Background: Over the past decade, many hospitals have adopted hybrid operating rooms (ORs). As resources are limited, these ORs have to prove themselves in adding value. Current estimations on standard OR costs show great variety, while cost analyses of hybrid ORs are lacking. Therefore, this study aims to identify the cost drivers of a conventional and hybrid OR and take a first step in evaluating the added value of the hybrid OR.Methods: A comprehensive bottom-up cost analysis was conducted in five Dutch hospitals taking into account: construction, inventory, personnel and overhead costs by means of interviews and hospital specific data. The costs per minute for both ORs were calculated using the utilization rates of the ORs. Cost drivers were identified by sensitivity analyses.Results: The costs per minute for the conventional OR and the hybrid OR were €9.45 (€8.60-€10.23) and €19.88 (€16.10- €23.07), respectively. Total personnel and total inventory costs had most impact on the conventional OR costs. For the hybrid OR the costs were mostly driven by utilization rate, total inventory and construction costs. The results were incorporated in an open access calculation model to enable adjustment of the input parameters to a specific hospital or country setting.Conclusion: This study estimated a cost of €9.45 (€8.60-€10.23) and €19.88 (€16.10-€23.07) for the conventional and hybrid OR, respectively. The main factors influencing the OR costs are: total inventory costs, total construction costs, utilization rate, and total personnel costs. Our analysis can be used as a basis for future research focusing on evaluating value for money of this promising innovative OR. Furthermore, our results can inform surgeons, and decision and policy-makers in hospitals on the adoption and optimal utilization of new (hybrid) ORs. Implications for policy makersIn an era where sustainable healthcare is an important policy issue, cost drivers and accurate cost estimates of hybrid operating rooms (ORs) are lacking. The costs of the hybrid OR were mainly driven by the inventory and construction costs and its utilization rate. As the hybrid OR is substantially more expensive (€18.84 vs. €9.45), decision-makers and surgeons should be aware of the increased costs of the use of highly advanced ORs and weigh the extra costs with the health gain. Besides providing insight in the costs of the OR, this paper can be used as a basis for future research focusing on evaluating the added value of the hybrid OR.  Implications for the public We performed a comprehensive bottom-up cost-analysis to inform clinicians and policy-makers on the costs and cost drivers of an (hybrid) operating room (OR), as a first step in evaluating the added value of the hybrid OR. As the hybrid OR is substantially more expensive (€18.84 vs. €9.45), decision-makers and surgeons should be aware of the increased costs of the use of highly advanced ORs and weigh the extra costs with the health gain. Besides providing insight in these costs, our analysis can be used as a basis for future research focusing on evaluating value for money of this promising innovative OR.Over the past decade, many academic and teaching hospitals have adopted a hybrid operating room (OR), and many others are considering it. The compound annual growth rate for the coming 5 years (2019-2023) of the hybrid OR market growth was estimated at 12.5%. Hybrid ORs are currently mainly used for cardiovascular surgery, but for neurosurgery and surgical oncology interest is increasing. The first observational studies in cardiovascular surgery verified this promising nature by showing a reduced length of stay and reduced operation time. Adoption of such an OR however is a large investment. Since surgical healthcare expenditures already account for a large part of the annual healthcare costs, these innovative ORs have to prove themselves in terms of value for money.The hybrid OR claims to improve efficiency by means of reducing secondary procedures and improve surgical performance which results in improved clinical outcomes. The variety can be explained by differences in study design such as the inclusion of different cost categories eg, expensive implants, medical devices, robotics, and site differences as being a (non)teaching hospital, the number of available ORs, the occupancy rate and healthcare system (country-specific). These site characteristics can especially have an influence as OR costs are mainly evaluated top-down. As this method is known to provide little insight into cost drivers and often results in inaccurate cost estimates, a bottom-up cost analysis is proposed for further research. This method is used to provide more insight into the cost drivers and enable optimization of processes which could result in cost reductions.In evaluating the added value or cost-effectiveness of the hybrid OR, it is crucial to have insight in its incurred costs. So far, the costs related to the hybrid OR have not been studied. Furthermore, to put suchcosts into perspective, it is important to also gain insight into the incurred costs related to the conventional OR setting. Current estimations on the costs of a conventional OR report a great variety, ranging from $7 to over $100 per minute.The aim of this study was to perform a comprehensive bottom-up cost-analysis to inform clinicians and policy-makers on the costs and cost drivers of an (hybrid) OR as a first step in evaluating the added value of the hybrid OR. The results are incorporated in a calculation model to enable usage and adjustment of the input parameters to a specific hospital or country setting . This guideline contains reference prices and formulas to estimate costs related to personnel, equipment, construction, and overhead. The analysis was conducted in five Dutch hospitals, all equipped with both a conventional and hybrid OR. Specific characteristics of the included hospitals are described in The bottom-up cost-analysis was conducted following the Dutch guideline for costing analyses. In this study, the conventional OR was defined as an OR where open procedures are performed to evaluate the costs of a basic OR environment. Endoscopic specific devices, to perform laparoscopic and robotic procedures, were thus left out of the analysis. The hybrid OR was defined as an OR in which an imaging technique – at least a fixed C-arm – is installed.2) of an OR based on Dutch key numbers presented by the Dutch advisory board on healthcare housing. Following these key numbers, constructing a standard m2 in a hospital costs €3479 in 2018. For the OR department, these costs should be differentiated with 160%, resulting in a cost of €5.595 per m2(Cm2 costs OR department).In constructing an (hybrid) OR, each hospital makes specific choices for its design because of, among other things, architectural preferences or limitations, preferences for ventilation systems, country-specific legislation, and budget constraints. To avoid such hospital-specific differences, we estimated total costs for constructing a square meter . To estimate the specific costs for a m2 of OR per hospital, we identified the total m2 of each of these specific functionalities within the OR department based on floor plans . The costs of these m2 were calculated using their specific differentiation based on key numbers and expert opinions, such as 140% for holding and recovery, 75% for corridors and offices as described in 2 price of an OR per hospital was calculated by:This differentiated cost results in the costs of a m2” to the square meters. An example of the calculation can be found in The “C” in this formula refers to costs and the “M2 costs of an OR were multiplied with the mean surface of a conventional OR and a hybrid OR including the control room, based on data from the participating hospitals. Yearly costs of interest and amortization were calculated by using a life span of 25 years and an interest rate of 4.2%. A5% maintenance cost over the construction costs was included.To estimate the construction costs of both ORs, the average m For each hospital the inventory for the conventional and hybrid OR was identified, comprising all equipment standing and hanging in the OR such as operating table, operating lights, (computer) screens, chairs, instrument tables, step stools, and closets. To evaluate the actual (negotiated) costs, the equipment and inventory were linked to the actual acquisition costs paid by each hospital based on their recent invoices . These costs were categorized as follows: general inventory, anesthesia equipment, OR lights, arm pendants, OR table, image routing system, X-ray radiation protection aprons, and fixed C-arm. Per cost category, average costs were calculated to determinethe average inventory cost for each OR. The yearly costs of interest and amortization were calculated using a depreciation period of 10 years, and interest rate of 4.2%. Yearly maintenance costs of 5% of the average acquisition costs for general inventory and 8% for imaging equipment were included  personnel. The total personnel costs were calculated by taking the mean of these hospital estimations.Per hospital, we identified the composition of the OR team that is available during a surgery in general in the conventional OR and the hybrid OR. The personnel costs per hour were calculated by dividing the total number of effective working hours per year of each function by the total annual loaded salary. The expected higher overhead costs for the hybrid OR  are incorporated by calculating the overhead over the average construction costs because the surface of a hybrid OR is larger than the surface of the conventional OR.Overhead expenses, the costs that are not directly attributable to a particular resource but are essential in providing care eg, electricity, water, cleaning service, and administrative tasks, were only calculated over the construction costs and personnel costs to avoid double counting. We used the general percentage for overhead on the direct costs for medical departments of 38%, as recommended by the Dutch guideline.The costs from the bottom-up cost analysis, except the costs for personnel, are expressed in yearly costs. Those are the average costs of the five consulted hospitals. To calculate the total costs per minute, it was needed to combine the average total yearly costs with the average utilization rate. Therefore, the available hours and occupied hours of a conventional OR were identified per hospital for 2018. This resultedin an average utilization rate of the conventional OR. As cross-functional use of the hybrid OR was encountered in several hospitals, we calculated the utilization rate only based on the procedures that were performed with the fixed C-arm, ie, the hybrid OR was used as such and not as a conventional OR. The utilization rate was obtained by dividing the annual number of procedures with the fixed C-arm by the total annual procedures in the hybrid OR, multiplied with the utilization rate of a conventional OR.The average yearly costs for both ORs were divided by the average occupied hours per year. The sum of these costs and the average hourly personnel costs resulted in a cost per hour for both ORs. Those costs were converted to costs per minute . To evaluate the cost drivers for the conventional and hybrid OR, a deterministic sensitivity analysis was performed. The main cost parameters were varied using the minimum and maximum values identified in the five hospitals. To evaluate the influence of personnel costs and overhead percentage, we used a 10% upper and lower limit. In addition, as a second sensitivity analysis, the utilization rate was varied from 30% to 