Residents of south Louisiana face a range of increasing, climate-related flood exposure risks that could be reduced through local floodplain management and hazard mitigation planning. A major incentive for community planning to reduce exposure to flood risks is offered by the Community Rating System (CRS) of the National Flood Insurance Program (NFIP). The NFIP encourages local collective action by offering reduced flood insurance premiums for individual policy holders of communities where suggested risk-reducing measures have been implemented. This preliminary analysis examines the extent to which parishes (counties) in southern Louisiana have implemented the suggested policy actions and identifies key factors that account for variation in the implementation of the measures. More measures implemented results in higher CRS scores. Potential influences on scores include socioeconomic attributes of residents, government capacity, average elevation and past flood events. The results of multiple regression analysis indicate that higher CRS scores are associated most closely with higher median housing values. Furthermore, higher scores are found in parishes with more local municipalities that participate in the CRS program. The number of floods in the last five years and the revenue base of the parish does not appear to influence CRS scores. The results shed light on the conditions under which local adaptive planning to mitigate increasing flood risks is more likely to be implemented and offer insights for program administrators, researchers and community stakeholders. Since Hurricanes Katrina and Rita of 2005, risk awareness has grown among stakeholders of coastal Louisiana communities facing increasing flood risks from sea-level rise, intense storms and land subsidence. Like many other coastal regions, population growth along the Louisiana coast combined with limited land use planning has exacerbated these risks. For example, by the end of the 21st century, annual flood costs in the United States could increase from $2 billion to $7–$19 billion because of climate change, urbanization and urban emissions ,4. GivenThe Community Rating System (CRS) of the NFIP provides incentives to local communities to enact collective measures to mitigate flood risks. This analysis builds on the earlier work of several studies that examined contextual factors that may explain variation in CRS participation and helped shed light on the conditions under which local collective action may be more likely. This is especially relevant for researchers and stakeholders of Louisiana, where no previous study has examined CRS participation and given the historical ambivalence among counties and local communities concerning planning and land use management efforts . AlthougThe objectives of this study are to examine the CRS participation rates and performance of parishes (counties) in south Louisiana and to identify key factors associated with greater implementation of the CRS flood risk-reducing measures.The CRS is a voluntary incentive program designed to encourage communities to implement structural and non-structural flood risk-reduction measures beyond minimum NFIP requirements. Participating communities are evaluated and given a score based on the number of planning milestones they have met. The CRS scores reflect a range of activities, including implementation of land-use controls, such as preservation of floodplain as open space, regulation of development in flood-risk areas and watersheds and development of a comprehensive floodplain management plan. These measures result in a discounted flood insurance rate for National Flood Insurance Program (NFIP) policyholders in that community. NFIP discounts flood insurance rates based on a point system that ranges from 5% to 45%, increasing in 5% increments, corresponding to the score, or total number of points received ,7.CRS communities vary in size and may include local municipalities and parishes. Each jurisdiction within a parish has the opportunity to participate in the CRS program and is not considered part of a county-wide CRS program. In other words, if decision makers of an incorporated municipality want CRS program discounts, they must enact their own separate CRS program, distinct from the county program. Thus, the county-level CRS programs cover residents and communities within the unincorporated areas of the county.The CRS program seeks to further three broad goals: to reduce and avoid flood damage to insurable property; to strengthen and support insurance aspects of the NFIP; and to foster comprehensive floodplain management. Following reorganization in 2013, the program focuses on six core flood-loss reduction areas: reduction of liabilities to the NFIP fund; improvement of disaster resiliency and sustainability of communities; integration of a “Whole Community” approach to address emergency management; promotion of natural and beneficial functions of floodplains; increased understanding of risk; and adoption and enforcement of disaster-resistant building codes . The CRSMeasures under this category include those that advise people about the flood hazard, encourage the purchase of flood insurance and provide information about ways to reduce flood damage. These activities also generate data needed by insurance agents for accurate flood insurance rating. They generally serve all members of the community.This series credits programs that provide increased protection to new development. These activities include mapping areas not shown on the Flood Insurance Rate Maps (FIRMs), preserving open space, protecting natural floodplain functions, enforcing higher regulatory standards and managing stormwater. The credit is increased for growing communities.These measures attempt to protect existing development, which is considered to be at risk within the participating jurisdiction. Credit is provided for a comprehensive floodplain management plan, relocating or retrofitting flood-prone structures and maintaining drainage systems.This series provides credit for measures that protect life and property during a flood, through flood warning and response programs. There is credit for the maintenance of levees and dams and also for programs that prepare for their potential failure.Community class rankings in the CRS range from 1 to 10. A Class 1 community can receive the highest insurance rate discount of 45%. A Class 9 community can receive a 5% discount. A Class 10 community either has failed to receive a minimum number of points or has become inactive in the program and does not receive a discount. In order for a community to become a member of the CRS program, it must be in good standing with NFIP regulations (has adopted and enforced NFIP floodplain management regulations that conform to NFIP standards) and appoint a CRS coordinator to handle all application work. Further, the CRS requires that communities actually implement these plans and monitor activities annually as a condition for renewal. Each year, communities must re-certify under the CRS program to ensure that the community is still performing the tasks for which it has received CRS points. Furthermore, a new CRS class will not be enacted until the next point tier is reached. Therefore, a community with 1000 points will have the same CRS class of 8 as a community with 1498 points. CRS class changes occur in May and October of each year. If the community does not renew each year, its residents will lose any NFIP rate discounts [As of October 2015, the CRS program had 1368 participating communities in the United States, or approximately 5% of the total NFIP communities present. Roseville, California, is the only Class 1 ranked community in the United States . LouisiaHistorically, Louisiana communities have been slow to adopt planning measures , despiteIn 2007, the United States Federal Emergency Management Agency (FEMA) ranked Florida, California, Texas, Louisiana and New Jersey respectively as highest risk for flooding based on a composite risk score derived from floodplain area, per capita housing and number of housing. Researchers found that “non-structural” methods, such as those measured by the CRS rating, were more than twice as effective as “structural” measures, such as dams, at reducing the level of damage from flooding ,18. FurtIn England, the Netherlands and Germany, strong flood mapping tools drive planning decisions, as flood management efforts focus increasingly on non-structural methods. However, these tools still run the risk of remaining just that: tools. These programs still see resistance between central and local governments, individuals and professional planners –23. FurtThe CRS program with its incentives to individual NFIP policyholders, prescription of collective risk-reducing measures and annual evaluation of participating jurisdictions is an important resource for local decision makers seeking to reduce flood exposure risks. Previous research has