Insecticide-treated nets (ITNs) are one of the most cost-effective measures for preventing malaria. The World Health Organization recommends both large-scale mass distribution campaigns and continuous distributions (CD) as part of a multifaceted strategy to achieve and sustain universal access to ITNs. A combination of these strategies has been effective for scaling up ITN access. For policy makers to make informed decisions on how to efficiently implement CD or combined strategies, information on the costs and cost-effectiveness of these delivery systems is necessary, but relatively few published studies of the cost continuous distribution systems exist.To address the gap in continuous distribution cost data, four types of delivery systems—CD through antenatal care services (ANC) and the expanded programme on immunization (EPI) , CD through schools (Ghana and mainland Tanzania), and a combined community/health facility-based distribution , as well as mass distributions —were costed. Data on costs were collected retrospectively from financial and operational records, stakeholder interviews, and resource use surveys.Overall, from a full provider perspective, mass distributions and continuous systems delivered ITNs at overlapping economic costs per net distributed , with two of the school-based systems and the mass distributions at the lower end of this range. From the perspective of international donors, the costs of the CD systems were, for the most part, less costly than the mass distributions . The 2015 school-based distribution (7.30 USD) and 2016 health facility-based distribution (6.52 USD) programmes in Tanzania were an exception. Mass distributions were more heavily financed by donors, while CD relied more extensively on domestic resource contributions.These results suggest that CD strategies can continue to deliver nets at a comparable cost to mass distributions, especially from the perspective of the donor. Malaria continues to represent a significant public health challenge, despite being a preventable and treatable disease. Malaria is responsible for an estimated 216 million cases and 445,000 deaths globally, each year . Since 2ITNs are the most widely used intervention for malaria control in Africa, representing the main vector control tool in nearly all malaria endemic African countries . They arIn 41 of 45 countries in the World Health Organization (WHO) African Region, the policy is to distribute ITNs free of charge . Mass diWhile mass distributions are a cost-effective way to quickly achieve high coverage over a particular area, coverage gaps begin to appear almost immediately post-distribution through net deterioration, loss of nets, and population growth, therefore, requiring complementary continuous distribution channels to sustain or “keep up” coverage over time , 26–28. Continuous distribution (CD) strategies employ channels other than mass distributions to deliver ITNs and comprise routine ITN delivery at ANC and EPI, schools, community-based , and sales within private sector, including social marketing. Despite the WHO recommendations for a combined approach utilizing both mass campaigns and CD systems to deliver nets, most countries are still relying heavily on mass distributions to distribute ITNs . Data reThe existing knowledge base on the costs and cost-effectiveness of CD systems is sparse. Only a few studies were identified that describe the costs of health facility-based CD , 42, 43.This manuscript describes a study of the costs of four types of delivery systems , schools, and a combined community/health-facility distribution) delivered at scale in various country contexts, utilizing a consistent method for cost collection and analysis for all programmes. The objective of this study is to compare the costs of the four delivery systems.The costs of four types of delivery channels were analysed in this study. These delivery channels were classified as either CD or mass campaigns. Distributions implemented at intervals of 3 years or greater with the goal of achieving universal coverage were classified as mass campaigns while all other distributions were classified as CD. Intermittent approaches, like the school distributions analysed in this study, while not purely continuous strategies (as nets were only made available once per year to students in eligible classes), were classified as CD strategies. Like other CD strategies, school distributions aim to boost coverage in the interval between mass campaigns and rely heavily on lower government levels to function effectively. Three school-based CD programmes were included in this study: two in Tanzania (2015 and 2017) and another in Ghana in 2016. Four health-facility based CD programmes were included: two in Ghana (2015 and 2016), one in Mali (2015), and one in Tanzania (2016). One combined community/health facility-based CD programme was included (Zanzibar 2015). Two mass campaigns were also included . The ITN programmes chosen for this analysis were deliberately selected to represent different ITN programs and delivery channels in sub-Saharan Africa.A description of each intervention was developed based on document reviews , and through key informant interviews with individuals involved in the management, supervision, and logistics of the distribution programmes at all levels; this includes donors, partner organizations, members of the National Malaria Control Programmes (NMCPs), and individuals at the local health and school levels . The intervention description guided all further data collection components by outlining and delimiting the appropriate resource inputs to be costed and partner organizations which had to be identified and contacted.Because the costs were analysed from the provider perspective, all costs associated with provision of the health care intervention are included from the perspective of the providers of the intervention . Household costs, whether direct or indirect, were not included. All direct costs of the program to the providers were included, including commodities, social and behaviour change communication, transport, payment of salaries, and volunteer time. No cost-savings to the provider due to reduced treatment were included.Additionally, costs were also analysed from the international donor perspective, meaning only the subset of costs incurred (financed) by international donors were included in these sub-analyses. Costs incurred (financed) by domestic government agencies  were not included in these sub-analyses.Cost data were collected retrospectively from the financial and operational records kept by partner organizations and through interviews with programme managers and implementers involved in ITN distribution activities. Data were collected by examining the agencies’ financial records, including budgets, expenditure records, reports, receipts and invoices; as well as operational records, such as reports and published documents. The ingredients approach was used for each of the programs analysed, however for certain line items and programme activities, if the information was deemed too sensitive to be released as inputs and prices , or was not available in adequate detail , aggregated expenditure was used. Aggregate costs were most commonly used for line items such as “indirect costs”, “other direct costs”, and national level salaries, and often components of training, planning, supervision, SBCC, equipment, storage, and transport costs at the national level. Aggregate costs were never used for nets and were rarely used for costs obtained at the lower levels (district and beyond), such as district or facility personnel and fringe costs, transportation costs, or storage costs.e.g. government human resource costs) information was collected through stakeholder interviews or, in the case of the third round of the Tanzania School Net Program (SNP3) and Ghana analyses, a resource use survey instrument was utilized.When costs or resource inputs were not reflected in financial or other available records  national and international, (2) the district, subdistrict, or circuit (the subdistrict level for schools in Ghana) (3) and, peripherally, at health facilities or schools (or other distribution points). Resources were generally valued using opportunity costs derived based on the reported expenditures or budgets and, in the case of personnel, on salary plus fringe benefits. Capital goods were valued based on their procurement costs or, in the case of building rents, on the average market value of similar properties. Where data was unavailable, the World Health Organization-CHOosing Interventions that are Cost-Effective (WHO-CHOICE) database was used for valuation purposes, after conversion to non-purchasing power parity-adjusted United States Dollars (USD) .Interviews were also conducted to collect resource use and expenditure information at lower governmental levels . Key individuals, such as members of the district health management teams, malaria focal persons, health facility staff and teachers responsible for distributing nets, and others that were involved in the management of the distribution programmes at the lower levels were interviewed. Data collected from these interviews were then used to make assumptions about estimated resource and time use, as well as actual expenditures and costs for specific distribution activities at lower health system levels. An average cost for all districts, all schools, and all health facilities interviewed or surveyed was calculated and then applied to all participating districts/schools/health facilities.Costs were divided into capital and recurrent costs, based on the lifetime of the goods or service being purchased. Capital costs are costs incurred to purchase goods or services with a life span longer than 1 year. Recurrent costs are costs incurred for goods or services lasting less than 1 year. Capital costs were discounted in the economic analysis using lifetimes and discount rates determined through stakeholder interviews, expert information, and past literature. Varying discount rates and lifetimes were examined in sensitivity analysis. Both financial and economic analyses were conducted. These two types of analysis show (1) financial costs—what the actual expenses of running a programme were, and (2) economic costs—the value of all resource use during the study period. In the financial analysis, capital costs were not discounted and were instead applied in full at the time of the purchase.The costs of the programmes included in this paper were collected in a range of international currencies over a period of time dating back to 2015. In order to draw meaningful comparisons between costs from different programmes, costs were adjusted to a common year and a single currency. Costs of interventions were first converted from local currency to US Dollars using the exchange rate at the year the initial costing analysis was performed. These costs were then split into tradable costs (e.g. nets) and non-tradable costs . The estimated costs of tradable goods and services were inflated or deflated, in the case of the 2017 Tanzania School Net Programme, to 2016 USD using the consumer price index (CPI) adjustment . The estThe primary output measure used in the analysis was the number of nets distributed. These data were collected from partner organization records. The number of nets distributed was used to calculate the cost per net distributed. A second combined output measure was also calculated—cost per person-year of protection (PYP), which assumes that, in the base case scenario, one net can cover two people for a period of 3 years. The primary output was also used to calculate a third combined output measure—cost per treated-net year (TNY), which assumes a lifetime of 3 years per net in base case scenario