100% for both ORs to show the influence on the costs per minute. As a final sensitivity analysis for the hybrid OR, the influence of different utilization rates of the fixed C-arm, varying from 10% to 100%, on the costs per minute were evaluated. In this analysis, it was assumed that the hybrid OR was optimally used (utilization rate of 92%) since the hybrid OR can also be used for surgeries without the use of a fixed C-arm .2 and 85.36 m2, respectively. For the conventional OR the estimated annual construction costs were €71.673 (range €47.141-€97.118), the annual inventory costs were €113.330 (€63.710-€153.379) and the hourly personnel costs were €328 (€324-€333). Over construction costs, the annual overhead was €27.236 (€17.914-€36.905) and over personnel, the hourly overhead costs were €125 (€123-€127).For the hybrid OR the estimated annual construction costs were €126.092 (€60.726-€180 945), the annual inventory costs were €433.241 (€329.938-€519.947) and the hourly personnel costs were €366 (€361-€371). The overhead costs over the construction costs were €47.915 (€23.076-€68.759) and €139 (137-€141) over personnel per hour. The fixed C-arm accounted for 40% of the total fixed costs. The mean utilization rate for the conventional and hybrid OR were 92% (range 87%-96%) and 48% (range 14%-55%), respectively. These mean utilization rates were based on four hospitals because of a registration problem in one hospital. The detailed costs per cost category can be found in As seen by  The identified costs per minute for the conventional OR were €9.45 (€8.60-€10.23) and for the hybrid OR €19.88 (€16.10-€23.07), measured in a setting of five hospitals in the Netherlands. The difference between the two ORs (€10.43) is mainly explained by higher inventory costs for the hybrid OR namely, €7.17. Noteworthy is that although advanced imaging technology is expensive, personnel costs remain an important element in the costs of both ORs . For the hybrid OR, the construction costs became an important element compared to the conventional OR (increase from 7% to 12% for the per-minute costs), which is explained by the larger room needed. This study incorporated costs for disposables and had a top-down perspective which presumably explains the difference to our results. Raft et al found a cost per minute of €10.80 for using the OR and post-anesthesia care unit by a top-down cost analysis. Since they also incorporated the costs of the post-anesthesia care unit, medicines, and disposables used during operations the results are hard to compare. As purchasing power parity differences are rather modest, this might reflect differences in norms for capital expenditure between US and European hospitals; it is however likely that the relative difference between conventional and hybrid ORs is comparable.Comparing our results with available results in the literature is challenging because existing studies include different input parameters and adopt different perspectives. For instance, a mean cost of $37.45 per minute was found for a conventional OR in the United States. Two studies showed that the hybrid approach reduced operating time, length of stay and resulted in less resource use for cardiovascular surgery. They however neglected the potential higher intervention cost for using the hybrid OR. Another cardiovascular study evaluated the additional costs for the hybrid approach and reported higher costs, but took a reduction of procedure time for the hybrid approach into account in reporting the total costs. Therefore, it is not possible to deduce the additional costs only for the use of the hybrid OR.To date, no studies that solely evaluated the costs of using a hybrid OR were identified. However, some studies compared a specific hybrid surgery with a non-hybrid surgery in which intervention costs were taken into account.To the best of our knowledge, this is the first bottom-up cost analysis that provides insight into the costs and cost drivers for both the conventional and the hybrid OR. The main strength of our study is that we evaluated the costs transparently by (1) performing a bottom-up cost-analysis, (2) specifically stating results for the different cost categories, (3) comprehensively describing the methods and calculations used in the analysis, and (4) enclosing a calculation model in which input parameters can be changed to specific settings for instance, to incorporate the costs of a mobile C-arm in the conventional OR or include costs for endoscopic devices and disposables . Another In addition, the size of the hybrid OR differed substantially in our analysis (range: 52.3-106.2) by using the average surface we may underestimate these costs as well. Third, as we only included Dutch hospitals, the generalizability of our results may be limited. Therefore, we contacted one additional hospital in a high-income country , which recently built innovative operating suites, to verify the construction and inventory costs. The costs of this hospital seemed comparable to the Dutch setting. Also, their construction costs, which were based on the actual construction costs, were in line with our estimates. Finally, overhead costs are very hard to obtain using a bottom-up methodology, therefore we chose a fixed percentage over the construction costs and personnel costs defined in the Dutch manual for cost calculations. This could result in an over or underestimation of the indirect costs. However, ranging the overhead over 34% and 42% (base case 38%) had a relatively minor influence on the total costs per minute. Also, the maintenance costs for imaging equipment were difficult to obtain, especially when considering that additional staff is necessary to support the maintenance activities within the hospitals. The estimated maintenance of 8%, which was obtained through expert consultation, might therefore be an underestimation. As in our sensitivity analysis the upper and lower values of the C-arm showed a relatively limited effect on the results, we expect that a different percentage for maintenance costs would not change our conclusions.The present study has several limitations. First, the base case analysis assumed that personnel had no idle time and a minimal team needed for running the (hybrid) OR was taken into account which underestimates the actual costs. Furthermore, the correction for irregular hours on personnel costs was assumed equal for the conventional and hybrid OR. This might underestimate the personnel costs for the hybrid OR, when this OR is often used during irregular hours .This underestimation could influence our results as personnel costs are an important factor driving the costs of both ORs . This to a minimum gain of 0.031 quality-adjusted life year is required. This means, using this simplified calculation, that for performing an endovascular aneurysm repair in the hybrid OR, at least 11 days in perfect health should be gained to be considered cost-effective compared to a conventional OR. This is based on the utilization rate of 43%. When calculating the incremental costs using a different procedure duration, an estimation can be made for other procedures as well. Based on this calculation it may seem difficult for the hybrid OR to become cost-effective, however by increasing the utilization rate or comparing the intervention to a different technique, eg, laparoscopy which results in lower incremental costs, it becomes more likely that the hybrid OR is cost-effective. “This suggestion is strengthened by a recent evaluation of a navigation system that is used during surgery, which requires the hybrid OR. In removing locally recurrent rectal tumors the use of a navigation system in the hybrid OR has the potential to become cost-effective. However, the level of cost-effectiveness of the navigation system is also strongly dependent on the utilization rate of both the navigation system and the hybrid OR.”The following example gives an impression on the added value per patient needed to consider the use of the fixed C-arm (in the hybrid OR) cost-effective. From the estimated costs for the conventional and hybrid OR, we can calculate the base case incremental costs for the hybrid OR per minute (€10.43). Assuming a surgical procedure of approximately 2.5 hours, for example, an endovascular aneurysm repair, results in an incremental cost of €1.565 per procedure. These incremental costs only incorporated the additional costs of using the hybrid OR, not additional materials that might be needed. When a cost-effectiveness threshold of €50.000 per quality-adjusted life year is assumed,This study identified that the main factors influencing the OR costs are: total inventory costs, total construction costs, utilization rate, and total personnel costs. Therefore, our results could inform surgeons, decision and policy-makers in hospitals on the adoption and optimal utilization of new (hybrid) ORs. Although it seems that hospitals have to strive to use the fixed C-arm as often as possible, one should keep in mind that using the hybrid OR should add value to the patient. To evaluate the added value, the calculation model  can be u, and those are likely to become more advanced  it is important to evaluate its cost-effectiveness. Therefore, in the near future prospective comparative studies have to be performed to evaluate the actual benefits of using these advanced ORs in terms of complication rates, efficiency, and survival. Those outcomes can be used to identify interventions that yield the most from the advances of the hybrid OR, as well as informing cost-effective usage of the hybrid OR in general. Finally, in order to assess the generalizability of our results to non-European or low- and middle-income countries, it would be valuable to validate our calculation model from a different perspective .As it is expected that the number of hybrid ORs will rise worldwide The authors would like to thank the all participating hospitals that contributed to this study by delivering their hospital data. Ethical approval was not required for this study, as no human participants were involved. All authors report no conflicts of interest. MR, JPCG, and SP have received an unrestricted research grant from Siemens Healthcare GmbH for other but related research projects. Conception and design: JPCG, VR, SP, and ML. Acquisition of data: SP and ML. Analysis and interpretation of data: SP, ML, JPCG, VR, MR, and WHvH. Drafting of the manuscript: SP and ML. Critical revision of the manuscript for important intellectual content: SP, ML, JPCG, VR, MR, WHvH, TR, and LP. Statistical analysis: SP and ML. Supervision: JPCG, VR, MR, and WHvH.1Department of Operating Rooms, Radboud Institute for Health Sciences, Radboud University Medical Center, Nijmegen, The Netherlands. 2Division of Psychosocial Research and Epidemiology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Amsterdam, The Netherlands. 3Department of Health Technology and Services Research, University of Twente, Enschede, The Netherlands. 4Department for Health Evidence, Radboud Institute for Health Sciences, Radboud University Medical Center, Nijmegen, The Netherlands. 5Department of Surgery, The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands. 6Department of Medical Technology, Isala Hospital, Zwolle, The Netherlands.Supplementary file 1. Calculation Model.Click here for additional data file.Supplementary file 2. Characteristics of Participating Hospitals.Click here for additional data file.Supplementary file 3. Example of the Calculation of the Construction Costs.Click here for additional data file.Supplementary file 4. Formulas to Calculate the Costs Per OR.Click here for additional data file.


