sub-basin of Site IRL1 had 65% undeveloped, 24% agricultural and 11% urban/residential land-use areas in 200928. The sub-basin of Site IRL2 had significantly higher urban/residential area at 65%, low agricultural (4%) and 33% undeveloped land-use areas in 200928.In the Indian River Lagoon, the study focused on two sampling sites located in two separate sub-basins of the northern Indian River Lagoon subject to frequent HABs: Site IRL1 in the northern Indian River Lagoon near Titusville, and Site IRL2 in the central Banana River Lagoon  were obtained from the South Florida Water Management District .Monthly total Kjeldahl nitrogen and total phosphorus data for Indian River Lagoon (1997–2018) were obtained from the St. Johns River Water Management District . Nitrate, soluble reactive phosphorus, total suspended solids and chlorophyll www.ncdc.noaa.gov/IPS). Flow data capturing discharge from Lake Okeechobee into the St. Lucie Canal (Site 02276877) were obtained from the U.S. Geological Survey. Satellite imagery captured by MERIS and Sentinel-3 OLCI were analyzed using the Cyanobacteria Index (CI)29 for cyanobacteria abundance and distribution in Lake Okeechobee. The CI was calculated from MERIS imagery for dates in 2005, and Sentinel-3 imagery in 2018.Rainfall data for the Stuart and Titusville (Florida) meteorological stations were obtained from the NOAA Climatological Data for Florida web site  at 1000× magnification31. Subsamples of seawater were filtered onto 0.2 µm Nuclepore filters and mounted between a microscope slide and cover slip with immersion oil. If not analyzed immediately, samples preserved with buffered glutaraldehyde were refrigerated and counted within 72 h.Fluorescence microscopy was used to enumerate picoplanktonic cyanobacteria  were estimated by using conversion factors for different taxonomic groups applied to biovolume estimates: i.e. 0.065× biovolume of diatoms, 0.22× biovolume of cyanobacteria or nanoplanktonic eukaryotes, and 0.16× biovolume of dinoflagellates or other taxa38.Cell biovolumes were estimated by assigning combinations of geometric shapes to fit the characteristics of individual taxaBasic statistical procedures  were carried out using SAS v9.2 .60 values (i.e. time in days for 60% water exchange), otherwise referred to as the e-folding time. Water residence time data for the inner St. Lucie Estuary used in this paper were provided by D. Sun of the South Florida Water Management District . The St. Lucie Estuary is strongly influenced by tidal water exchange rates as well as freshwater flushing rates, therefore both factors are incorporated into the model formulations of water residence time20. Water residence time estimates for the study period were based on linear regression relationships developed between historical E60 values derived from a hydrodynamic model for 1997–199920 and rainfall integrated over a period of two weeks prior to the date (i.e. X) of the E60 model estimate. Regression for North Fork was E60 = −0.2534X + 17.028, R2 = 0.77. Regression for South Fork was E60 = −0.0257X + 2.9239, R2 = 0.21. A number of factors contribute to the lower R2 of the relationship for South Fork, including the morphology of the estuary and the implications for tidal mixing, very shallow mean depth, small volume compared to North Fork and direct impacts of the flow-regulated discharges from the St. Lucie Canal to South Fork.Water residence times are expressed as E39. The largest biomass peaks of marine species were observed in late summer/early fall (August-October) of 2006  were lower than during the same period in 2007 . These observations indicate that periods of high rainfall and discharge from the watershed can restrict marine phytoplankton biomass due to diminished water residence times39, highlighting the potential importance of water residence time in modifying the potential intensity of HABs involving marine species. The impact of water residence time can be compounded by elevated colored dissolved organic matter and tripton  in watershed discharge, which can reduce light availability in the water column for phytoplankton. This is reflected by lower mean CDOM and turbidity values in the August-October period of 2006 (i.e. 59 Pt-Co and 7.8 Ntu), than the same period in 2007 (i.e. 127 Pt-Co and 18.5 Ntu). However, the magnitude of potential light limitation for phytoplankton production may be partially mitigated by the shallow mean depths in the St. Lucie Estuary (i.e. mean depths of 2–2.5 m).The results of a three-year study of the St. Lucie Estuary provide insights into two different ways storms affect HABs, i.e. internal blooms (i.e. autochthonous) of marine species and externally introduced blooms  of freshwater species006 Fig. , when sa006 Fig.  due to bies Fig.  and loweies Fig. , despite42 , built in the early 1900’s to prevent flooding in south Floridaake Fig. , resultiake Fig. . The bioels Fig. , providi54. Hurricanes enhance the potential for blooms by elevating nutrient loads to the lake from the watersheds north and west of the lake, which in combination with long water residence times and mandated discharge from the lake, create a “perfect storm” of conditions for the potential introduction of intense HABs into the estuary. Most recently these conditions have led to re-occurrence of intense cyanobacteria blooms in the St. Lucie estuary in 2016   further demonstrates the relationship between