calculations.In this analysis, the base case scenario relies on the following set of assumptions: a discount rate of three percent has been applied to economic capital costs, and each ITN provides 3 years of protection for two people and assumes 100% of nets distributed are being used. One-way sensitivity is then used to test these assumptions on the economic cost per person year of protection from the full provider perspective.Table The mass distributions in Mali aimed to cover the entire population in Bamako (an urban setting) and Segou  by providing one ITN for every two persons. Before distributing the ITNs, advocacy and planning meetings were held with regions and districts, as well as trainings and microplanning. The population targeted for mass distribution was enumerated and registered. During the household registration, the heads of each household were provided vouchers that could be exchanged for a net at the time of distribution. The ITNs for the mass distributions were stored in several large warehouses in Bamako before being transferred to several health districts, in the case of the Segou mass distribution; or directly to the health facilities, in the case of the Bamako mass distribution. At all levels  local government officials identified and paid for the storage warehouses and set up a system for securing the ITNs throughout the storage period before their distribution. Logistics plans were developed for the transportation, warehousing, and pre-positioning of the ITNs for all levels. Commercial haulers transported the ITNs via road to the health districts in Segou and Bamako. After the ITNs were delivered to the county seats of the selected districts—or directly to the health areas, in the case of Bamako—the district health authorities were responsible for pre-positioning the ITNs at the distribution points. Social and behaviour change communication (SBCC) plans were also developed and implemented. During distribution, community liaisons handled the distribution of the nets, in collaboration with community health associations. Heads of households travelled to specific distribution points to exchange their vouchers for nets. If more or fewer nets than were originally planned for were needed, additional personnel were sometimes hired to assist with the process of redirecting nets to meet requirements. Local civil society organizations were also encouraged to support some of the costs.The Ministry of Health (MoH) provided free nets to pregnant women at their first ANC visit and to infants when they complete their national immunization series. Nets were distributed to health facilities within all regions of Mali. Because this process has been ongoing for an extended period, little refresher training or additional planning was necessary. Nets are stocked year-round in central storage warehouses in Bamako. Transportation of the ITNs to the health districts from central storage took place via road, on a semi-annual basis, using commercial haulers selected by the implementing partner. After the ITNs were delivered to the districts, the district health authorities were responsible for allocating the ITNs to the health facilities, based on requisition forms that health facilities submitted monthly. Typically, health facilities store a 3-month supply of nets; however, this varied depending on the facilities’ storage capacity and was, therefore, inconsistent across health facilities. While other health facility distributions programs often suspend distributions during mass distribution years, Mali’s health facility distributions continued operating in Bamako and Segou. CD utilized human resources at the district and health facility levels throughout the year. These personnel assisted with the transport, storage, and issuing of ITNs; supervision, planning, training, and data reporting. Health facility personnel retrieved nets from district warehouses and transported them  to health facilities for storage and they provided nets to patients along with counselling and advice on ITN use as part of the ANC/EPI visit.For the CD of ITNs through health facilities in 2015–2016, nets were procured internationally, after which they were shipped to Ghana, cleared through customs and delivered to central medical stores (CMS). Before CD activities began, nets for all three channels of distribution  were delivered to either regional or district stores. The central/regional medical stores department typically distributed the nets with reimbursement for transportation costs, paid for with funds provided to the NMCP by international NGOs. Once nets arrived at the district or regional stores, they would then be delivered to health facilities based on demand forecasts. Occasionally, sub-districts helped facilitate this process (particularly in larger districts). They also provided additional storage space, assisted with the transport of nets to the health facilities and reporting. MoH personnel assisted with the transport, storage, and issuing of ITNs; supervision, planning, training, and data reporting. Health facility personnel retrieved nets from district warehouses and transported them  to health facilities for storage and they provided nets to patients along with counselling and advice on ITN use as part of the ANC/EPI visit.en masse once per year to students enrolled in primary school classes two and six. Nets were distributed as soon as possible after delivery (often the next day) to avoid protracted storage at the school. Students were enumerated using concurrent enrollment registers or through the use of Education Ministry Information System data and nets were directly distributed by teachers. Implementing partners and the Ministry of Education supervised the school distribution through SHEP officers and school principals. Implementing partners also provided additional communication, health promotion, and behaviour change communication support, with assistance from other international NGOs. Implementing partners and the NMCP also provided training of trainers and support for regional and district-level training for net distribution activities.For the school distribution in 2016, the nets were procured internationally, shipped to Ghana, cleared through customs and delivered to regional health stores, district governments, circuit government, or directly to schools. Once nets arrived at the district or regional stores, they were then delivered to schools based on assessment of past enrollment records that had been validated by School Health Education Programme (SHEP) officers. Nets distributed to schools are typically distributed i.e. health centres and dispensaries) was taken on by a number of international stakeholders and a logistics firm was sub-contracted to physically transport the ITNs. The project began with a “Smart Push” strategy in which each health facility received an initial supply of ITNs. The numbers that each facility received were planned based on patient volume data. Following the “Smart-Push,” facilities report quarterly on the number of nets they have distributed and are re-stocked accordingly. The nets are given free-of-charge to pregnant women at their first ANC visit, and to children at 9 months when they receive their first measles rubella vaccination.For the 2016 continuous health facility-based distribution channels in mainland Tanzania, ITNs were imported to Tanzania through a procurement agent, which contracted a local logistics firm to transport them from the port to one of four private warehouses. The process of managing nets from their arrival at the central warehouses through their transport to health facilities  were empowered to issue coupons to any eligible resident of their area to receive an ITN. A resident could then present the coupon at a health facility  in exchange for an ITN. The system at the shehia for determining eligibility consisted of a committee working with the sheha to determine whether the applicant for an ITN met pre-specified eligibility criteria. Eligibility criteria were as follows: absence of an adequate number of ITNs in the home during reactive case detection activities, the resident is an orphan, resident is a widow/widower, resident is disabled, resident’s net was lost or destroyed in a disaster (such as flood or fire), the resident has recently moved/returned to the area, the resident’s ITN is damaged/severely worn, or the resident’s household does not have sufficient ITNs. Some coupons for ITNs were issued directly during reactive case detection by district malaria surveillance officers.The 2015 combined community-based and health facility CD in Zanzibar distributed the vast majority of ITNs through a community mechanism (61%) while the remainder of the nets were distributed through the ANC and EPI systems . The community system functioned through a mechanism by which shehia mechanism, all other nets in the CD system were distributed through health facilities. Eligible patients at facilities were issued coupons which can be redeemed at the same facilities in exchange for an ITN at no cost. Eligibility in this system was determined by the following criteria: for ANC, at a woman’s first ANC visit for each pregnancy, or for EPI, measles vaccination.Besides those nets given through the community/shehias play no role in transport or storage of ITN. Reporting on net distribution proceeded from shehias and health facilities through districts to the national level. Districts additionally played a role in supervision and training. SBCC and other communication activities could also be coordinated through districts, but all mass media activities were coordinated at the national level. Support for quantification in this system was provided by international consultants.ITNs were distributed directly from the national level to the health facilities, where all CD nets were stored and all coupons for CD nets were redeemed. Districts and ITNs for the 2015 Tanzania school distribution were procured and delivered to a hired warehouse in Dar es Salaam. Before distributing the ITNs, advocacy and planning meetings were held with regions and districts, as well as trainings and microplanning. Students were enumerated by local education authorities using enrollment registers of the target classes and a validation exercise was conducted to ensure the accuracy of these numbers. After re-quantification was completed and verified, and before distribution activities began, nets were delivered to the districts and then transported to all schools in each district, based on the approved, validated quantifications for each school. At the school level, teachers were trained prior to ITN delivery. Nets were distributed as soon as possible after delivery, ideally the next day, to avoid prolonged storage at the school. ITNs were distributed to schoolchildren in primary school classes 1–3, 5, and 7 in Mtwara and Ruvuma and classes 1–5 and 7 in Lindi. Members from the national, regional, district, and ward levels formed supervision teams who oversaw the trainings, transportation, and distribution of ITNs in the selected districts. Additional behaviour change communication support was provided in the form of mass media and print materials.SNP4 scaled up the programme from three regions in 2015 to seven regions in 2016 and SNP5 targeted 14 regions in 2017. The number of classes targeted varied by region in relation to previous distributions. Selection of classes was based on provisional enrollment data from a President’s Office Regional Administration and Local Government (PO-RALG) system (Basic Education Management Information System (BEMIS)) as of June 30, 2017, earlier NetCALC universal coverage modelling, and consultation with NMCP. Engagement and coordination meetings to involve key stakeholders in the implementation of SNP5 were conducted