Black carrot (BC)with its potential health benefitsdue to thegreater amount of anthocyanins and the potent antioxidant activitycould be utilized as a natural colorant. The objective of this studywas the entrapment of BC anthocyanins by external ionic gelation techniquewithin the biopolymer matrix including pectin, alginate, and the mixtureof both. Beads were characterized in terms of entrapment efficiency(EE), morphology, total anthocyanin content, and antioxidant capacitymeasured by the 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonicacid assay. Furthermore, the color of the beads as well as yoghurtsamples fortified with BC-containing beads were evaluated during storageat 4 °C for 4 weeks. While the EE for anthocyanins ranged between47.3 and 96.6%, the antioxidant capacity changed from 50.4 to 97.7%.The maximum anthocyanin retention was found as 91.7% for 1% BC containing1% pectin (P) + 1% alginate (A)-based beads after 4 weeks of storage.In addition, anthocyanin protection reached up to 62% and antioxidantcapacity up to 55.6% in the fortified yoghurt samples containing A-basedbeads during storage. It is concluded that external ionic gelationcould be a feasible method for BC anthocyanins due to its protectiveeffect against acidic environment. We have alsofortified the yoghurt samples with beads to investigate the stabilityof BC anthocyanins in an acidic environment. Moreover, anthocyaninprotection ability of those bead matrices was determined during storageat 4 °C for 4 weeks.22.1Sodium azide, calcium chloride,methanol, acetic acid, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid (Trolox) were obtained from Sigma Chemical Co. (St. Louis. Mo.U.S.A.). The other chemicals and solvents used in this experimentwere of analytical grade.BC extract was obtained from DÖHLER—NaturalFood & Beverage Ingredients (Turkey). GENU Pectin LM-101 AS wasreceived from Kelco—Food, Beverage & Nutrition, USA, andManugel DMB alginate was received from FMC Corporation (USA).2.226 P, A, andthe mixture of PA stock solutions as a carrier material were preparedby dispersing powders into separate beakers at 2% (w/v) in 0.02% sodiumazide containing 100 mL of deionized water. Then, the dispersionswere stirred constantly at room temperature (20–25 °C)overnight for complete hydration and used for gel preparation thefollowing day. The carrier–active material mixture was preparedby mixing the stock polymer solutions  withthe BC extract at different ratios  by stirringfor at least 30 min to ensure complete mixing. Then, the polymer–BCmixture was dropped into 200 mL of mildly agitated 3% (w/v) calciumchloride (CaCl2) solution using a 5 mL syringe with a 21G (0.8 × 38 mm) needle. The beads were allowed to cross-linkwith Ca2+ for 30 min at room temperature and collectedby filtration and washed with distilled water to remove excess Ca2+ on the surface. The beads were not dried to avoid any changein their properties and stored prior to usage.Thepreparation of beads by external ionic gelation was adaptedfrom Córdoba et al.2.32.3.127 The EE was calculated on wet basis by TheEE was calculated as the ratio of bioactive mass in the beads andthe mass of bioactives in the initial mixture of BC extract and coating.2.3.228Outerstructural featuresof beads were analyzed using an optical microscope  with 40X magnifications.2.3.33 with some modifications. 1 g of sample was treatedwith 25 mL of MeOH–HOAc–H2O (50:8:42) solventsystem. The treated samples were homogenized by a homogenizer (IKAT18 Basic Ultra-Turrax) at 10,000 rpm for 30 s. Then, the sampleswere centrifuged  at 4500g for 20 min,and the supernatants were collected. This extraction protocol wasrepeated two times for the pellet, and the supernatants were pooledto a final volume of 50 mL. The prepared extracts were stored at −20°C until analysis.Priorto spectrophotometric analysis, for each sample, three independentextractions were prepared according to Ersus and Yurdagel2.3.429 Anthocyanin contents are expressed as mg of cyanidin-3-O-glucoside equivalents per 100 g of sample.The obtainedextract for each sample was used for the determination of the totalanthocyanin content by measuring the absorbance at 530 nm, which isthe maximum absorbance value of anthocyanins.2.3.530 In all assays, Trolox was used as a standard, and the resultswere expressed in terms of mg Trolox equivalents (TE) per 100 g dryweight of the sample.Totalantioxidant capacity was estimated by the 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonicacid (ABTS) assay according to Miller and Rice-Evans.2.3.6L*, a*, and b* values of samples was performed by placingthe samples in Petri dishes. The readings were made in triplicate,and the equipment was calibrated with the white calibration platebefore any reading.Color determinationswere conducted by using a CM-3600d chromameter . The reading of 2.4A stability study of the BC extract-containingbeads was conductedin glass containers in a refrigerator at 4 °C for a period of4 weeks. The color parameters (CieLab), the total anthocyanin content,and the total antioxidant capacities were evaluated. The aim of thestorage was to investigate the effect of time on the physical andchemical properties of the BC extract-loaded beads under constantstorage conditions.2.5Commercial yoghurt products were purchased from localsupermarkets on the day of fortification. Yoghurt formulations weremade by adding 15 g of 1% BC extract-loaded beads on the basis ofA, P, or PA into 50 g yoghurt samples separately and stored in glasscontainers in a refrigerator at 4 °C for further analysis. Physico-chemicalproperties were evaluated over a month at 4 °C (weekly). Plainyoghurt, 1% BC extract-containing yoghurt, and blackberry-containingcommercial yoghurt were also analyzed over 4 weeks at 4 °C (weekly).2.6P < 0.05). Error bars on figures represent standarddeviations. The differences between all samples and among the sampleswere evaluated. Each analysis was performed in triplicate, and theresults were reported as mean value ± standard deviation.Statisticalanalysis was applied using processed samples as independent factorversus analysis results  by utilizationof one-way analysis of variance with Tukey’s HSD post hoc test, alginate (1–2%),and CaCl2 (1.5–5%), and EE was improved by enhancingthe alginate concentration. Besides, Li et al.37 reported the EE of two proanthocyanidin fractions obtainedfrom Choerospondias axillaris fruitpeels as 43.4 and 62.2%, in which EE of a fraction with higher molecularweight was found to be greater than that of a fraction with lowermolecular weight. In addition to this, there were much more molecularinteractions between proanthocyanidins in a fraction with higher molecularweight, indicating enhanced retention of the active substances withinthe gel network. In another research, the effect of fillers in orderto improve the encapsulation efficiency of tea polyphenols withinalginate beads was studied. According to the outcomes, EE of tea polyphenols(3 mg/mL) was found to be 38.51, 36.48, 48.56, and 57.76% for plainalginate (2%), alginate (2%) + inulin (2%), alginate (2%) + arabicgum (2%), and alginate (2%) + chitosan (1%) based beads, respectively,indicating enhanced structured network by using suitable filler substances.38 Flamminii et al.39 reported the EE of olive leaf polyphenols as 21% by incorporatinginto the alginate beads, which was improved to 78, 56, and 52% forthe alginate–pectin, alginate–whey protein, and alginate–sodiumcaseinate systems, respectively.While the EE of alginate-based beads was found to be 47.3%for1% BC loading, it was improved up to 72% with the incorporation ofpectin. Similarly, Pasukamonset et al.40 in which the recovery of antioxidant capacityof dandelion polyphenols within alginate–whey protein-basedmatrix produced by emulsification/internal gelation as well as alginateor pectin-based matrix obtained by external hydrophilic gelation wasobtained to be more than 80%. On the other hand, the maintenance ofantioxidant capacity of hibiscus extract measured by the DPPH assaywas found to be in the range of 55.2–60.6% for microcapsulesfabricated by ionic gelation-atomization using pectin as a carriermaterial, which is lower than the outcomes of the present study.41Regarding the maintenance ofantioxidant capacity after bead formation,it was found to be higher (88.2–97.7%) for 0.25% BC loadingfor each polymer matrix rather than others. These results are in agreementwith the study of Belščak-Cvitanovic et al.,3.241 obtained a more rigid structure with alginate-based microcapsules,rather than pectin, chitosan, psyllium, and carrageenan-based microcapsules.Furthermore, Flamminii et al.39 indicateda more regular structure with alginate and alginate–pectin-basedbeads in comparison to alginate–whey protein and alginate–sodiumcaseinate-based beads. Due to the fact that the shape of the beadsis one of the most important determinants, especially in complex drug-deliverysystems,42 the beads should be tested interms of morphology.3.3L*, a*, and b* values in which L* is the lightness of color ; a* value,  and b* value, . The color values of fabricated beads were also determined during 4 weeks of storage. In detail,the minimum retention (21.9%) was obtained with 0.5% BC-containingPA-based beads at the end of the storage period. On the contrary,the maximum retention was 91.7% for 1% BC-containing PA-based beadsafter 4 weeks of storage. The loss of total anthocyanin content ofthe beads at the end of storage was found to be 48.4, 36.7, and 36.1%with P-based beads, 58.8, 67.6, and 60.7% with A-based beads, and68.1, 78.1, and 8.3% with PA-based beads for 0.25, 0.5, and 1% BCextract loading, respectively.The effectof active material concentration and type of polymer solution on beadstability was evaluated during 4 weeks of storage under refrigerationconditions at 4 °C. The effect of storage on the total anthocyanincontent of BC extract-containing beads is shown in 2 Li et al.38 studied the recovery of tea polyphenols within alginate-basedhydrogel beads during storage at room temperature in the dark for30 days. The protection ability of the bead matrices was quantifiedas 77.35, 80.32, 86.58, 83.65, and 85.73% for free polyphenols, alginate,alginate + inulin, alginate + gum Arabic, and alginate + chitosansystems, respectively. The results showed that the recovery of polyphenolsduring storage was increased with the inulin addition. In anotherstudy, the storage stability of anthocyanins within microparticlesproduced by dripping-extrusion method was evaluated at 5, 15, and25 °C in the dark. The outcomes of this study indicated thatanthocyanin retention was decreased by increasing the storage temperature.While maintenance of the anthocyanins was determined as 97% after35 days at 5 °C, it decreased to 85% after 30 days at 15 °Cand 26% after 