shown that the CRS program does in fact promote discount-seeking activities ,25. The Participation in the CRS program may encourage and support several key attributes of more resilient communities, as identified by resilience theorists. For example, Adger  and collDespite the opportunity to learn and adapt following large disturbances, lack of available information on flooding, inundation, land use and growth patterns can present challenges for community stakeholders to participate in informed decision making and for decision makers to formulate and implement proactive disaster management planning . FurtherThus, the CRS program has an important role to play in Louisiana as community stakeholders work to reduce flood exposure risks. What factors may explain variation in parish-level measures for floodplain management and hazard mitigation evaluated under the CRS program? We turn to recent related research that considers the preconditions and attributes of more resilient communities, and the specific influences on CRS participation in particular, to select variables to include in our analysis.In recent years, researchers have attempted to identify the most suitable indicators to assess disaster resilience. For example, in 2010, Cutter and colleagues  introducSeveral studies have examined influences on community and household-level disaster planning. First, experience with recent floods has been found to be associated with greater community interest in and acceptance of collective planning efforts . RegardiOther potential influences on parish-level adaptive planning in general are the capacities and resources of the parish government. Since planning occurs at the sub-federal and sub-state level ,43, the Finally, recent studies examining specific influences on CRS participation and implementation of risk-reducing measures point to the importance of hydrological conditions, the socioeconomic attributes of residents and government capacity. Our study builds most directly on the work of Landry and Li (2012) in which they examined the CRS participation of 100 counties in North Carolina from 1991 to 1996 . They teThese studies suggest that socioeconomic attributes of residents, county government capacity, physical factors, such as elevation, and experience with recent flood events may influence the level of CRS planning. Thus, we include indicators of these conditions and attributes of the parishes (counties) within the south Louisiana study area.As stated previously, this analysis examines the extent to which southern Louisiana parishes have taken steps to exceed NFIP requirements to reduce local flood risks and identifies the factors that account for variation in the CRS scores among the parishes.The sample selected consists of the 35 parishes of South Louisiana, listed below in The dependent variable is taken directly from each parish’s CRS score. We used this score (as opposed to the CRS class level) in order to be able to statistically analyze a continuous variable. The scores range from a low of 0 to the highest parish score of 2213.The independent variables included in this analysis are summarized below in We began by conducting a Pearson correlation analysis among the variables to identify any potentially highly correlated independent variables. Next, we conducted a multiple regression analysis to determine the relative statistical associations between the independent variables and the CRS scores. We conducted the analysis in SPSS Version 21. The choice of the analysis was appropriate given that the dependent variable, the CRS score, is a continuous variable. The descriptive statistics for the dependent variable and the independent variables included in the study are shown in Our first research objective is to examine the level of participation in the CRS program among counties (parishes) in south Louisiana. Of the 35 parishes in the study area, 15 have achieved CRS class rankings. The second research objective is to identify key factors that may explain variation in the CRS scores. We conducted a Pearson bi-variate correlation analysis to identify statistically-significant associations between the variables in the analysis before constructing the multiple regression model. We considered a significant correlation value of 0.7 or higher to indicate a high degree of multicollinearity. We found that the percentage of college-educated residents was significantly and positively associated with the average housing value within the parishes, with a Pearson R of 0.770. Therefore, we did not include both variables in the regression analysis. We selected the housing value variable for further analysis, because it provides an indicator of not only economic resources, but also tangible assets that may be damaged by floods. Since none of the other independent variables were found to have Pearson R values of greater than 0.7, these five were retained for inclusion in the regression analysis. The results of the Pearson analysis are summarized below in Next, we conducted a multiple regression analysis using the five selected independent variables. The results of the analysis are summarized in et al., 2015, and Cutter et al., 2009 [The regression analysis yielded an adjusted R squared of 0.571, indicating that these five independent variables explained 57% of the variation in the parish CRS scores. Higher CRS scores are associated most closely with higher average housing values. The distribution of housing values within the study area is illustrated in l., 2009 ,47, for Regarding the capacity of the county governments, the presence of more municipalities participating in the CRS program within a parish is significantly associated with higher CRS scores at the parish level. This finding is not surprising and supports the conclusions of Landry and Li in their study of North Carolina counties’ CRS participation from 1991 to 1996 . The preThe finding that average county elevation is not significantly associated with CRS planning activities is consistent with previous research of Florida counties conducted by Zahran in 2010 . In LouiThe objective of the analysis was to examine the context under which coastal parishes (counties) may be more likely to take steps to make themselves safer through floodplain management and other measures encouraged by the CRS program. The results of the regression analysis indicate that higher CRS scores are found in parishes with higher housing values and with a higher number of municipalities within the parish that also participate in the CRS program. Surprisingly, indicators of greater exposure to flood risks, including lower mean elevation and past flood events, were not found to be significantly associated with greater participation.The CRS program is an important effort by the federal government to encourage local governments to become more proactive and adaptive in flood hazard mitigation planning. As the National Flood Insurance Program (NFIP) faces major deficits, this incentive-based approach to spur more collective floodplain management activities among county and local jurisdictions is compelling. Are there key contextual factors that may affect the extent to which local jurisdictions are willing or able to participate?This analysis of southern Louisiana parishes indicates that acceptance of the incentives offered through the CRS program to move toward more collective hazard mitigation efforts may be influenced to a large extent by the socioeconomic attributes of the parish. These findings are consistent with prior related research in suggesting that more affluent communities with more valuable housing and property are more likely to achieve higher CRS scores. Furthermore, consistent with the Landry and Li study of counties in North Carolina, this analysis found that the presence of more local jurisdictions within the parish that also are participating in the CRS program is associated with higher county CRS scores . The pre


