Ethical challenges of genetic counselling for schizophrenia include effective communication of critical scientific information in an easily understood manner by patients and relatives, and the ability to ensure communication is unencumbered by medical jargon. Levels of literacy in the target population may limit this process, making it difficult for patients to attain the desired levels of informed consent to make crucial decisions during genetic counselling. Multilingualism in target communities may further complicate such communication. This paper outlines the ethical principles, challenges and opportunities facing clinicians when conducting genetic counselling for schizophrenia and how these might be met, drawing on lessons from South African studies. The paper draws on reflections of clinician and researcher experiences gained from clinical practice or research on the genetics of schizophrenia and psychotic disorders in South Africa. The context of genetic studies in schizophrenia is used to illustrate the ethical challenges in genetic counselling for schizophrenia, both in clinical and research settings. Attention is also drawn to multicultural and multilingual populations, particularly where the preferred language lacks a well-developed scientific language of communication for some of the genetic concepts that have to be presented during the genetic counselling process. The authors describe the ethical challenges and how to address these to empower patients and relatives to make well-informed decisions despite these obstacles. Principles applied by clinicians and researchers during the genetic counselling are described. Potential solutions, including the establishment of community advisory boards to address potential ethical challenges inherent to the genetic counselling process, are also shared. Genetic counselling for schizophrenia still faces ethical challenges which require a balance of principles of beneficence, autonomy, informed consent, confidentiality and distributive justice, while striving to present accuracy in the science that guides the process. Evolution in language and cultural competency therefore needs to occur alongside scientific advances in genetic research. Key stakeholders need to partner and build capacity and expertise in genetic counselling through the provision of funding and resources. The goal of partnerships is to empower patients, relatives, clinicians and researchers to share scientific information in a manner guided by empathy while retaining scientific accuracy. Genetic counselling in schizophrenia is an essential part of clinical practice but needs to be evidence-based and guided by developments in genomic research. Research into the genetic causes of schizophrenia, and other serious illnesses, may include relatively large sample sizes and data sharing across international borders . EthicalA relevant question that arises when considering the process of genetic counselling is whether the ethical framework that governs this type of counselling and the sharing of information on genetic results is robust enough to ensure the ethical conduct of clinicians while protecting the rights of patients. The Human Heredity and Health in Africa Consortium (H3Africa) Ethics and Community Engagement Working Group identified some of the principles pertaining to genomics research in Africa and established guidelines for consideration of feeding back genetic results of health importance . As muchGenetic research plays an important role in producing results that support the scientific evidence on which sound genetics counselling is based. An approach proposed by Moher, Bouter, and Kleinert to the ethics of research is that, for society’s benefit, research and knowledge must be trustworthy . This imAccess to trained genetic counsellors who have the expertise to interpret complex genetic information and to provide feedback in a manner that empowers families to take decisions about their health outcomes and the management of their conditions, becomes critical , 4, 7. TIn this paper, we examine several studies on the ethics of genetic counselling and the learnings from three large-scale multicentre studies on mental health and on the genetics of schizophrenia, to explore the ethical challenges that may confront clinicians. Linking the parallels between communication when conducting research on genetic causes of schizophrenia and communication when conducting genetic counselling for schizophrenia is done to illustrate similar challenges and how these might be addressed. This paper focuses on how to enhance the robustness of the ethical framework that governs such communication in both genetic counselling and genetic causes of schizophrenia research. This interrelatedness is important for building the evidence that forms the basis of knowledge to support genetic counselling.Some of the challenges in genetic counselling include balancing of the well-being and interests of the patient and family (beneficence) while avoiding causing harm  through ensuring information sharing on genetic and hereditary risks is shared in a non-stigmatizing and empathetic manner. This balance may be further complicated by the lack of standardized guidelines; ambiguities related to variant interpretation due to a lack of translational studies; and underrepresentation of people of some communities in the reference genome . AlthougClinicians thus need to weigh how much of the known factual information about heredity is shared for the benefit of patients while ensuring non-maleficence by supporting patients and families to manage the angst that comes with the uncertainty of not knowing the risk for developing schizophrenia in the offspring. Clinicians also carry the responsibility of supporting patients and families to balance the facts shared about the genetic risks while mitigating against the stigma that may be associated with the implication of passing on a chronic mental illness like schizophrenia to their offspring. A possible mitigating strategy that is proposed by Matimba et al.  is for cChallenges that may be faced by clinicians in an African setting may include difficulties in how to present complex ideas in a more simplified manner that is understood by patients and relatives during genetic counselling , 13. ForOther challenges when it comes to informed consent for sharing and processing information about genetic causes of illnesses include the problem of simplifying the language of communication to make the jargon of genetic concepts like genes, DNA, and the different types of analysis or sequencing accessible to patients in general . FactorsIn a country like South Africa, where there are 11 official languages and where only two of the 11 languages have a well-developed scientific language that includes commonly used terms to describe genetic concepts, these challenges are further amplified , 14, 15.Striking a reasonable balance between the contrasting approaches of sharing the minutest details about a study or genetic risk and glossing over such details to provide only the simplest information that participants or patients can understand during genetic studies or the process of genetic counselling may be difficult if the process is not guided by firm ethical boundaries. This is more so in the case of schizophrenia and other similar or severe mental illnesses, where capacity to give informed consent may not always be intact and confidentiality has to be balanced with the need to consult with the patient’s families or relatives. An example of how this was handled is presented by Campbell et al. , who werMultisite, cross-country research, as a basis for evidence to support genetic counselling for schizophrenia, may also have implications with regard to access to resources for collaborating partners per country. The capacity to use multisite derived data in order to write and publish scientific papers drawn from these sites may not be feasible for all participating research units, depending on their resources . This woCultural competency in mental health means the ability to engage with communities from different cultural backgrounds efficiently and in an appropriate and relevant manner. A clinician’s understanding of the socio-cultural factors balanced with the relevant medical issues is important to empowering patients to make sense of information shared on genetic aspects of the causes of schizophrenia , 17.As in the consideration of distributive and social justice described above, regional nuances and cultural competence frameworks need to be taken into consideration when conducting research and interpreting data in multisite genetics and genetic counselling studies , 4, 11. When it comes to the language, especially in multilingual settings, the sharing of genetic counselling using relevant and accessible communication methods becomes important. Some studies have shown that basic terminology that may be used even during psychoeducation may not be understood by patients and relatives in multilingual setting such as South Africa , 19. MotThere have been a number of multicentre studies on the genetics of schizophrenia and the genetics of psychotic disorders  which have included study sites in South Africa . There hEach of these research projects has provided an opportunity to examine the process that researchers followed in addressing information sharing on genetic studies, informed consent issues for genetic research  and the setting up of governance frameworks for gaining access to large scale data across country boarders , 2.The SAX study is a case–control study that was conducted among Xhosa-speaking participants in South Africa . The goaRecruitment took place in both rural and urban settings. A Community Advisory Board (CAB) was set up to represent community interests. A Community Advisory Board (CAB) was