20 days at 25 °C.2 Theeffect of storage on the total antioxidant capacity of BC extract-containingbeads is shown in There are limited number ofstudies in the literaturerelated tothe storage stability of anthocyanins entrapped within beads. Forinstance, hibiscus anthocyanin retention within pectin (2%)-basedbeads obtained by dripping-extrusion method was found to be 97% after35 days storage at 5 °C. Moreover, it has been noticed that thestability of anthocyanins may be affected by storage condition-relatedparameters (extrinsic) and microparticle composition-related factors(intrinsic).P < 0.05) after 4 weeks of storage. While the minimumretention (10.6%) was obtained with 0.25% BC-containing A-based beads,the maximum retention was found (84.9%) for 1% BC-containing PA-basedbeads after 4 weeks of storage. The loss of total antioxidant capacityof the beads at the end of storage changed in the range of 17.1–75%for P-based beads, 43.7–89.4% for A-based beads, and 15.1–88.8%for PA-based beads. Our results also showed that the retention ofthe total antioxidant capacity was increased with the ascending concentrationlevels of BC extract to be encapsulated.The ABTS results achieved inthis work have shownthat the totalantioxidant capacity of beads generally decreased significantly  during storage. On the other hand, there was no tendency toincrease or decrease regularly regarding the a* and b* indices, but generally there was a correlation betweenanthocyanin retention and a* index.The beads obtained in this study were also evaluated in termsoftheir color stability during storage. Color values of beads duringstorage are reported in 3.5Yoghurt,a highly nutritional quality product, was selected toevaluate bead stability in the current pH value and acidity. The protectionability of beads was investigated by measuring the anthocyanin content,antioxidant capacity, as well as color values of the samples during4 weeks of storage periods at 4 °C. However, plain yoghurt samplesand BC extract-containing yoghurt samples after 3 weeks of storageand blackberry-containing commercial yoghurt after 2 weeks of storagecould not be analyzed due to deterioration. The changes in the totalanthocyanin content of all yoghurt samples are shown in P < 0.05) during 4 weeks of storage. The decrease intotal anthocyanin contents was found to be 50.7, 38.0, and 40.5% forP, A, and PA based beads containing yoghurt samples, respectively.The results showed that the total anthocyanin contentof yoghurtsamples including BC-loaded beads decreased significantly (P > 0.05)in fortified yoghurt samples with beads during 3 weeks. It was clearthat fortification of yoghurt by entrapped BC extract within polymer-basedbeads showed a protective effect on antioxidant activity against acidicconditions.The change in the total antioxidant capacity for all yoghurt samplesis shown in L* values were found to beso low for plain yoghurt, yoghurt colored by extract from red bellpepper, yoghurt with inclusion complexes, and yoghurt colored by artificialcolorant stored for 60 days.44When the extract- and BC-containing beads wereadded to yoghurt,they could simulate the color of blackberry-containing commercialyoghurt. Results also indicated that all colorants including beadsand extract evaluated in this work had a tendency to lighten the colorof the yoghurt samples during 30 days of storage 4a. Howeva* indexfor all yoghurtsamples is shown in P > 0.05) in a* indexat the end of the storage of yoghurt samples fortified by anthocyanin-containingbeads and commercial blackberry-containing samples, whereas the retentionof a* value for the BC extract-containing yoghurtsamples was found to be 49.5% at the end of 3 weeks.The variation in the b* index for all yoghurtsamples is shown in P < 0.05)during storage of all yoghurt samples except for plain yoghurt. Withregard to the yoghurt samples fortified with anthocyanin-containingbeads and commercial blackberry-containing samples, there was no significantdifference (P > 0.05) between 0 and 1 week. Itwashighlighted that the BC-containing beads produced by external ionicgelation may be useful as a natural colorant in an acidic environmentsuch as yoghurt.The variationin the 4In thisstudy, pectin,alginate, and their mixture were used forthe entrapment of BC extract using external ionic gelation in orderto improve the functionality and stability of the bioactive compounds.It was observed that the concentration of BC and the type of the polymeraffected the physico-chemical properties of beads as well as theirstorage stability. The EE was found to be the highest as 96.6% for0.25% BC-loaded P-based beads. Moreover, after 4 weeks of storage,the maximum retention level for anthocyanins and the antioxidant capacitywere calculated for 1% BC-containing PA-based beads as 91.7 and 84.9%,respectively. On the other hand, regarding the fortified yoghurt samples,the maximum anthocyanin recovery and antioxidant capacity were determinedfor the A-based bead. The results of this study showed that BC anthocyaninscould be successfully entrapped in different polymer matrices includingpectin, alginate, or mixture of both. These beads have the potentialto be used as food colorants and food additives by incorporating intodietary supplements, functional foods, and pharmaceuticals.


Anoplolepis gracilipes is a widespread invasive ant that poses significant threats to local biodiversity. Yet, compared to other global invasive ant species such as the red imported fire ant (Solenopsis invicta) or the Argentine ant (Linepithema humile), little is known about the diversity of RNA viruses in the yellow crazy ant. In the current study, we generated a transcriptomic database for A. gracilipes using a high throughput sequencing approach to identify new RNA viruses and characterize their genomes. Four virus species assigned to Dicistroviridae, two to Iflaviridae, one to Polycipiviridae, and two unclassified Riboviria viruses were identified. Detailed genomic characterization was carried out on the polycipivirus and revealed that this virus comprises 11,644 nucleotides with six open reading frames. Phylogenetic analysis and pairwise amino acid identity comparison classified this virus into the genus Sopolycivirus under Polycipiviridae, which is tentatively named “Anoplolepis gracilipes virus 3 (AgrV-3)”. Evolutionary analysis showed that AgrV-3 possesses a high level of genetic diversity and elevated mutation rate, combined with the common presence of multiple viral strains within single worker individuals, suggesting AgrV-3 likely evolves following the quasispecies model. A subsequent field survey placed the viral pathogen “hotspot” of A. gracilipes in the Southeast Asian region, a pattern consistent with the region being recognized as part of the ant’s native range. Lastly, infection of multiple virus species seems prevalent across field colonies and may have been linked to the ant’s social organization. The yellow crazy ant,  Solenopsis invicta) [Linepithema humile), a polygynous, supercolony-forming ant species [Several life history traits common to invasive ants, such as polygyne  and supercolony , have been believed to contribute to their ecological dominance in their introduced ranges ,2,3. Howinvicta) ,8,9. Inf species ,11,12.Anoplolepis gracilipes, is a globally distributed invasive ant; yet, the native range of this ant species remains debatable as multiple datasets failed to reach consensus [A. gracilipes is relatively low. More efforts are therefore required to better understand the viral diversity of this invasive ant species. The yellow crazy ant, onsensus . Howeveronsensus ,15,16. Honsensus . While aonsensus  and threonsensus . Compareonsensus ,20, the A. gracilipes, which was then used to identify and characterize new virus genomes and other virus-like sequences. We also surveyed the presence of these viruses across the ant’s geographical range and examined if colonies collected from Southeast Asian regions harbor higher levels of virus diversity. Lastly, genetic analyses were carried out to assess the genetic diversity and selection signals for one of the discovered viruses  associated with the yellow crazy ant, which is expected to facilitate the understanding of evolutionary dynamics of virus-ant interactions.This study aims to explore the virus diversity in this ant species with a focus on RNA viruses and their genetic diversities and evolution. We used high throughput sequencing to generate a transcriptomic database for A. gracilipes colonies were collected from the roadside of semi-natural habitat, with one from Penang, Malaysia  and the other from Okinawa, Japan . Penang is considered part of the putative native range of A. gracilipes, while Okinawa is located in the ant’s known invasive range [® Stranded mRNA Library Prep  with an average 200 bp library insert size. The library was sequenced in the pair-end mode (2 × 100 bp) using the Illumina HiSeq 4000 platform.Two ve range . A singl−40. The virus-like transcripts were identified using MEGAN Community Edition v6.18.8 [The pair-end reads obtained from Illumina sequencing were trimmed to remove adaptors and low-quality sequences  using Trimmomatic v0.36 . The de  v6.18.8 . The ope v6.18.8 , and con v6.18.8  against  v6.18.8  database v6.18.8 , and the v6.18.8 .The potential viral ORFs were subjected to a preliminary taxonomic inference using BLASTp  that comA. gracilipes colonies, ten worker ants from each of the two colonies were randomly selected for RNA purification. To investigate the prevalence of these viruses in the field, additional 107 yellow crazy ant colonies were collected from Malaysia (n = 35), Indonesia (n = 4), Taiwan (n = 40), Okinawa (n = 18), and Hawaii (n = 10). Malaysia and Indonesia have been considered part of the putative native range of A. gracilipes, while Taiwan, Okinawa, and Hawaii are located in the ant’s known invasive range [To verify the occurrence of the identified viruses in the two original ve range . The totTM 1st strand cDNA Synthesis kit  under conditions as follows: 30 °C for 10 min., 42 °C for 60 min., followed by a final inactivation step at 95 °C for 5 min. The cDNA then served as a template for PCR reaction amplifying the partial sequence of RNA-dependent RNA polymerase (RdRp) or Capsid protein region using respective virus-specific primer sets  and Agr_EF1a.rev (5′GCTCCTTCACCGAGATGTTC-3′) (The virus-specific primers were designed using Primer3 . First smer sets . The PCRGTTC-3′) .Sopolyciviruses, and additional ten unclassified polycipiviruses, were used to reconstruct the phylogeny with three iflaviruses as outgroup. Amino acid sequences of the entire non-structural polyprotein (ORF5) were aligned using MUSCLE implemented in MEGA 7 [Taxonomic placement of the identified virus was performed using a phylogenetic approach. We