Pakistan’s Maternal, Newborn and Child Health (MNCH) Program is faced with multiple challenges in service delivery, financial and logistic management, training and deployment of human resources, and integration within the existing health system. There is a lack of evidence on managerial aspects of the MNCH program management and implementation.This study used qualitative methods to explore the challenges national, provincial and district program managers have faced in implementing a community midwifery program in province of Punjab while also exploring future directions for the program under a devolved health system. While the program had been designed in earnest, the planning lacked critical elements of involving relevant stakeholders in design and implementation, socio-demographic context and capacity of the existing health system. Financial limitations, weak leadership and lack of a political commitment to the problem of maternal health have also had an impact on program implementation.Our study results suggest that there is a need to re-structure the program while ensuring sustainability and collaboration within the health sector to increase uptake of skilled birth attendance and improve maternal health care in Pakistan. Since the introduction of the primary health care (PHC) concept , community based health worker (CBHW) programs flourished substantially, especially in developing countries –3. ThesePakistan’s first community health worker program, National Program for Family Planning and Primary Health Care (NPFPPHC), commonly known as the Lady Health Worker (LHW) Program was initiated in 1994 . This prIn 2006, Pakistan initiated another community health worker based program, titled Maternal, Newborn and Child Health (MNCH) program with a bid to improve maternal health indicators, particularly those related to the MDGs. Under this program, midwives were trained and licensed to practice maternal health care services  in rural communities, with the aim to increase skilled birth attendance and reduce maternal mortality . So far,The government systems in Pakistan underwent a major restructuring under the democratic reform of devolution in 2011 resulting in a redefinition of the health related mandates at federal, provincial and district levels –17. TheoThe existing literature on Pakistan’s community midwifery program focuses primarily on service delivery, distribution and access aspects of the deployed midwives. There is a lack of evidence on managerial aspects of the MNCH program management and implementation. The present paper’s objective is to explore the challenges national, provincial and district program managers have faced in implementing a community midwifery program in a rural district of Punjab and the future directions for the program under a devolved health system.The data presented in this paper are part of a larger mixed-methods study that aimed to assess challenges faced by the Pakistani CMWs in providing services to women in rural Punjab, Pakistan. Data for this study were collected over a 3-month period in 2011 in district Attock, Punjab. To explore the range of challenges involved, qualitative data were collected from program managers, community midwives and local community members . Alongside, a comprehensive review of relevant policy and planning documents was also conducted.District Attock was selected for this research as it was among the first regions in which MNCH program was launched and had sufficient numbers of CMWs deployed, for a significant time period , to assess coverage. The overall development, geographical and social characteristics of the district matches that of Northern Punjab and Khyber Pakhtunkhwa provinces of Pakistan. Furthermore, MNCH program in District Attock is funded and managed by the Government of Pakistan with no donor support. The program hence has resource constraints reflective of the situation in most of the districts of Pakistan. This unique perspective of the district made this area an ideal location for exploring the challenges faced by community based health workers in providing maternal health care services.Data for this manuscript has been drawn from program policy documents reviewed and interviews conducted with program managers. The reviewed policy documents included Planning Commission documentation (PC– 1) and Deployment guidelines for Community Midwives. These policy documents detailed program objectives, interventions to be adapted and services to be provided to improve infrastructural and behavioral issues associated with low utilization of maternal health services in rural areas of Pakistan. Deployment guidelines developed for community midwives were also reviewed in detail to develop an understanding of CMWs’ scope of work and responsibilities.Open ended, in-depth interviews were conducted with purposefully selected key actors involved in program planning and implementation process at the federal, provincial and district level, namely nine national, provincial and district program managers. Group interviews were conducted with seven (07) participants .All the interviews were conducted in interviewees’ offices at a time convenient to them. Interview guides were used to obtain information from the interviewees. Interview questions developed and refined by the core research team were aimed at soliciting experiences and opinions of the participants about the study question.All interviews were recorded and transcribed into English for further analysis. A random sample of the transcripts was checked by the authors for translation accuracy.A thematic content analysis approach was adopted for analyzing content of the interviews, as an on-going iterative process through which issues and challenges regarding program management and implementation were identified early on. This allowed for a rigorous probing in subsequent interviews . Open coThe study observed various procedures to enhance the credibility of the research findings. All the policy makers involved in the implementation of the program were included in the study. Data triangulation included use of policy documents and primary data collection through individual and group interviews. A peer coded two interviews independently and codes were reviewed for consistency. Throughout the data collection and analysis debriefing within research team , peer discussions and respondent validation were undertaken, thereby ensuring interpretative accuracy of the results.Ethical clearance for this study was obtained from bioethics committee of Pakistan Medical Research Council. A formal letter was sent to each interviewee to explain objectives of the study while requesting an appointment for conducting the interview. Interviewees were assured anonymity. Written, informed consent was obtained from all participants prior to the interviews, while ensuring complete anonymity and confidentiality.In depth interviews with nine (09) main actors in Pakistan’s MNCH program, namely the national, provincial and district managers, development partner and representative of the midwifery training regulatory authority (Pakistan Nursing Council–PNC) were conducted. The interviews were analyzed using the policy analysis framework developed by Walt and Gilson . ResultsStakeholders are at the heart of policy and program development, influencing program design, content, strategies, and implementation modalities; however, they cannot be separated from the organizations within which they work. This theme attempts to identify the different actors involved in the MNCH program development process, the level of influence they exercised and their authority within the context of this program.The Maternal, Newborn and Child Health program for Pakistan was developed in the wake of an MNCH strategy, launched in 2005. The main actors involved in the development of the MNCH PC -1 were the then Ministry of Health (MoH) and Department for International Development (Dfid).The respondents shared that during development of the program policy and implementation plans, relevant personnel were not involved. Although the Director General for Health was part of the team developing the program but inputs were not taken from provincial and district Health Departments. Principals of Nursing Schools where midwives were to be trained were also not engaged in this process; especially planning pertaining to training and deployment of midwives for community services.‘DG Health was involved in planning, but not the EDOs and Principals of Nursing Schools. We have Nursing instructors with facility-based training skills, but we do not have a single trained midwifery tutor in Punjab to teach midwifery skills for community bases services.’ (MNCH Manager)Community based Midwives, a major component of MNCH program were envisaged to provide home based maternity care in rural areas. This was similar to the flagship program titled National Program for Primary Health Care and Family Planning (NPPHCFP) implemented for providing primary health care and family planning services through lay community health workers  initiated in 1994; however, the national manager for this program was also not involved in planning and development of the MNCH program.MoH developed the PC-1 detailing objectives, implementation strategies, operational arrangement and estimated costs. MoH and Dfid each committed to fulfill fifty percent of the program costs; UNFPA, USAID, UNICEF and other international organizations financed selected components in the program, through MoH. UNFPA was the main sponsor for deployment of CMWs in selected districts; USAID, through its program PAIMAN sponsored supplies for CMWs in its ten adopted districts while the remaining were supported through the funds available to MNCH from MoH.All the respondents reported external influences from donors and prominent local political figures in program design and implementation. The national and provincial program managers highlighted that international partners’ agendas influenced program design, its components, and the services package to be included. Allocation of budget for a preferred program component was hence driven by the donors.