by international stakeholders and the NMCP at the national, regional and district levels. Trainings were also held in the seven regions that had implemented SNP in the previous round (SNP4), as well as the seven new regions.Nets were provided by two international donor sources and procurement was handled differently for the different net sources. Nets from one source were manufactured in Arusha and transported to regional centres and then transported by a logistics agent directly to the schools for distribution. The remaining nets were manufactured internationally and shipped to Dar es Salaam. A clearing agent cleared them through customs, and the same logistics agent used for the other nets, transported the nets directly to the schools for distribution.Prior to distribution international stakeholders and the logistics agent developed detailed microplans and desk validated ITN quantities. Prior to delivery to schools, the logistics agent used mobile phone applications to ensure that deliveries were made to the right locations in the right quantities. Original shipping containers were transported to a central point in each of the regions where nets were offloaded and delivered as soon as possible  to schools. This avoided the need for storage at regional level or at the schools. Supervision teams included representatives from the national levels from the NMCP and international stakeholders, regional and district-levels, with some involvement from the wards. A local marketing and promotion agent was contracted to conduct SBCC activities prior to and during distribution in selected regions.Total financial and economic costs and cost per net distributed, along with the total number of nets distributed for all programmes analysed are presented in Table Unit financial and economic costs for all programmes analysed are presented in Fig. Overall the financial costs per net ranged from 6.02 to 22.08 Int$ and 3.43 USD to 8.87 USD. The financial costs per net (Int$) were generally much higher for nets delivered in Tanzania compared to those delivered in Ghana and Mali due to price level differences between countries. The financial costs of nets delivered through continuous systems via health facilities and schools were generally higher than those delivered through mass distributions, with Ghana’s school distribution and Tanzania’s 2017 school distribution as the exceptions. The financial costs per net delivered through the health facility-based continuous systems were relatively consistent for each of the programs analysed. Economic cost per net ranged from 6.20 Int$ to 25.79 Int$, and 3.56 USD to 9.90 USD. The economic costs per net (Int$) were generally higher for nets delivered in Tanzania (including Zanzibar) compared to those delivered in Ghana and Mali. The economic costs of nets (USD) delivered through continuous systems via health facilities and schools were, in general, higher than those delivered through mass distributions, with Ghana school distribution and Tanzania’s 2017 school distribution as the exceptions. The economic costs per net (USD) delivered through the health facility-based continuous systems were relatively consistent for each of the programmes analysed, with Zanzibar’s combined community + health facility programme incurring the highest cost.Figure The proportion of costs provided by international donors was higher than that provided by the government for both mass distributions, Ghana’s school-based CD, Tanzania’s school-based distributions and Tanzania’s health facility-based CD in both the economic and financial analysis. The proportion of contribution from domestic sources was greater than 50% in Mali and Ghana’s health facility-based continuous distributions and Zanzibar’s combined community and health facility-based CD.Overall, CD systems required substantially more investment from domestic sources compared to mass distributions. While international donors generally covered the costs of important line items such as nets, transportation, and central storage, the government provided financial and economic resources in the form of personnel, vehicles, storage space at local levels, as well as additional ITN transportation costs locally in many settings.When only the international donor perspective is considered, the Tanzania mainland CD programmes incur the highest economic and financial costs per net, especially when adjusting for price level in the Int$ conversion.Table Because the same base case scenario has been applied to all programmes, the relative economic cost per person year of protection and the relative economic cost per treated net year reflect same ordering as when the comparison is based solely on unit costs (per net distributed).The line item breakdown of the financial and economic costs for all programmes analysed are presented in Fig. The proportion of the total financial and economic costs made up by ITNs were generally highest in mass distributions, followed by the school net distribution in Ghana and the most recent school distribution in Tanzania. The other distribution systems had a similar proportion of costs attributed to ITNs. For the total costs made up of personnel and fringe (human resources) alone, the health facility-based distributions generally had the highest proportion of costs attributed to human resources. All the continuous systems, however, incurred substantial personnel and fringe costs, compared to mass distributions. The proportion of the total financial and economic costs made up by transport, were comparable for all distributions systems. However, the Zanzibar combined community/HF-based programme had the lowest proportion of costs attributed to transport in the economic analysis. The proportion of the total financial and economic costs made up by storage were also generally comparable for all distribution programmes, with Ghana school-based distribution and Tanzania HF-based distribution having the highest share of storage-attributed costs. The CD programmes generally had higher proportions of costs attributed to supervision and monitoring evaluation compared to the mass distributions. Tanzania’s 2015 school-based distribution had the highest proportion of costs attributed to supervision and M&E; this is likely due to the aforementioned re-quantification and validation exercises that were required after issues with enumeration. The proportion of costs made up by training activities were relatively consistent for all programmes, however, Mali’s HF-based distribution had the lowest share of costs attributed to training. Tanzania’s health facility-based distribution had the highest share of costs attributed to training, compared to the other programmes, likely due to the fact health facility distribution was completely overhauled and substantial changes were introduced. The proportion of the total financial and economic costs made up by planning and coordination costs were generally higher in mass distributions, while they were lowest in Mali’s health facility-based system. The proportion of the total costs made up by SBCC costs was substantially higher for Tanzania’s school-based distribution programmes compared to the other programmes.One-way sensitivity analysis testing base-case assumptions on the economic cost per person year of protection from the full provider perspective is shown in Table ITN use, obtained from population surveys, is defined as the proportion of the population that slept under an ITN the night before the survey. The use:access ratio gives an estimate of the proportion of the population using nets, among those that have access to one within their household. This indicator provides data on the behavioural gap for net use—rather than a gap because not enough nets are available . The mosThe base case analysis assumes that 100% of ITNs distributed are used. When the use:access assumption is adjusted to reflect actual use:access estimates from the countries in which these programmes were implemented , substantial increases in the cost of the programmes per person year of protection were observed. A reordering of the cost per PYP was also observed. Tanzania’s 2017 school-based distributions remained the lowest cost per PYP, however the mass distributions replaced Ghana’s school-based distributions as the next lowest cost per PYP, followed by Ghana’s school-based distributions, followed by Mali’s health facility-based distribution, followed by Tanzania’s health facility-based distribution, followed by Tanzania’s 2015 school-based distribution, and the health facility-based distributions in Ghana replace Zanzibar’s combined community and health facility-based distributions as the most expensive programme per PYP. Use:access is, therefore, a very significant determinant in the efficiency of these programmes in the provision of PYP.Net durability, or the useful life of an ITN, is assumed to be 3 years in the base case scenario. In reality the lifespan of ITNs may actually be closer to two rather than 3 years –33. In tThe base case analysis also assumes that two people sleep under a net each night. A study presenting survey data from several sub-Saharan African countries between 2008 and 2012 showed that the mean number of users per net is significantly lower when enough ITN were available (at least one net for every two people) ) compared to if the household did not have enough ITN ) . TherefoThe cost of the net itself makes up a substantial portion of the total cost of net programmes. This cost is also slightly variable from programme to programme, with a range of 2.02–3.64 USD per net, and has decreased substantially in recent years, therefore, the effect of using a constant net price is presented to remove potential variation in unit costs arising only from net purchase price. When a consistent net price is used, a reordering of the cost per PYP is observed. The mass distributions replace Ghana’s 2016 and Tanzania’s 2017 school-based distributions as the lowest cost per PYP, these school-based distributions drop to second lowest cost per PYP, followed by the health facility-based distributions, and then Tanzania’s 2015 school-based distributions, and then followed by combined community/HF distribution in TZ.This study compared the cost of ITN distribution through mass campaigns, health facilities (ANC and EPI), schools, and community-based approaches. Costs for systems of these types were collected in three different countries. Overall, findings from the study showed that when the full provider perspective is considered, ITNs distributed through mass campaigns had a lower economic unit cost than those distributed through ANC/EPI-based systems. CD systems tend to distribute fewer nets compared to mass distributions, therefore their total costs are typically much lower than mass campaigns even though mass campaigns might have lower unit costs.ITNs distributed through school-based CD also, generally, had a higher economic cost per net distributed compared to the mass distributions in Mali, when adjusted for price level, though the school-based net distributions in Ghana (2016) and in Tanzania (2017) had a slightly lower unit economic cost than these mass campaigns when reported in USD.When the costs of domestic contributions are excluded, and donor financed components of the programmes are considered, the costs of the CD systems appear to be less costly than mass distributions. The 2015 school-based distribution and 2016 health facility-based distribution programmes in Tanzania were an exception. This was largely due to the fact that in Tanzania, distribution through these systems involved extensive international donor involvement at the time of the cost data collection, compared to the systems operating in Mali and Ghana. Health facility-based distribution in Tanzania was overhauled