collected sequences of multiple polycipiviruses containing full non-structural polyprotein (ORF5) sequences from GenBank. A total of 28 polycipiviruses, including three chipolyciviruses, one hupolycivirus, 14 n MEGA 7 . The besn MEGA 7 , and then MEGA 7  with 1,0n MEGA 7  with 1,0n MEGA 7 , as estin MEGA 7 . The conn MEGA 7  and rootn MEGA 7 .A. gracilipes colonies . RNA of a single randomly-selected worker from each colony was purified, and cDNA was synthesized following the same procedure mentioned above. The synthesized cDNA was then used as a template for PCR that amplifies partial sequences of RdRp region, using the following primers, AgrV3_RdRp.for (3′- CAAAGTGAATGACCCCGAGT-5′) and AgrV3_RdRp.rev (3′-ATGCGAGAAATCGTGTTTCC-5′) primers , and up to ten clones were subjected to Sanger sequencing. Two individuals  were found to have two different haplotypes among the ten sequenced clones, whereas only a single haplotype was recovered from the sequenced clones for the rest of the individuals. Six Trinity assembled transcripts  and Myrmica scabrinodis virus 1 (GenBank: ASK12206.1), were also included in the reconstruction of the phylogenetic tree. These sequences were aligned using MUSCLE implemented in MEGA 7 [To examine whether our Trinity contigs originate from the same or different host individuals , we reconstructed the virus phylogeny based on 13 partial sequences of RdRp region from 11 field-collected  primers . PCR pron MEGA 7 . The besn MEGA 7 , and then MEGA 7  with 100n MEGA 7  with 1,0To examine whether quasispecies exist in the identified polycipivirus and characterize the selection pattern, we estimated Tajima’s D on the RdRp region from 13 sequences of field-collected colonies using MEGA 7 . The non−40 out of a total of 70,145 trinity transcripts assembled by Trinity. The preliminary taxonomic inference with BLASTp and fast minimum evolution phylogenetic tree placed these virus-like transcripts into one of four virus families, including 11 Dicistroviridae, 3 Iflaviridae, 18 Polycipiviridae, and 2 unclassified Riboviria . For the other 16 transcripts, pairwise comparisons of any two transcripts revealed >80% nucleotide identity. These two transcripts contained the same genomic feature that possesses five main ORFs: ORF1 (nucleotide (nt) 218–1063; 281 amino acids (aa)), ORF2 , ORF3 , ORF4 , and ORF5 . Moreover, a short ORF2b  was located within the ORF2 (Anoplolepis gracilipes virus 3 (AgrV-3)” (GenBank: MW078933).Two of 18 the ORF2 . The aliDicistroviridae sp. 1; GenBank: MZ394715, MZ394716) comprised two contigs  was 413 nt and possessed a partial putative RdRp sequence  were derived from two contigs (744 nt and 1226 nt), respectively, and were joined by four pairs of reads. The conserved domain in the 5’ end contig of YCA-associated putative Dicistroviridae sp. 3 was a partial putative RdRp region. A putative capsid protein was identified in the following contig  was 732 nt, containing a partial putative RdRp region , 70.72% , 66.42% (Dicistroviridae sp. 2), and 75.22% (Dicistroviridae sp. 4) with Wuhan arthropod virus 2, respectively. The YCA-associated putative dicistrovirus 3 was closely related to Solenopsis invicta virus 1A  contained two contigs  that were connected by two pairs of cross contig reads. Both contigs were the partial region of a putative RdRp (Iflaviridae sp. 2 (GenBank: MZ394723) was 2006 nt, which contained a partial region of a putative RdRp . The protein identity between YCA-associated putative Iflaviridae sp. 1 and sp. 2 were 79.49% (5’ end contig) and 81.94% (3’ end contig), respectively. The phylogeny of Iflaviridae sp. 1 and 2 were within the branch of Iflaviridae (The YCA-associated putative ive RdRp . The topive RdRp f. YCA-asaviridae .Riboviria sp. 1 (GenBank: MZ394724) contained a partial putative 560 nt RdRp region (Riboviria sp. 2 (GenBank: MZ394725) was 1636 nt and contained a putative partial Capsid protein region  with 55.43% identity. No overlapping region was found in the two Riboviria sp. contigs. The fast minimum evolution phylogenetic tree supported two contigs both were picornaviruses  [Hupolycivirus: Kandapolycivirus  [Malus domestica, Honeycrisp G.935) [The Bayesian inference method produced a tree topologically identical to the maximum likelihood tree, showing AgrV-3 is most closely related to viruses in the genus of ycivirus . Our phyipponii) , the gens lylei) , and Appp G.935) .RT-PCR verification of viral sequences from the original two colonies indicated that most of the viruses were only found in the Malaysian colony. We also conducted a field survey for the presence of all viruses and found that most of the viruses were only detected in their putative native ranges  with low infection rates but completely absent in the ant’s known invasive range  of six  Okinawa . This isPicornavirales especially in the families of Dicistroviridae, Iflaviridae, and Polycipiviridae, other RNA viruses  and dsDNA viruses  have also been reported in ants [Anoplolepis gracilipes virus 3, AgrV-3) as well as several unmapped  viruses in the yellow crazy ant through a transcriptomic approach. Anoplolepis gracilipes virus 3 represents the first Polycipiviridae virus and also the third complete virus genome that has been discovered in this invasive ant species.While most viruses discovered from ants are single-stranded RNA viruses belonging to the order  in ants . Methods in ants ,45, and, in ants ,41,42,43 in ants , and dsD in ants  in ants Polycipiviridae is a relatively new virus family (2017) of Picornavirus [Polycipiviridae, including Sopolycivirus, Hupolycivirus, and Chipolycivirus [Sopolycivirus under the family Polycipiviridae. Based on the species demarcation criteria established by ICTV [Polycipiviridae [Anoplolepis gracilipes virus 3” (GenBank: MW078933).The rnavirus ,49 with ycivirus ,49. Our iviridae ,51,52,53Anoplolepis gracilipes virus 3 (hereafter: AgrV-3) with putative ORF functions and orientation  length  and 16 partial contigs that possess high nucleotide identity (>80%) against each of the two full-length contigs. As our assembly derived from a pooled RNA , we constructed a phylogeny tree that includes six RdRp containing transcripts  is 0.4988, a pattern that is comparable to another ant-infecting Sopolycivirus  [Dicistroviridae) and Linepithema humile C-virus 1 (unclassified Riboviria), in Argentine ants [A. gracilipes suggests that a purifying selection is potentially acting on the RdRp region for optimizing the virus’ fitness in the yellow crazy ant. The similarity in the genetic diversity and evolutionary pattern between the two polycipiviruses raises an interesting question of whether the presence of diverse variants is a common characteristic in Polycipivirus and highlights the need of further research on the evolutionary dynamics of viruses in this newly established virus family. The presence of diverse variants has been thought to contribute to rapid adaptations of viruses to dynamic environments in the hosts, such as escaping from the host immune or even transmission to a novel host . Multipline ant) . Interesine ant) , LhuPcV1ine ants , providiDicistroviridae , two inmonogyne ,8,9, andmonogyne ,7. The pmonogyne ,5. A. gracilipes across southeast Asia. Unlike these data deriving from the ant host, our study provided evidence from the perspective of the viral pathogens infecting the host. The pattern of virus species diversities placed the pathogen “hotspot” of A. gracilipes in this particular geographical region. This result, together with the finding that the RdRp sequences recovered from Malaysia are ancestral to those recovered from the known introduced ranges , was found infecting the red imported fire ant [Pathogens are often found to be co-introduced with their hosts via human-mediated transportation network . Arrivalfire ant , and (2)fire ant . These ffire ant ,18,63 haLastly, our study also reveals potential limitations in short-read sequencing technology. The eight unmapped viruses were only partially characterized due to difficulties in assembling and recovering full-length transcripts associated with short-read sequencing . This ef


The world is continuously facing the threat of emerging infectious diseases. The COVID-19 pandemic has affected over 200 countries and territories of the world, and has also severely affected the world’s healthcare and financial systems [The current COVID-19 pandemic has caused more than 661 million infections with over 6.6 million deaths worldwide . ApproxiApproximately, 38.4 million people are living with HIV worldwide . In the Dengue virus is the causative agent of dengue fever, and multiple other health complications. Approximately 100–400 million dengue cases occur annually, of which 80% remain mild and asymptomatic, while 20% cause severe infection, leading to 22,000 deaths annually . The US The Zika virus is an emerging infectious disease of great public health importance. Most of the infections caused by Zika virus remain asymptomatic or mildly symptomatic. While some Zika infections are associated with microcephaly in children born from mothers infected with Zika during pregnancy, in adults, Zika infection is responsible for Guillain Barre Syndrome. Several Zika virus vaccines are at different stages of development, but none of the vaccines have been approved by the FDA . In 2022, more than 83,000 cases of Mpox (Monkeypox) have been reported from 110 countries around the world [Human papillomavirus (HPV) is one of the most potent viruses, and it causes sexually transmitted diseases on a large scale worldwide. Approximately, over 150 types of human papillomavirus have been identified, and nearly 40 of them lead to infectious cycles in the host . Many HPPolio is a highly contagious virus that is responsible for causing disability and life threating disease. Due to unified global polio eradication efforts, there has been a 99.9% reduction in global polio cases. Polio is endemic in only two countries, but recently some single polio outbreaks have been reported in high-income countries . It is iGlobally, 1.1 million people are dying each year from viral hepatitis. The World Health Organization has developed a strategy to eliminate hepatitis by 2030. Luckily, a highly effective vaccine is available for the Hepatitis B virus. The coverage of the Hepatitis B virus vaccine is 90% among children, but there is a need to increase the birth dose of HBV vaccination in newborns to prevent the vertical transmission of the Hepatitis B virus . There iThere have been several outbreaks of the Ebola virus in the last ten years. Several vaccine development techniques, including inactivated vaccines, DNA base vaccines, virus-like particles, virus-like replicon particles, recombinant viral vector vaccines, and plant-based vaccines were used to develop vaccines for the Ebola virus . The onlUltimately, viruses are continuously evolving and some of them have high mutation rates. Multiple variants of COVID-19 have shown different disease transmission and infectivity patterns ,21. The