‘The partner organization and Government of Pakistan were to contribute 50% each to the total cost of the program. The partner however, did not follow through with the payments on time’. Political interference was reported at all levels, affecting allocation of funds, program implementation, hiring of staff including candidates’ selection for midwifery training.“We asked the district officials to conduct a mapping study of their districts to identify number of villages and midwives required for each district but, unfortunately, a tug-of-war started there for control of finances, and due to political interference the districts started haphazard and immediate recruitment’ (Development partner)In analyzing the program implementation process, it was noted that the important stages of the health policy development  were followed but not completed and program planning experienced problems that affected implementation process. Although the program was developed to cater to current maternal health indicators of Pakistan but in this process involvement of provincial and district governments in agenda building and program planning was found to be almost nonexistent.The specific problems, which had an effect on the program planning and implementation process, were centralization, shortage of trained public health professionals , influence of a narrowly focused biomedical model of health in midwifery training and financial resource constraints. Centralization also hindered a wider participation from other important stakeholders such as district health departments, NGOs, and professional groups of nurses and midwives.Interviewees at provincial and district levels expressed frustration with limited financial and administrative authority delegated to them. The provincial MNCH department was dependent on quarterly release of funds from the national MNCH for managing procurements and staff salaries for all districts.Similarly, the district MNCH officials did not have any financial authority with limited administrative powers. Managers found this limiting authority to be frustrating as they could not purchase requisite materials when needed nor did they have power to hire or layoff district program personnel. Similarly, funds transferred from national to provincial to district program offices were frequently delayed resulting in delayed transfer of staff salaries. The provincial and district management countered this problem by re-appropriating funds from other budgetary heads for managing routine affairs of the program.Considering that devolution of the health ministry was imminent at time of data collection, there were no post devolution plans for transfer of authority, financial management and long-term sustainability. The Punjab health department was at the time developing plans for an integrated reproductive health and nutrition program. However, it has been four years post devolution and till date an integrated program is yet to materialize. It was the general opinion at district level that all vertical programs should be merged and integrated within district health department. The development partner raised concerned with the limited capacity of the provincial health departments for managing this program after devolution.‘If after the devolution, Program is devolved to the provinces, then I don’t know how they will run it’ (Development partner)‘After the devolution, there will only be funding flow form the federal government. There is a lot of confusion, things are not clear yet. But we understand that the federal funded programs will stay on and Punjab will have an integrated RH program’. (Program Manager)There was a considerable delay in hiring key program personal at all three administrative levels. National manager for the program was hired three years after initiation of the program and in interim period the program was run on ad-hoc basis. Punjab Provincial manager revealed that several districts still did not have a regular Public Health Specialist for managing the program at district level and this responsibility was given as an additional charge to either a district health official or the national program’s district official. Most of the district officials working for the program were on deputation from the health department. This led to the issue of frequent staff transfers thereby affecting implementation of program activities in the districts.Regarding training and management of community based midwives, all respondents highlighted registration and deployment, key component of the MNCH program, as a particularly weak area requiring much improvement for achieving intended targets.A common concern raised by provincial and district respondents was that of registration of the trained midwives prior to their placement in rural communities. At the time of data collection, 4700 midwives had been trained but only 2800 had been registered and issued license to practice. The delays in registration were primarily due to inability of graduates to pay registration fees and incomplete documentation sent by the MNCH program to PNC. The Registrar PNC informed that registration fee issue was resolved through donor funds. However, issue of missing requisite documents was still causing delays in registration.Respondents at National program management level highlighted that midwifery component of the program took three to four years to establish with the result that although midwives were trained but due to delays in registration and absence of any deployment guidelines, several trained midwives had either gotten married and relocated to some other locality or taken up a private sector job. This resulted in considerable loss for the program given the considerable costs involved in training CMWs.‘The program needs a strategy to keep the CMWs attached to the program as it will be difficult to bring back those lost to the private sector’. (Development partner)A monitoring and evaluation process for assessing progress of the program had been defined and devised but not implemented. Delivery of supplies to strengthen health system for providing maternal and childcare was recorded but this information was not associated with service delivery targets. Similarly, the program had plans to integrate with national Health Management and Information System (HMIS) but it had not been linked up with it. Although the district officials continued to send a monthly report to provincial head quarters, this information was not reflected in HMIS.Reference to field supervision and monitoring of midwives, the program had divided it into technical and administrative components; technical field monitoring of CMWs’ performance was responsibility of the district tutors and administrative supervision was domain of a Lady Health Supervisor (LHS), an employee of the National Program. Issues highlighted by district program officials were that this fragmented mode of monitoring and supervision was ineffective and not comprehensive. Neither of the two intended field supervisors had been given a formal training or a job description of their task. The tutors at times were not available for making monthly visits due to their routine job activities. The LHS, on the other hand, was not given a salary or other support for field visits and hence considered such supervisory visits additional burden.The CMWs were trained to refer complicated and emergency cases to a health facility providing obstetric care. They were given a referral form for such situations; however, the referral sites had not been defined and no mechanism had been defined to solicit feedback from the facilities regarding referred cases. These lacunae in program design made it difficult to monitor progress and performance of a CMW and subsequently make requisite improvements.In analyzing the content of MNCH program, an analysis of the program documents and deployment guidelines developed for CMWs was undertaken. PC-1 of MNCH program defined the objectives of the program which were geared to increase skilled birth attendance for achieving MDGs 4 and 5. The CMW were proposed to be a cadre of community-based health workers, who would meet the international definition of skilled birth attendants. However, in practice, there are several shortcomings in the implementation, as explained in the previous section.The training curriculum for CMWs was adapted from international sources, based on a biomedical model and was hence not context specific. This was compounded by a dearth of CMW tutors across the country thus affecting teaching, which was done primarily by nursing tutors and obstetricians in a clinical setting. Service structure and career path for the inducted CMWs was also not defined. The program intended to increase SBA but development of training institutes for CMWs had not been addressed. The PC-1 had not defined mechanisms for the financial sustainability of the program beyond 2012.Training curricula of midwives falls under the domain of Pakistan Nursing Council (PNC), the regulatory body for nursing and midwifery practice in Pakistan. PNC revisits and revises nursing and midwifery curricula every three years based on recommendations of teaching faculties; however, the midwifery curricula had not been revised prior to initiation of midwifery trainings under MNCH program. The PNC respondent highlighted that irrelevant teaching faculty was training the midwives and their curriculum had more of theoretical content rather than practical and field based training. Also teaching program was not standardized across the country resulting in varied level of training conducted within the resources available to different midwifery schools. In schools where medical doctors taught, training was deficient in terms of field practice. Similarly, the midwifery curriculum did not specify practices that a community midwife should adopt and avoid in a community setting.