peak P. bahamense biomass and high rainfall El Niño periods  than for phosphorus loads (i.e. 4–13%) (Gao 2009).For the Indian River Lagoon ecosystem, a long-term continuous 20-year time-series of phytoplankton composition and biomass dating back to 1997 provides an opportunity not only to examine hurricane effects, but also more general trends in climatic effects on HABs. The northern Indian River Lagoon has repeatedly experienced intense HABs since 1997  and damaged terrestrial biomass in the watershed which can potentially be transported into the estuary. Because of long water residence times in the northern Indian River Lagoon  storm impacts can extend for months. For example, the effects of storm events in July-October  can have both short-term and long-term impacts, including elevated nutrient concentrations which can extend into the following year. The phytoplankton biomass time-series for Site 1 in the northern Indian River Lagoon provide an example of the latter phenomena , resulting in transport of nutrient rich sediments into the eastern bay, increased nutrient load from the bay’s watersheds and destruction of mangrove habitat. The hurricane period was followed by intense marine picoplanktonic cyanobacteria blooms from 2005–2008, in part aided by the very long water residence times in the bay73.The two estuaries highlighted in this study illustrate how the impacts of hurricanes and El Niño periods on HABs not only depend on direct effects on nutrient loads that drive blooms, but also differences in the structure and function of individual ecosystems, such as water residence time, flushing rates, as well as indirect  introduction of blooms from the watershed. For ecosystems with long water residence times and shallow depths, like Lake Okeechobee and the northern Indian River Lagoon, enhanced watershed nutrient loads caused by hurricane and El Niño-related rainfall can directly enhance the potential for autochthonous HAB events. Physical disturbance of sediments by storm events can also enhance internal nutrient loads, as observed in Lake Taihu, China39, and the linkage to bloom-prone Lake Okeechobee. As a result, the greatest potential for autochthonous marine algal blooms occurs during periods of comparatively low rainfall, watershed discharge and nutrient levels, but longer water residence times which permit the accumulation of phytoplankton biomass. Conversely, periods of high rainfall, watershed discharge and nutrient inputs from the watershed can be associated with lower peak phytoplankton biomass levels due to short water residence times. Similar relationships have been observed in two other ecosystems in Florida, the Guana, Tolomato, Matanzas estuary75 and Lake George76. In both ecosystems the strong hurricane seasons of 2004 and 2005 yielded reduced peaks in phytoplankton biomass due to reduced water residence times, despite elevated concentrations of nutrients. The exception to this trend in the St. Lucie Estuary is high rainfall periods associated with high discharges from Lake Okeechobee during intense freshwater HAB events in the lake, leading to freshwater HABs of allochthonous origin in the estuary39. Similar relationships have been observed in other estuaries77, such as the Caloosahatchee estuary in Florida78 and San Francisco Bay79.By contrast, the St. Lucie Estuary presents a different picture, in part because of the shorter and more variable water residence times (i.e. 1–16.5 days)82. The toxic dinoflagellate bloom, dominated by Karenia brevis, extended over a broad reach of the inner shelf near several major freshwater outflows from the watershed, including large inputs from the Caloosahatchee River. The red tide resulted in serious impacts to aquatic animal and human health, as described for earlier red tide events in the region, including mass mortalities of marine animals and human health impacts related to exposure to aerosolized neurotoxins produced by K. brevis (i.e. brevetoxin)83. As in the case of the St. Lucie estuary, the Caloosahatchee River was subject to large discharges from Lake Okeechobee in response to the strong hurricane season in 2017 and high rainfall in the spring of 2018. Recent research has shown that periods of high discharge result in significant elevation of nutrient levels in the estuary82. It may be hypothesized that such discharges contribute to the nutrient supplies that support red tide events, such as the event observed in 201882, highlighting the need for further research on land-sea interactions in relationship to coastal blooms.Potential impacts of hurricanes and El Niño periods on HABs are not limited to coastal estuaries and inland lakes, but can extend into nearshore and open ocean environments, particularly in shallow shelf regions. The potential is illustrated by the intense red tide event experienced along the southwest coast of Florida in the summer of 201884. Depending on the species involved, HABs can also pose threats to human and animal health, particularly as it relates to toxin exposure85. Beyond these direct harmful effects, there are indirect side effects to HAB phenomena, which can have important economic and life-style consequences87. Except for some inquisitive and committed phycologists, most people find the types of intense algal scums encountered in the St. Lucie Estuary  and alteration of food web dynamicsry Figs.  and 5 anry Figs.  disturbiDataset 1.