Whilst involvement of the motor cortex in the phenomenon of freezing in Parkinson’s disease has been previously suggested, few empiric studies have been conducted to date. We investigated motor cortex (M1) excitability in eleven right-handed Parkinson’s disease patients  with verified gait freezing using a single-pulse transcranial magnetic stimulation (TMS) repetitive finger tapping paradigm. We delivered single TMS pulses at 120% of the active motor threshold at the ‘ascending (contraction)’ and ‘descending (relaxation)’ slope of the tap cycle during i) regular tapping, ii) the transition period of the three taps prior to a freeze and iii) during freezing of upper limb movement. M1 excitability was modulated along the tap cycle with greater motor evoked potentials (MEPs) during ‘ascending’ than ‘descending’. Furthermore, MEPs during the ‘ascending’ phase of regular tapping, but not during the transition period, were greater compared to the MEPs recorded throughout a freeze. Neither force nor EMG activity 10–110 s before the stimulus predicted MEP size. This piloting study suggests that M1 excitability is reduced during freezing and the transition period preceding a freeze. This supports that M1 excitability is critical to freezing in Parkinson’s disease. More than 60% of PD patients experience some form of freezing with disease progression, and the incomplete understanding of the pathophysiology and circuit mechanisms limits effective treatment5. Freezing most commonly occurs while walking (FoG) but also during swallowing6, speech3, and particularly during repetitive movements of the upper limbs (ULF)9. Like FoG, ULF significantly impairs daily activities such as handwriting, tooth brushing, typing, or bimanual coordination11. The similar spatiotemporal characteristics of FoG and ULF12 prompted research interest in ULF to generate neurophysiological hypotheses on FoG and freezing phenomena in general.Freezing phenomena in idiopathic Parkinson’s disease (PD) represent a significant source of disease-related disability14. The production of internally generated movement strongly depends on movement planning in frontostriatal projections to M115. The desynchronization of sensorimotor beta-band oscillatory activity during the acceleration phase of a repetitive movement cycle has previously been related to activity across the subthalamic nucleus, the primary motor cortex (M1), and prefrontal areas16. Furthermore, impaired movement-related modulation of beta-band activity over the sensorimotor area has been associated with freezing behaviour19, and can be observed for up to three taps before a freeze8. Combined EEG-TMS studies have established a direct pathophysiological link between oscillatory activity and M1 excitability, showing that M1 excitability was enhanced during beta-band desynchronization but reduced during beta-band synchronization21.Ultimately, ULF represents the failure to produce an intended movement sequence effectivelyHere, we studied M1 excitability in PD freezers during regular tapping, transitions, and ULF. Specifically, we investigated the transitions between regular tapping and episodes of ULF using single-pulse TMS time-locked to the tap cycle. Based on the available neurophysiological evidence, we hypothesized that M1 excitability would be modulated throughout the tap cycle with higher excitability during the contraction (ascending) phase and lower excitability during the relaxation (descending) phase. Secondly, we hypothesized that M1 excitability would be reduced during ULF episodes compared to ascending periods of regular tapping. Finally, we anticipated that M1 excitability during the transition period between regular tapping and freezing would show reduced excitability in ascending compared to freezing and lower difference of M1 excitability between ascending and descending.SD = 5) with a mean tapping frequency of 2.53 Hz (SD = 1.29). Within these blocks, we detected 237 freezing episodes across all patients, lasting on average 0.83 s (SD = 0.13). In two patients, no TMS pulse coincided with a freezing episode , 447 during regular tapping , and 58 during the transition phase . For the descending slope, 503 pulses were triggered , 425 during regular tapping , and 78 during the transition phase . Another 59 pulses coincided with ULF . Finally, 123 of the pulses (36 ascending (29.27%), 80 descending (65%), 7 ULF (5.69%)) did not evoke a MEP (<200µV) and were thus set to value zero.Overall, 1,067 single pulses were delivered. Of these, 505 were applied during the ascending slope  and greater compared with ULF  . During the transition phase, MEPs during the ascending cycle were still greater than for descending  but no longer differed from periods of ULF .We found that during regular tapping, ascending MEPs were greater compared with descending MEPs (mean ± SEM) compared to descending  phases  or descending and ULF  contrasts.As expected, right FDI EMG activity outside the MEPs of both regular and transition time segments was modulated across the tap cycle and was slightly greater for ascending , or for transition , or between regular tapping ascending and ULF .There was no correlation EMG activity 110–10 ms before a single TMS pulse and MEP size. This held true for both normalized and un-normalized MEP size Fig. . AdditioP = 4.764e-07), as expected. However, this was driven by the differences in the behavioural state between “regular tapping ascending” vs. “descending” (P < 0.001) and “regular tapping ascending” vs. “freezing” (P = 0.0374). However, neither EMG activity (P = 0.511) nor force (P = 0.883) were predictive in this model.Nevertheless, to further exclude any confounding effects of pre-stimulus EMG activity or force on MEP size, we added a linear regression analysis with MEP size as dependent, and behavioural state, EMG activity, and force as independent variables (refer methods for details). Overall, the model showed predictive value on MEP size =8.894, We used adaptive single-pulse TMS to study M1 excitability in PD freezers during the ascending (contraction) and descending (relaxation) cycles of regular finger tapping, the transition period, and actual freezing episodes. As expected, cortical excitability fluctuations in M1 were preserved during regular tapping and transitions depending on the movement phase. However, M1 excitability was reduced during freezing compared to the ascending phase of regular tapping, despite the intention to move. During ascending transition taps, M1 excitability was no longer increased compared to freezing and pointed to a premonitory abnormality of M1 in increasing excitability.22. In control analysis, we did not find a correlation of background EMG with MEP size, nor did we find predictive value of force or EMG background activity when we included these variables to a multiple regression model. However, the effect of behavioural state  persisted. Thus, it is unlikely that background EMG or force explained our findings23. Instead, phase-specific modulation of corticospinal responsiveness has been observed during lower24 and upper limb cyclic movements23 in younger, healthy subjects, and our findings support that such excitability fluctuations aligned to the movement cycle are preserved in PD freezers. However, PD freezers were impaired when increasing cortical excitability during the freeze and the transition period. In the light of previous research, the insufficient increase of M1 excitability of the Parkinsonian M1 does not necessarily constitute a general abnormality but rather emerges during voluntary movement. Whereas the PD resting motor thresholds were found to be mostly similar to those reported in healthy controls25, the Parkinsonian M1 has shown reduced cortical excitability increases during isometric contraction and in reaction time tasks. This was reflected by flattened input-output curves in incremental isometric contractions26, and by abnormalities in pre-movement facilitation in a reaction time motor task, such that the Parkinsonian M1 showed an earlier but lower excitability increase prior to externally cued voluntary movements28. Our findings suggest that the freezing phenomenon may critically depend on such rapid and sufficient adjustments of cortical excitability to stabilize repetitive movement sequences. In turn, failure of these excitability adjustments – as observed during the transition period – may give rise to freezing, which could represent a generalized pathophysiologic mechanism common to the symptoms observed in upper and lower limbs and speech. Indeed, motor deterioration in our experiment became first kinematically visible during freezing but was neurophysiologically measurable in the transition phase already, when cortical excitability decreased. This may have been related to increasing performance difficulties and reflect a “timing problem” when modulating M1 excitability over the tap cycle. In this sense, increasing demands on motor performance such as fatigue or high tapping speed9 might have deranged the physiological time course of excitability modulations and preparation of M1 to increase excitability when needed16.In our experiment, background EMG was slightly higher comparing the ascending to the descending slope of tapping, whereas the force levels did not differ between conditions29. Particularly the generation of self-initiated sequential (finger) movements – movement most susceptible to freezing – depends on intact signal transmission from the frontoparietal executive control network30 and the BG-thalamic-motor loop, involving the supplementary motor area, M1, putamen, globus pallidus, and ventrolateral thalamus31. In PD, reduced effective connectivity between these areas and the loss of automaticity32 lead to compensatory over-reliance on the cognitive striatum for effective movement production. The competition for limited processing resources with increasing motor task difficulty is then believed to cause transient striatal dysfunction and STN overactivity, ultimately resulting in freezing33.In general, M1 excitability and successful motor output rely heavily on afferent inputs from diverse cortical and subcortical areas to M128. These models embedded M1 and other gait-related structures in an “inhibited” or “hypoactive” state33 and indeed, we found that M1 was less excitable such that the insufficient “facilitation” of M1 excitability paralleled the freeze. From the present experiment, however, we cannot infer if M1 was actively inhibited or lacked afferent input (or both). This distinction is pathophysiologically meaningful since