‘Most of the trainers have no concept regarding the role of CMWs so they teach them biomedical models of reproductive health, genetic abnormalities, sexology. A midwife does not need that. They should have trainers from the same field (nursing) so that they develop the concept of theoretical, clinical and practical training required for CMWs’. (PNC representative)Practical training of midwifery students is conducted at the District Headquarter Hospital (DHQ) where nurses also get trained. The current practice did not provide them with the requisite time for learning and practicing maternal and delivery care skills. Furthermore, the lack of trainers was also highlighted by the district program officials.‘The DHQ is small with a lot of students attached i.e. nurses, LHVs and now CMWs. Number of trainees in DHQ Attock is more than the trainers can cover (more than 200 students in each class). Nurses and LHVs dominate the training so the CMW can only observe (in the DHQ)’. All program managers were concerned about the high attrition and drop out of trained midwives and midwifery students as this was a waste of program’s limited funds. One of the factors leading to drop out during training was lack of appropriate facilities in hostel accommodation and arrangements for married students and their children.‘There are funding provision in PC1 for civil work, but surprisingly there is nothing for support staff  or utility bills. In most places students are cooking themselves. Should they concentrate on training or cooking?’ (Program Manager)Another factor highlighted as leading to drop out was lack of proper orientation regarding job description, large catchment population, lack of transport for field visits, a low pay of PKR 2000 (approx. USD 20) and lack of a career path.‘We do not give them proper orientation (information) either, before start of training. We should inform the families and candidates about the scope of work before hiring so that only those should join who can willingly work in community setting’ (Program Manager)Other concerns raised by national and provincial program managers were lack of a retention policy for the trained midwives and long-term financial sustainability of the program.The trained midwives were required to serve under MNCH program for a period of three years (mandatory service bond) during which time they were given supplies and a retaining fee. After this time period, the program had no planned policy for long term follow up and options for retention in program.Similarly, with the devolution imminent, the interviewees reported that there were no final plans for program continuity. The PC– 1 for all vertical programs were planned till the year 2014 during which time they would receive an annual budget from the federal budget. However, after that time, provinces would be responsible for managing all aspects of the programs including procurement, human resources management, field operations, etc.‘Long-term sustainability of the program is shaky as government has no money’ (Development partner)Provincial and district program managers identified a general lack of ownership by the health department as a major factor, which could impact long-term sustainability of the program. They were of the view that in the current scenario of donor funds, the program was continuing its operations within the resources made available to them but there should be more focus on results based financing‘There is no clarity on the future modalities of Program management and directions / guidelines form the federal level. The structural integration as proposed in Punjab PC1 is also unclear.’ (MNCH Manager)The MNCH program had two main components, the first of which was to strengthen district health department through technical and managerial capacity improvements, streamline services for provision of basic and comprehensive emergency obstetric and newborn care (EmONC) and integration of MNCH services at district level. The second major component of the program was to launch a cadre of community based skilled birth attendants .In analyzing program context, it was revealed that political instability influenced all levels of program leading to weak institutions, low priority for health services provision and a delayed program implementation. The prevalent financial crunch impacted overall governmental expenditures on health with the result that release of funds to provincial MNCH program was delayed. Despite provision of requisite inputs  for provision of EmONC service at primary health care level , district health system was unable to follow through. However, systemic public sector health department problems of vacant positions, absenteeism and deficient infrastructure compromised service delivery. All these factors hindered effective provision of quality services.Respondents at national level flagged the concern of deficient service delivery in districts and their compromised capacity to deliver services at par with those proposed in the program PC-1. They further admitted that they were forced to work with whatever resources were available to them.‘By now, we should have spent almost 55–60% of the allocated funds, as opposed to 30% that we have received so far. Overall, in this Program we have not moved as it was supposed to’. (Development partner)The community based skilled birth attendant (CMW) component of the program was not entirely context specific. The defined selection criteria (as per PNC regulations) required a candidate to be between the ages of 18 to 45 years, preferably married, have ten years of formal education with 45% score in secondary school exam. These selection criteria were compromised in several districts. Young married women were not permitted by their family to join the program and the older married women had compromised knowledge. In many cases institutes had to teach the midwifery students basic reading and writing skills as well. Students were required to reside in hostel accommodation during training period but because of lack of facilities, lack of accommodation for married women, many of the students dropped out of the program. The students who remained in training were mostly unmarried young girls. Furthermore, there was no policy in the program pertaining to maternity leave and benefits for midwifery trainees.‘Most of the students are from poor families, and have no support system to leave their children at home while attending classes. According to PC-1, this (midwifery) has to be residential training; now what would a married woman with children do? We have given relaxation in Punjab; come in the morning and leave at 3:00 p.m. but patient load is more in evening or at night. So their training suffers. Tutors complain that married students are frequently absent, sometimes their kids are not well, sometimes, mother-in-law is sick, etc. etc. The young unmarried students are so young that in our culture it is not acceptable that they talk about reproductive health. To expect women to accept them and have them conduct a delivery is a big challenge. These are strange problems; unless you experience these, you don’t foresee them and hence don’t plan to address them’. The midwifery regulatory authority highlighted that they have been requested to revise selection criteria so that single women and those with lower grades may also apply thereby increasing the pool of applicants. Registrar PNC highlighted that some of the districts had relaxed the selection criteria at their own behest. This had caused problems for graduates in setting up a practice in community settings.