in 2016 and reintroduced in a new format and therefore required substantially more training, supervision, and M&E costs compared to the other programmes. The 2015 School Net Programme was also still very much in its pilot phase and, therefore, required substantially more initial investment and donor involvement, as well. Between 2015 and 2017, cost-cutting measures were introduced that resulted in the Tanzania’s school-based distribution becoming the least expensive programme per net distributed.The costs of the two mass distributions per net distributed and person year protected are comparable to those in the school-based CD system in Ghana and, more recently, Tanzania. These costs reflect a full provider perspective including both international donor funded activities as well as in-country government contributions of staff and resources. Donors and other planners of ITN distribution systems need to consider in-country contributions in the planning process of identifying value for money or other measures of efficient ITN distribution systems. However, they need not necessarily consider in-country contributions as explicit costs from their budget perspective. Furthermore, many of the in-country contributions, while not strictly in-kind contributions, represent use of staff time and infrastructure that are pre-existing, non-fungible expenses. The realization of these elements as costs of ITN distribution occurs only because the Ministry has directed its resources  to be used in the distribution of ITNs rather than in any alternative manner, or by utilizing spare capacity in the system to conduct these activities. When planning for future programmes, it is important to consider whether it is feasible for the government to direct their already paid for resources towards CD or whether implementing CD will overload already weak health systems with new activities. A clear example of this was the implementation of a community-based distribution programme in South Sudan. In this case, costs were substantially higher compared to similar programmes, because the programme was implemented in a context where government input, like vehicles, was not possible or was extremely limited .The overall economic costs per net distributed through the CD systems were generally higher than that those of the mass distributions from a full provider perspective, with the exception of Ghana’s and Tanzania’s most recent school-based distribution. However, there are some key differences between the systems that may explain this. First of all, mass distributions tend to benefit from some returns to scale due to the fact that huge quantities of nets are distributed over a such short period of time, compared to routine systems that operate year-round and rely on health workers to issue ITNs to individuals visiting an ANC or EPI clinic or presenting a coupon from a community-based scheme. Additionally, due to the continuous nature of health-facility-based and community-based CD and the level of commitment and cooperation required from the government at all levels for this programme to function effectively, ANC and EPI distributions required more substantial domestic contribution. The government provided the largest share of resources in both the economic and financial analyses for nearly all of the health facility-based CD analysed, with the exception of those in Tanzania, where international donors took on additional cost responsibilities for which the Malian and Ghanaian governments had been responsible in their respective programmes. While international donors covered the costs of important line items such as nets, transportation, and central storage, the government provided financial and economic resources in the form of personnel, vehicles, storage space at the lower levels, and additional ITN transportation costs at the lower levels. While the school-based CD models analysed in this study were not purely continuous systems, but rather intermittent systems with annual delivery, they still required substantial cooperation and commitment from all levels of government, particularly the lower levels (districts/circuits and schools), to function effectively. The intermittent nature of this system is reflected in the generally lower cost to the government per net compared to ANC/EPI and community-based programmes (fewer human resources required for this system to operate compared to purely continuous systems seen in ANC/EPI/community-based).When compared to studies that have previously attempted to ascertain the costs of CD programmes, the costs reported in this study are generally higher , 42, 43.ANC and EPI distributions directly target groups with special biological vulnerabilities to malaria and might provide additional value per ITN compared to mass distributions targeting the entire population. Mass distributions are typically conducted every 3–5 years. Mass distributions provide high coverage and high equity, however, pregnancies and births that occur between mass distributions represent vulnerable populations potentially unprotected without effective CD programmes . AdditioThe school channel delivered ITNs at a lower economic cost per net distributed, compared to ANC/EPI-based distribution systems. Pilot programmes in Tanzania have demonstrated that this system is a feasible method for rapidly and equitably distributing large quantities of ITNs , 37. IniZanzibar’s combined community/health facility-based distribution had the highest unit cost, overall. However, this is not surprising, as other programmes have cited higher costs for this type of channel . Pilot pThe economic cost per PYP for all programmes were sensitive to net use, lifespan of the net, and the number of users per net. Use is an important determinant of the efficiency of these programmes. While there is substantial information available about how CD programmes affect net coverage and access, there is little information available on how the source of net affects net use. Understanding who uses nets distributed through CD programmes, and how often they are being used, will be critical for decision-making.Surveys were used to obtain local level costs in the Ghana and Tanzania analyses (with the exception of the 2017 school-based distribution), however due to resource limitations and travel restrictions, a survey could not be used to obtain costs at the local levels in the Mali analysis and the 2017 Tanzania school-based distribution, therefore key informant interviews at local levels were used to make estimates for these costs. Thus, key differences in the in-country contributions (mostly incurred at the lower government levels) within these two settings may potentially be attributed to differences in methodology. To some extent the international donor analysis may mitigate this potential bias by removing most domestic resource contributions. In that sub-analysis the unit cost results appear largely comparable across countries.While the survey instruments were useful for capturing resource use and expenditure information at the lower levels for the programmes in which they were used, the results at each unit observed varied. Because these units were not a representative sample, they could lead to biased estimates, and because there were only a small number of sampled units, it is not possible to extrapolate to the full programme with high levels of precision. Additionally, it is possible that recall or social desirability bias could lead to over- or under-reporting of resource use, which in turn, may result in biased or imprecise estimates of government contributions. In future cost evaluations, care to directly observe, measure, and estimate these parameters (particularly personnel time contribution) may help to measure government contributions to the delivery systems more precisely, as well as to estimate the overall financial and economic costs of the distribution systems more accurately.This study also assumes that the costs associated with CD through health facilities at the lower levels were fixed rather than related to the number of nets distributed. Therefore, average costs were obtained using either the questionnaire data or informant interviews and applied to each district/sub-district/health facility/school to obtain an overall cost for each of the specified levels. Therefore, it is likely that the true cost of health facility-based distribution at these lower levels could differ from reported costs based on whether this assumption holds true. This could also have programmatic implications. When making this assumption, the overall costs of health facility-based CD seem to be driven by the number of health facilities, sub-districts, and health facilities served, as these levels require substantial resources . However, it is important to reiterate that the major share of these local level costs is provided by local government rather than international donors.shehias) therefore the travel time and travel-associated costs would have been minimal. Additionally, many of those costs would have been incurred anyway as children receiving nets through school distributions would have likely attended schools regardless and mothers receiving nets through ANC/EPI would have likely travelled to these clinics regardless for their antenatal care and their children’s immunizations. Furthermore, as nets were provided free of charge, households were not expected to make any contribution towards the cost of the net itself.It is also necessary to note that household costs (both direct and indirect) were excluded from this analysis. Therefore, the estimated cost of these programmes in terms of total societal resources is likely to be downwardly biased. However, it is unlikely that these household costs would have been substantial. Generally, ITNs were distributed within close proximity to the households  and mass distributions had the lowest economic cost per net distributed (3.56–4.31 USD for school-based and 4.37–4.61 USD for mass distributions), compared to ANC/EPI (7.95–8.59 USD), Tanzania’s 2015 school-based distribution (9.08 USD), and combined community/HF programmes (9.90 USD). However, from the international donor perspective, there was little difference between the four channels studied (3.30–4.55 USD), with the exception of Tanzania’s 2015 school distribution (7.30 USD) and Tanzania’s 2016 health facility distribution (6.52 USD). Mass distributions are heavily financed by donors, while CD relies more heavily on domestic contributions. Due to the continuous nature of community and health facility-based CD and the level of commitment required from the government, community and health facility-based CD required more substantial domestic contribution. Donors and other planners of ITN distribution systems need to consider in-country contributions in the planning process, even though these may not have explicit budget implications. When planning for future programmes, it is important to consider whether it is feasible for the government to direct resources  towards CD, or whether implementing CD will overload already weak health systems with new activities. Additionally, it is important that countries are able to sustain investment in CD, and that the policy environment is amenable to the incorporation of CD into policy when piloted programmes prove successful. The results suggest that CD strategies can continue to provide nets for a similar unit cost to mass distributions from the perspective of the donor but will require additional  domestic contributions (per net) compared to mass distributions. While CD may prove more costly than mass campaigns in some instances, government buy-in and investment in CD results in a sense of ownership of the programme and can lead to long-term sustainability under the right conditions.