inhibition versus facilitation of M1 rely on different intracortical circuits and neurotransmitters, which both reflect separate active processes35. We found that excitability during freezing did not differ significantly from excitability during the transition. In fact, M1 excitability was lowest overall at relaxation during the transition – when motor output was still intact. Motor arrest during freezing, however, occurred involuntarily, suggesting that the abnormally excited M1 may indeed result from activation failure (lack of excitatory afferences) rather than active inhibition. Even though this observation is based on a relatively small sample, we believe it is still worth noting since it highlights the importance of the underlying movement intention (voluntary vs. involuntary motor stops) and the reliance on the cognitive control network to compensate for deficiencies in internal movement generation and motor automaticity15. Nonetheless, it remains for future studies to investigate inhibitory plasticity measures during a freeze.As such, our observations are compatible with existing pathophysiological models of freezing that have postulated a ‘dysexecutive’ communication failure between frontostriatal and primary motor areas with a paroxysmal failure to produce effective motor output36, or combined stimulation of the dorsolateral prefrontal cortex and M1 with anodal transcranial direct current stimulation37 and further corroborate the notion of an hypoactive M1 during freezing.Finally, our findings parallel clinical observations made on freezing of gait, in which TMS protocols increasing M1 excitability have led to an improvement in symptoms, i.e., 10 Hz repetitive TMS of M1There are a few points to consider when interpreting our results. First, the presented data of our pilot study is based on a small sample size, which naturally increases variability and may limit statistical power. Hence, we used a statistical approach focusing not on individual patients but on the pooled number of evoked MEPs per experimental condition. This enabled us to collect sufficient data (regular tapping asc (447 MEPs)/desc (425 MEPs), transition asc (58 MEPs)/desc (78 MEPs), freezing (59 MEPs)) to perform meaningful analyses.24 and combined TMS-EEG studies showed that beta-band desynchronization over the sensorimotor area related to increased M1 excitability and greater MEP size38. In PD patients, our work8 and the work of other groups16 found that during repetitive finger tapping beta activity modulation was preserved and modulated along the tap cycle in a similar fashion albeit differences have been observed regarding peak beta amplitudes19. Thus, we concluded that modulation of M1 excitability relative to the tap cycle would be preserved in PD patients, and this is indeed one finding of the present study.Second, as healthy controls do not freeze, we did not add a healthy control group, even though the logical foundation of our experiment is based on available literature on repetitive movements in health. We know from healthy subjects, for example, that MEP size is modulated along cyclic upper and lower limb movements39. On-state freezing, on the other hand, would have provided heterogeneity in terms of L-Dopa resistant freezing vs. paradoxical on-freezes, both of which are considered different phenomena40. Also, previous TMS experiments found no meaningful differences in MEP amplitudes (during isometric contraction) before and after L-Dopa intake42 and particularly during finger tapping, cortical beta band modulations did not differ between medication on- and off-states in an EEG experiment with PD patients43.Third, we chose to study patients in the off-medication state only as it allowed to observe the genuine disease-related pathophysiology of freezing and L-Dopa withdrawal significantly increases freezing likelihoodIn conclusion, freezing reflects an episodic event of ineffective motor output. Our findings suggest that the Parkinsonian M1 is impaired in increasing excitability during a freeze and, critically, already during the transition phase preceding a freeze. Therapeutic interventions modulating M1 should be investigated as a treatment approach to ameliorate freezing phenomena in Parkinson’s disease.44  or psychiatric disorders  that would impact interpretability, performance, and/or compliance. Patients participated with written informed consent and the study protocol followed the Declaration of Helsinki, which was approved by the ethics committee of the University of Tübingen (protocol number: 916/2018BO1).We studied eleven right-handed patients with akinetic-rigid iPD and clinically verified FoG44 Table . Exclusi46, and determination of the individual active motor threshold (aMT) following standardized procedures published elsewhere47. The EMG was recorded from the right first dorsal interosseus (FDI) in the belly-tendon montage . Patients placed their right index finger on a force sensor and were instructed to perform internally generated taps as fast and accurately as possible up to a 2 N peak maximum while keeping the finger in permanent contact with the sensor. Real-time visual feedback was provided on tapping force and accuracy. A tapping block lasted 30 s, followed by a 20 s pause to prevent fatigue. Overall, one session included ten blocks of continuous, self-paced tapping or more, if possible.Patients were investigated during their “medication off” state having withdrawn overnight from their dopaminergic medication. Preparation included the mounting of the EMG electrodes, identification of the individual motor hotspot46. A custom-made real-time signal processing algorithm based on Simulink Real-Time  was used to trigger single TMS pulses in response to specific pressure sensor force conditions during the tap cycle. We phase-locked the pulses to 25% (ascending) and 75% (descending) of the tap cycle  in randomized order  at 120% aMT stimulus intensity50 and customized MATLAB® scripts . Each patient’s kinematic data was segmented into single taps and ULF episodes using MATLAB function ‘findpeaks.m’. One tap cycle consisted of a downward press (trough to peak) and an upward release (peak to trough). A trough-peak-trough section was considered a full tap if it lasted <2 s and if its amplitude deflection from trough-to-peak and peak-to-trough was ≥1 N (50%)8. We then calculated mean tapping frequencies  and average EMG activity . ULF episodes were detected visually based on the following criteria: (i) reduction of amplitude deflection >50%, (ii) Freezing Index (FI) > 1, and (iii) duration ≥0.5 s51.Preprocessing was done with FieldTrip open-source toolbox47. Lower amplitudes were set to 0 µV and considered ‘no MEP’ for statistical analyses. The mean MEP size per individual and tapping block served as reference for normalization to account for intra- and interpersonal drifts in cortical excitability52. MEPs were classified as either ‘ascending’ or ‘descending’ during regular tapping and in the transition period, which was taken as the last three taps preceding a freeze8. MEPs during freezing episodes were not divided into ascending or descending due to the lack of effective motor output when freezing.Peak-to-peak MEPs were extracted from the EMG signal 20–40 ms after the TMS pulse and stored for analysis if >200 µV55. Therefore, we deliberately chose the right FDI muscle to record MEPs due to its partial agonist activity during finger tapping to reduce the magnitude of these influences.It is known that force levels and background EMG activity at or just before single-pulse TMS may influence MEP size(i) recording time and adherence of PD patients in “medication off” was limited and (ii) the number of freezes and transitions that could be recorded in the laboratory setting and with it the number of MEPs evoked per condition could not be predicted. This also meant that we planned to perform non-parametric pairwise comparisons between contrasts of interest as introduced above, i.e. we had neurophysiological hypothesis as introduced above comparing regular ascending vs. descending, regular ascending vs. ULF, transition ascending vs descending, and transition ascending vs. ULF.This piloting study aimed to investigate real-time cortical excitability in PD patients during repetitive finger movements, transitions and freezing. We decided on an exploratory strategy, as 23.Further, we used a linear multiple regression model to predict MEP size from different independent variables, i) behavioural state , ii) background EMG activity (rooted mean square 10–110 ms prior to stimulation) and force at the time of stimulation. MEP size was treated as dependent outcome variable to ensure that EMG activity and MEP sizes were not linearly relatedShapiro-Wilk test) and thus all statistical tests were two-tailed nonparametric , and a p-value of < 0.05 was considered statistically significant. MEPs are presented as percent (%) change from the block average.Statistical analyses were performed with SPSS® version 25 (IBM), Prism 8 (GraphPad), and Matlab® ; graphs were created with Prism 8. MEP amplitudes were not normally distributed (Further information on research design is available in the Reporting Summary