“Even if a woman is 40 years of age but is not married, omen do not want to avail maternity and delivery services from them as they have a pre-conceived notion that they are not aware of maternal care issues.” (PNC representative)Local cultural values had also not been incorporated in deployment guidelines whereby each CMW is allocated a catchment population of 10000, with no transport facility to move about in designated area. The patriarchal culture and religious values practiced commonly in Pakistani society, especially rural areas, have particularly hindered mobility of young CMWs in their catchment areas.Existence of private maternity care providers, which include both trained and untrained professionals, has also not been factored in design of the CMW program. Preference for private care providers , 26 has The study results provide valuable insights from program managers involved in development and implementation of a community health worker based, vertical program for maternal health care. The program failed to achieve its desired impact primarily by not involving key stakeholders from within the health system, midwifery training institutes and parallel program in the design stage. This resulted in implementation struggles at all levels. The MNCH program had been designed for a centralized health system with the result that following devolution, the program is struggling to regain its already weak footings. The main reason for this appears to be a lack of health policy makers and planners ‘capacity for managing health services in a devolved system. Furthermore, financial crisis and lack of a sustainability plans appear to have affected optimum program functioning.Financial sustainability of programs with pre-defined targets needs an uninterrupted support to enable the program to achieve its goals. Major reasons for low and interrupted financial support of the program appear to be lack of funds, devolution of the health services and the financial crisis in health sector. A lack of funds and delayed disbursement has been cited as the major reason for the MNCH program not being able to establish and function optimally. A chronological dissection of the program management reveals breaches in financial flows by all the development partners, namely government and international development partners. The program also lacked a sustainability plan; it was a planning presumption that CMWs would be assimilated within the existing health infrastructure and be accepted by the community without any resistance from other health care providers. However, considering this as a handicap due to the program being in ‘infancy’ stage, the program had started functioning as per the program plan, albeit with deficits. The successes achieved by the program in terms of streamlining financial management received a major setback due to the devolution of health services management to the provinces. At time of devolution, financing mechanisms for the provinces were yet to be developed, with the result that financial disbursements for the programs have been retained at the federal level till the year 2016. This has added a further complexity; implementation and administrative management at provincial health department with financial control at federal level is still a hurdle for achievement of program targets.Historically, health sector in Pakistan has been allocated a budget of less than 1% annually; primarily due to focus on defense and development. Lack of a political commitment for social sectors, natural and manmade disasters and a global financial crisis have been cited as the common reasons for a low budgetary allocation for the health sector.th constitutional amendment, health institutions were decentralized to provincial level and made Autonomous Medical Institutes in 1990 under the World Bank funded Social Action Program [th Constitutional Amendment, which has effectively devolved the health services to the provinces in its entirety [The government of Pakistan has been ‘experimenting’ with administrative reforms of decentralization and devolution since the mid 1990s . Prior t Program . The aim Program , 28. Theentirety . DespiteThis confused state of affairs is apparent at the MNCH program management level as well, with national, provincial and district managers struggling for effective implementation but not unable to achieve due to lack of administrative and financial controls. Considering the extent of MNCH Program services and the nature of its functions, it is imperative that financial as well as administrative authority for program management be entrusted to the district health department. This would give the program an operational fiscal and administrative space for improving service delivery within the district. Dependence on a centralized system for management will continue to have an impact on overall efficiency of the program.The MNCH program, in theory, relies heavily on collaboration with the National Program and the public sector health system at district level. However, there is a need to clearly define the parameters for this collaboration, identifying the roles and responsibilities for each of the partners and their stakes. Lack of clearly assigned roles is resulting in the program not functioning at its optimum. The CMWs, an instrument of the program for achieving an increase in uptake of skilled birth attendance, are faced with multiple setbacks in establishing their services due to a lack of support from the district health system, fragmented supervision and vague feedback . ProgramThere is also a need to define clearly the ambit of the program, taking care to avoid duplication of interventions already in place for improving maternal health and increasing uptake of skilled birth attendance. At present, two other public sector organizations/programs are concurrently providing maternal care services–the NPFPPHC and the district health system. The LHWs are required to provide ANC care and refer pregnant women to the local Rural Health Center (RHC), where women are given an incentive for a facility based delivery along with free delivery services , under tth Constitutional Amendment. The program was then handed over to provincial managers for regional management. This lack of a committed leadership resulted in the program facing challenges in implementation including financial lapses, provincial implementation, deployment of personnel, partnerships and coordination within and between the other government run programs and health system. Success in achieving the targets of vertical programs is as much dependent on the program planning and implementation as it is on the management and leadership [The MNCH program has been subject to insufficient . The national manager for the program was appointed three years after the program plan had been developed and implemented; in the interim period, the then Ministry of Health officials managed program as an additional charge. However, the national manager worked with the program for a limited time period of two years due to the changes in health sector following the 18adership . The othadership , 32. ConThe administrative instability resulting from absence of a national program manager and health sector reform of devolution has had an impact on effective program implementation, delayed deployment of service providers and lack of integration within the existing health system. Health policy makers and program managers should continue strengthening the services delivery under this program by ensuring availability of trained midwives, improved quality of services, and adaptation to the contextual factor arising from the health sector devolution using context specific interventions. Renewed focus on ensuring sustainability and collaboration within the health sector is needed to increase uptake of skilled birth attendance and improve maternal health care in Pakistan.


