Up to now, the genetic evaluation of Spanish dairy goats has been based only on milk production traits. However, fertility is also an important economic trait for goats and is vital for maintaining the profitability of dairy farms, and the estimation of its genetic parameters and selection responses is, therefore, crucial for the genetic improvement of dairy goats. In this study, we estimated the genetic parameters of various fertility traits in the Florida and Payoya goat breeds, which are prototype breeds exploited under different production systems in Spain. Next, we assessed selection indices to determine the most suitable one to produce the highest number of expected selection responses. The overall interval between kiddings gives the highest genetic response but cannot be recommended as early selection criteria because it is expressed late in the female’s life, especially in the Florida breed, in which a high early culling is carried out in females with low fertility. Nevertheless, an index including the age at first kidding and the interval between the first and second kiddings can be used as a precocious selection criterion in both breeds. The results from this study can be used as a basis for the future genetic improvement of fertility traits.The aim of this study was to estimate genetic parameters for several female fertility criteria and to choose the most suitable selection index in Spanish Florida and Payoya goat breeds. In this study, we analyzed as fertility traits, the age at first kidding (AgFiKid), and the interval between the first and second kiddings (Int12Kid), between the second, third, and remaining kiddings (Int3toKid), and between all kiddings (IntAllKid) in 51,123 and 22,049 Florida and Payoya females, respectively. Genetic parameters were estimated by fitting animal models using restricted maximum likelihood (REML) methodology. We proposed six selection indices to compare the genetic responses for all traits included, based on a new selection index theory. The heritability and repeatability estimates of the traits were low, as expected. The genetic correlations among fertility traits covered a wide range of values from 0.07 (AgFiKid-Int12Kid) to 0.71 (Int3toKid-IntAllKid) in Florida and from −0.02 (AgFiKid-Int12Kid) to 0.82 (Int3toKid-IntAllKid) in Payoya. Overall, the results of this study indicate that IntAllKid gives the highest genetic responses in both breeds but is expressed late in a female’s life. However, AgFiKid and Int12Kid could be recommended as early selection criteria for female fertility in both breeds. Spanish dairy goat breeds are farmed under different production systems varying from extensive, semi-extensive, and semi-intensive to intensive systems, depending on their productive aptitude and performance. The Florida and Payoya breeds are among the autochthonous dairy goats with the widest range of geographic distribution and production systems. The Florida goat can be found in different countries in the world and several regions of Spain , but especially in Andalusia, where it is raised under semi-extensive to semi-intensive production systems, while the Payoya breed is present only in Spain in the region of Andalusia, under semi-extensive to extensive production systems.Nowadays, the genetic evaluation of Spanish dairy goats is based mainly on milk production traits , and studies have focused on estimating the genetic parameters of these traits ,2,3. HowMost studies of the genetic and environmental association between milk production and reproductive traits in various species showed an unfavorable relationship between them not only in dairy cattle ,5 and inThus, the main objectives of the present study were to estimate the genetic parameters of fertility traits for the first time in Spanish dairy goats, using Florida and Payoya breeds as models, and to find the best selection index for the desired expected genetic response.In this study, we used reproductive records and pedigree information from ACRIFLOR  and ACAPA (Association of Payoya Goat Breeders), which contained information on age at first kidding and the different intervals between the kiddings of Florida and Payoya females, respectively. To estimate the genetic parameters, herds with few records or scarce genetic connections were discarded. The genetic links were due to artificial insemination sires. Artificial insemination (AI) has been implemented in the Florida and Payoya breeds since 2005 within the selection scheme carried out by ACRIFLOR and ACAPA, respectively. In the last years, approximately 1808 AI with 5 bucks (from a set of 25 semen donors) and 600 AI with 3−4 bucks (from a set of 8 semen donors) were realized in Florida and Payoya, respectively. Connectedness was estimated by calculating the coefficient of similarity between herds (SG), which is the number of daughters of a connector male shared between two herds in relation to the total number of animals that have been evaluated in those two herds, establishing an SG equal to 5% as a limit for a herd to enter in the genetic evaluation process. Animals from the Payoya breed are reared under semi-extensive to extensive production systems, whereas the Florida breed presents a higher level of intensification. The following traits were considered in this study as measures of a goat’s fertility: the age at first kidding (AgFiKid), the interval between first and second kiddings (Int12Kid), the interval between second, third, and remaining kiddings (Int3toKid), and the interval between all kiddings (IntAllKid). All traits were expressed in months. After data editing, the final data set contained information on 130,849 and 67,478 reproductive records from 51,123 and 22,049 Florida and Payoya females, respectively. In total, the pedigree included 56,305 animals for Florida and 26,392 for Payoya.The following model was fitted to estimate the genetic parameters for all the fertility traits for Florida and Payoya breeds:programs , applyinp′ for IntAllKid was five times greater than that of AgFiKid. Weights in vector f′ to be used for weighting the expected breeding values (EBVs) on v were calculated by f′ = −1p′C′G, where C′ is the covariance matrix between the objectives in vector u and the EBV used as criteria in vector v, and −1G is the inverse of the (co)variance matrix for the selection criteria v. Matrices C′ and G were obtained from the genetic parameters by assuming all the additive genetic variances to be standardized f′ varied, and matrices C and G also changed. When objective and criteria are the same traits, the (co)variance matrix between the objectives and the criteria C becomes a genetic additive (co)variance matrix, in which the diagonals are equal to one [C′:The genetic responses using different selection objectives/criteria were computed and compared in the Florida and Payoya breeds according to the classic selection index theory  reformull to one . Off-diaG and C′ are directly dependent on the genetic parameters, these matrices can be derived directly from genetic parameters to build the desired index. Next, the genetic responses were obtained by weighting, for each of the traits, all those responses obtained in the correlated selected traits, including the direct genetic response to itself. Thus, assuming the EBVs are known and applying the assumption given above about all the additive genetic variances being one, the direct genetic response would be the selection intensity (i) reduced by the accuracy of the EBV. The correlated response would be the additive genetic correlation times the selection intensity reduced by the accuracy of the EBV. Gathering this information into a matrix expression, the cumulated genetic responses will be obtained by:k in t is the cumulated genetic response in trait k and hk the squared root of the heritability of trait k. For the comparison of the expected responses, a constant selection intensity of one was assumed, leading to comparable relative results.As This study estimated for the first time the genetic parameters related to female fertility traits in Florida and Payoya dairy goat breeds , and expected to find the genetic responses by combining these traits in selection indices as a step towards their incorporation into the routine genetic evaluation.The basic descriptive statistics for Florida and Payoya female fertility traits are presented in The values for Int12Kid, Int3toKid, and IntAllKid were similar in both breeds and ranged between 5.7and 17.1 months in Florida and 6.3 and 16.7 months in Payoya. Their averages varied from 10.78 months ± 1.74 (for Int12Kid in Payoya) to 11.46 months ± 1.72 (for Int3toKid in Payoya) and were lower than the ones mentioned in US dairy goats ,20, but The range of solutions for the fixed effects: herd–year–season of kidding, lactation length, and herd production level affecting fertility traits are shown in 2), and repeatabilities (t) for the evaluated traits are shown in 2 values for AgFiKid were lower than the estimates in other dairy goat breeds , heritabilities . The h2± 0.007; ; 0.00 to± 0.007; ; 0.06; [± 0.007; ). Other ± 0.007;  in a metThe repeatability for Int3toKid and IntAllKid showed very low estimated values, of the same magnitude as their corresponding heritabilities in both breeds , and similar to the values obtained by García-Peniche et al. (2012) , dos Sang between AgFiKid and Int12Kid in Payoya, and covered a wide range of values. The genetic correlations between AgFiKid and kidding interval traits ranged from 0.07 (AgFiKid-Int12Kid) to 0.19 (AgFiKid-Int3toKid) in Florida and from −0.02 (AgFiKid-Int12Kid) to 0.07 (AgFiKid-Int3toKid) in Payoya. Estimated rg among kidding intervals oscillated between 0.24 (Int12Kid-Int3toKid) and 0.71 (Int3toKid-IntAllKid) in Florida, and between 0.12 (Int12Kid-Int3toKid) and 0.82 (Int3toKid-IntAllKid) in the Payoya breed.The estimates of genetic correlations among fertility traits are shown in Correlations between age at first kidding and kidding intervals in other studies were reported to be negative  of fertility traits were higher in most cases in this breed. In general, the selection indices with combined criteria give greater selection responses than when a single criterion was used, and index 5 and the full index (index 6) presented the highest genetic responses. Moreover, it should be noted that the responses were higher when IntAllKid was included in the selection objective/criteria. In the selection indices with IntAllKid as a selection objective, the maximum expected genetic response was achieved in indices 3, 5, and 6 in Florida (−0.0106 months per generation) and Payoya (−0.0088 months per generation). In the case of a combined selection objective (IntAllKid and AgFiKid), the maximum response was obtained in indices 5 and 6 in Florida (−0.0710 months per generation) and Payoya (−0.0498 months per generation).The expected genetic responses for the different fertility indices in Florida and Payoya breeds are presented in The expected responses varied in magnitude depending on the defined selection objectives, the traits included as criteria in the selection indices, and their genetic parameters (heritability and genetic correlations). The best global responses were achieved when both IntAllKid and AgFiKid were combined in the selection objective and when the indices included IntAllKid as a selection criterion. This can be explained by the fact that IntAllKid provides the maximum amount of information on fertility traits, which increases selection accuracy and optimizes genetic response. However, in practice, this trait is expressed later in the female’s life, and direct selection for this trait will reduce this gain in accuracy by delaying the generation interval. Poor fertility is one of the most common reasons for culling females , even at an early age, especially in the Florida breed, where 24.7% of females are eliminated between the first and second parities (14.8% in Payoya). Using precocious selection criteria, such as AgFiKid and Int12Kid, separately (indices 1 and 2), will generate a very slow genetic response, especially for the Payoya breed. Nevertheless, in both breeds, it could be possible to select females based on these two criteria together under a combined selection goal , further research is needed into other fertility criteria that are expressed early in the animal’s life.