convened, and consisted of, patients and relatives advocacy groups, members of hospital facilities boards and other members of the local Xhosa communities. The CAB met once per quarter to discuss various issues arising from the project, including stigma. The approving Human Research Ethics Committees were updated annually, or when appropriate, on the proceedings of the CAB. The CAB also reviewed informed consent forms, where appropriate, for the purposes of guiding an update process based on the CAB input. Additional CAB activities included psychoeducation sessions about mental illness and local services to address the needs of people living with mental illness and their relatives.The main challenges had to do with communicating study information to potential participants effectively due to the need to translate the main study concepts around genetic research and the specific information regarding the DNA analysis that was to be conducted on collected samples. Limitations included the fact that the scientific language and everyday language to express the concepts and study information did not exist at the time of conducting the study. An added nuance was that grammatically correct translation from English to Xhosa was not always the most accessible language that could be easily understood by the community from which the participants would be recruited. Linked to the accessibility of information, when translated into Xhosa, was the issue of low literacy levels in some of the communities targeted for recruitment. This required alternative ways of communicating information other than the written format and also different ways of expressing informed consent beyond a signature on a consent form.Using professional translators did not meet the required needs of the language challenges. The initial study information leaflets had to be reworked to adapt the first translated documents into more accessible language formats. This complicated the forward and backward translation in that it required a few rounds of translations to get to the desired quality and accuracy of information between the English and Xhosa participant information leaflet.The issue of low levels of literacy was addressed through the verbal delivery of the study information and this was undertaken by research assistants and fieldworkers during outpatient clinic visits. The verbal deliveries allowed for a seamless introduction of the study and the opportunity to address questions in group settings. The group work was followed by a sharing of written information and an initial invitation that those who were interested in taking part in the study could indicate their interest to the fieldworkers. The next stage was to offer an appointment for enrolment where more detailed information about the study was shared, to those who were interested. Another important finding was that for a subsection of the study that sought consent for storage of cell line samples the recruiter who sought consent was a strong predictor of participant consent . This waThis is a large scale case-control, multicentre study, across 4 countries in Africa namely, Ethiopia, Kenya, South Africa, and Uganda, that was still running at the time of the writing of the current paper. Participants were drawn from psychiatric inpatient and outpatient facilities. Genome-wide association studies were performed on DNA extracted from saliva. Data sharing occurred across country boundaries governed by country-specific pieces of legislation and regulations.Informed consent procedures were affected by language limitations. Mitigation strategies included translation of the participant information leaflets, application of a formal assessment of capacity to consent tool and the use of outpatient waiting room engagements in group format to first lay the ground work for sharing information to potential participants.Adding an iterative element during the participant recruitment phase of the study enabled participants’ understanding of the study information to improve enough to be able to give informed consent for themselves instead of requiring proxy consent. Feedback on study results and findings on the genetic causes of schizophrenia was given to the community advisory board, the institutional managers and other stakeholders linked to the psychiatric services, also.The MindKind study researched how to include the participant voice by involving them in decision-making about the governance framework of the research project and about who should access the data bank for research purposes . The parSetting up advisory boards across the different countries differed according to access to resources, specifically smartphones and data for connectivity. This required specific remuneration for costs of data toward participants to be included within the proposal for the study and the budget. Study costs. The translation of jargon-filled technical terms having to do with the sharing and storage of data also posed a challenge.Critical to the inclusion of participants in the decision-making process adopted in the MindKind study was the upskilling of participants to empower them with the knowledge that enabled them to take an active role in such governance processes . ParticiThe link between genetic counselling and genetic research in schizophrenia, as highlighted in the three studies described in this paper, seems to suggest that the ethical challenges clinicians face, in both contexts, share similarities. Concepts of beneficence and non-maleficence, distributive justice, informed consent, balancing accessible information against the potential for stigmatization, broad vs. specific content, and proxy consent, all require a balancing act that clinicians have to undertake in consultation with patients and relatives to ensure volition. When one adds the need for trustworthiness and reliability of the data shared with patients on the genetics of schizophrenia, then the clinical utility of the data being shared also comes into play. Persistent challenges include the lack of equity in the availability of and the access to resources, as well as the limited access to clinicians with expertise in genetic research and genetic counselling. Although these persistent challenges may imply that clinicians may continue to struggle to meet community needs on genetic counselling for schizophrenia, there are some encouraging signs of potential for growth and capacity building. Evidence of opportunities is further outlined next, using a global and regional perspective as well as evidence from genetic counselling conducted across other medical specialities.The possibility of stigmatization when informing individuals and families about their genetic risk for schizophrenia has already been alluded to and remains a significant factor to consider . FurtherUse of the Genetic Counselling Outcome Scale (GCOS-24), which is a Patient Reported Outcome Measure (PROM), has been advocated to help provide evidence to support service development in genetic counselling. This measure was developed for use in the National Health Service (NHS) system of healthcare in The United Kingdom and has since been validated across other countries like Denmark and The Netherlands . The othPollard et al.  also notJenkins and Arribas-Ayllon  describeIn African societies, genetic counselling for schizophrenia may be faced with additional unique ethical challenges. These may include issues of access due to resource limitations, informed consent, and the impact of collective values and practices on individual autonomy . The colIn South Africa, genetic counselling for schizophrenia is similarly also faced with additional unique ethical challenges , 29. TheAnother area for future development in populations with diversity in culture and languages, as found in South Africa, is the issue of translating key genetic concepts into indigenous languages. The lessons learnt from the studies like the NeuroGap and SAX, present opportunities to amend practice and training and make it work for the scientific community by ensuring that gaps are addressed and plugged. To maximize on lessons learnt from the studies illustrated above, clinicians and researchers can use these gaps as opportunities to amend practice or training and improve access to genetic counselling by ensuring that such gaps are addressed and plugged through training. Learnings from the NeuroGap Study indicate that one way around the difficulty of translating information on genetics may be to deliberately conceptualize and write participant info leaflets in indigenous languages first and then translate these to English, which may at times be an easier process to undertake than the other way round .Useful examples of genetic counselling may be found in other specialities and conditions like pulmonary arterial hypertension as described by Morris et al. . They inRecommendations and opportunities are presented at systemic and clinical practice level. Training of genetic counsellors is required to improve capacity and expertise in the field of genetic counselling in schizophrenia and other psychotic syndromes. Given the many challenges that have already been outlined as risks to access to care and culturally appropriate and locally relevant information, guidelines specific to genetic counselling also need to be developed to fit regional and national community need , 7, 15. Training in genetic research and genetic counselling needs to be expanded into undergraduate medical and other health professions training programs, to further expand expertise, especially in under-resourced communities. The diversification of training is important especially in under-resourced communities, as the liberty to have standalone genetic counsellors is not a luxury all can afford , 33 The Kim et al.  further There are limited clinical practice guidelines