Drosophila, glucose can activate genes via the transcription factor carbohydrate-responsive element-binding protein (ChREBP) to induce glucose utilization and de novo lipogenesis. Here, we identify a termite orthologue of ChREBP and its downstream lipogenic targets, including acetyl-CoA carboxylase and fatty acid synthase. We show that all of these genes, including ChREBP, are upregulated in mature queens compared with kings, sterile workers and soldiers in eight different termite species. ChREBP is expressed in several tissues, including ovaries and fat bodies, and increases in expression in totipotent workers during their differentiation into neotenic mature queens. We further show that ChREBP is regulated by a carbohydrate diet in termite queens. Suppression of the lipogenic pathway by a pharmacological agent in queens elicits the same behavioural alterations in sterile workers as observed in queenless colonies, supporting that the ChREBP pathway partakes in the biosynthesis of semiochemicals that convey the signal of the presence of a fertile queen. Our results highlight ChREBP as a likely key factor for the regulation and signalling of queen fertility.Termites are among the few animals that themselves can digest the most abundant organic polymer, cellulose, into glucose. In mice and  The basal stage . Becausefat body ,17, the fat body .Prorhinotermes canalifrons , have an alternative reproductive strategy involving secondary kings and queens . This reproductive option is open to immature stages and castes, such as workers (stricto sensu pseudergates) and nymphs, and is accompanied by the rapid development of reproductive organs and onset of reproductive activities following the moult of the immature termite.An important fact that has not been taken into consideration for the study of ChREBP so far is that glucose is the basic component of cellulose. Although cellulose is the most abundant organic polymer on Earth, only few animals can digest it. Among them are termites (Insecta: Blattodea: Termitidae), whose ecological success stems partially from their adaptation to use recalcitrant plant lignocellulose as their primary food source through symbiotic associations. Termites consume an estimated 3–7 billion tonnes of lignocellulose annually, with 74–99% of the ingested cellulose being hydrolysed . AnotherNasutitermes sp. , the reproductive strategy is usually shifted towards the production of a high number of winged dispersers by a highly fecund long-lived physogastric (hypertrophied abdomen) primary queen and her mate (the primary king) assisted by numerous permanent workers and soldiers. This sophisticated colony organization, characterized by large, stable, well-defended and provisioned nests, is primarily maintained through pheromone communication that plays a major role in maintaining reproductive monopoly by the queen. In three eusocial insects , a conserved class of fatty-acid derived pheromones (saturated hydrocarbons), overproduced by queens or fertile individuals, functions as sterility-inducing pheromones and as a signal of fecundity [Cryptotermes secundus , queen-specific pheromones act as a fertility signal to indicate queen presence and to prevent nest-mates from reproducing [In the honeybee, a specific diet and, more generally, the nutritional status influence caste determination and behavioural development ,24. The ecundity . This reecundity . In the roducing .Because termites use cellulose as their main carbohydrate source, we hypothesized that this dietary specialization may have placed a selective pressure on the ChREBP pathway in termites. This prompted us to investigate the role of ChREBP as a putative control factor involved in nutritionally driven regulation of the phenotypic plasticity of termite caste systems. We report here the caste-dependent expression pattern of ChREBP and its lipogenic target genes in eight different termite species. We also describe ChREBP expression during caste differentiation. Moreover, we show the impact of the diet on ChREBP gene expression, and finally, via a combination of a pharmacological approach and behavioural assays, we identify ChREBP as a fertility-signalling factor in termites.2.2.1.Zootermopsis nevadensis [Macrotermes natalensis [Homo sapiens, Mus musculus, Drosophila melanogaster and Apis mellifera were aligned and compared with those of the termite sequences. The comparison indicated an evolutionarily-conserved termite ChREBP of 1111 amino acids with a molecular weight estimated to be 112 kDa . The structural organization of regulatory and functional domains, such as the glucose-sensing module (GSM), was evolutionarily conserved among all ChREBP orthologues [H. sapiens and M. musculus) and termite  GSM ChREBP amino acid sequences exhibited 45–46% sequence identity.We performed a BLAST search using the mammalian ChREBP coding sequences as bait, and this identified a single homologous gene in the two available published termite genomes: vadensis  and Macrtalensis . To evalhologues . MammaliUsing Bayesian inference (BI) and maximum-likelihood (ML) methods of tree reconstruction, we performed a phylogenetic analysis of the GSM amino acid sequences. The alignment contained 331 amino acids from 45 sequences from vertebrates (human and mouse), arthropods including a xiphosuran, eusocial insects  and solitary insects , and a mollusc. The two methods of phylogenetic reconstruction produced ChREBP trees with very similar topologies  analysis using specific primers showed that ChREBP was upregulated in mature primary queens and in mature neotenic queens compared with young primary queens, alate females, male reproductives and sterile individuals (a). Prorhinotermes canalifrons workers are totipotent and can rapidly develop into male and female secondary reproductives (neotenics) when the primary queen or king disappears [b). This suggested that ChREBP might play a role in the regulation of caste differentiation, specifically into the queen phenotype.Because of its high developmental plasticity, we investigated ChREBP expression in ividuals a. Prorhisappears . When thP. canalifrons, the queen exhibits a prodigious fecundity due to a gradual increase in the maturation of ovaries and continuous growth over time. This exceptional growth of an adult insect, known as physogastry, is unique to the termites [c). Furthermore, the difference in ChREBP expression observed between young and mature queens represents a clear link between ChREBP and fertility, since young primary queens (4 months old) were only slightly physogastric, while mature primary queens (4 years old) had reached maximum physogastry (a). In addition, in all other termite species studied, the highest ChREBP expression was detected in the most physogastric and hence the most fertile queens based on the observed number of ovarioles (data not shown).In Termitidae, and to a lesser extent in termites . We compsogastry a. In addP. canalifrons using a commercial antibody generated against the human ChREBP peptide. As seen in the electronic supplementary material, figure S3, indeed this antibody showed a high specificity for the ChREBP termite protein. Consistent with the mRNA profile, the ChREBP protein was detectable in protein extracts from queens (d), and we also detected the protein at the cellular level using high-resolution panoramic colourimetric and immunohistological imaging (e) of different metabolic tissues, but was absent in muscles , which are ChREBP target genes necessary for de novo synthesis of fatty acids [The localization of ChREBP in termite queen tissues, its function in sugar tolerance in the fat body  and its fat body  stronglyty acids ,5,7,9.2.2.Drosophila [P. canalifrons female neotenics and Nasutitermes sp. queens compared with workers. Indeed, we found a two to threefold increase in ChREBP mRNA levels in queens that were re-fed carbohydrates, in contrast to no changes in ChREBP mRNA levels in workers ) compared with controls . This effect was absent when kings were fed TOFA . Thus, activation of the ChREBP target gene ACC appears to be necessary for a queen to be perceived as a queen and to suppress the reproductive potential of sterile nest-mates.In order to test one of the hypothesized downstream effects of ChREBP, i.e. the regulation of synthesis of pheromones produced by mature queens to signal fertility, we combined a pharmacological approach and behavioural assays in -butting ,35,47. T-butting ,49. This3.P. canalifrons workers, which become reproductive females, and when the ChREBP pathway is experimentally silenced dominant workers engage in head-butting behaviours as part of establishment of who takes over the reproduction. Collectively, these findings, in combination with the tissue localization of ChREBP expression in fat bodies and ovaries, strongly suggest that ChREBP is involved in fertility signalling and maintenance of reproductive dominance by the resident queen. We further document that dietary carbohydrates in the abdomen of mature female reproductives affect queen physiology, by inducing ChREBP expression and thus its lipogenic target genes.We have shown that the transcription factor ChREBP, an evolutionarily conserved glucose sensor that regulates gene expression to drive fatty-acid biosynthesis in mice and in the fruit fly, has a unique expression profile in termites, which are the oldest social insects. ChREBP is highly expressed in mature reproductive females of eight different termite species, compared with reproductive males and sterile worker and soldier individuals. ChREBP expression is increased in et al. [D. melanogaster, lipid homeostasis influences pheromone production, since fat-body gene inactivation encoding lipid metabolic effectors, such as FAS, decreases the amount of pheromones [Termites use cellulose as their main carbohydrate source, and glucose metabolism and its regulation are especially important as a signal of prodigious fecundity in reproductive females. Indeed, glucose acquired through adult feeding is especially important for females of longer lived species, who have to mature eggs throughout their adult life ,51. Receet al.  demonstreromones ,54. In teromones . In termeromones . Using Teromones ,47. ThisZootermopsis (Archotermopsidae), JH and ecdysteroid titres were measured during ovarian