Herpes simplex virus has affected two-thirds of the global population at one time or another. It can affect the eyes when the patient is first infected, or years later when the latent virus is reactivated.According to the World Health Organization (WHO), more than 3.7 billion people under 50 years – 67% of the global population – have been infected with herpes simplex virus at some point in their life.Herpes simplex virus is categorised into two distinct types: HSV-1 and HSV-2.HSV-1 is transmitted via direct contact, usually via saliva, and commonly presents as cold sores or fever blisters. It is also a cause of eye infection and significant visual impairment.HSV-2 is transmitted via sexual contact or from mother to child during birth . It causes genital herpes infections and, occasionally, ocular neonatal infection.Herpes simplex virus is usually acquired in childhood or adolescence. After the initial infection, the virus can enter nerve cells in the dorsal ganglia and lie dormant, or latent.Primary ocular herpes infection is often asymptomatic. The virus can present with cold sores or fever blisters , usually in response to a ‘trigger’ such as fever or stress. Clinically, ocular infection can be subdivided into keratitis, uveitis and retinitis, each of which will be discussed in this article.This can manifest asInfectious epithelial keratitisNeurotrophic keratopathyStromal keratitisEndotheliitisClinical features. The earliest presentation is corneal vesicles which later coalesce to form a dendritic ulcer. The dendritic ulcer .Treatment. The usual treatment recommended is topical aciclovir eye ointment 3% five times/day until the ulcer has healed. Trifluridine is an alternative that is used in the USA. Topical ganciclovir 0.15% five times/day is an alternative treatment. If there are many recurrences, consider prescribing oral aciclovir 200 to 400 mg twice/day as prophylaxis. This drug is considered safe for long-term use with 6-monthly evaluation of renal function tests.Clinical features. There is impaired corneal sensation with a superficial punctate keratitis that can form an epithelial defect with a smooth margin . A grey-white stromal infiltrate (neurotrophic ulcer) with well-defined, heaped-up epithelial margins may develop; this can lead to progressive stromal thinning, corneal perforation or secondary infection.Diagnosis. Microbiological evaluation with bacterial culture is recommended to rule out secondary infection.Treatment. Intense lubrication and prophylactic antibiotics are recommended. The antibiotics should be kept to a minimum to reduce toxicity to the epithelium. Antivirals are not indicated. Options for non-resolving cases include bandage contact lens or amniotic membrane grafting with tarsorrhaphy if the expertise and resources are available.There are two main types of inflammation of the corneal stroma due to herpes simplex virus.Immune stromal keratitis presents with corneal stromal oedema  or PCR. It is important to also exclude secondary bacterial infection.Treatment. This depends on the type of inflammation. Immune stromal keratitis is managed with topical low-dose corticosteroids 4–6 times/day with gradual tapering for 4–6 weeks, along with either topical aciclovir ointment 5 times/day or topical trifluridine for 2–3 weeks. For recurrent cases, provide prophylactic cover by giving oral aciclovir 200–400 mg 2 times/day.Necrotising stromal keratitis is treated using 400–800 mg oral aciclovir 5 times/day. After complete healing of the epithelial defect, topical corticosteroids may be added to reduce inflammation, but only with regular slit lamp examination to look for any recurrence of infection or corneal thinning, which could lead to corneal perforation.Endotheliitis is presumed to be immunogenically mediated, but the presence of live HSV has been postulated in some cases.Clinical features. Endotheliitis presents with stromal oedema, similar to immune stromal keratitis , characterise HSV anterior uveitisRecurrent episodes of unilateral anterior uveitisPast history of herpes simplex infectionRaised IOPDiffuse KPs Patchy or sectoral iris atrophy Diagnosis. Diagnosis can be confirmed by an anterior chamber (AC) tap and PCR for herpes simplex virus, or the Goldmann–Witmer coefficient (GWC) test . The GWC is a test that compares the levels of intraocular antibody production to that of serum, as measured by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay.Treatment. Management includes oral aciclovir 400–800 mg, 5 times/day combined with topical steroids and topical cycloplegics. Raised IOP is treated using anti-ocular hypertensive therapy. If there is any active keratitis, topical steroids should be used with caution.Herpes simplex retinitis is caused when the virus infects the retina. It is seen more commonly in immunocompromised patients. It can occur in neonatal herpes simplex infection in association with herpes encephalitis. Some cases of acute retinal necrosis (ARN) are caused by the virus.Clinical features. There are large, white retinal infiltrates, sheathing of the retinal vessels  for 5–10 days; followed by oral aciclovir  for 4–6 days. Retinal detachment following acute retinal necrosis (ARN) can be managed with a pars plana vitrectomy and silicone oil tamponade.


Ciona intestinalis in Prince Edward Island  has been causing devastating losses to the local blue mussel farms. In order to gain first insights into so far unexplored factors that may contribute to the invasiveness of C. intestinalis in PEI, we undertook comparative microbiome and metabolome studies on specific tissues from C. intestinalis populations collected in invaded (PEI) and native regions . Microbial community analyses and untargeted metabolomics revealed clear location- and tissue-specific patterns showing that biogeography and the sampled tissue shape the microbiome and metabolome of C. intestinalis. Moreover, we observed higher microbial and chemical diversity in C. intestinalis from PEI than in the native populations. Bacterial OTUs specific to C. intestinalis from PEI included Cyanobacteria  and Rhodobacteraceae , while populations from native sampling sites showed higher abundances of e.g., Firmicutes (Helgoland) and Epsilonproteobacteria (Kiel). Altogether 121 abundant metabolites were putatively annotated in the global ascidian metabolome, of which 18 were only detected in the invasive PEI population , while six  or none were exclusive to the native specimens from Helgoland and Kiel, respectively. Some identified bacteria and metabolites reportedly possess bioactive properties  that may contribute to the overall fitness of C. intestinalis. Hence, this first study provides a basis for future studies on factors underlying the global invasiveness of Ciona species.Massive fouling by the invasive ascidian  Sargassum spp., since herbivores preferably graze on seaweeds from the invaded population [Bonnemaisonia hamifera, inhibits the settlement of indigenous algae [Pacifastacus leniusculus transported the fungal pathogen Aphanomyces astaci to Europe, which significantly reduced native North Sea crayfish populations [Biological invasions represent the second largest cause of biodiversity loss and is only surpassed by anthropogenic species extinction ,2,3. Thepulation ,9. Accorpulation . 1,1,3,3us algae  and is tus algae . The “bius algae . For insulations ,14.Ciona intestinalis and its sister species C. robusta are among the most notorious invasive ascidians [C. intestinalis is native to the NE Atlantic and its adjacent seas , where it occurs in moderate abundances [C. intestinalis has spread globally and is generally regarded as a pest in invaded habitats in the NW Atlantic and Bohai and Yellow Seas (China) [Ciona intestinalis reportedly reduces species richness of the sessile macrobiota in invaded habitats [C. intestinalis causes high mussel mortalities (up to 50%), leading to significant economic losses for the aquaculture industry [C. intestinalis’ adaptive capacity  is considered as a major factor for its invasiveness [C. intestinalis [C. intestinalis are still not fully understood.Tunicates are sessile filter-feeders with a protective outer coating, the tunic ,16. The scidians ,17. The undances . In rece (China) . Ciona ihabitats . It is ahabitats . Foulingindustry . Its subindustry . PEI’s aindustry . Togethestinalis ,24. HoweC. intestinalis. Therefore, integrated metabolome and microbiome studies on C. intestinalis specimens from native  and invaded  habitats were performed. The microbiome of the tunic and the gut was characterized, comparatively, by amplicon sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. Likewise, the tunic and inner body of individual C. intestinalis specimens were comparatively profiled by a UPLC-MS/MS-based (ultra-performance liquid chromatography-tandem mass spectrometry) untargeted metabolomics approach. Furthermore, the global metabolome of the three ascidian populations was investigated by untargeted metabolomics using state-of-the-art tools  dereplication workflow [Current evidence suggests a prominent role for defensive secondary metabolites and associated  microbiota in globally successful MIS ,13,25. Hworkflow , in-siliworkflow , moleculworkflow ).C. intestinalis specimens was conducted in September 2017 in Helgoland  and Kiel Fjord . Invasive specimens were sampled in PEI  in October 2017. Seawater samples (1 L each) were collected aseptically in triplicate. Ascidian and water samples were immediately transported to the laboratory and processed at the same day. For individual genetic and chemical analyses of C. intestinalis, 10 intact individuals were chosen per sampling location, transferred into sterile 50 mL reaction tubes and promptly frozen at −80 °C. For chemical extractions at population level, approximately 0.5 kg of ascidian material were collected per sampling site in plastic bags and directly frozen at −80 °C. Seawater samples were sterile filtered as described in Parrot et al. 2019 [2 piece of the tunic was cut for extraction of microbial DNA. Subsequently, the gut was removed from the inner body and the gut content was removed by flushing it with sterile ultrapure water (sterile filtered and UV-treated) by using an injection needle attached to a syringe. A subsample of the gut (0.04 cm2), which was stored at −20 °C until further processing, was taken for genotyping the C. intestinalis individuals. From individuals with a very short gut , a small part of the tunic was frozen