to help clinicians to weigh the role of genetic testing in mental disorders like Schizophrenia. What complicates this is the complex interaction between genetic and environmental factors . This liNorms need to be developed to address the translation of basic concepts on genetic information into indigenous languages. The maintenance of good quality translation for diverse populations and end-users could be extended beyond a condition such as schizophrenia and psychiatry but could encompass other disciplines within which training and accreditation bodies like the Health Professions Council of South Africa (HPCSA) and similar bodies across the global South and the North, could expand . Such noAt a glance, prevailing challenges that face clinicians in genetic counselling for schizophrenia, and the persistent resource limitations may seem discouraging. However, opportunities exist for progress to be made in building ethically sound technical capacity and expertise in genetic counselling for schizophrenia and other mental disorders. Commitment from key stakeholders will be essential to ensure availability of funding and resources to advance and support development of expertise in genetic counselling. Collaborative opportunities to improve access to resources could translate to increased capacity that may address challenges and help develop more expertise in this field. Funding and creation of training opportunities could further strengthen accessibility when such opportunities are leveraged to target under resourced communities. If one maximizes on all these opportunities successfully, the field of genetic counselling in schizophrenia and other mental disorders has significant potential and possibilities for expansion to ensure the goal of accessibility for all who need this service is realized.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Plasmodium falciparum genomic surveillance more cost-efficient and convenient in sub-Saharan Africa. We compared the genetic structure of parasite populations sampled from 289 first ANC attendees and 93 children from the community in Mozambique between 2015 and 2019. Samples were amplicon sequenced targeting 165 microhaplotypes and 15 drug resistance genes. Metrics of genetic diversity and relatedness, as well as the prevalence of drug resistance markers, were consistent between the two populations. In an area targeted for elimination, intra-host genetic diversity declined in both populations (p=0.002–0.007), while for the ANC population, population genetic diversity was also lower (p=0.0004), and genetic relatedness between infections were higher (p=0.002) than control areas, indicating a recent reduction in the parasite population size. These results highlight the added value of genomic surveillance at ANC clinics to inform about changes in transmission beyond epidemiological data.Routine sampling of pregnant women at first antenatal care (ANC) visits could make  However, routinely collected genomic data from ANC has not been evaluated for its suitability for sentinel surveillance.For continuous genomic surveillance of malaria, samples must be collected regularly, and, especially critical for low-resource settings, cost-efficiently.Plasmodium (P.) falciparum parasite population circulating in pregnant women at their first ANC visit and in the community are genetically similar, including similar genetic diversity (intra-host and population-level), relatedness between infections, and prevalence of antimalarial resistance markers. To test our hypothesis, we analyzed the parasite population in ANC users in southern Mozambique, and compared it to parasites found in children aged 2–10 years sampled in household surveys. Furthermore, we compared the parasite populations in three areas with declining transmission between 2015 and 2018. Manhiça and Magude were low-transmission areas, with Magude recently targeted for elimination with a package interventions,24 while Ilha Josina is a historically high-transmission setting.17We hypothesized that the P. falciparum-positive dried blood spot (DBS) samples from ANC users (n=378) and children sampled in population-representative household surveys (n=180) were attempted sequenced. 241 amplicons were targeted, including 165 microhaplotypes informative about genetic diversity in the parasite population. 68.5% (382/558) of the samples were successfully sequenced and passed the filtering criteria , which incorporates intra-host relatedness between clones, was lower at 1.8, while 1-Wright’s inbreeding coefficient (1-Fws) was 0.39, both indicative of inbreeding. Parasite density was associated with measured intra-host diversity, with higher diversity observed for women with higher-density infections. eMOI showed an overall declining trend from 2017 to 2018–2019, and was highest in Magude. 1-Fws showed trends but did not reach statistical significance. Primigravid women had higher eMOI compared to multigravidae in the univariate analysis, but the effect disappeared when adjusting for parasitemia, time, and area (Supplementary Table 1). No statistically significant differences were observed between seasons or human immunodeficient virus (HIV)-status groups. Among children, 62.4% carried polyclonal infections, the average eMOI was 2.3, and 1-Fws was 0.55. Similar to ANC users, children with higher-density infections showed higher eMOI.Half of the pregnant women attending ANC consultations carried polyclonal infections . On averws and odds of infections being polyclonal showed declining trends . No temporal changes in intra-host diversity were observed in Manhiça, while in Ilha Josina, there was an increasing trend over time in polyclonal infections. Intra-host diversity among 47 children from Magude sampled cross-sectionally were compared with samples from ANC users in Magude . In Magude, eMOI declined by 50% per year  across the 165 microhaplotype loci ranged from <0.01 to 0.90, with a mean of 0.57 . Mean HE did not differ between populations when accounting for locus-to-locus variability . ANC infections generally showed low relatedness, with a mean pairwise identity-by-descent (IBD) of 0.026 . IBD was slightly but significantly higher between infections in Magude compared to within and between other areas . Infections in children tended to be more related compared to infections in ANC attendees, and between the two populations. Restricting the comparison to samples from overlapping years (2017–2020) and temporal windows (April 15 to June 30), mean IBD between ANC infections was 0.018, similar to the mean IBD of 0.017 observed for infections from children Genetic relatedness between pairs of dihydrofolate reductase and dihydropteroate synthetase (pfdhfr-pfdhps) genes were highly prevalent in both populations (>90%). In particular, sulphadoxine-pyrimethamine (SP) resistance-associated polymorphisms in the pfdhfr gene had almost reached fixation in the population, with 98.6% carrying the triple 51–59-108 mutant. No A581G nor I431V mutations in pfdhps were detected. Three quarters of the study population carried a multidrug resistance 1 (pfmdr1) F184Y gene mutation associated with amodiaquine resistance, while 1.2% carried the N86Y, and 0.3% carried the D1246Y mutations. The chloroquine resistance transporter (pfcrt) 72–76 CVIET mutant genotype was observed in four individuals, three of them children. No mutations in the kelch 13 propeller gene, pfkelch13, associated with artemisinin resistance, was observed in either population.Prevalence of all markers of antimalarial resistance researched in this study was similar between ANC users and children from the community . ParasitP. falciparum genomic surveillance. We found that genetic diversity and pairwise inter-host relatedness, as well as prevalence of drug resistance markers, were consistent between first ANC users and children aged 2–10 years, representing the community. In Magude, which was subject to an eliminating campaign, similar declining trends in intra-host diversity were observed for both ANC users and children. Our findings demonstrate the potential of ANC-based malaria genomics as a straight-forward and cost-efficient approach to assess the impact of antimalarial interventions and genetic variants of public health concern.This study applied a multiplexed amplicon sequencing approach targeting microhaplotypes and drug resistance markers to assess the representability of pregnant women attending their first ANC consultation for sentinel 17 A few studies have also compared the genetic diversity of parasite populations in pregnant women and the community,22 but these were based on small sample sizes, only one took place in Africa, and, importantly, none accounted for parasite densities. With this study, we expand the potential scope of ANC-based surveillance to include genomic surveillance of P. falciparum genetic diversity and resistance markers. We find that both primigravid and multigravid first ANC users, regardless of HIV status, can be included in a sentinel population. Since no differences were observed between seasons, sampling could take place throughout the year. However, other studies did find seasonal differences8, indicating that this might depend on the setting. Furthermore, it may not be realistic to reach sufficient sample sizes at ANC facilities alone at very low transmission, and it would be necessary to combine ANC sampling with other sampling strategies, such as health facility surveys. ANC sampling would also not be ideal if the goal is to identify finer relatedness patterns, including transmission networks, because of the temporal sparsity of samples. Consistent with previous observations that parasite populations are at least partially structured in time,25 relatedness was higher among cross-sectionally sampled children than among continuously sampled ANC users, with