Urbanisation in Latin America (LA) is heterogeneous and could have varying implications for infant mortality (IM). Identifying city factors related to IM can help design policies that promote infant health in cities.We quantified variability in infant mortality rates (IMR) across cities and examined associations between urban characteristics and IMR in a cross-sectional design. We estimated IMR for the period 2014–2016 using vital registration for 286 cities above 100 000 people in eight countries. Using national censuses, we calculated population size, growth and three socioeconomic scores reflecting living conditions, service provision and population educational attainment. We included mass transit availability of bus rapid transit and subway. Using Poisson multilevel regression, we estimated the per cent difference in IMR for a one SD (1SD) difference in city-level predictors.Of the 286 cities, 130 had <250 000 inhabitants and 5 had >5 million. Overall IMR was 11.2 deaths/1000 live births. 57% of the total IMR variability across cities was within countries. Higher population growth, better living conditions, better service provision and mass transit availability were associated with 6.0% (95% CI −8.3 to 3.7%), 14.1% (95% CI −18.6 to −9.2), 11.4% (95% CI −16.1 to −6.4) and 6.6% (95% CI −9.2 to −3.9) lower IMR, respectively. Greater population size was associated with higher IMR. No association was observed for population-level educational attainment in the overall sample.Improving living conditions, service provision and public transportation in cities may have a positive impact on reducing IMR in LA cities. The accelerated urban population growth that occurred over the last three decades resulted in rapid expansion of many cities with inadequate urban planning.6 Cities can have hazardous levels of air pollution,7 which has been linked to premature birth, and higher morbidity and mortality among infants.8 9 In addition, almost 20% of the population currently lives in poverty.6 10 This creates a particularly vulnerable environment for infants as a result not only of poorer access to care but also because of greater exposure to social and environmental conditions hazardous to health.10 This heterogeneity in urban environment observed within and across urban areas in LA challenges the idea of ‘urban advantage’, by which cities are believed to have lower maternal and IMR compared to rural areas.11LA is one of the most urbanised regions in the world.12 13; however, analyses at the city level have been mostly restricted to a few big metropolitan areas.14 15 Examining how characteristics of cities affect mortality and health outcomes is important to identify actions and policies to improve infant health and promote health equity in the context of a rapidly urbanising world. We used data from a unique multinational urban health collaboration to quantify differences in IMR across a wide range of cities in LA and to examine how key features of cities are related to IMR.Social inequalities linked to child mortality have been extensively described at the national or subnational levels16 For this study, we included cities for which vital statistics registries were available from 2014 to 2016 and presented good quality of death registry based on a separate analysis of adult mortality.17 We assumed that cities with good levels of registration for adult deaths (coverage of 90% or above) also have good coverage of deaths among infants. We included 286 cities in Argentina, Brazil, Chile, Colombia, Costa Rica, Mexico, Peru and Panama. Eleven cities  were excluded due to lack of vital registration for the years of study. A total of 74 cities  were excluded because the estimated coverage of adult mortality was considered of low quality. Mean level of mortality coverage in all excluded cities was below 85% and excluded cities had poorer living conditions and lower provision of water connected to public network compared to cities included in this study , which includes all cities of 100 000 or more inhabitants in 2010 in 11 countries for a total of 371 cities. Each city was defined geographically by administrative units  that encompassed the urban extent of the city in 2010 using satellite imagery.is study .10.1136/jech-2020-215137.supp1Supplementary dataIMR. We calculated IMR (deaths less than 1 year of age per 1000 live births) for the period 2014–2016. Deaths and live birth were retrieved based on deceased’s and maternal place of residence, respectively. Three years were pooled to increase the stability of the estimates.6 10 13We explored several characteristics of cities that we hypothesised would be related to IMR based on prior literature.City population: we obtained population data from national censuses that provided estimates or projections for years 2010 and 2015. We investigated population size in 2010 as a continuous variable and categorised as (1) below a quarter million, (2) greater or equal to quarter million to less than half million, (3) greater or equal to half million to less than one million, (4) greater or equal to one million to less than five million, and (5) greater or equal to five million.Annual population growth rate (APGR): this was calculated based on differences in population size between 2015 and 2010.Urban social environment: we retrieved census indicators and harmonised them across countries for 12 socioeconomic (SE) variables describing education, housing, water, sanitation and employment. Using principal component analyses, we identified three components that incorporated seven variables. Variables related to employment (per cent of unemployed and per cent of labour force participation) and housing materials  did not load onto any of the three components.Each indicator was standardised to a mean of 0 and SD of 1, and indicators that loaded onto each component were summed to create three SE scores. Each score may be related to IMR through different mechanisms:18 and exclusion from productive systems.19 We reverse coded the overcrowding indicator so that higher score values signify better living conditions.Score of living conditions (related to housing and poverty), including (1) per cent of households with piped water inside the dwelling; (2) per cent of households with overcrowding ; and (3) per cent of population aged 15–17 years attending school, as a marker of social inclusion, since low school attendance among adolescents has been linked to povertyScore of service provision (related to public services that cities provide to dwellings), including (1) per cent of households with access to water from a municipal public or private water network, and (2) per cent of households with sewage system connected to a municipal public or private sewage network. Higher score values signify better service provision.20 Higher score values signify better educational achievement in the population.Score of educational attainment (as a marker of SE context) including (1) per cent of population aged 25 years or above that has completed high school level or above, and (2) per cent of population aged 25 years or above that completed university level or above. This score characterises SE aspects of cities different from living conditions and service provision captured in the other two scores.Urban built environment: we created an indicator of mass transit availability based on the presence of subway and bus rapid transit (BRT) networks in the city. Mass transit availability was considered as present if the city had either subway or BRT network available, and absent when none of these options were present.Healthcare access: access to healthcare services for infants was proxied by the city coverage of first dose of triple viral vaccine  among the population of 1-year-olds. MMR1 presents a schedule that is similar across countries making it suitable for harmonisation. Data for the year 2016 were provided by WHO.21National gross domestic product per capita (GDP per capita): we retrieved the national real GDP (output-based) per population for 2015 for each country from Penn World Tables22 and use the median value of the sample (US$15 530.7) to categorise countries in two groups: above and below the median GDP per capita. We stratified on GDP because country economic conditions could modify the importance of the city-level factors, we studied for IM.We described the distribution of city characteristics by categories of city population size. We examined the distribution of IMR and city-level predictors in the overall sample and by countries. Since IMR was approximately normally distributed in our sample, we decomposed the total variance in IMR across and within countries using a linear mixed model with a random intercept for each country and no covariates. We then estimated the association of city-level variables with IMR using a Poisson multilevel model. City-level predictors were first explored separately and then combined. To determine whether associations of city variables with IMR were independent of access to healthcare, we further adjusted for MMR1 coverage.We explored effect modification by country GDP per capita by repeating analyses in countries with GDP per capita above and below the median value of the sample, and by testing for statistical interactions between GDP per capita and city-level predictors.Selected characteristics of the cities are shown in When countries were grouped by GDP per capita , better 23) also yielded similar results.To explore the influence of outliers, we carried out sensitivity analysis excluding cities above the 99th or below the 1st percentile of IM and found similar results. The exclusion of Peru  was within countries. Higher population growth rate, better living conditions and services provision and availability of mass transit were associated with lower IMR, independently of access to healthcare. Greater population size was associated with higher IMR. GDP per capita modified these associations, such that services provision was associated with lower IMR only in countries with GDP per capita below the median.24 which remain the main causes of death among infants.25 Prior work has shown that urban slum prevalence is related to IMR across countries,26 27 but these studies did not identify the features of slums that may be most important and ignore heterogeneities across cities. We show that specific living conditions and service provision at the city level are related to IMR in cities, highlighting the potential role of city-level policies affecting these factors in reducing IMR in