maturation in young reproductive females following their release from inhibitory stimuli produced by mature queens. Four days after disinhibition, JH released by corpora allata and its titre in haemolymph decreased while ecdysteroid titre increased. Fully mature queens had the highest rate of JH production, the lowest ecdysteroid concentrations and the highest number of functional ovarioles [Drosophila, the sterol regulatory element-binding protein, a key regulator of lipid synthesis that functions in synergy with ChREBP to control lipogenic target genes [In all animals, digestion and absorption of carbohydrates in the diet induces profound hormonal changes, such as in the concentration of insulin. In most insects, including termites, two major hormones are essential to elicit major behavioural and physiological events. Juvenile hormone (JH) controls metamorphosis in immature insects –58 and mvarioles . Intereset genes , is actiet genes . As ChREIn conclusion, ChREBP is a transcription factor upregulated in mature queens that, through a carbohydrate-rich diet, links nutritional status and endocrine control with the reproductive status of queens through the regulation of lipid metabolism. Our preliminary results also suggest that regulation of the lipid metabolism might be important in controlling termite phenotypic plasticity. The conservation of ChREBP in all termite species studied suggests that ChREBP has remained an important regulator over the course of millions of years of termite evolution.4.4.1.P. canalifrons (Rhinotermitidae) containing hundreds of individuals each were collected with pieces of wet wood on the Réunion Island in the Indian Ocean. Colonies were brought to the laboratory and reared at 28°C, 80% relative humidity and a 12 L : 12 D regime. Degraded birch wood was used as food. Cross-breeding of emerging alates was done in 2010 and in 2014, so that primary queens used in this study were 4 years or 4 months old, respectively. Colonies from Termitidae termite species  were collected in French Guiana in March 2014. After removing their legs and antennae, individuals were put directly in RNAlater (Life Technologies) and maintained at −20°C.In 2001, colonies of 4.2.We obtained 45 amino acid sequences of ChREBP  for two vertebrates (human and mouse), one mollusc and 42 arthropods  and amino acid sequences of Mondo A for two vertebrates (human and mouse) from the database at NCBI. Sequence searches were performed using ‘ChREBP alpha’, ‘WBSCR14’, ‘MLXIPL’, ‘MLX interacting protein-like’, ‘MondoB’, ‘Mondo’, ‘dMondo’, ‘dChREBP’ and ‘Mio’ keywords.Zootermopsis nevadensis [Macrotermes natalensis [eaview software [Mus musculus GSM region boundaries (accession no. NP067430) [Amino acid sequences from the termites vadensis  and Macrtalensis  and fromsoftware , and thesoftware ,32. OwinP067430) .rotTest v. 3.3 software [rotTest was JTT + G (Jones Taylor Thornton model [hyML [rBayes [racer v. 1.5.0 [The best model of evolution was selected using Psoftware  and follon model ). ML anael [hyML  using an [rBayes , each cov. 1.5.0 . A conse4.3.Total RNA was extracted from 3 mg of nitrogen-frozen crushed individual termites and purified using miRNeasy Micro kit (Qiagen) and RNAse-free DNAse according to the manufacturer's instructions (Qiagen). Reverse transcription was performed with 500 ng of total RNA using the iScript™ cDNA synthesis kit according to the manufacturer's protocol (Bio-Rad). Quantitative PCR was performed with 5 ng of reverse-transcribed total RNA, 0.5 µM of each primer (Eurogentec) in 1× Power SYBR Green PCR Master Mix (Life Technologies) using LightCycler StepOnePlus (Applied Biosystems). All samples were normalized to the threshold cycle value for actin mRNA, chosen as an invariant control . Primer 4.4.−1; Abcam). Liver and total fat protein extracts from glucose re-fed mice were used as positive controls of the experiments and efficiency of the antibodies (relative molecular weight 95 kDa). Blots were developed with ECL SuperSignal West Pico chemiluminescent reagents (Pierce).Termites, mice liver and adipocyte total extracts (40 mg each) were prepared using lysis buffer as described elsewhere . ProteinP. canalifrons ChREBP cDNA was generated from queen poly(A) RNA and cloned in pT7CFE1-CHis vector (Thermoscientific). The protein was synthesized using in vitro TNT T7 Quick Coupled Transcription/Translation in Reticulocyte Lysate System (Promega) and analysed by western blotting. In vitro translated proteins (2 µl) were subjected to SDS-PAGE analysis on 12% gels and transferred to nitrocellulose membranes. In vitro synthesized ChREBP proteins were detected with rabbit polyclonal antibody raised against a peptide mapping at the C terminus of ChREBP of human origin . We realized an immunizing peptide blocking experiment as recommended by Abcam. ChREBP antibody is neutralized, i.e. incubated with five to eight times excess blocking peptide  that corresponds to the epitope recognized by the antibody, to antibody by weight during hybridization overnight at 4°C. Blots were developed with Clarity™ Western ECL Blotting Substrate on ChemiDoc MP (BioRad).To determine the specificity of binding of ChREBP antibody to ChREBP termite protein, 4.5.Prorhinotermes canalifrons and Nasutitermes sp. mature queens were fixed for 24 h in 4% paraformaldehyde and embedded in paraffin. Serial sections (4 µm) were immunostained for ChREBP overnight at 4°C using rabbit polyclonal anti-ChREBP antibody  followed by a fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody . Sections were mounted using Vectashield mounting medium with DAPI , scanned using the Pannoramic Lamina multilabel slide scanner (Perkin Elmer) and observed with Pannoramicviewer and ImageJ. To determine the specificity of binding of ChREBP antibody to ChREBP termite protein, we also realized immunizing peptide blocking experiments, as recommended by Abcam, on sections of P. canalifrons and Nasutitermes sp. mature queens. Before proceeding with the staining protocol, ChREBP antibody  is neutralized, i.e. incubated with five times excess blocking peptide  that corresponds to the epitope recognized by the antibody, to antibody by weight overnight at 4°C.4.6.Nasutitermes sp. and physogastric neotenic female reproductives, neotenic male reproductives and 30 workers of P. canalifrons from six colonies per species were placed in Petri dishes on humidified sand, fasted for 24 h, and then fed for 24 h (Nasutitermes sp.) and 30 h  with a piece of wood impregnated with 10% glucose. Individuals were weighed before to start the experiment and after the diet protocol to observe losses and/or gains of weight. Nitrogen-frozen crushed female neotenic and queen abdomen and worker extracts were prepared and analysed for actin, ChREBP and FAS mRNA levels.Physogastric queens, kings and 100 workers of 4.7.P. canalifrons from three colonies were isolated in Eppendorf tubes pierced with numerous small openings and filled with humidified sand. Each tube was placed in a Petri dish (ø = 5 cm) containing 30 workers from the same colony. This set-up allowed antennation among workers and the diffusion of volatiles emitted by the reproductives into the Petri dish. Reproductives were fed for 3 days with 0.5 cm2 square paper (Whatman 3MM) loaded with 10 µl of TOFA  suspended in ethanol at a concentration of 3 mg ml−1 or with 10 µl of ethanol for control experiments. It is known that TOFA is converted into 5-tetradecyloxy-2-furoyl-CoA exerting an allosteric inhibition on ACC that prevents fatty-acid synthesis in adipocytes and hepatocytes [Physogastric secondary queens and kings of atocytes ,49. Workatocytes . Queens atocytes ) in eachet al. [n = 30) compared with queenright  conditions, similar to what has been observed in Z. nevadensis [Cryptotermes secundus [A control experiment, adapted from Penick et al. , was convadensis  and Crypsecundus .Drosophila [To determine if inhibition of fatty-acid synthesis by TOFA caused a change in other semiochemical profiles of the workers, we recorded trail-following pheromone bioassays as described previously . We obseosophila , we diss4.8.P. canalifrons were collected from three colonies and kept in three Petri dishes containing humidified sand and supplied with pieces of birch wood. The groups were monitored daily. Three weeks of absence of reproductives induced development of new reproductives, male and female neotenics, in all nine experimental groups. Eggs were observed in all groups as well. Total RNA was extracted and purified from individual termites and ChREBP expression was quantified as reported above.Nine groups of 20 old workers of 4.9.t-test. For statistical analyses with non-normal distributions or unequal variances, analyses were carried out using Mann–Whitney U-test. Differences were considered statistically significant at p < 0.05.Quantitative results are expressed as means ± s.e. The comparison of different groups was carried out using unpaired two-tailed Student's