the difference disappearing when restricting the comparison to similar temporal windows.Pregnant women seeking ANC have previously been shown to mirror trends in malaria prevalence in the general population, although with a delay, and with more heterogeneity between gravidity groups at higher transmission settings.12 In line with this and previous studies,11 we found the highest population diversity in the highest-transmission setting, Ilha Josina. Conversely, we also found the lowest intra-host diversity in Ilha Josina (both eMOI and 1-Fws). This might be explained by importation of parasites to low-transmission Magude and Manhiça from areas with higher transmission. A study from nearby low-transmission Eswatini observed similarly high diversity, which was attributed to frequent importation.26 Genetic diversity on its own might, therefore, not always be a suitable proxy for local transmission intensity, and stratification based on genetic metrics should be carefully validated against other epidemiological data, including assessing the potential role of importation.Genetic diversity has been proposed as a surrogate marker of transmission intensity.ws, increased mean IBD, and lower HE) highlight how parasite genomics can complement clinical and epidemiological data to evaluate the impact of control interventions. Between 2015 and 2017, Magude was targeted with biannual rounds of mass drug administration (MDA), followed by reactive focal MDA in 2018, and three rounds of indoor residual spraying (IRS).24 Even though parasite positivity rates declined in all three areas during the study period, and at similar levels and rates in Magude and the control area Manhiça ,27 we only observed evidence of declining intra-host diversity in Magude. Furthermore, Magude showed significantly lower population diversity and higher mean IBD compared to the other areas. A study from Zambia found a similar reduction in the complexity of infections following an MDA trial.28 These findings reveal programmatically important changes to the parasite population structure, i.e., recent reductions in the parasite population size, not apparent from prevalence and incidence estimates.The genetic indicators of reduced transmission observed within Magude -based methods, microhaplotypes allow for higher resolution and consequently more accurate estimates of diversity and relatedness, while being more convenient than microsatellites.15 to affect the parasite population. To confirm the generalizability of this approach for routine surveillance, more studies should be carried out in different epidemiological settings and include larger community sample sizes. Finally, we observed a clear dependence of sequencing coverage on parasite density, which may be explained by technical limitations. When only few, if any, parasite genomes are present in DNA extracted from a DBS, it will be difficult to amplify the parasite DNA for sequencing. This limitation applies to all genotyping techniques,9 and we reached comparably high sensitivity with the protocol applied here. The relationship between density and intra-host diversity may also be affected by biological processes, such as competitive stress and host immunity30, and future studies are needed to investigate this. Regardless of underlying causes, parasite density is an important confounding factor to adjust for when studying intra-host diversity.This study is limited by the number of samples available to sequence from children, particularly when stratifying by site and year, restricting comparisons with ANC attendees. For intra-host diversity, we therefore focused on Magude, where most samples from children originated. We did not consider the potential issue of parasite importation from neighboring regions, nor reasons for ANC non-attendance, although we would not expect any potential selection biasP. falciparum collected from ANC users and children representing the community. In both ANC users and the community, we found genetic indicators of a recent reduction in the parasite population in an area targeted for elimination, demonstrating the added value of genomic data for impact evaluation. Multiplexed amplicon sequencing has great potential to support decision-makers with genomic intelligence, and adopting a cost-effective and convenient ANC-based sampling strategy would be a valuable step towards making genomic surveillance more feasible in malaria-endemic areas.In conclusion, this study extends the scope of ANC-based sentinel surveillance to include genomic malaria surveillance. We did not observe differences in genetic diversity, relatedness or resistance markers between 17 Magude district was subject to a package of interventions in 2015–2018 including MDA with dihydroartemisinin-piperaquine and IRS with dichlorodiphenyl-trichloroethane and pirimiphos-methyl, resulting in a 85% reduction of in all-age positivity rates.24 (S methods p 3)This genomic surveillance study took place between 2015 and 2018 in three malaria-endemic areas in Maputo province in southern Mozambique. Transmission intensity ranged from low in Manhiça and Magude, to moderate-to-high in Ilha Josina, and it declined in all three areas during the study.27 The main reason for exclusion was not residing in the area. Women donated a finger-prick drop blood onto filter paper (dried blood spot), and HIV status, date, age, gravidity, area of residence, and recent movements were recorded. 3,933 children aged 2–10 years were sampled for annual age-stratified household surveys in the study area. The surveys were conducted around May every year (following the rainy season) from 2015 to 2019. DBS were obtained together with basic sociodemographic, clinical, and vector-control information.24 Self-reported gender was evenly represented in the surveys, with 50.3% girls and 49.2% boys (unavailable information for remaining 0.5%).Samples were collected from pregnant women at ANC clinics and children participating in household surveys. 10,439 pregnant women were recruited when attending their first ANC visit at Manhiça District Hospital, Ilha Josina Health Center, or Magude Health Center between November 2016 and November 2019. For 8,910 of the visits, informed consent to participate was obtained, and 8,745 visits were included in the study.All study protocols were approved by CISM’s and Hospital Clínic of Barcelona’s ethics committees, and the Mozambican Ministry of Health National Bioethics Committee. All study participants gave written informed consent, or in the case of minors, written informed assent and consent by a parent/guardian.P. falciparum-positive DBS samples (from 378 ANC users and 180 children) using a Tween-Chelex based protocol (S methods p 3). A multiplex panel of PCR primers targeting 241 P. falciparum amplicons of 150–250 bp was developed . Amplicons included 165 microhaplotypes informative about genomic diversity and relatedness in southern Africa31, and markers of drug resistance in 15 genes4, including polymorphisms associated with resistance to artemisinin (pfk13), SP (pfdhfr and pfdhps genes), and amodiaquine (pfcrt and pfmdr1 genes). We followed the manufacturers’ instructions as in Tessema et al 202029. DNA was amplified for 15 or 20 cycles for multiplexed PCR, depending on parasitemia and ability to amplify, and for 15 cycles for indexing PCR. A randomly selected subset of resulting libraries was assessed by capillary electrophoresis using a TapeStation . Libraries were pooled accounting for differences in yield due to parasitemia, and the pool was bead-cleaned using CleanMag® Magnetic Beads at 1X ratio to remove primer dimers. Pooled libraries were run on an agarose gel from which the amplicon-sized band was excised, and DNA extracted using Monarch® DNA Gel Extraction Kit . Library pools were quantified and assessed using a TapeStation and a Qubit fluorometer (S methods p 3). The purified libraries were sequenced in either a MiniSeq, or NextSeq instrument .DNA was extracted from 558 available 32 (version 0.1.5), to infer alleles. Briefly, reads were demultiplexed for each locus using cutadapt33, and DADA234 was used to cluster reads using an error-inference model. Cutadapt and DADA2 were also used to filter and truncate reads based on quality and length. Finally, homopolymers and tandem repeats were masked. Subsequently, alleles with fewer reads than the maximum observed reads in any locus for negative controls (14 reads) were removed, along with alleles with <1% within-sample frequency. Samples with a coverage of <50 diversity loci with a read depth of 100 were filtered out. Finally, diversity loci with <100 samples covering them with a read depth of 100 were also removed.FASTQ files were run through a Nextflow-based pipelinest to April 30th, and the remaining year as dry season.27 Years were defined based on transmission season, i.e., from November 1st to October 31st. When comparing time periods for ANC attendees, 2018 and 2019 were combined to balance sample size with 2017, where more cases were sampled due to higher transmission. Only children were sampled in 2015 and 2016, and these years were also combined. Primigravidity was defined as a first pregnancy, while multigravidity was defined as having had at least one previous pregnancy. Population diversity was measured as th allele, with allele frequencies estimated statistically using a Multiple Chain Monte Carlo (MCMC) algorithm from MOIRE v3.0.0 (R package)35. Intra-host diversity was measured using the following metrics: MOI, eMOI, MOIRE MCMC algorithm. eMOI takes within-host relatedness into account, and can be interpreted as the expected MOI if population diversity was infinite Rainy season was defined as November 1th locus of a given sample. Individual mean Dcifer v1.2.0 (R package)36, accounting for the presence of polyclonal infections and the probability