urban areas.The large heterogeneity in IMR observed across cities (even within the same country) highlights the importance of examining city-level factors as predictors of IMR. In our study, city-level measures of living conditions  and services provision (water and sanitation) were independently associated with lower IMR. Quality housing as well as access to water and sanitation services are known to reduce the transmission of respiratory and enteric diseases,28 Increased population growth at the expense of migration from rural areas may reflect the influx of disadvantaged populations and may create demands that cities are unable to face resulting in higher IMR.29 However, we found that greater growth was associated with lower IMR suggesting that growth may be proxying other unmeasured factors related to lower IMR.We also found that higher population growth was associated with lower IMR while greater population size was related to higher IMR, after accounting for other city predictors. Further analyses are needed to examine how the population dynamics of cities may affect IMR. Population growth could reflect migration and/or natality rates. Decreased fertility and natality associated with improved status of women have been linked to lower IMR.30 We found that availability of BRTs and subways was associated with lower IMR independently of city population size and other city-level predictors. Availability of timely, rapid and extensive public transportation network may be key to reduce inequalities in access to healthcare related to IMR, as well as in access to employment opportunities and consequently better SE position for dwellers living in impoverished city areas. To our knowledge, this is the first study exploring the relationship between city transport and IMR.30 Given limitations in the information, we used to characterise public transportation these preliminary findings deserve further investigation.A novel aspect of our study was the investigation of public transportation, which has been hypothesised to impact health.2Country economic development appeared to modify some of the associations between city-predictors and IMR. We found that better service provision was only associated with lower IMR in cities of countries with GDP per capita below the median. This may be related to greater variability in these predictors across cities in countries with lower GDP income or to the fact they are relevant predictors in these contexts because of the causes of death driving IMR.31 32 few if any studies have examined these factors at the level of cities. The study of city-level factors is especially relevant to the development of local interventions. A growing body of work has documented how local interventions in cities can improve infant health.33 For example, housing improvements and water and sanitation provision,34 35 or more comprehensive urban redevelopment interventions36 have been linked to reduction in respiratory and enteric infections34 35 as well as in IM36 in urban slums in LA.Our study is unique and unprecedented in that we have compiled and harmonised data on urban environment, and IMR across nearly 300 cities in LA. We were able to describe heterogeneity in IMR across cities and study how a range of city-level factors are related to IMR. Although many studies have focused on IMR in urban areas, to our knowledge, this is the first investigation examining the influence of city social and built environments on IMR across multiple cities. Although several previous studies have documented associations of social and economic factors with IMR at the country level,37 This measure clearly has important limitations and does not adequately capture the complexity of healthcare access. A better representation of healthcare access may require the combination of several indicators related to family planning, antenatal care, multiple vaccine coverages and access to treatment during disease episodes like diarrhoea or pneumonia.37 Lastly, our city-level analyses, although informative, ignore heterogeneity across neighbourhoods within cities.Because of the number of cities studied, some of the measures available had limitations. Measures of public transportation were limited to subways and BRTs. These have advantages to traditional buses, but they do not fully capture transit availability. We did not include other potentially relevant physical environment features such as air pollution or density of green areas. The characterisation of healthcare access for infants was restricted to coverage of MMR1, a single-dose vaccine that tends to be more equally distributed in the population than interventions requiring specialised personnel or complex technology.23 but sensitivity analyses excluding Peru resulted in similar findings. If more under-registration of infant deaths is associated with lower SE conditions, the associations we report may be underestimates of true associations. Future studies involving IM need to address under-registration through specific approaches that estimate IMR, like indirect demographic methods or statistical methods designed for small-area estimations.Finally, we restricted our study to cities with good quality of mortality registries based on adult mortality estimations with the purpose of generating unbiased estimates of IM. However, it is possible that registration of infant deaths and live births could be conditioned by other determinants of under-registration different from the ones related to adult mortality. There is evidence that undercounting of child deaths may be especially problematic in some areas of PeruIn an increasingly urbanised world, it is critical to identify which urban policies are necessary to improve population health. Our city-level analyses show that features of urban social and physical environments are related to important differences in IMR across cities. Prioritisation of urban policies and interventions related to improving living conditions, sanitary services and public transportation availability may be necessary to have positive and sustained impacts on infant survival in urban areas.Although many studies have described differences in infant mortality across countries or regions, little research has explored heterogeneity across cities within countries. This heterogeneity may be especially important in nations where rapid urbanisation has been heterogeneous. Identifying which aspects of the urban environment contribute to differences in infant mortality is important to inform policies and programmes to improve infant health.We found that in Latin America, although infant mortality rates (IMR) differ across countries, large heterogeneity is observed across cities within countries. Higher IMR were observed in cities with higher proportions of residents experiencing social exclusion, inadequate housing conditions and insufficient municipal provision of sanitary services. Availability of mass transit in cities was associated with lower IMR.Achieving further reductions in infant mortality in Latin America will require focusing on reducing social inequities in urban populations and prioritising interventions related to improving living conditions, sanitary services and public transportation.


Research in the field of genomics and genetics has evolved in recent years and so has the demand of consumers who are increasingly interested in genomic prediction of diseases and various traits. The aim of this study is to identify genetic service delivery models, policies governing the use of genomics medicine, and measures to evaluate genetic services in the province of Quebec, Canada.ad hoc questionnaire was designed and administered online in 2017 to healthcare workers with good knowledge or experience in the provision of BReast CAncer genes 1 and 2 (BRCA1/2), Lynch syndrome, familial hypercholesterolemia, inherited thrombophilia genetic tests, engaged in policy planning or evaluation of genetic services. A quali-quantitative analysis of the survey results was performed.An Thirty professionals participated in the study. The delivery models are classified in five categories according to the leading role of healthcare professionals in patient care pathways: i) the geneticist model; ii) the primary care model; iii) the medical specialist model; iv) the population screening program model; and v) the direct-to-consumer model. Barriers to genetic services are the coverage of genetic tests by the publicly funded healthcare system, the availability of qualified personnel, and the number of genetic centers. Regulatory oversight concerning the provision of genetic services appears to be insufficient.Integration between genetics and the overall healthcare system in Quebec is in an early phase. Current models of genetic services require good level of genetic knowledge by all medical specialists, collaboration among different healthcare personnel, and work redistribution. The proper implementation of genomics into healthcare can be achieved through education and training, proper regulatory oversight, genomic policies, and public awareness. Personalized medicine has been adopted worldwide after the completion of the first sequence of human genome in 2003 . This haCurrent organizations of genetic services in Europe and in selected countries, i.e., the US, Canada, Australia, and New Zealand, form the basis for study by the Personalized PREvention of Chronic Diseases (PRECeDI), a European multicenter project on personalized medicine . The proCanada is one of the countries outside of Europe that was included in the PRECeDI multicenter project. The core facilities of genetic services in Canada consist of genetic centers affiliated with universities or healthcare institutions. Professional resources delivering genetic services mainly include genetics staff  and other healthcare providers involved in delivering genetic services as part of multidisciplinary teams , medical specialists, nurses, psychologists, and social workers) . AlthougAt the time of the survey, the legislative framework governing the delivery of genetic tests in Quebec and Canada was based on guidelines from local ethics committees, on the voluntary accreditation and participation of genetic laboratories in external quality assessment (EQA) schemes, and on certification of non-medical staff trained in genetics, such as the one provided by the Canadian Association of Genetic Counsellors (CAGC). The Genetic Non-Discrimination Act (GNDA) was adopted by the Canadian Parliament in May 2017, after the collection of the survey