Parthenolecanium corni (Bouché) (Hemiptera Coccidae). Our objective was to characterize the transmission biology of grapevine leafroll-associated viruses (GLRaV) and grapevine virus A (GVA) by this soft scale species in order to evaluate its ability to spread these viruses. In transmission experiments with nymphs sampled from different vineyards infected with GLRaV 1, 2, 3 and GVA, P. corni transmitted only GLRaV 1 and GVA to healthy vines. GVA was predominantly transmitted along with GLRaV 1, whereas the latter could be transmitted alone from single or co-infected vines. Vineyard-sampled second instar nymphs were more efficient than first instars at transmitting GLRaV 1, whereas both instars displayed similar transmission rates for GVA. Short virus inoculation access periods and the absence of virus in eggs of females living on infected grapevines fulfilled the criteria of non-circulative semi-persistent transmission mode. Grapevine-infecting ampelo- and vitiviruses are transmitted by scale insects belonging to several species, among which is the European fruit lecanium,  Vitis vinifera L), ‘grapevine leafroll disease’ (GLD) occurs in all major grapevine-growing areas. In New York State, USA, mean yield losses are estimated at 15–20%, but can reach up to 40% [Closteroviridae, namely, grapevine leafroll-associated virus (GLRaV) 1, 2, 3, 4, 7 and 13 have been associated with GLD [Ampelovirus, and are naturally transmitted to grapevine by scale insects (Hemiptera Coccoidea) belonging to the families Coccidae  and Pseudococcidae  [Planococcus ficus (Signoret) to the herbaceous host Nicotiana benthamiana Domin [Vitivirus: grapevine virus A (GVA), grapevine virus B (GVB) and grapevine virus E (GVE) [Nicotiana benthamiana and N. clevelandii Gray by scale insects of three species: Pl. ficus [Pseudococcus longispinus (Targioni Tozzetti) [Parthenolecanium corni (Bouché) [One of the most important viral diseases of grapevine  ,7. Notabna Domin . Moreovel. ficus , Pseudocozzetti)  and Part(Bouché) . As thes(Bouché) ,12,13,14(Bouché) ,18,19,20(Bouché) ,27,28,29(Bouché) , the phlP. corni, the European fruit lecanium, is the most widespread species in vineyards and is monovoltine. Adult females lay eggs mainly on canes in spring, and neonates, called ‘crawlers’, migrate to basal leaves. Once fixed on leaves, the nymphs move only to find a new feeding place or when unfavourable conditions of survival  force them to leave their attachment point, and when they molt from first (L1) to second (L2) instar nymphs during summer [P. corni L1 can be spread by wind and thus transmit GLRaV 1 and GVA to neighboring vineyards [P. corni natural populations dwelling on infected vines to effectively transmit the viruses is poorly known and evaluated. Therefore, our purpose was to investigate the transmission potential of vineyard-resident P. corni depending on its developmental stages. For this purpose, we used transmission experiments, consisting of sampling L1 and L2 nymphs, in spring and summer–autumn, respectively, from leafroll-infected grapevines in a vineyard, and transferring them onto healthy vine cuttings in the laboratory [Four of the aforementioned viruses are known in French vineyards: GLRaV 1, 2, 3 and GVA. In northeastern France, the palearctic soft scale g summer . The maig summer ,32,33. Pineyards ,35. Howeboratory ,25. SuchP. corni populations originated from five commercial grapevine vineyards located at Bennwihr, Kientzheim, Nothalten, Ribeauvillé and Turckheim , as well as from an experimental vine vineyard located at Ihringen . Distribution of viruses among the vines tested in each vineyard is given in P. corni colonies. GLRaV 2 being rare in our vineyards, associations with this virus were underrepresented. IAP experiments were performed from 12 June to 26 July for L1, and from 23 July to 30 October for L2, depending on annual climatic conditions.A multiplex RT-PCR procedure was used for the detection of GLRaV 1, 2, 3 and GVA in insect samples at different growth stages  before transmission experiments, using the protocol developed by Beuve et al. . In addiNymphs were collected and stored in 1.5 mL Eppendorf tubes with 500 μL RLT buffer  containing 1% β-mercaptoethanol. Tubes were kept at −20 °C before total RNA extraction and virus detection by multiplex RT-PCR. The content of the Eppendorf tubes was ground with a PolyLabo™ electric mill, then incubated for 3 min at 56 °C. A quantity of 450 μL of supernatant from the tubes was centrifuged for 2 min at 14,000 rpm in an RNeasy column tube to remove cellular debris and precipitates. Then, 400 μL of filtrate was poured in a 1.5 mL tube, to which 200 μL ethanol was added. The mix was poured in an RNeasy column tube and centrifuged for 30 s at 10,000 rpm to fix the RNA. The column was then washed with 700 μL RW1 buffer for 5 min and centrifuged for 30 s at 10,000 rpm, and then with two successive washes with 500 μL of RPE buffer and respective centrifugations of 30 s and 2 min at 10,000 rpm. Residual ethanol in tubes was removed by centrifugation for 1 min at 10,000 rpm. Total RNAs were eluted during 1 min with 50 μL RNase-free water; then, column tubes were centrifugated for 1 min at 10,000 rpm, and eluates were stored in 1.5 mL tubes at −20 °C for subsequent RT-PCR. All centrifugations were performed at room temperature. RNase-free water and RNAs extracted from leaves of the accession Y258, an Armenian cultivar Liali Bidona co-infected with GLRaV 1, 3 and GVA sampled from our collection of accessions , were usFor PCR, in each sample, 12.5 µL of “Master Mix”, 2.5 µL of “Factor Q”, 7.8 µL of sterile ultra-pure RNase free water and 1.2 µL of the mix of primers-PCR  at 25 µMIn a set of source grapevines from Nothalten, the viral content of the leaves and of the L1 nymphs was analysed by RT-PCR on different canes of the same plant. A quantity of 100 mg of leaf tissue was ground at room temperature with pestle, mortar and Fontainebleau sand in 1 mL RLC buffer , to which was added 10 μL β-mercaptoethanol and 20 mg polyvinylpyrrolidone 40. The extract was transferred to a 1.5 mL Eppendorf tube, incubated for 3 min at 56 °C and centrifuged for 1 min at 4000 rpm. RNA extraction was then performed with approximately 400 μL of supernatant, as described previously.V. vinifera cv. Pinot noir, a cultivar frequently planted in Alsace, Champagne and Burgundy (clones P114 and P115), or from germinated seeds of Pinot noir, Pinot blanc and Muscat Ottonel. Mother plants of these cuttings were regularly tested for the absence of GLRaV 1, 2, 3 and GVA by ELISA and RT-PCR. Plants were grown individually in pots under greenhouse until the 6–12 leaves stage and then used in transmission experiments. They were regularly sprayed with an insecticide to ensure the absence of insects. This spraying was stopped at least one month before transmission experiments. Samples of ten recipient plants were tested for their virus-free status with ELISA prior to experiments.Grapevines free of leafroll viruses and GVA were obtained either from rooted cuttings of n = 10, mean number L2/vine ± sd = 17 ± 13). Each recipient plant was isolated under a 0.1 mm–mesh perforated plastic bag . Transmission experiments were conducted at 20–23 °C, 16 h/8 h (L/D) under artificial light. After IAP, source leaves were removed and recipient plants were immediately sprayed with mevinphos  to kill the insects. After two days, the treated plants were checked for possible surviving insects, then transferred into a glasshouse compartment dedicated only to insect-free recipient plants. Recipient plants with nymphs from healthy grapevines and conducted under the same conditions were used as negative controls. In late November, the recipient plants were pruned back to two buds and stored under a glasshouse in cold conditions for hibernation. In spring, they were transferred into a heated glasshouse. All recipient plants were periodically sprayed with insecticide and fungicide, and pruned to avoid overgrowth until the end of the study.Leaf pieces bearing ca. 100 L1, or 50 to 100 L2, were cut from source plants and clipped onto different leaves of recipient grapevines (distributed on 3–4 leaf pieces per plant) for an inoculation access period (IAP) of 5–7 days. Within 3–4 days, as the leaf fragments dried out, most of the insects crawled off to the recipient plant. For sampling overwintering L2, twigs of infected vines bearing L2 were collected in January in the Riesling vineyard of Nothalten. The L2 were allowed to wake up at room temperature and then placed with a fine paintbrush into cages . The cages were then attached with hairclips to leaves of recipient vines grown under a heated glasshouse (Infection of recipient grapevines was checked by double-antibody sandwich (DAS)-ELISA. The accession Y258, multi-infected with GLRaV 1, 3 and GVA, was used as positive control for the three viruses. The accession Chardonnay V38 , infecteP. corni L2 nymphs sampled on grapevines from Nothalten infected with either GLRaV 1, or GLRaV 1 and GVA, or GLRaV 1, 3 and GVA. Our previous transmission experiments from these virus associations showed the best transmission rates , with a higher transmission rate for L2. No difference in infection rate was observed for GVA . Whatever the growth stage, transmission rates between GLRaV 1 and GVA were not significantly different whether the source plants harboured GLRaV 1 and GVA, or GLRaV 1, 3 and GVA  when it was alone or associated with any other virus in the same source. Thus, GLRaV 1 transmission rate was lower when the source plant was co-infected with only GLRaV 3  after inoculation. For GLRaV 1, 64% transmissions were first detected ca. 4 months, 32% ca. one year, and 4% 15 months, after inoculation. For GVA, 67% transmission were first detected ca. 4 months, 28% ca. one year, and 5% 15 months, after inoculation. Overall, detection delay of GVA appeared similar to that of GLRaV 1, although GVA could sometimes be detected earlier than GLRaV 1.L2 nymphs collected on infected vines in Nothalten vineyard transmitted GLRaV 1 to five out of fifty-two inoculated grapevines, giving an overall transmission rate of 9.6%. The shortest IAP was 2 h 40 .P. corni in relation to grapevine-infecting ampelo- and vitiviruses, by performing transmission experiments of nymphs sampled in infected vineyards. Our experiments aimed at better understanding the vector biology of First (L1) and second (L2) instar nymphs were sampled on infected grapevines in vineyards. When batches of L1 and L2 were tested by RT-PCR, most of them were found harbouring viral RNA of at least one virus among GLRaV 1, 2, 3 and GVA , confirmWhen L1 and L2 similarly sampled were subjected to inoculation to healthy recipient plants, they transmitted GLRaV 1 and GVA to grape cuttings, but neither GLRaV 3 nor 2 see  Table 4Table 4. P. corni L2 transmitted GLRaV 1 to 29% of recipient plants. With L2, we obtained a maximal transmission rate of 48% for GLRaV 1  and experimental conditions . Further work should quantify virus titer in mixed infections using qRT-PCR to evaluate the virus ratio in transmission experiments.Sforza et al.  previous GLRaV 1 . Transmificiency . TransmiP. corni populations sampled from several vineyards, GLRaV 3 was never transmitted , are transmitted by aphids [Aphis illinoisensis Shimer might contribute to the spread of GLRaV 2 among American wild Vitis spp. Unintentional propagation of this virus is actually due to diffusion of infected scions and/or rootstocks.Even though the number of samples was too small to draw a conclusion, y aphids ,44, no vy aphids ,45. Klaay aphids  suggesteIndeed, virus detection in individuals (viruliferousness) is not linked to their transmission ability. GLRaV 2 and GLRaV 3 were detected in nymphs, even if these viruses were not transmitted. GLRaV 1 was ingested with sap, and rejected in honeydew in which it was also detected .P. corni is a vector of GLRaV 1 and GVA but not of GLRaV 3, and that GVA was predominantly transmitted along with GLRaV 1 and rarely alone. Virus inoculation occurred within a few hours, in agreement with a semi-persistent transmission mode, as shown for mealybug species [In conclusion, our transmission experiments with vineyard-sampled viruliferous nymphs showed that  species . Further species . Using a species ,19 and i species . To conf species . Progres


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