that regions of the genome are shared by chance. Prevalence of resistance markers was calculated as the number of individuals carrying a mutated allele out of all individuals with a valid genotype for the respective locus. In case of both wildtype and mutant alleles present in one individual, the individual was considered mutant carrier if the infection was polyclonal by eMOI (eMOI>1.1), otherwise only the major allele (wildtype or mutant) was considered. For genotypes involving multiple amplicons, only samples with a single allele present were included to avoid issues with phasing.Where ws. The effect size of continuous time on intra-host diversity was estimated from multivariate regressions with an interaction between time and area. To compare intra-host diversity between ANC users and children, only samples from Magude were included due to low sample sizes for children in Manhiça and Ilha Josina. HE was compared between populations with Linear Mixed Models (R package nlme) fitting locus as a random effect. Random subsampling matching populations by area and year was performed to compare groups of similar sample size. Differences in mean relatedness were assessed with permutation testing. Prevalence of resistance markers was compared with Pearson’s chi-square test or Fisher’s exact test. Multiple comparisons were corrected for using the Benjamin-Hochberg procedure with a q-value of 0.05, resulting in a final alpha of 0.0062 applied to indicate significance. All analyses were performed using R version 4.3.0.Univariate and multivariate regression analyses were used to estimate intra-host diversity and assess the effect of factors of interest. P-values and confidence intervals for eMOI were obtained from zero-truncated Poisson regressions. Logistic regression was used for percentage polyclonal and 1-F


Mycteria cinerea) and painted stork  occurs frequently in captivity. Dusit Zoo is a captive breeding facility where storks with phenotypically ambiguous patterns have recently been observed, and their status remaining inconclusive. Here, we used a combination of phenotypic characters and genetic markers (cytochrome b and 14 microsatellite markers) to distinguish and identify hybrids from the two parental species (n = 114). Haplotype analysis revealed asymmetric mtDNA introgression from M. cinerea to M. leucocephala, with twelve morphologically classified M. leucocephala individuals carrying heterospecific mtDNA. Comprehensive biparental genetic assessments identified 33% of all three genetic clusters as admixed individuals, of which most were either F2 hybrids, backcrosses with M. leucocephala, or hybrids of unknown generation, implying weak premating isolation with the absence of intrinsic postzygotic isolation between parentals. Morphological analysis demonstrated that the absence or indistinctness of a black bar across the breast is the most noticeable trait to identify these hybrids. The endangered M. cinerea was found to have genomic contamination from M. leucocephala and vice versa, with at least 41 hybrid individuals being identified. These findings provide critical information for detecting hybrids and identifying suitable breeding stocks with genetic purity for future reintroduction and conservation management.Hybridization between milky stork ( This process is regarded as one of the major threats to species that are highly threatened or have a small population, as it can lead to the decline or eventual extinction of the pure species’ genetic background (genetic swamping). Accordingly, the emergence of hybrids tends to be an issue because they appear to have detrimental consequences for pure species6.Hybridization and its impact on the evolutionary consequences for hybridizing species are heavily reliant on the fate of their hybrid offspring, because fertile hybrids can mediate gene flow or introgression of genetic materials between species9. However, unintentional human-mediated hybridization may detrimentally influence the success of breeding and reintroduction, raising serious concerns about the genetic integrity of breeding stocks or reintroduced individuals12. Detecting introgressive hybridization is, therefore, an urgent need for preserving the genetic integrity of the remnant pure species.Ex situ management, in terms of captive breeding, has become a conservation strategy for the rescue and restoration of an endangered species in preparation for future reintroductionMycteria cinerea) is a large waterbird in the Ciconiidae family with an almost entirely white plumage that is patchily distributed in tropical wetland ecosystems across Southeast Asia 15. The expansion of habitat degradation and environmental pollution, primarily caused by anthropogenic activities, has dramatically decreased M. cinerea populations, leading them to be listed as an endangered species on the IUCN Red List, with approximately 1500 mature individuals remaining in the wild17. In Thailand, M. cinerea was once found in the south, but was thought to be locally extinct in the past few decades18. Thus, most of the storks found nowadays in Thailand are vagrants, typically found in the central region.The milky stork (M. cinerea in captivity prior to future reintroduction in Thailand20. The primarily captive breeding of M. cinerea took place in 1998 at Dusit Zoo (DZ), Bangkok, where 19 founders were raised together with its sister taxon, the near threatened painted stork , for which captive breeding had already been commenced in 1997 with an initial 70 founding individuals. The two stork species shared a large aviary for 16 years until all captive bred M. cinerea (n = 21) were relocated to Nakhon Ratchasima Zoo (NRZ: Nakhon Ratchasima Province) in 2014 for further breeding while M. leucocephala were still maintained in the DZ.The Zoological Park Organization of Thailand has initiated a project to breed 23. Morphologically, M. leucocephala can be distinguished from M. cinerea by plumage patterns and colorations, including black and white markings on the wings, a black bar across the breast, and a deep pinkish on the tertials23. These three distinct traits are present only in M. leucocephala but not in M. cinerea. To date, the captive breeding program at DZ has increased the number of M. leucocephala to 173 individuals, while at NRZ the M. cinerea population has been expanded to 48 individuals.The two storks exhibit similarities in their appearance, behavior, and have a close evolutionary relationship with little genetic divergence between themM. cinerea and M. leucocephala) and could not be clearly identified through their morphology. Therefore, these stork individuals were suspected to be hybrids of the two species, as previously reported in the Malaysian National Zoo  and Jurong Bird Park (Singapore)25. Here, we combined phenotypic characteristics and genetic analyses  to identify and discriminate captive hybrids from M. cinerea and M. leucocephala. The findings will provide fundamental information for developing conservation management and reintroducing the stork population.It has recently been observed that some stork individuals at DZ showed phenotypically ambiguous patterns and colors  plot generated by discriminant analysis clearly showed three distinct groups consisting of phenotypically ups Fig. . The corM. leucocephala and M. cinerea groups, with the M. leucocephala group predominantly on the negative side of CDF 1 and M. cinerea group showing the opposite. In contrast, the intermediate individuals exhibited a wide range of phenotypic traits between the two pure species and were positioned closer to the M. leucocephala group than the M. cinerea group , 53 M. cinerea , and 37 intermediate individuals (17.45%).The plot revealed a separation between the M. leucocephala, 37 intermediate individuals, and 53 M. cinerea, from the morphological identification, were sequenced for the mitochondrial cytochrome b (cyt b) gene, yielding a 1029 bp length sequence with no internal stop codons. Both Bayesian inference (BI) and maximum likelihood (ML) phylogenetic trees revealed similar topologies and clearly demonstrated two major lineages between M. leucocephala and M. cinerea groups with significant support values  individuals.A total of 190 individuals, comprised of 100 M. leucocephala  and two morphologically M. leucocephala individuals (PS57 and PS167) had the M. cinerea haplotype, confirming that these individuals were hybrids of the two stork species  that were morphologically identified as M. cinerea carried the M. cinerea mtDNA haplotype, implying that they are more likely pure M. cinerea.Interestingly, 10 intermediate individuals whose morphology was closer to that of M. leucocephala, 37 intermediate individuals, and 53 M. cinerea according to morphological identification) whose genomes were amplified successfully by all primer pairs in cross-species amplification. However, we found that locus Cc06 was unreliable for allelic reading, and three loci  were monomorphic; thus, these loci were excluded from the analyses (see Supplementary Table P < 0.05). However, they were not excluded from further analyses because the existence of HWE deviation could be linked to a recent genetic admixture between two populations in DZ, as evidenced by the presence of potential hybrid individuals.We genotyped 14 microsatellite loci for each of the 114 individuals  for K = 3, indicating that the individuals could be divided into three genetic clusters  were classified as F2 hybrids, one individual (PS30) was classified as a backcross with M. leucocephala, one individual (PS57) was classified as M. cinerea, and four individuals  were classified as hybrids of unknown generation. From 37 individuals morphologically identified as intermediate; 13 individuals were classified as pure M. leucocephala, eight individuals were classified as F2 hybrids, four