data has been completed, and therefore was not addressed in the survey questionnaire. Since then, the Quebec Court of Appeal’s reference decision (December 2018) to the effect that the GNDA is unconstitutional has been appealed to the Supreme Court of Canada and, for now, the Act remains in effect. The GNDA makes a criminal offense to require a person undergoing a genetic test, or obtaining the access to, or forcing someone to disclose the results of such a test as a condition to the provision of goods and services ,. HoweveThe study was carried out through an online survey available from January to April 2017. The survey targeted healthcare workers, researchers with different backgrounds , and policy makers with good knowledge of and/or practical experience in the provision of at least one of four selected genetic tests , assessment of genetic service delivery models, and policy planning of genetic services in Quebec. To be considered eligible for the study, participants had to be currently practicing in the province. The participants were contacted by email through the Quebec Network of Applied Medical Genetics, the Quebec Association of Genetic Counsellors, and the Canadian Association of Genetic Counsellors. To increase the response rate, participants received an email reminder 6 weeks after the initial invitation email.Ethics approval for this study was given by McGill University’s Faculty of Medicine Research Ethics Board (study code A01-E02-17A). The informed consent form was administered as the online questionnaire’s first page. The form gave information about the study’s purpose, procedure, risks and benefits of participation, confidentiality, withdrawal, compensation, and contact information of the research coordinators. Participants were informed that there were no foreseeable risks to them and that they could withdraw from the survey at any point. If they chose to participate, their answers could be published in anonymized form. Any identifying elements are only visible to the authors of the study and will be destroyed 5 years after the end of the survey. The recording of informed participant consent was accomplished by participants ticking a checkbox that indicated that they had read the consent form and agreed to participate in the study.The questionnaire items were generated based on literature review and through counseling with European experts in clinical genetics, evaluation of genetic services, and policy making. Further modification of the questionnaire and its translation from English into French was carried out to adapt the tool to the Quebec context.A bilingual French–English questionnaire was distributed through the online platform Survey Monkey . The firThe second part of the survey was on assessment of genetic service delivery models and addressed healthcare workers engaged in health data collection and analysis at local, regional, and provincial levels. It was composed of six sections: a) evaluation of activity (eight questions); b) quality assessment (three questions); c) evaluation of health outcomes (two questions); d) electronic records and genetic information (three questions); and e) genetic services and coverage .The third part of the survey focused on policy regarding genetic testing and related services; it addressed experts in policy planning/research on genetic services employed in provincial institutions , universities or clinical research centers. It was composed of the following sections: a) policy (nine questions); b) access and availability of genetic services ; and c) professional education and training (two questions).Descriptive statistics, median, and interquartile range (IQR) were calculated for quantitative variables, while frequencies were generated for qualitative variables. Where possible, the differences between groups of respondents  were calculated with the chi-square test with level of significance set at p < 0.05. The IBM software, Statistical Package for Social Sciences (SPSS) version 25.0 for Windows , was used for data analysis. Two researchers independently analyzed responses to the four open-ended questions. Each researcher identified the items pointed out by the respondents by eliminating and grouping together sub-items in an Excel spreadsheet. Any discrepancies in individual evaluations were resolved through consensus discussion with a third researcher.Thirty individuals participated in the study Table 1,via other medical specialists, nurses, and midwives. Although the counselors of pretest genetic counseling are mainly medical geneticists and genetic counselors  of experience in clinical genetics. According to respondents, individuals at increased risk of one of the four selected genetic disorders are referred to genetic counseling by various healthcare workers  is mostly managed by medical geneticists, genetic counselors, or GPs in the case of FT . The genetic team is responsible for risk assessment, counseling, and testing of individuals or families affected by or at risk for genetic disorders. Depending on the case, the team collaborates with other medical specialists  who could be part of the genetic service . The access of patients to this model of genetic services may occur through two different pathways: a) Patient → GP or medical specialist → Counselor → Lab ; and b) Model II: Primary care model. A prominent role is played by primary care units, which may include primary care physicians (GPs or family physicians), a nurse practitioner, or a physician assistant. In these units, GPs have some training in genetics and can undertake an initial risk assessment using standardized referral guidelines. In some cases, GPs refer at-risk patients to genetic services, while in other cases, they care for patients without consulting medical geneticists or genetic counselors. The pathways associated to this model are: a) Patient → GP → Lab and b) Patient → GP → Counselor → Lab . Under tModel III: Medical specialist model. Genetic tests can be requested directly by medical specialists  who may be able to manage patients with or at risk of genetic disorders without consulting medical geneticists or genetic counselors. The possible pathways in Model III are: a) Patient → Medical specialist → Lab and b) Patient → Medical specialist → Counselor → Lab . The fouModel IV: Genetic services integrated into population screening programs. In this model, genetic services are provided within organized population screening programs . There are three possible patient pathways in Model IV: a) Patient → GP or medical specialist → Counselor → Lab; b) Patient → GP or medical specialist → Lab; and c) Patient → Counselor → Lab. The IVa pathway occurs when a patient takes part in a population-based screening program; a healthcare professional involved in the screening program can perform an initial risk assessment and refer the patient to genetic counseling. The genetic counselor or medical geneticist can suggest genetic testing and, based on results of the test, can make recommendations as to monitoring and/or clinical intervention. In the IVb pathway, healthcare personnel involved in a population-based screening program can perform risk assessment and suggest genetic testing. Based on results of the test, the healthcare personnel can make recommendations as to monitoring and/or clinical intervention. In the IVc pathway, a patient contacts a genetic counselor or a medical geneticist who can suggest genetic testing and, based on results of the test, can recommend monitoring through available population-based screening programs and/or clinical intervention . Only BRIn Model V: DTC model, genetic testing services are offered online by private companies. Healthcare professionals are not usually involved in the process, nor are medical referrals required for genetic testing. The main pathway associated with Model V is Patient → Lab . AlthougThe implementation of genetic services for predictive genetic testing has resulted in the development of new roles, namely, genetic nurses and genetic care coordinators. The main factors that led to the current genetic service delivery models in Quebec Table 3 Three participants with expertise in health data collection and analysis completed the second part of the survey on the assessment of genetic services. The respondents consisted of two physicians and a genetic counselor with a median of 20 years (IQR not computed) of experience in clinical genetics. According to these respondents, two health facilities usually collect, store, and retrieve data on genetic services through electronic processes. Despite the adoption of electronic processes, a link to provincial-level patient data is only available in one facility. Among the measures of activity of genetic services available in Quebec, the respondents did not indicate the number of new and follow-up appointments (The respondents declared that electronic records currently implemented in Quebec [Dossier Santé Québec (DSQ)] include genetic information but were not aware of the possibility to use DSQ information to evaluate the appropriateness of care according to a patient’s condition or diagnosis. The genetic conditions that are met with an adequate provision of genetic services in Quebec, as indicated by the respondents, are BRCA1/2, cardiovascular diseases, Huntington disease, and neurological conditions .Of 30 respondents, 26 completed the third part of the survey on policies of genetic services. Three respondents are fully engaged in policy planning and/or research on genetic services, who are physicians  and researchers with a median of 20 years (IQR not computed) of experience in clinical genetics. More researchers  are aware of guidelines from local ethics committees for the evaluation of research protocols involving biobanks. However, few specified that the guidelines originate from the Tri-Council Policy Statement. Genetic counselors  are more aware that, in Quebec, the accreditation and participation of genetic laboratories in EQA schemes are not mandatory but are promoted. Most respondents are aware of legislations governing the practice of non-medical health professionals, such as genetic nurses and technical staff trained in genetics (15/26). The certification system in Quebec for non-medical staff trained in genetics is also well known by the respondents (21/26), of which seven indicated the CAGC and the American Board of Genetic Counselling (ABGC). The respondents declared that no specific legislation addresses DTC genetic testing. They indicated that provincial or local guidelines