for genotyping instead. Another ca. 2 cm of gut tissue was immediately subjected to microbial DNA extraction. For individual metabolomics analysis, the remaining gut, mantle, and inner body tissues (hereinafter referred to as “inner body”) were placed into a sterile 50 mL reaction tube and the remaining tunic into a sterile 15 mL reaction tube. Samples were stored at −80 °C until metabolomic analysis.Sampling of native al. 2019  and filtC. intestinalis for all three sampling locations, 30 individuals (10 per sampling site) were genotyped with the mitochondrial marker fragment COX3-ND1 [xl capillary sequencer. Sequences were aligned in BioEdit [C. intestinalis , gut (n = 30), and seawater samples  was performed on OTU counts, which were rarefied and square root transformed for standardization (Bray-Curtis similarity index). In order to consider phylogenetic relatedness of OTUs, an additional nMDS plot based on weighted UniFrac distances [Subsequent data analysis was done with R v. 3.5.2  using thphyloseq , vegan [q [vegan , and pic[picante . The OTUistances  was calcistances . The Maristances  was usedistances .n = 60, i.e., 10 replicates per sampling site and tissue; n = 3 per sampling site).For individual metabolome analyses, single ascidian specimens were dissected into inner body and tunic  and ultra-purified water was prepared with an Arium Lab water system . Prior to UPLC-QToF-MS/MS measurements, extracts were diluted with MeOH to a final concentration of 0.1 mg/mL and blank MeOH samples were used as solvent controls. Analyses were performed with an Acquity UPLC I-Class System coupled to a Xevo G2-XS QTof Mass Spectrometer , which was equipped with an Acquity UPLC HSS T3 column  operating at 40 °C. The injection volume was 5.0 µL. A binary mobile phase system  was pumped at a flow rate of 0.6 mL/min by applying the following gradient (% of A given): initial, 99%; 2.5 min, 50%; 12.5 min, 0%; followed by washing and reconditioning of the column. The total run time was 16 min. The MS and MS/MS spectra were recorded in positive mode as previously described by Parrot et al. .m/z tolerance: 0.01 (or 10 ppm). Chromatogram deconvolution was done with the baseline cut-off algorithm at a baseline level of 500, a minimum peak height of 1000 and a peak duration range of 0 to 0.5 min. Grouping of isotopic peaks was performed by applying the isotopic peaks grouper algorithm with the following parameters: m/z tolerance: 0.01 (or 10 ppm), retention time (Rt) tolerance: 1.0 min, maximum charge: 3. In a final step, all samples were combined into one peak list by using the join aligner algorithm (m/z tolerance: 0.01 (or 10 ppm), Rt tolerance: 0.5 min, weight m/z:Rt: 75:25). Compounds detected in MeOH or extraction blanks were removed from this list. The resulting data were saved in the GNPS compatible data format MGF and the comprehensive peak list was exported to Microsoft Excel 2010 for further analysis. The peak area of population level extracts was used to investigate whether a peak was homogenously distributed across all sampling locations or showed an enhanced (at least 10-fold increase compared to other sampling sites) or exclusive production at one sampling site.Recorded data were converted with msconvert  to mzXMLhttp://dnp.chemnetbase.com, MarinLit: http://pubs.rsc.org/marinlit/ and Reaxys: https://www.reaxys.com). Putative hits were validated by biological origin. The detected fragmentation pattern was verified with the in-silico fragmentation tool CFM-ID [Pre-processed MS/MS data were submitted to the Feature-Based Molecular Networking (FBMN) workflow available at the online platform GNPS . Brieflyl CFM-ID  for putan = 10 per sampling site and tissue) and population extract level (n = 3 per sampling site), nMDS plots were compiled (Bray-Curtis similarity index). Peak areas of individual level extracts were normalized by dry weight .The average yields of population level extracts were statistically compared across the three different sampling locations via one-way ANOVA and a subsequent Tukey’s HSD test as described above. For statistical comparison of the metabolite profiles at individual  and two native habitats of C. intestinalis, i.e., Helgoland (H) and Kiel (K). From each location, three different sample types  were analyzed . The Shannon and Simpson diversity indices were similar for both tissues (Shannon: 3.9 (G), 3.7 (T); Simpson: 0.89 (G), 0.88 (T)). Location-wise, all alpha diversity measures were on average highest for Canadian C. intestinalis specimens. For instance, the diversity measures were on average higher in the Canadian population  when comparing to Helgoland  and Kiel Fjord populations . One-way ANOVA verified significantly different OTU richness and PD when comparing all samples , but was insignificant for the Shannon and Simpson diversity indices . The subsequently calculated Tukey’s HSD test revealed for some compared groups significant differences, e.g., the number of detected OTUs and the PD was significantly higher in Canadian tunics compared to that of Helgoland (p < 0.01), while comparison to Kiel tunics was insignificant  and rare OTUs (99.8% with <1% relative abundance) were detected. According to the distribution of singletons, rarefaction curves indicated sufficient coverage of gut and not yet completely saturated curves for tunic and seawater samples . Averagep = 0.001), but also tunic and gut tissues had different microbiome profiles . In order to verify the robustness of the beta-diversity results, we applied an additional ordination based on weighted UniFrac distances. In accordance with the nMDS plot based on the Bray-Curtis similarity index, the UniFrac-based ordination plot showed clustering by sample type and sampling location while being less apparent (p < 0.001) and revealed stronger impact of the sample type  compared to the sampling locations .Phylogenetic analysis assigned OTUs to 39 different bacterial phyla. Although differential abundances across the different samples were observed, the phyla Proteobacteria (53.8%), Bacteroidetes (16.6%), Cyanobacteria (8.5%), and Actinobacteria (5.1%) were the most abundant across all sample types . Multivaapparent . ANOSIM Synechococcus sp.) were present in all tunic and gut samples and are hence regarded as core OTUs , while Helgoland specimens showed a higher proportion of Actinobacteria  and various Firmicutes  in their gut (Leptolyngbya sp. OTU14 (Acrophormium is the heterotypic synonym of Leptolyngbya) and unclassified Oxyphotobacteria OTU80) and Alphaproteobacteria  were elevated in tunics or guts from Canadian specimens, respectively. Interestingly, the cyanobacterial genus Leptolyngbya  was only detected in ascidians from PEI, while the actinobacterial genus Bifidobacterium was exclusive to guts of ascidians from Helgoland specimens and Pseudomonas sp. were only identified in Kiel Fjord samples.With regard to the geographic location, the phylum Epsilonbacteraeota was specifically associated with all Kiel samples  and sample type  and differed from the surrounding seawater.Consequently, the microbiome of C. intestinalis populations. Ascidian samples were extracted at (i) population level (pooled samples of 13 g freeze-dried whole ascidian specimens per sampling site and replicate) to examine the ascidians’ global chemical profile at different locations and at (ii) individual level for comparing tunic and inner body tissues . Manual peak picking yielded 121 abundant metabolites, which were annotated by manual and automated dereplication tools, i.e., DNP, MarinLit, GNPS [5, 35, 41, 43, 49, 53, 58, 87, 89, 90, 96, 103, 105, 108, 117), lipids , peptides (55), polyketides , and terpenoids . The high abundance of lipids and terpenoids was confirmed by MN, since the three largest clusters were assigned to lipids and tetrapyrrole-type pigments (Population extracts from Canadian samples yielded significantly lower quantity of extracts (0.3 g ± 0.01) compared to those from Helgoland (0.4 g ± 0.02) and Kiel Fjord  was only present in the inner body of C. intestinalis and two carotenoids  were exclusively detected in its tunic  or tunic samples . Most of the putatively identified compounds, e.g., unsaturated fatty acids , indole alkaloids , and polyunsaturated amino alcohols , were detected in both tissues. Comparative metabolome analysis of individual ascidian samples proved tissue-specific metabolite profiles  showed distinct clustering relating to the three different sampling locations a. HoweveC. intestinalis from different locations revealed that most compounds (80%) were common to all population level extracts. However, 24 metabolites appeared to be unique to one sampling location while 19 metabolites had higher quantities in one sampling site , the sesquiterpenoid antibiotic YM 47525 (60), and the anthracycline polyketide rubomycin M (100), plus six pigments belonging to carotenoid  and tetrapyrrole  classes (12) and d-erythro-4,8,10-sphingatrienine (15), the alkyl sulfate sodium 10-(hydroxymethyl)-2,6,14-trimethylpentadecyl sulfate (79), and the chemical family of tetrapyrroles (41). The two compounds with enhanced production in C. intestinalis from Kiel Fjord could not be linked to any known NP and remain therefore unknown.Of the 29 compounds that showed enhanced or exclusive production in Canadian extracts, nine were putatively identified as the peptide MIP-A3 . Geograsalinity . Firmicudium sp.  that is C. intestinalis. A recent study on the microbiome of colonial ascidian Clavelina oblonga revealed lower haplotype-diversity in the invasive populations, suggesting that lower genetic diversity correlates with the decreased microbial diversity detected in the invasive specimens [In addition to environmental factors shaping the microbiome of the three different populations, haplotype-diversity of the host may also have impacted the microbial diversity of pecimens . HoweverC. intestinalis, we focused on bacterial taxa conveying potential beneficial effects for C. intestinalis , Ruegeria sp. (mainly Canada and Helgoland), and Kiloniella sp. (mainly Helgoland and Kiel) and the Cyanobacterium Synechococcus sp.  are known producers of bioactive compounds such as antimicrobial and antifouling agents [Arcobacter sp. (mainly Kiel), Roseobacter sp. (mainly Canada), and Ruegeria sp. (mainly Canada and Helgoland) play a role in nitrogen cycling [Vibrio spp., observed at all three sampling sites) may be beneficial for C. intestinalis, especially in marine areas with high