individuals  were classified as M. cinerea, one individual (IN101) was classified as a backcross with M. leucocephala, and 11 individuals were classified as hybrids of unknown generation. From 53 individuals morphologically identified as M. cinerea; 49 individuals  were classified as pure M. cinerea, two individuals (MS26 and MS47) were classified as hybrids of unknown generation, one individual (MS21) was classified as an F2 hybrid, and one individual (MS172 from DZ) was classified as a backcross with M. leucocephala. Thus, a total of 114 individuals could be identified according to their genotype classes as 54 pure M. cinerea (47.37%), 29 pure M. leucocephala (25.44%), 17 hybrids of unknown generation (14.91%), 11 F2 hybrids (9.65%), and three backcrosses with M. leucocephala (2.63%), but with no F1 hybrids or backcrosses with M. cinerea . Conversely, four DZ individuals  showed congruence between the morphological and genetic data, indicating they were neither pure M. leucocephala nor hybrids , which were classified as admixed by STRUCTURE but as pure 27. This study shows the first evidence of introgressive hybridization between M. cinerea and M. leucocephala in Thailand, where the two storks were historically kept and raised together in the same captivity in DZ. The DZ was made aware of the problem and is the only zoo actively attempting to prevent hybridization by keeping the two species separate, even if their efforts were not completely successful. The presence of hybrids among pure parental populations as a result of past hybridization raises concerns about the genetic integrity of breeding stock, which needs to be explicitly investigated.Detecting hybrids from the pure species is a critical first step in developing a breeding program for endangered or extinct in the wild species for subsequent reintroduction in the futureM. cinerea and M. leucocephala could be distinguished by six phenotypic characters, whereas intermediate individuals (putative hybrids) were placed in a middle position between the two groups. According to the plot in Fig. M. leucocephala and the intermediate groups showed more data dispersion than M. cinerea, which could be attributed to the wide variation in the BBB, MOW, and PTF traits. In contrast, M. cinerea lacks these three characteristics, they exhibit AWB and an absence or indistinct MUW.Morphological based CDF analysis highlighted that 30. In this study, haplotype analysis detected M. cinerea haplotypes in several morphologically similar M. leucocephala but not vice versa, inferring asymmetric introgression of mtDNA from M. cinerea to M. leucocephala populations. Thus, the F1 hybrid is most likely produced successfully by M. cinerea females. However, we found either species’ mtDNA haplotype in F2 hybrid genomes, raising the possibility of successful reciprocal hybridization across species. Since no first-generation hybrid was identified in this study, the direction of hybridization success between these incompletely isolated species remains uncertain. However, there appears to be no intrinsic postzygotic isolation; F1 hybrids are viable and not sterile, as evidenced by the presence of F2 and backcross hybrids; and the low sequence divergence of the cyt b gene supported this21.Without explicit examination of hybrid production, genomic introgression may possibly reflect the direction of hybridization success between species and help us understand the evolutionary history and mechanisms of reproductive isolation between hybridizing speciesM. leucocephala population and two in the M. cinerea population. Although we do not have enough information to draw conclusions about their origins, it is probable that the founders of M. cinerea came from two distinct sources. STRUCTURE analysis with various threshold values was performed. A threshold of < 0.95  lead to an underestimation of hybrids due to mtDNA data inconsistency. In contrast, with a qi of 0.9925, all birds were classified as admixed , because the highest qi values of pure M. cinerea and M. leucocephala were 0.983 and 0.984, respectively. We assured that the STRUCTURE analysis of 10 microsatellite loci with a qi of 0.95 was quite effective in distinguishing between admixed and pure individuals, whereas using criteria of a qi of 0.8, 0.95, or 0.99 proved ineffective in separating backcross hybrids from pure individuals, as demonstrated by samples PS57, IN74, and IN166. This exemplifies the limitations of the STRUCTURE program in identifying backcross hybrids31.STRUCTURE analysis revealed three genetic clusters (K = 3) among the examined storks, one in the M. leucocephala (~ 9%), respectively, implying that F1 hybrids are more likely to mate with each other than with the parental species. In other words, morphologically intermediate F1 hybrids appear to be selected infrequently by pure parentals. Furthermore, some hybrids are more prone to backcrossing with M. leucocephala than M. cinerea. There are two possible explanations for this: (1) pure M. cinerea exhibit strong premating isolation and so do not choose to mate with morphologically intermediate F1 hybrids; (2) M. leucocephala outnumbers M. cinerea in DZ, increasing the likelihood of encountering and mating between the F1 hybrid and M. leucocephala. The latter point is in accord with a greater number of backcrosses with M. cinerea  than with M. leucocephala  being previously detected at Jurong Bird Park, where M. cinerea predominated over M. leucocephala25. As a result, this data supports the second explanation, where both M. cinerea and M. leucocephala exhibit weak mate recognition and discrimination.Based on the NEWHYBRIDS results, the majority of the hybrids that could be class-identified were F2 hybrids (~ 35%) and backcrosses with M. leucocephala phenotypic characteristics, but the genetic data represented M. cinerea, emphasizing the limitations of morphological data in distinguishing hybrids from pure species. This individual is probably a backcross with M. cinerea, which carries the genes responsible for the formation of M. leucocephala plumage traits. Since morphological traits may be regulated by several genes associated with the selection and fitness of the parental species32. Therefore, further genetic studies are required to consider which genes control the morphological characteristics of hybrids and the two stork species33. In addition to the above example, morphological-genetic incongruities were discovered in nine intermediate individuals  that were genetically identified as M. leucocephala, but phenotypically they do not appear to be pure M. leucocephala. We suggest that analyses of a large number of genetic markers is required to resolve these ambiguous results36.We discovered cases of discrepancy between the morphological and genetic data, as demonstrated by sample PS57 that exhibited M. leucocephala was greater than that of hybrids that resembled M. cinerea. Among the morphologically intermediate individuals detected in the DZ population, F2 hybrids and backcrosses with M. leucocephala showed similar morphological features that are intermediate between the pure parentals; all of them had a few very pale pinkish tertial feathers, five (56%) individuals had unclear black and white striped markings on the wings [the estimated MOW areas were less than the lowest value (366 cm2) detected in pure individuals], and none of them had a clear black bar across the breast; while three and one individuals of F2 hybrids and backcross with M. leucocephala, respectively, lacked this trait.Overall, the morphological and genetic results were relatively consistent, and the proportion of hybrids that morphologically resembled M. leucocephala, except that none of them had a clear black bar across the breast. As a result, the absence or appearance of an unclear black bar across the breast may be linked to a genetic admixture in hybrid genomes, and so this trait can be used to initially visually distinguish hybrids from pure individuals. Note that the diagnostic phenotypic traits used in the study are only applicable at the adult stage because juveniles of the two stork species  exhibit a similar appearance of a brownish plumage and look different from adults24. To preserve the genomic integrity of the pure species, the multilocus markers obtained in this study could be utilized as diagnostic markers to detect hybrids of M. cinerea and M. leucocephala at any stage of age  in both captive and wild populations.In contrast, hybrids of an unknown generation had a similar appearance to pure 37. This study revealed that the endangered M. cinerea had genomic contamination from M. leucocephala and vice versa during its 16 years in captivity in Thailand through repeated backcrossing. Three (6.25%) of the 48 storks from NRZ and 38 (57.58%) of the 66 storks from DZ had hybrid identities. These hybrids are now kept in zoos and can be individually identified through mark recapture rings. To prevent crossbreeding, we recommend that hybrid storks be kept separate from pure storks and that they should not be used as stocks in future breeding or reintroduction programs. If hybrids must be raised in the same enclosure as their pure parental species , sterilizing both the male and female hybrids is another option for preventing genetic contamination and preserving the genetic integrity of pure species. Moreover, the discovery of such backcrosses serves as a warning that the remaining individuals of M. leucocephala at DZ should urgently be genetically investigated to ensure each individual’s genomic purity.Human disturbance is regarded as one of the major factors causing hybridization and subsequent genomic contamination between closely related species, particularly in captivity, such as in zoos38. It is possible that these birds are natural hybrids, or they are captive hybrids that have been