that could help health departments organize genetic services to serve also as research and educational resources are currently under development.Regarding access and availability of genetic services, genetic laboratories are mostly in the public sector (23/26) and public health insurance covers genetic tests of proven efficacy and related services  (26/26). Generally, genetic tests requested by physicians according to clinical guidelines are accepted in health facilities across the province and are covered by public health insurance. However, the current provision of genetic services does not adequately meet the population’s needs in Quebec according to the majority of the sample . Key needs include the availability of and access to genetic services, qualified personnel , and genetic centers  are facing with respect to the provision of genetic services in Quebec  that actively support genetic teams in Quebec, as in other settings . This isThere were no differences in the delivery of genetic tests with considerable evidence of efficacy and cost-effectiveness   when comMost facilitating factors for the implementation of genetic service delivery models, such as the provision of genetic tests, availability of qualified personnel, and specialized centers, are also identified as the barriers for the appropriate provision of genetic services in Quebec, which seems counterintuitive. However, this indicates that the rate of genetic specialists and specialized centers have not kept pace with the increase of genetic discoveries and the related increase in demand for genetic testing and counseling services in the general population. Regarding the provision of genetic tests, new models of delivery have to be devised within the healthcare system to enhance the access to genetic testing. The integration of genetic services into population screening programs  is a justifiable option and complementary to the more established model led by geneticists. It uses already available services and healthcare providers to enhance referrals to genetic testing, which in turn facilitates the identification and follow-up of those at high risk of inherited disorders. Unfortunately, it is a relatively new model of delivery and not yet widely implemented. Among Canadian provinces, the integration between screening and genetic testing services is under development in Quebec and guidelines are implemented only in the context of breast cancer screening programs in Alberta , BritishToo many non-geneticists claim to be geneticists, too many professionals do genetic counseling without proper training.” In light of this, policies aiming at the improvement of undergraduate and postgraduate training in medical genetics, including ethics, are of paramount importance to enhance knowledge and competency of various healthcare workers in providing genetic services. Certifications of national associations, such as those provided by the CAGC and the ABGC, are equally important to guarantee the proper provision of genetic services by qualified healthcare providers. Indeed, standards defining training, practice, and registration requirements for genetic counselors have been developed in Canada by the CAGC and in the US by the ABGC  and international  associations should be mandatory.Genomic applications need to be evaluated before integrating genomic information into the healthcare system. The implementation of genetic discoveries should be governed by appropriate legislative frameworks that can ensure quality by setting standards, evaluating performance, and monitoring outcomes of services. In light of this, a global approach involving national and international professional organizations working with government agencies is necessary to ensure that genetic testing and related services are of a high quality and consumers are protected.In conclusion, current delivery models require good level of genetic knowledge, adequate funding, public policies, and public understanding of genetics and genomics applications. Moreover, different healthcare personnel need to take responsibility for the provision of genetic services to guarantee the sustainability of the genetic service delivery models.To our knowledge, this is the first study that identifies and classifies genetic service delivery models in Quebec. The study has highlighted elements of good practice in genetic service provision and areas for improvement of current genetic service delivery models that may be of use to healthcare providers and policy makers actively involved in personalized medicine.The datasets generated for this study will not be made publicly available because they include information that could compromise research participant privacy/consent.The studies involving human participants were reviewed and approved by McGill University’s Faculty of Medicine Research Ethics Board (study code A01-E02-17A). The patients/participants provided their written informed consent to participate in this study.All authors contributed to the conception and design of the study. BU, JH, MZ, and BK reviewed and translated the survey instrument. BU organized the database. BU and CV performed the statistical analysis. BU and PV wrote the manuscript with input from all authors. All authors read and approved the final manuscript.The study is funded by the PRECeDI project (Marie Sklodowska-Curie Research and Innovation Staff Exchange-RISE), Grant agreement no: 645740.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Schistosoma japonicum is a waterborne parasite that causes schistosomiasis in humans and in more than 40 animal species. Schistosoma japonicum shows distinct genetic differentiation among geographical populations and multiple hosts, but the genetic diversity of different developmental stages of S. japonicum from is less studied. Such studies could elucidate ecological mechanisms in disease transmission by analysing feedbacks in individual physiology and population state.S. japonicum were genotyped with microsatellite loci, including 346 cercariae, 701 adult worms and 393 miracidia. Genetic diversity and molecular variation were calculated at different population levels. Kinships (I′) among cercariae at intra-snail and inter-snail levels were evaluated. Genetic distance (Dsw) was compared between paired and unpaired worms, and partner changing was investigated through paternity identification for miracidia.After infection using cercariae from a pool of snails shedding together (Method I) and infection using mixed equal numbers of cercariae from individually shed snails (Method II), different developmental stages of P < 0.001) than that among different snails after deleting near-identical multi-locus genotypes (niMLGs). The allelic diversity of worms in Method I was lower (P < 0.001) than that in Method II, and allele frequency among mice in Method I was also less consistent. The parents of some miracidia were worms that were not paired when collected. The Dsw between each female of paired and unpaired males was much larger (P < 0.001) than that between the female and male in each pair.The cercaria clones in individual snails varied from 1 to 8 and the kinship of cercariae within individual snails was significant higher (S. japonicum miracidia in individual snails and the unbalanced distribution of miracidia among snails suggests a non-uniform genetic distribution of cercariae among snails in the field. This further influenced the genetic structure of adult worms from infections with different cercariae sampling methods. Schistosoma japonicum in mice can change paired partner, preferring to mate with genetically similar worms. These characteristics provide implications for understanding the balance in genetic diversity of S. japonicum related to the transmission of schistosomiasis.Most of the infected snails contained multiple miracidia clones. The aggregation of genetically similar  Oncomelania hupensis, remains large in China, with only a recent 5.6% decrease over 10 years [O. hupensis snails and schistosomiasis patients in some transmission interrupted areas [Schistosoma japonicum [S. japonicum populations in China has been observed using a variety of markers, including nuclear genes [S. japonicum was thought to result from geographical separation, habitat isolation [S. japonicum among geographical populations or among different species of hosts. The genetic mechanism on biology is less studied both at the individual physiological level and at the population level, including the changed distribution pattern and genetic structure of Schistosoma during different developmental stages in the life-cycle, which may highlight important ecological mechanisms driving schistosomiasis.Schistosomiasis is one of the most prevalent parasitic diseases and causes severe socio-economic losses and public health problems worldwide . Althoug10 years , 3. The ed areas  and anthaponicum . Geograpar genes , 10, mitar genes , 12, micar genes , 14, andar genes , 16. Thesolation , co-evolsolation –20, and solation , 21, 22.Biomphalaria glabrata snails could be infected with multiple miracidia, previously implied by the detection of multiple genotypes of S. mansoni cercariae shed from individual snails [S. japonicum genotypes have been detected from a single O. hupensis snail when ten cercariae were genotyped [Schistosoma in snails.Miracidia invade specific species of snails and develop into sporocysts that eventually generate cercariae, which are shed out into water to seek and penetrate a compatible host, leading to human schistosomiasis infection. l snails , 24. Froenotyped . Howeverenotyped , which mS. japonicum [S. japonicum from water buffalo, cattle, and humans were differentiated significantly from those from goats, pigs, dogs, and cats. Rudge et al. [S. japonicum among these hosts. This inconsistency may mainly relate to the genetic differences originating from cercariae in different snails redistributed by flood and limited sample size, in addition to the selective pressure under different definitive hosts. Since it is challenging to collect adult worms from naturally infected hosts in the field, artificial infection of laboratory hosts has been essential, especially for studies in population genetics, immunology, and pharmacology. The different methods of sampling or infection, including cercariae release or collection from snails and infection using different cercariae populations, may affect the genetic distribution of the parasite in definitive hosts.There are more than 40 domestic and wild mammals that serve as