anthropogenic activity [C. intestinalis. Further investigations analyzing several invasive populations are needed to verify the role of these microbes, in particular those specific to Canadian ascidians, for the global expansion of C. intestinalis.To investigate the contribution of the microbiome to the overall fitness of stinalis . Cyanobad fishes . For insbya spp. ,68, a cyg agents ,70,71,72 cycling ,72 and h cycling ,61. Addiactivity ,61. SpecPrevious studies suggested that both symbiont recruitment strategies, vertical and horizontal transmission, play crucial, complementary roles for survival, but also establishment of invasive ascidians in new ecosystems ,60,61,73C. intestinalis showed a diverse metabolome. Many putatively annotated metabolites were previously isolated from other marine invertebrates indicating a microbial/microalgal origin, as reported for 10-hydroxyphaeophorbide a  [Moorea sp. and Bacillus sp.) of several putatively identified compounds  in the tunicate microbiome. However, we did not observe a clear correlation between the individual tunic microbiomes and metabolomes  . We puta ,81. Furtabolomes , which mC. intestinalis [C. intestinalis [Ruditapes philippinarum [Remarkable location- and tissue-specific patterns were also observed in the metabolome of individual and population samples . Locatiostinalis . Notablystinalis . Salinitppinarum  and ther43, 87, 89, 103, 105) or have antimicrobial activities . Notably, while most bioactive compounds were present in all three populations , two metabolites  were only detected in the invasive population from Canada. The putatively identified antibiotic YM 47525 (60) has been shown to be fungicidal against Candida albicans [C. intestinalis with protection against pathogenic fungi. Likewise, the anthracycline polyketide rubomycin M (100) that was putatively annotated in the Canadian population is a potent antibiotic [C. intestinalis, a finding previously reported from various other ascidians [Similar to the microbiome, we inspected putatively annotated compounds with regard to their known bioactivities. Several putatively annotated metabolites are reportedly cytotoxic  significantly impacted the microbial community composition and metabolite profiles of different C. intestinalis populations. Stable core OTUs and tissue- or location-specific bacterial taxa and metabolites suggest a high degree of flexibility when adapting to new environmental conditions. While additional sampling is needed to verify the role of microbes and metabolites during invasion, our results give first evidence that invasive C. intestinalis contain a richer microbiome and metabolome that may enhance its adaptive capacity in the new environment. Several ascidian-associated microbes have reported bioactivities  or other ecologically relevant functions , potentially contributing to the health and fitness of C. intestinalis. Some putatively annotated metabolites may provide beneficial bioactivities  supporting the ascidian’s chemical defense. Hence, beneficial microbiota and chemical weapons may be relevant factors in addition to other reported characteristics  regarding the global expansion of Ciona species. Additional studies on several invasive and native populations across broad geographical scales will be necessary to understand the contribution of the microbiota and metabolome to its global invasion success.In this study, we simultaneously examined for the first time the metabolome and microbiome of native and invasive specimens of


ArabidopsisrbohD and rbohF transposon knockout mutants and analyzed ultrastructural changes after TuMV inoculation. The development of the TuMV infection cycle was promoted in rbohD plants, suggesting that RbohD plays a role in the Arabidopsis resistance response to TuMV. rbohF and rbohD/F mutants display less TuMV accumulation and a lack of virus cytoplasmic inclusions were observed; these observations suggest that RbohF promotes viral replication and increases susceptibility to TuMV. rbohD/F displayed a reduction in H2O2 but enhanced resistance similarly to rbohF. This dominant effect of the rbohF mutation could indicate that RbohF acts as a susceptibility factor. Induction of hydrogen peroxide by TuMV was partially compromised in rbohD mutants whereas it was almost completely abolished in rbohD/F, indicating that these oxidases are responsible for most of the ROS produced in this interaction. The pattern of in situ H2O2 deposition after infection of the more resistant rbohF and rbohD/F genotypes suggests a putative role of these species on systemic signal transport. The ultrastructural localization and quantification of pathogenesis-related protein 1 (PR1) indicate that ROS produced by these oxidases also influence PR1 distribution in the TuMV-A.thaliana pathosystem. Our results revealed the highest activation of PR1 in rbohD and Col-0. Thus, our findings indicate a correlation between PR1 accumulation and susceptibility to TuMV. The specific localization of PR1 in the most resistant genotypes after TuMV inoculation may indicate a connection of PR1 induction with susceptibility, which may be characteristic for this pathosystem. Our results clearly indicate the importance of NADPH oxidases RbohD and RbohF in the regulation of the TuMV infection cycle in Arabidopsis. These findings may help provide a better understanding of the mechanisms modulating A.thaliana–TuMV interactions.Turnip mosaic virus (TuMV) is one of the most important plant viruses worldwide. It has a very wide host range infecting at least 318 species in over 43 families, such as Brassicaceae, Fabaceae, Asteraceae, or Chenopodiaceae from dicotyledons. Plant NADPH oxidases, the respiratory burst oxidase homologues (RBOHs), are a major source of reactive oxygen species (ROS) during plant–microbe interactions. The functions of RBOHs in different plant–pathogen interactions have been analyzed using knockout mutants, but little focus has been given to plant–virus responses. Therefore, in this work we tested the response after mechanical inoculation with TuMV in  Potyviridae), a virus with a single (+) RNA of approximately 10 kb encoding a polyprotein, which is processed by three virus endogenous proteinases into 12 proteins [Brassicarapa subsp. pekinensis and chinensis (L.), H. Bailey, Brassicarapa (L.), Sinapis sp. and Raphanus sativus (L.). It also attacks Beta vulgaris (L.), Spinacia oleracea (L.) and Nicotiana tabacum (L.) [B. napobrassica crops in Southern Ontario [B. napus in England and Wales [B. oleracea var. capitata [Turnip mosaic virus (TuMV) belongs to the genus Potyvirus (family cum (L.) ,4,5. Exa Ontario  and 70% N. benthamiana and Arabidopsis, together with viral proteins. Secondly, important ultrastructural changes observed during TuMV infection are due to the presence of filamentous viral particles (VPs) and viral cytoplasmatic inclusion (CI) [Tu, was identified in lettuce cv. Cobbham Green [TuRB01 was the first TuMV resistance gene to be mapped in brassicas, in the Brassica napus line N-o-1 [B.napus lines used by Jenner and Walsh [Brassica napus and Brassica juncea plants, TuMV resistance genes TuRB01, TuRB03, TuRB04 and TuRB05. In the case of Arabidopsis, resistance is acquired mostly by the generation of different types of mutants. TuMV infection is not supported efficiently after silencing of HSP70 [DNA-binding protein phosphatase 1 (DBP1) gene does not influence plant growth in Arabidopsis, but does result in resistance to two potyviruses—TuMV and plum pox virus (PPV) [Potyvirus particles are organized as a flexous rod in length and are between 11 and 15 nm in diameter. Akin to other (+) RNA viruses, at the ultrastructural level TuMV causes remodeling of the host cellular membranes to create viral structures called “viral factories” . In the ion (CI) . Generalion (CI) ,14. Howene N-o-1 . Among tnd Walsh ,17, linend Walsh  and othend Walsh . Coutts nd Walsh  identifiof HSP70 ,21. Knocus (PPV) . DespiteNicotiana-Tobacco mosaic virus pathosystem [2-) in the apoplast which are rapidly dismutated to hydrogen peroxide (H2O2). The model plant Arabidopsis thaliana presents a multiple family of 10 Rboh genes [Rboh genes control a wide range of developmental as well as environmental processes. Two of these genes, RbohD and RbohF, are pleiotropic and function together, mediating diverse functions in abiotic stress signaling as well in pathogen responses. These two genes present a different expression pattern during plant development and are differentially modulated during the immune response [The formation of reactive oxygen species  is a regular process in the metabolism of aerobic organisms . These Rhosystem . Plant Nhosystem . RBOHs chosystem . They geoh genes . Analyseoh genes  demonstrresponse . Moreoveresponse ,34. Furtresponse .RbohD and RbohF have been extensively documented in many plant–pathogen interactions, the potential function(s) of Rboh-mediated responses during plant–virus interactions remain poorly investigated. The aim of this research was to perform Turnip mosaic virus inoculation on Arabidopsis rbohD and rbohF mutant plants to assess the role of these NADPH oxidases in this pathosystem. We want to examine whether RbohD and RbohF contribute to signal transduction crosstalk in TuMV–Arabidopsis interactions.Even though the roles of Arabidopsis thaliana wild type Col-0 and the three rboh mutants at 3 and 7 days post-inoculation (dpi). However, OD405 values were higher in the Col-0 and rbohD plants than in rbohF and double rbohD/F mutants , the rbohF and double mutant rbohD/F OD405 values significantly decreased at a later time  clearly showed the presence of TuMV in all  mutants . Moreoveter time . Analyse rbohD/F . MoreoverbohD, rbohF and double mutant rbohD/F at 0 to 7 days after TuMV inoculation, as well as in mock-inoculated plants, were examined. In Col-0, inoculated plants’ virus particles were first noticed starting from 3 dpi, and were mainly located inside the vacuoles of mesophyll cells  and virus cytoplasmic inclusion present in cytoplasm , in contrast to PR1 rbohF and rbohD/F, which displayed the lowest induction with TuMV at 3 dpi . However, the level of detectable PR1 decreased by 1.309- to 2.317-fold between 3 and 7 dpi in all mock- and virus-inoculated plants, with the lowest decrease observed in rbohF and rbohD/F. The amount of PR1 protein in the different lines correlates with the level of TuMV proliferation and the number of virus particles, suggesting that PR1 is a