released or escaped from captivity. So far, no genetic evaluation is available to confirm that those individuals are hybrids of M. cinerea and M. leucocephala or not, and so it is imperative that a thorough and urgent genetic investigation of these birds be conducted. Besides, other studies have shown that both M. cinerea and M. leucocephala can hybridize with other related stork species in captivity, such as the lesser adjutant Leptoptilos javanicus24. Zookeepers should be aware of this and avoid rearing them with L. javanicus or other related species. More importantly, habitat loss is one of the major threats and causes of the decline or extinction of wild large waterbirds40. In situ preservation and protection of wetland habitats is also recommended, not only for the future sustainable conservation of M. cinerea and M. leucocephala, but also to reduce the chance of hybridization in inevitable overlapping areas.In addition to the detection of hybrids in captivity, there have also been occasional reports of storks with intermediate trait detections in some wetland habitats in Thailand, such as at Bueng Boraphet (Nakhon Sawan Province), Bang Pu recreation center (Samut Prakan Province), Khlong Tamru salt pans (Chon Buri Province), Bang Tabun (Phetchaburi Province), and Laem Phak Bia (Phetchaburi Province)M. cinerea × M. leucocephala) were identified, comprised of three individuals from NRZ (n = 48) and 38 individuals from DZ (n = 66). Most hybrids could be classified according to their morphology as follows: a few pinkish tertial feathers, unclear black and white markings on the wings, and an absence of/or unclear black breast band. The latter trait is plainly apparent in hybrids and so can be used to visually distinguish hybrids from pure species. Combining morphological identification with genetic assessment (both mtDNA and nDNA) provides a more efficient approach for detecting and distinguishing hybrids from the parental species, and these findings can serve as an important foundation for conservation efforts and long-term management of M. cinerea and M. leucocephala populations.A total of 41 hybrids  and 48 M. cinerea were collected in 2018 during regular veterinary examinations at two captive breeding locations, Dusit Zoo (DZ), Bangkok, and Nakhon Ratchasima Zoo (NRZ), Nakhon Ratchasima Province, respectively. Before blood was collected, all birds were weighed, rung with a mark recapture ring, and photographed for morphological analysis. Approximately 300 μL of blood was taken from the subclavian vein under the wing by the zoological park veterinarian team using a 1-mL syringe. Blood samples were immediately stored in a 1.5-mL tube coated with EDTA for prevention of blood coagulation and later preserved at − 80 °C in the laboratory until DNA extraction. This study was performed in accordance with relevant guidelines and regulations, including the ARRIVE guidelines (http://www.ARRIVEguidelines.org) for the ethics of animal research. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Science, Chulalongkorn University (Protocol Review No. 1823015).Blood samples of 173 Total genomic DNA was extracted from dried blood spots on filter paper using a FavorPrep™ Tissue Genomic DNA Extraction Mini Kit (Favorgen Biotech Corp.) following the manufacturer’s instruction.24 and Nabhitabhata et al.41: 1; all-white body (AWB), 2; pink/coral markings under the wings (PMUW), 3; black and white markings under the wings (MUW), 4; black bar across the breast (BBB), 5; black and white markings on the wings (MOW), and 6; pink tertial feathers (PTF)  were morphologically analyzed because juveniles of the two species and their putative hybrids appear similar in their plumage patterns and colorations, making species discrimination implausible. To differentiate pure storks from the hybrids based on their morphological characters, we photographed each stork individually and compared their major differences on six distinguishable phenotypic traits, following with modification from Yee et al.M. leucocephala and putative hybrids. We, therefore, examined these three phenotypic traits in detail by calculating the marking/bar areas of those three traits from digital images using Adobe Photoshop CC 2018 and ImageJ v.1.5142, and then recording the area values instead of “absent” or “present” for each specimen. To ensure the same quality of the all-digital images, we standardized the scale bar of each image as 2 cm per one subdivision. The image was cropped to only cover the BBB. The width of each image was then determined to be equal to the width of the breast depending on each individual (approximately 50 cm), and the resolution of each image was set to 72 pixels/centimeters. The black area of the breast in a JPEG file was converted to the red area by adjusting the brightness using the threshold color tool in ImageJ. The resulting red area of the breast was then measured using the ROI Manager tool. Each BBB area was measured twice for each digital image and the “average” values used for further analyses. The same method was applied for measuring the black area of the MOW. For the PTF, the shades of pink were estimated using the Commission internationale de l’e´clairage L* a* b* (CIELab) color space technique in the Adobe Photoshop program. The CIELab color uses three values  to specify color: the L-axis (lightness), the a-axis (green to red), and the b-axis (blue to yellow)44. We converted the RGB image to the Lab color image, then selected five locations covering the pink area of the feathers, and used the “average” of five positive a-axis values. In this case, the brightness and darkness of images were not standardized prior to estimation because these parameters have no effect on shades of pink values on the a-axis.The AWB and PMUW traits were scored as 0 or 1 according to their absence or presence, respectively. For MUW, it was scored as 0, 1, or 2 for absent, systematic markings, and nonsystematic markings, respectively. For BBB, MOW, and PTF, these traits exhibit wide variations among individuals and could not be easily identified between morphologically pure ® version 26 (IBM Corporation).For data analysis, we used canonical discriminant function (CDF) analysis to predict the groups to which each individual was assigned based on the six phenotypic variables listed above, and to visualize the relationships between the group positions. The statistical analysis was performed using IBM SPSS Statistics for window b gene (cyt b) was PCR amplified and sequenced to identify the haplotype of each stork individual, as well as the maternity of the hybrids. This PCR amplification and sequencing used the L14990 and H16065 primers45 with thermal cycling performed in a 45 µL reaction volume containing 2 µL of DNA template (approximately 30–50 ng), 0.5 µM of each forward and reverse primer and 1 × premix of EmeraldAmp® MAX PCR master mix (Takara). The PCR cycling conditions were as follows: initial denaturation at 93 °C for 3 min, followed by 35 cycles of denaturation at 93 °C for 30 s, annealing at 52 °C for 1 min, and extension at 72 °C for 1.15 min, and then followed by a final extension at 72 °C for 10 min. The PCR products were electrophoresed using a 0.8% (w/v) agarose gel, stained with SYBR® Safe DNA gel staining dye (Invitrogen™), and visualized under a blue light transilluminator. The desired PCR products were sent to Bionics Inc., South Korea for commercial purification via gel extraction and sequencing services.A portion of the mitochondrial cytochromeM. leucocephala (n = 24), intermediate individuals (n = 37), and pure M. cinerea (n = 53), were genotyped at 14 microsatellite loci that were originally developed from (i) the oriental white stork Ciconia boyciana: Cbo108, Cbo109, Cbo133, Cbo151, Cbo168, and Cbo23546, and (ii) the white stork Ciconia ciconia: Cc05, Cc06, Cc07, Cc10, Cc42, Cc50, Cc58, and Cc7248. The 5’- end of all forward primers was labeled with one of two fluorescent dyes  to facilitate automated genotyping. Some markers were amplified in a multiplex, whereas others were amplified independently. PCR amplifications were performed in a reaction volume of 15 µL, containing 1 × premix of EmeraldAmp® MAX PCR master mix (Takara), 0.5 µM of each forward and reverse primer and 30–50 ng of genomic DNA. Amplification profiles for all primers were conducted as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing for 30 s .A total of 114 stork specimens, which were phenotypically classified as pure 49 implemented in MEGA v.11.0.650, and the sequences were translated into amino acids to check for gaps or internal stop codons. We generated the phylogenetic trees by using both Maximum likelihood (ML) and Bayesian inference (BI) methods to analyze phylogenetic relationships and mtDNA haplotypes. The best fit model of nucleotide substitution for the data set of cyt b sequences was estimated using jModelTest v.2.1.1051 based on the Bayesian Information Criteria (BIC). jModelTest indicated the best model of nucleotide substitution was GTR + G. The ML tree was performed in PhyML v.3.052, using the GTR + G model and 1,000 bootstrap replicates. The BI was conducted in MrBayes v.3.2.153, performing 10,000,000 generations with sampling every 100 steps and a burn-in of 2,500 generations. The results were visualized in Figtree v.1.4.454. The cyt b sequences of M. ibis (Genbank accession: U72784.1) and M. americana (AF082066.2), the two closely related species of our interest, were used as outgroups. We additionally included two retrieved cyt b sequences of M. cinerea (U72778.1) and M. leucocephala (U72777.1) from GenBank in the phylogenetic analyses.All obtained nucleotide sequences were edited and aligned using CLUSTAL W55. The extent of linkage disequilibrium between pairs of loci was evaluated using FSTAT v.2.9.456 with Bonferroni correction