definitive hosts of aponicum , 27, 28.aponicum . It is haponicum . Wang etaponicum  considere et al.  found liS. japonicum are located mainly in the portal and mesenteric veins of definitive hosts. Worms show genetic differentiation among groups from the liver, hepatic-portal-vein, and mesenteric blood vessels [S. japonicum, respectively. In addition, the pairing of adult worms is a prerequisite for egg production and contributes to the genetic diversity of eggs. Partner changing has been reported for schistosomes, for example, S. mansoni females were more likely to switch mates when the newcomer male was more dissimilar to themselves than their first mate [S. mansoni and S. intercalatum tend to change partners and revert to homo-specific pairing [S. japonicum which is homo-specific mating?Adult worms of  vessels . This dirst mate , and het pairing , 34. TheS. japonicum at different life stages was investigated, including cercariae from snail hosts and adult worms and eggs from definitive hosts. Cercariae were further investigated by identifying the number of genotypes per individual snail  and by estimating kinship of cercariae within and among snails. After mice were infected with two methods that collected cercariae either by pooled snail shedding or mixing equal numbers of cercariae from each snail shed individually, the genetic structure of the adult worm population from the mice was compared with the original cercarial population, and the consistency in allelic distribution of adult worms among mice was evaluated. Furthermore, the phenomenon of partner changing of S. japonicum in mice was confirmed, and pairing preference of adult worms was investigated with genetic distance between worms.In this study, the genetic variation of Sample collection and experimental design are summarised in Fig. Oncomelania hupensis snails were collected in March and September 2016 from the eastern area of Dongting Lake , Hunan province, China. Snails were located in ditches in 3 villages at a distance of 10 km. The collection site is a marshland and not a national park or other protected area or private land. Collections were carried out with the permission and assistance of the Institute of Schistosomiasis Control of Hunan Province, China, which supervises snail control in the area. Oncomelania hupensis is not an endangered or protected species. Snails were brought back to the laboratory and classified as Group A for those collected in March and Group B for those collected in September. After a month of captivity, snails were washed and transferred to individual water-filled vials for 3 h under light at 25 °C, to stimulate the emergence of cercariae for identifying S. japonicum infection. Finally, 20 snails from Group A and 25 snails from Group B were identified as infected with S. japonicum.2, 0.2 g/l MgSO4·7H2O, and 1 g/l glucose) under microscopy. After 3 washes, cercariae were pipetted individually with 2 μl sterilized washing solution onto a Whatman™ FTA card . After drying, cards were stored in a sealed plastic bag in a desiccator at room temperature. At least 50 cercariae per snail were collected individually and stored onto Whatman™ FTA cards.After cercariae were released from individual snails, some were transferred to a dish using a loop and washed in cercariae washing solution , 484 worms from mice by Method II (Group B) were collected.For subsequent infections, 17 of the 20 snails from Group A and 16 of the 25 from Group B remained alive and able to produce sufficient cercariae. Two different cercariae pooling methods were used for the two groups of snails. For the 17 remaining snails of Group A, all snails were pooled together in a flask containing water to stimulate the emergence of cercariae (Method I). Thirty-one mice were then infected percutaneously with 34 cercariae for each mouse. For the infection using 16 snails of Group B, 2 cercariae from each snail shedding individually were pooled together, and the mixed 32 cercariae were used to infect a mouse (Method II), replicating to infect 22 mice in total. All mice were sacrificed to collect 2, and 0.20 g/l NaHCO3). After 3 washes, miracidia were pipetted individually with 2 μl sterilised washing solution onto a Whatman™ FTA card. After being dried, the card was stored in a sealed plastic bag in a desiccator at room temperature. At least 50 miracidia per mouse stool were collected.Before all infected mice were sacrificed to collect adult worms, stools from each mouse were collected for 7 days, to concentrate eggs excreted through the intestine for miracidia hatching. Egg sediment from each mouse was exposed to dechlorinated water in a flask under light at 25 °C to hatch miracidia. Miracidia were harvested from the upper water of each flask by a pipette and transferred into a dish with miracidium washing solution . The disc was placed in the base of a 96 well, 1.2 ml U-bottom plate. After one wash with 100 μl FTA purification reagent  for 5 mins followed by 100 μl TE buffer , 14 µ of 0.1 M NaOH with 0.3 mM EDTA, pH 13.0 was added to each well and the solution left to incubate for 5 mins, after which 26 µl of 0.1 M Tris-HCl buffer, pH 7.0 was added. Then the plate was mixed by pulsing with a vortex mixer 3 times for approximately 10 s each. The solution was then left at room temperature for 10 min, pulse vortexed 10 times, and the eluate was transferred to a clean 96-well plate, and stored at − 20 °C for further use.As for individual adult worms, the anterior part, including the uterus, was cut off under microscopy from each adult female worm to avoid the influence of eggs in the uterus on the genotype of the female worm. While the whole male or immature female worms were used for DNA extraction. The total genomic DNA was extracted individually from each worm using a standard sodium dodecyl sulfate-proteinase K procedure  as follo28S ribosomal DNA gene fragment was amplified from DNA solution of a single cercaria, miracidium, or adult worm, using following primers: forward (5′-GTG GAG TTG AAC TGC AAG C-3′) and reverse (5′-GCT CAA CAW TAA TAG TCA AAC CTG-3′). PCRs were performed using the Illustra PuReTaq Ready-To-Go PCR Beads in a 96-well plate . For each bead, 2 µl of FTA elution from single larva or 0.5 µl of crude extract from a single worm were added, with 1 µl of each primer, and water to a final volume of 25 µl. The amplification was implemented with the following program: 95 °C for 5 min; followed by 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 40 s at 72 °C; and a final extension step at 72 °C for 7 min. Reactions were then checked by running 5 µl on a 2% agarose TAE gel.To confirm genomic DNA existed in FTA card elution or crude extract from ethanol stored sample, the 2O, then carried out in a Bio-Rad thermocycler with the following program: 95 °C for 5 min; followed by 35 cycles of 30 s at 95 °C, 90 s at 60 °C, and 3 mins at 72 °C; followed by a final extension step at 60 °C for 45 min. PCR reactions were checked by running 5 µl on a 2% TAE agarose gel, and successful amplifications were sent to Sangon Biotech  for fragment analysis on an ABI3100 genetic analyzer . Genotypes were scored using a microsatellite plugin of Geneious 11.0 [Small scale genotyping was done for 30 cercariae, 30 miracidia, and 30 worms which were chosen randomly, to construct an optimal multiplex microsatellite loci panel for PCR amplification. Twenty loci developed and validated in previous studies , 36 and ous 11.0 . All samNa), the effective number of alleles (Ae) in GenAlEx 6.5 [Ar) and gene diversity (Hs) in hierfstat ver. 0.04–22 [Genetic diversity of each locus was estimated in all genotyped cercariae by calculating the observed number of alleles  and 122 cercariae from 16 snails of Group B (used in Method II) were evaluated. After genotyping and filtering low-quality data, at least 5 pairs of adult worms (when present) and all unpaired worms from each mouse were successfully genotyped. In total, 357 worms from mice in Method I (using cercariae from 17 pooled snails of Group A), 344 worms from mice in Method II  were involved. Additionally, the genetic diversity of adult worms in each mouse was estimated to check the allelic consistency of adult worms among mice in Method I and II separately. For miracidium populations, genetic diversity of miracidia from liver and stool was calculated at supra-population level , due to only 3–4 miracidia from liver or stool in each mouse were genotyped, including 93 miracidia from livers (3 per mouse) and 124 miracidia from stools (4 per mouse) in Method I, and 88 miracidia from both of livers and stools (4 per mouse separately) in Method II.The The genetic differentiation of all genotyped cercariae from Group A and Group B was estimated using AMOVA in Arlequin ver. 3.5.2.2 . After eallelematch ver. 2.5 [The number of cercarial genotypes from each snail was counted. Since it is difficult to distinguish typing errors or somatic mutation during sporocyst multiplication from the original miracidium genotype, a group of cercariae genotypes with fewer than 2 mismatched alleles were identified as niMLGs and assigned to a lineage derived from the same miracidium in each snail. Finally, the number of miracidia that established genetic clones in each snail was evaluated based on the lineage number using the R package ver. 2.5 .amCluster in allelematch, to produce the filtered dataset for kinship analysis between miracidia. Then, kinship among singletons (miracidia) was estimated using the allele size correlation coefficient (I′) according to Streiff [Since cercariae with niMLGs in one snail were presumed to be derived from one miracidium , one of  Streiff  in SPAGe Streiff  at intraNa, the basic index for indicating gene abundance of a population [Na was then estimated for each locus with the function nb.alleles in R package hierfstat. The relativity between genetic diversity and snail number was evaluated by calculating the smallest sample size of snails when 50% and 95% of total Na was set.The non-uniform genetic distribution of cercariae among snails implies that sample size of snail hosts would affect the allele distribution of the cercaria suprapopulation . pulation , 46, wasS. japonicum during mating, the inter-individual genetic distance Dsw [http://www.bioinformatics.org/~tryphon/populations/), and compared with that between the paired female one and unpaired worms  in the same host.To detect a potential genetic preference for ance Dsw  between Allele Frequency Analysis function in Cervus 3.0 [Simulation of Parentage Analysis