good marker for virus infection and that it could play a role in stopping the virus.The green fluorescence signal localizing PR1 was mainly detected in the epidermis cell wall, but also in the trichomes cells. PR1 was also detected in vascular bundles regardless of genetic background . Analyzi2O2 by cerium (IV) perhydroxide labeling in all four genotypes after TuMV infection and determined the corrected total electron density (CTED) based on the density of cerium perhydroxide precipitates. Analyses of the electron micrographs indicate that the H2O2 was induced by TuMV at 3 dpi as compared to mock-inoculated plants, and decreased subsequently at 7 dpi  in the apoplast near necrotized areas , especially at later times  to upper (non-inoculated) leaves [RbohD in a potato–PVYNTN system [rbohD interactions, it seems clear that RbohD has an important contribution to the resistance response to Potyvirus. In agreement, high levels of RbohD transcript were associated with resistance and hypersensitive response (HR) to PVY [RbohD contribution to resistance is not general to all pathosystems—e.g., rbohD supports less Alternaria brassicola biomass growth than in wild type or rbohF, suggesting that RbohD does not develop a specific antifungal activity and does not act as a resistance factor, but rather could play a role as a cell death regulator [Botrytis cinerea, the responses in rbohD (or rbohF) were not accompanied by an enhanced growth of the pathogen [RbohD.Plant NADPH oxidases have emerged as important regulators of many responses to the environment, and many studies document the role of plant NADPH oxidases in different plant–pathogen interactions ,37,38. Hathogens ,32,36. Wer times . Ultrasts spread A–D. Mores spread A–D. Cons of TuMV . Thus, tbacteria . Moreove) leaves . In viewN system  and comp) to PVY . Howeveregulator . In othepathogen ,44. TherrbohF and rbohD/F display less TuMV accumulation at 3 dpi than Col-0  application, here we quantified in situ the location of H2O2 by histochemical densitometry on the electron micrographs of infected tissues  as the usual source of ROS defense-related ROS generation [2O2 in rbohD correlate with the lower level of necrosis and the greatest susceptibility to TuMV, suggesting that RbohD-dependent ROS contributes to restrict TuMV infection. Similar findings, where downregulation of RBOH-dependent ROS drove the suppression of the HR, have been documented in other plant–pathogen interactions such as in response to the hemibiotrophic bacterium Pseudomonas syringae or the hemibiotrophic oomycete Phytophthora infestans [rbohF also displayed lower ROS production compared to Col-0, this genotype exhibited extensive necrosis and was more resistant, which indicates that RbohF-dependent ROS contributes to negatively regulate the HR and promotes susceptibility to this virus. This reduction in Rboh-dependent ROS with an increase in the HR has also been observed in response to some pathosystems such as to an avirulent P. syringae strain or to the biotrophic oomycete Hyaloperonospora arabidopsidis [Arabidopsis-A. brassicicola interaction [2O2 in rbohD and rbohF mutants suggests that RbohD and RbohF drive ROS generation at different locations, and that these different pools of ROS could be responsible for these opposed functions. This may be related to their differential pattern of expression. Although both oxidases are transcriptionally upregulated by pathogens, they present a differential spatio-temporal expression patter that contributes to fine-tune Rboh-dependent ROS production [rbohD/F displayed additive reduction in H2O2 but presented the same enhanced resistance than rbohF. This dominant effect of the rbohF mutation could indicate that RbohF acts as a susceptibility factor. It has also been documented that Red clover necrotic mosaic virus (RCNMV), a Dianthusvirus, hijacks the host generation of reactive oxygen species during infections [rbohD/F (and rbohF) plants displayed HR-like necrosis, the enhanced dominant resistance in rbohD/F could be associated with the compensatory activation of other defense pathways leading to cell death/necrosis [In contrast to other works, where the H tissues . This H2athogens ,43. Thisathogens . rbohD/Fathogens . Interes present A–F. By cenotypes ,53 and/oenotypes . In addienotypes . H2O2 hall death , with thneration ,36,56,57neration ,48. Evenneration . In our nfestans ,38. Howedopsidis ,59. Thuseraction . The difoduction . Intrigufections  while RCfections . Alternanecrosis .2O2 is able to upregulate the defense gene PR1 [Arabidopsis PR1 protein do not support these ideas. PR1 protein quantification indicated that PR1 deposition was induced as an effect of TuMV inoculation compared to mock-inoculated plants in all genotypes analyzed  presented strong PR1 depositions in the cell wall at the sites where virus particles and inclusions were present. However, in atrbohF or atrbohD/F, the lines where the virus infection cycle was limited, PR1 appeared around multivesicular bodies and rebuilt cell wall areas. These data suggest that more than the amount of PR1 protein itself, the areas where PR1 deposition occur are more important for the limitation of the virus infection cycle. Immunofluorescence localization of PR1 indicates the preferential accumulation of PR1 in the leaf epidermis, within the basis of trichomes, and in vascular tissues (Pathogenesis-related proteins (PRs) are usually induced during pathogen infection and biotic stresses and are classically associated with resistance . It has gene PR1 ,63. Howeanalyzed . Howeversistance . The expat 4 dpi . Noticeato Col-0 . In addi tissues . This obArabidopsis thaliana (L.) Heynh wild type (Col-0) plants and specific mutants—Arabidopsis thaliana rbohD (knockout mutant carrying a single dSpm transposon insertion in RbohD), Arabidopsis thaliana rbohF  and double Arabidopsis thaliana rbohD rbohF  [To determine changes in viral infections induced by Turnip mosaic virus (TuMV) and related to RBOHD and RBOHF, we used mutants) . All hom405nm values were acquired after 60 min in duplicate, 3 and 7 days after inoculation. The mean OD405nm values were statistically assessed with a one-factor analysis of variance (ANOVA), as described in Kozieł et al. [405nm values were computed as presented in [Fifty plants of Col-0 and fifty plants of each type of mutant were mechanically inoculated as described by Tomilson 1970) and Walsh and Jenner (2002) ,70 by us and Walsł et al.  with theented in  and usedented in :Cut-off 405 were compared to the calculated cutoff point and all OD405 values greater than 0.1738 were considered positive (confirmed presence of virus) [Arabidopsis thaliana plants.This calculated cut-off point value was 0.1738. The readings of ODf virus) . SignifiApproximately 15 fragments near the main vein of leaves from mock-inoculated . Slides were imaged under a PROVIS AX70 fluorescent microscope with an Olympus UP90 high-definition camera  and the photos were analyzed using Olympus Cell Sense Standard Software .The virus- and mock-inoculated leave tissues were treated as previously described by Kozieł et al. . To asse2O2 was accomplished using CeCl3 as described by Bestwick et al. [3, washed for one hour with the same buffer, and fixed according to the procedure of Otulak and Garbaczewska [w/v) OsO4 in a cacodylate buffer and dehydrated in a series of ethanol–water washings, as described in Kozieł et al. [2O2, a CTED method was used as presented by Kozieł et al. [2O2. Then, CTED values were analyzed for statistical significance according to methodology presented by Kozieł et al. [Detection of Hk et al.  with chak et al.  and Kozik et al. . The leaaczewska . Samplesł et al. . The matł et al. . Observał et al. . To quanł et al.  which enł et al.  with ANOA.thaliana Col-0 and NADPH oxidase rbohD and rbohF transposon mutants and performed in situ localization and quantification of virus particles, H2O2 and PR1 proteins. TuMV infection in rbohD led to a systemic development of virus infection. The virus concentration and localization significantly increased between 3 and 7 days after inoculation in rbohD, even more dynamically than in Col-0. In this mutant, in situ localization of H2O2 correspond with virus particles and was observed in vacuoles and between the cell wall and plasmalemma. Moreover, quantification of H2O2 showed that rbohD-inoculated plants displayed a considerable reduction in ROS accumulation compared to Col-0. ROS derived from RbohD plays a role in the resistance response to TuMV in this pathosystem. Opposite to rbohD, a significant decrease in virus infection cycle was observed in rbohF and rbohD/F compared to Col-0 or rbohD. Interestingly, rbohD/F response was accompanied by local necrosis, typical for HR-like reactions, with the characteristic rebuilding of cell walls, phenolic-like compounds deposition inside xylem elements, together with the rare occurrence of virus particles and complete lack of virus inclusions. Noticeably, in situ localization of H2O2 in these more susceptible genotypes revealed that the deposits of hydrogen peroxide were differentially linked to the cell wall, near plasmodesmata and in the area around necrotizing cells. This pattern of H2O2 deposition evidenced a possible longer distance transport of these reactive species in these genotypes that could contribute to the enhanced resistance observed. Deposition of PR1 protein was activated in all A. thaliana variants compared to mock-inoculated plants, but the more susceptible rbohD plants displayed a higher level of PR1, whereas rbohF and rbohD/F displayed a lower accumulation. Our qualitative and quantitative analyses indicated that PR1 location strictly parallels the virus alterations, although it is not clear that the PR1 induction acts as an antiviral factor. The important role of ROS produced by NADPH oxidases in regulating the Arabidopsis response to TuMV and a differential role of RbohD and RbohF in this interaction were revealed.In this study we compared the ultrastructural effects of TuMV infection on A. thaliana–TuMV pathosystem. It has been reported that nitric oxide signaling and calcium protein kinases (among other kinases) can modulate NADPH oxidase activity; these analyses may help provide a better understanding of the mechanisms modulating A. thaliana–TuMV interactions.Further molecular and cellular studies are needed to elucidate the possible role of other signaling molecules  that may potentially serve as active components in the