was applied based on allele frequency, to estimate the resolving power of codominant loci and critical values of the log-likelihood (LOD) with assumptions as follows: 10,000 offspring (miracidia) for each individual mouse; 10 candidate mothers and fathers in each mouse (estimated sample size according to the number of adult worms collected in a host); the proportion of candidates was set to 100% of genotyped adult worms; other options were set by default. Finally, both of the outputs from the Allele Frequency Analysis and Simulation of Parentage Analysis were referred in the Parentage Analysis module, to assign each offspring to its most likely parents. The parents that have more than 2 alleles mismatched with offspring were excluded, and those that had the biggest LOD were considered most likely as the parents.The allele frequencies of all adult worms and miracidia in each mouse were analysed and calculated using the rvus 3.0 . All 9 lNa, Ae, Ar and Hs) of loci, kinship coefficient (I′), and inter-individual genetic distance (Dsw) between two groups were evaluated with the significance level of 0.05. First, the normal distribution of datasets was checked with Sharpiro-Wilk test. Paired-samples/independent t-test was then applied when datasets met normal distribution. Otherwise, the Wilcoxon signed-rank test and Mann-Whitney U-test were applied for the related/independent samples. Bonferroni correction was used to correct the P-value in multiple comparisons.All data used in statistical analysis in this study are continuous data. Significance of difference on genetic diversity indices (r2 < 0.3), and deviated from the Hardy-Weinberg equilibrium (P < 0.001). Nine microsatellite loci showed high polymorphism in cercariae, including 156 cercariae from the 20 snails (7.5 ± 0.5 (mean ± standard deviation) cercariae checked per snail) of Group A and 190 cercariae from the 25 snails (7 ± 1 cercariae checked per snail) of Group B. Allele number (Na), the effective number of alleles (Ae), allelic richness (Ar), and gene diversity (Hs) for each locus are given in Additional file t(8) = 0.978, P = 0.357 for Na; t(8) = 2.125, P = 0.060 for Ae; t(8) = 1.179, P = 0.272 for Ar; Z = − 1.866, P = 0.062 for Hs)  = 3.277, P = 0.011 for Na; t(8) = 4.312, P = 0.003 for Ae; t(8) = 3.416, P = 0.009 for Ar; Z = − 2.524, P = 0.012 for Hs, with corrected P < 0.025 as significance) than those for 357 worms from Method I  = − 1.265, P = 0.242 for Na; t(8) = 1.116, P = 0.297 for Ae; t(8) = − 1.603, P = 0.171 for Ar; Z = − 0.896, P = 0.370 for Hs) with that for 344 adult worms from Method II  = − 4.034, P < 0.001 for Ae), which suggested that adult worms in each mouse from Method II contained more alleles than worms of each mouse from Method I.For comparison between populations related to mice infections, cercariae from snails not used for infection were excluded in the genetic diversity analysis and AMOVA. The AMOVA showed limited differentiation as 2.32% between two cercariae groups from snails used to infect mice (132 cercariae of Group A and 122 cercariae of Group B). The differentiation is also limited between the adult worms and the original population of cercariae . The differentiation of worms among mice were 0.49% and 0% for worms among 31 mice in Method I and worms among 22 mice in Method II, respectively. It revealed that worms among mice in Method II have higher consistency in allelic distribution than worms among mice in Method I.Na and Ar of miracidia from stools were similar with that from livers of mice  in Method I but Ae and Hs are different (t(8) = − 4.650, P = 0.02 for Ae; Z = 2.442, P = 0.015 for Hs)  = 1.492, P = 0.174 for Na; t(8) = 0.558, P = 0.592 for Ae; t(8) = 1.472, P = 0.174 for Ar; t(8) = 0, P = 1.0 for Hs). The genetic differentiation is limited between miracidia from livers and stools, with 1.02% in Method I and 0% in Method II.The S. japonicum that could originate from one or multiple miracidia infection. Among them, the ratio of female- to male-containing snails was 6:7 and 11:12 for the two groups, respectively, showing no parasite sex-ratio bias between snails. The other snails, carrying both sexes of adult worms, must have been infected with more than one miracidium. Furthermore, multi-locus genotypes (MLGs) of cercariae ranged from 6 to 8 (7.75 on average) in 20 individual snails of Group A, and from 5 to 8 (6.65 on average) in 25 individual snails of Group B. After near-identical multi-locus genotypes (niMLGs) were filtered, the number of clones was inferred as from 3 to 8 in individual snails of Group A and from 1 to 6 in individual snails of Group B, with an average of 6.6 and 4.2, respectively. This multi-clonal distribution even after filtration further implied that the multiple infections with more than one miracidium in a snail is common in the field.Of the infected snails, 65% (13 of 20) snails of Group A, and 92% (23 of 25) snails of Group B shed a single-sex of I′ of miracidia was 0.01 on average at the inter-snail level in both Group A and B  at intra-snail levels of Group A and increased to 0.27 (from − 0.33 to 1.91) in snails of Group B, both were higher (t(409.4) = − 14.649, P < 0.001 for Group A; U = 765784.5, Z = 12.836, P < 0.001 for Group B) than that between snails  of cercariae reach the plateau (where Na has no significant difference (P < 0.05) with the total Na of the corresponding locus) separately, while 23 or 24 (92%) snails need to be sampled in Group B when the Na of 4 loci  could reach the plateau. Na of other loci could not reach the plateau until all snails were sampled, which implied that 9 loci had varied allelic frequency among snails . The deviation of ted Fig. . More alsed Fig. . The numSimilarly, 150 different alleles were identified for 9 loci of cercariae from 25 snails of Group B, with an average of 16.7 per locus. Five snails (20%) was minimal to retrieve 50% total alleles (8.3 per locus) of cercariae, with a deviation of 9.0%, and 23 snails (92%) was necessary to retrieve 95% total alleles (15.8 per locus), with a deviation of 1.5%  between each paired male and unpaired females in the same host. It showed no significant difference (t(7) = − 2.197, P = 0.064) with Dsw between male and female in each pair . While for 32 mice that contained unpaired males, Dsw ranged from 1.5 to 20.6 (9.2 ± 2.8) between each paired female and unpaired males in the same host, larger than that between the female and male in each pair  (t(32) = − 4.435, P < 0.001, with a corrected P < 0.025 as the significance level)  were successfully assigned to their most likely parents Table , even whS. japonicum from over 8000 snails collected in the field. Through MLGs evaluation, most of the snails were confirmed to be infected with more than one miracidium of S. japonicum, with 6.6 and 4.2 cercariae clones on average per snail in the two groups, respectively. Of 45 snails, 80% (32/45) contained single-sex S. japonicum, and most of them were infected with multiple cercaria clones. Only one (3.1%) snail was infected with a single miracidium. This overdispersion is common for parasites within hosts in the field [I′) derived from miracidium genotypes in individual snails was significantly higher than those among different snails, indicating that miracidia clustering together in the same intermediate snail host have closer kinship than in different snails. This aggregation of genetically similar S. japonicum miracidia in individual snails and disequilibrium distribution of miracidia among snails may be explained with the following circumstances: (i) snail immune pressure: miracidia selected by the snail immune system and only those that have compatible alleles (and being more related) can survive when encountering a particular snail genotype; (ii) genetics: miracidia could exclude those with lower genetic similarity through the competition when a snail is multi-infected; (iii) snail control: snail populations were isolated into several subpopulations by strict snail control, which facilitates inbreeding of S. japonicum in separate populations, then amplifies the aggregation of S. japonicum with closer kinship and separate distribution among snails.A total of 45 snails were found to be infected with he field . The unphe field , the spahe field  have allS. japonicum at the population level. In this study, 20% of snails is enough to retrieve 50% of the total alleles of cercariae. It reflects that many alleles are shared among cercariae in different snails. However, sampling 95% of total alleles is difficult, as approximately 90% of snails were needed to cover the lower-frequency alleles. It is necessary therefore to understand the genetic diversity and distribution of alleles in cercariae among snails, to get the appropriate sample size for cost-effective population studies, especially for regions at low prevalence, where the degree of parasite overdispersion is higher [This non-uniform genetic distribution of cercariae among snails further implies that the sample size of snail hosts would affect the genetic diversity of the cercarial suprapopulation. Therefore, the sample size of snails should be evaluated to make sure cercariae from selected snails can cover most of the alleles of s higher , and allThe adult worms in mice obtained with two infection methods both showed no significant genetic differentiation from the corresponding original cercarial population in the AMOVA. However, the two methods have a different ability to maintain the genetic diversity of cercariae through the process of infection. The genetic diversity of adult worms in 22 mice from Method II  was similar to that of the original cercarial population, and the genetic diversity of adult worms in 31 mice from Method I (using cercariae from pooled snails) decreased when compared to that of the original cercarial population. Furthermore, the variation of adult worms among mice in Method I was larger than that in Method II. It implies that infection with equally pooled cercariae from individual snails (Method II) transfers more alleles from cercariae to adult worms in each mouse. The allele distribution among mice in Method II was more consistent than that in Method I. It is reasonable that cercariae from different snails in Method I do not have an equal chance to infect a mouse, which creates a larger genetic differentiation of adult worms between mice in Method I than that in Method II. The uneven distribution and release of cercariae on the surface of the water from each snail would make the cercariae sampling replicates more variable from a pool of snails using Method I, thereby altering the allele frequency of the adult worm population. Method II is therefore recommended for artificial infection, which can transfer more alleles from cercariae to adult worms and keep the consistency in the genetic structure of adult worms in mice.Ae and Hs in Method I which may result from the unequal sample size (3 vs 4), and no significant differentiation was shown. These data indicated that eggs of S. japonicum were transferred to the liver or intestine randomly.Referring to the genetic difference discovered between adult worms in different organs , we suspSchistosoma. It has been suggested that S. mansoni could separate and re-pair to a genetically dissimilar partner, which may increase the adaptive ability of offspring [S. japonicum may cause deviation from the allele sizes of original adult worms, which can differ among loci [S. japonicum adult worms. Previous studies reported that hetero-specific pairs always intend to re-mate to homo-specific pairs in a mixed infection of S. mansoni and S. intercalatum [S. japonicum, the genetic distance (Dsw) between unpaired worms  and paired females, was higher than that between the male and female in each pair. This indicates a tendency for S. japonicum to mate with a genetically similar partner, contrary to S. mansoni. Huang et al. [S. japonicum eggs in definitive hosts and hatching rate of eggs. It is known that egg-laying capacity and pathogenicity to definitive hosts of S. japonicum is more significant than that of S. mansoni [Schistosoma mansoni tends to produce less pathogenic eggs and cause less damage to hosts, even with the same number of cercariae as S. japonicum. Such adaptations are suitable for continuation of adult worm population of S. mansoni in the field, where few mammalian species can be used as definitive hosts. However, S. japonicum tends to produce many more eggs, which produce severe pathological damage, accelerating host death, while using more than 40 mammalian hosts. Considering that S. japonicum prefers a genetically similar partner to produce more eggs, we speculate that the different preference of pairing between S. japonicum and S. mansoni is driven by the difference in egg productivity, pathological damage, and available definitive hosts species in the life-cycle. How the distribution of S. japonicum in snails and pairing of adult worms affect the balance between adaptation by maximizing genetic diversity and virulence or transmission by minimizing genetic diversity deserves further investigation.Mating is an essential process in the life-cycle of ffspring . Only 9.ong loci , thus marcalatum , 53, malrcalatum . This mag et al.  reported mansoni . SchistoS. japonicum miracidia (cercariae filtered niMLGs out) in intermediate snail hosts in the field, the genetic structure of different developmental stages in mice by different sampling methods, and pairing preference of adult worms in mice. We found that infection with multiple miracidia is common in individual snails, with the aggregation of genetically similar miracidia in individual snails and disequilibrium distribution among snails. The sampling method during the infection has an impact on the genetic structure of adult worms. Partner changing and paring with genetically similar adult worms helps to balance the genetic diversity of S. japonicum for adaptation and transmission. All of the results give better understanding of the genetic variation of different developmental stages of S. japonicum. The genetic distribution characteristics and pairing preference of S. japonicum may provide an insight into genotype-related forces that affect the survival and transmission of this species.This study investigated the genetic diversity and kinship of Additional file 1: Table S1. The information for nine microsatellite markers used in this study.Additional file 2: Table S2. Genetic diversity of nine microsatellite loci in all genotyped cercariae from snails of two groups.Additional file 3: Table S3. Genetic diversity of each locus in adult worms from mice infected with two methods.Additional file 4: Table S4. Genetic diversity of combined nine loci in worms from each mouse in Method I and Method II.Additional file 5: Table S5. Genetic diversity of each locus in miracidia from the liver/stool of mice in Method I and Method II.


Ex situ seed banking was first conceptualized and implemented in the early 20th century to maintain and protect crop lines. Today, ex situ seed banking is important for the preservation of heirloom strains, biodiversity conservation and ecosystem restoration, and diverse research applications. However, these efforts primarily target microalgae and terrestrial plants. Although some collections include macroalgae , they are relatively few and have yet to be connected via any international, coordinated initiative. In this piece, we provide a brief introduction to macroalgal germplasm banking and its application to conservation, industry, and mariculture. We argue that concerted effort should be made globally in germline preservation of marine algal species via germplasm banking with an overview of the technical advances for feasibility and ensured success. Seaweed germplasm banking is an important resource for biodiversity conservation, human food security, and industry innovation. This Perspective article maintains that an international, coordinative initiative is needed to fully develop and capitalize on this resource. The mar2 ,19, thei2 ,20,21. T2 ,17,20–272 . Given tWild harvest of macroalgae has a longstanding history in many cultures, particularly in Asia, Polynesia, and South America. Modification and enhancement of wild stocks began as early as the 17th century in Japan. Today, over 200 species of macroalgae are wild harvested for various industries , but onlPyropia/Porphyra is the optimal stage for its long-term storage [Gracilaria and Agarophyton species that are well maintained as meristems or mature thalli [Chondracanthus chamissoi [Porphyra yezoensis [Saccharina spp. [Ex situ seed banking preserves the diploid, embryonic stage of the embryophyte plant life cycle. This stage is optimal for preservation because of its evolved traits that facilitate long-term dormancy in natural conditions  . Convers storage ,45. Pres storage ,46,47. Oe thalli . Live tihamissoi , Porphyrezoensis , Saccharina spp. ,51), butP. ubilicalis and Undaria pinnatifida as part of a top-50 foods list that people should prioritize eating for a healthier planet and global food sustainability [As the human population continues to grow, it is important to explore alternative food sources, like macroalgae, for sustainable nutrition. In collaboration with Knorr Foods, the World Wildlife Fund published a 2019 report supporting nability . As relinability . Ex situnability , althougnability . Germplanability . Lastly,nability , and thenability . StrainsMacrocystis pyrifera for alginate isolation and Ecklonia maxima for biostimulant production, for example, are still exclusively wild harvested and have yet to have well-developed mariculture methodologies for large-scale farms . Although M. pyrifera has germplasm collections that represent its distribution and genetic diversity in both hemispheres [E. maxima has yet to be genetically characterized in its limited distribution or developed to any degree for aquaculture purposes. Thus, economically important species should be prioritized in order to not only preserve optimized strains currently used but also conserve their genetic diversity in order to maintain their utility over time. Secondly, species that provide crucial ecological services  should be prioritized for germplasm banking. Particularly canopy- and habitat-forming species in the brown algal orders Laminariales and Fucales should be targeted for research and sampling of genetic diversity [M. pyrifera [G. lemaneiformis [Given the breadth in germplasm bank utility and value, one of the greatest challenges is identifying which species to prioritize; with estimates in algal diversity ranging from 72,500  to 170,0ispheres ,66, a briversity . Furtheriversity , should iversity  should beiformis ). Althoueiformis ,72. Lasteiformis ,74. AlthDespite a fast-growing interest in commercial aquaculture, corresponding with a rapidly increasing global demand for nutrition supplementation and industry raw material, few researchers outside of Asia have explored sustainable domestication programs, including the creation of macroalgae germplasm banks ,39,75,76A major obstacle to the creation of an extensive and viable macroalgal germplasm bank rests on the documentation and understanding of the genetic variation in coastal macroalgal communities and how genotype influences each organisms’ phenotype and its functional importance within the ecosystem. Maintaining biodiversity within a marine macroalgal ecosystem necessitates observing key interactions within a community to identify those species that provide invaluable ecological services  and mainMacrocystis [Porphyra [Saccharina [Major challenges of documenting genetic diversity across distributions have included not only considerable collection costs but, historically, impeding sequencing costs as well. However, work done over the past 30 years with increasingly economical sequencing approaches has made important progress in our understanding of genome evolution, genetic diversity, population structure, and ecosystem dynamics in commercially and ecologically important macroalgae . ccharina . These gIn closing, we emphasize that international and national programs that are currently supporting terrestrial plant seed banks  should equally support macroalgal germplasm banking efforts. Building a global network and coordinated initiative to build and maintain these collections is vital to supporting the Blue Revolution and continued mariculture advancement. By incorporating macroalgal germplasm banks into these efforts, we support not only human food security and industry but also the ecological function and services provided by marine primary producers. We also recognize that the ideas presented here are not necessarily novel; it is important that recognition of documents like PEGASUS be given. But given the very recent interest in seaweed mariculture outside of Asia and Europe, it is important that conversations surrounding cooperative and thoroughly characterized macroalgal germplasm banking begin as soon as possible, and the ideas presented here continue in their development as a living concept.