at a significance level of 0.01. To test for hybridization, we first used the Bayesian clustering procedure implemented in STRUCTURE v.2.3.331 to identify genetically distinct clusters  and potential admixture between species by calculating the posterior probability. In addition, STRUCTURE was used under the admixture model with correlated allele frequencies and without including sampling location as a prior. We used 10 replicates for each value of K, ranging from K = 1 to K = 10, with 2,000,000 MCMC replications after burn-in following a burn-in period of 500,000. The best value of K was selected following the Delta K (ΔK) proposed by Evanno et al.57 method, as calculated in STRUCTURE HARVESTER. A threshold membership coefficient (qi) value of 0.95 was used to distinguish between pure individuals (0 < qi < 0.05 or 0.95 < qi < 1) and hybrids (0.05 < qi < 0.95).The microsatellite data was tested for departures from Hardy–Weinberg equilibrium (HWE) of loci and within the population using GENEPOP v.4.735 to identify pure individuals and to classify hybrids into one of the six distinct frequency classes: pure M. leucocephala (PS), pure M. cinerea (MS), first generation hybrid (F1), second generation hybrid (F2), backcross of F1 with PS (BxPS) and backcross of F1 with MS (BxMS). The NEWHYBRIDS was run for five replicates with 1,000,000 sweeps and values after a burn-in period of 500,000 sweeps using Jeffreys-type prior for Theta and Pi. Individuals were assigned to NEWHYBRIDS genotype classes based on their posterior probability values (P), with P > 0.5 indicating that they belonged to that class. If no value reached 0.5 but the total of all hybrid classes was greater than this threshold, individuals were classified as hybrids of unknown generation.To considered if an individual had genomic admixture between two species (a hybrid), Bayesian posterior probability was applied using NEWHYBRIDS v.1.1Supplementary Tables.


Protein-based nanocarriers have demonstrated good potential for cancer drug delivery. Silk sericin nano-particle is arguably one of the best in this field. In this study, we developed a surface charge reversal sericin-based nanocarrier to co-deliver resveratrol and melatonin (MR-SNC) to MCF-7 breast cancer cells as combination therapy. MR-SNC was fabricated with various sericin concentrations via flash-nanoprecipitation as a simple and reproducible method without complicated equipment. The nanoparticles were subsequently characterized for their size, charge, morphology and shape by dynamic light scattering (DLS) and scanning electron microscope (SEM). Nanocarriers chemical and conformational analysis were done by fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD) respectively. In vitro drug release was determined at different pH values . The cellular uptake and cytotoxicity were studies using breast cancer MCF-7 cells. MR-SNC fabricated with the lowest sericin concentration (0.1%), showed a desirable 127 nm size, with a net negative charge at physiological pH. Sericin structure was preserved entirely in the form of nano-particles. Among the three pH values we applied, the maximum in vitro drug release was at pH 6, 6.5, and 7.4, respectively. This pH dependency showed the charge reversal property of our smart nanocarrier via changing the surface charge from negative to positive in mildly acidic pH, destructing the electrostatic interactions between sericin surface amino acids. Cell viability studies demonstrated the significant toxicity of MR-SNC in MCF-7 cells at all pH values after 48 h, suggesting a synergistic effect of combination therapy with the two antioxidants. The efficient cellular uptake of MR-SNC, DNA fragmentation and chromatin condensation was found at pH 6. Nutshell, our result indicated proficient release of the entrapped drug combination from MR-SNC in an acidic environment leading to cell apoptosis. This work introduces a smart pH-responsive nano-platform for anti-breast cancer drug delivery. Notwithstanding significant advances in understanding and treating BC over the past decade, the incidence and mortality rate related to BC continue to increase at an alarming rate accounting for approximately 2.26 million deaths by 20203. The International Agency for Research on Cancer (IARC) Global Cancer Observatory (GLOBOCAN) predicts 27.5 million new cancer cases per year by 2040, a 60% increase compared to the current statistics4. Current treatment modalities include any or a combination of surgery, radiation, immunotherapy, gene therapy, photodynamic treatment, and chemotherapy. Although these procedures can alleviate breast cancer, they have numerous disadvantages, including non-specificity, high toxicity, and multidrug resistance5. Thus, there is a need for alternative treatments for BC. Antioxidants have been widely used as chemo preventive agents to counteract BC progression. They can also be used as adjuvants in treating BC. Combining multiple antioxidants is one of most effective strategies in cancer management6.Breast cancer (BC) is the most prevalent malignancy and the leading cause of cancer death among women globally, accounting for 14.7% of all cancer-related deathstrans-trihydroxystilbene, Fig. 7. Polyphenols are a large category of natural phenol group with therapeutic and preventive effects on neurodegenerative disorders, cardiovascular disease and cancer, due to the beneficial properties of phenolic and polyphenolic compounds including anti-inflammatory, antioxidant and anticancer activities9.Resveratrol  generation, and inhibits cancer initiation and cancer development11.Resveratrol cancer preventive properties were first reported in 197712. It also has anti-tumor properties in different types of cancer, including breast, colorectal, pancreatic, endometrial, kidney, skin, and others19. Of note, melatonin lowers estrogen receptor expression, inhibits DNA replication, prevents metastasis, regulates angiogenesis, and apoptosis21.Melatonin an indoleamine (N-[2-(5-methoxy-1H-indol-3-yl)ethyl]acetamide or N-Acetyl-5-methoxytryptamine, Fig. 22. Recent data strongly suggest that multifunctional nanocarriers offer enhanced efficacy, with limited side effects by modulating the local internal milieu such as redox conditions, temperature, pH, and actions of specific enzymes; or by external stimuli, such as ultrasound, light, and magnetic field23.Recent advances in nanomedicine have ushered in a new era in cancer treatment by targeting cancer cells without affecting normal cells, thus potentially overcoming multidrug resistance. However, cancer heterogeneity and the tumor microenvironment complexity may attenuate the anti-cancer properties of nanocarriers25. The other reason is their protein-based nanocarrier drug delivery systems (P-NDDS) properties. P-NDDSs can have a significant role in BC treatment due to their beneficial characteristics27. For example, they are generally recognized as safe (GRAS)28, meaning there isn't any risk in using these carriers. They are also biodegradable—so over time, they break down inside the body. Another advantage of P-NDDSs that makes them attractive is their lack of immunostimulatory activity27. Thus, they don't trigger an immune response when administered28.For two reasons, protein-based-charge-reversal nanocarrier development is one of the most promising strategies in this area. The most important reason is their pH-triggered charge-reversal property. Thus, they can absorb the negative-to-positive charge transition in response to changes in ambient pH. In fact, after accumulation in the tumor's acidic microenvironment, they obtain a positive charge that facilitates their cellular uptake29. They give it a hydrophilic property and a high antioxidant activity via scavenging free radicals. In addition, sericin is a stable, non-toxic, biodegradable, and biocompatible protein that interacts with other molecules easily. Besides its high antioxidative properties, it has a high potential to form a soluble and stable nanoparticle to wrap various compounds. The wrapping provides a long blood circulation time for entrapped compounds, prevents their decomposition, and releases them at specific targets31.Silk sericin is one of the best proteins for developing a protein-based pH-triggered charge-reversal nanocarrier because of the pH-responsive nature of its building blocks. It is a 20–400 kDa protein extracted from dried silk cocoons, constituting about 25% of the total cocoon weight. This globular protein has 18 polar amino acids with amino, hydroxyl, and carboxyl groups33. In fact, nanoprecipitation is a modest "bottom-up" approach that depends on the supersaturation of a hydrophobic solute upon adding water that has lately emerged as a general and flexible technique to produce nanoencapsulation with precise control of its physico-chemical characteristics34.Previously published articles have shown that the nanoprecipitation method, also known as flash-nanoprecipitation, is a facile, mild, and low-energy input technique for nanocarrier preparation. It is also one of the most useful and safest strategies for encapsulating active molecules at sub-micron and nanoscale levelsTherefore, this study focused on developing a novel pH-triggered charge reversal–protein-based nanocarrier using silk sericin employing the nanoprecipitation method. This nanocarrier was used to co-deliver resveratrol and melatonin as an antioxidant combination therapy to MCF-7 breast cancer cells. After the physico-chemical investigations of the fabricated nanoparticles, MR-SNC in vitro drug release behavior was studied at three pH values . In addition, the MR-SNC potential to decrease the MCF-7 cells’ viability was investigated at different pHs . The intracellular uptake of MR-SNC in MCF-7 cells and the DNA fragmentation were also studied.trans-Resveratrol—3,4′— powder  and all other chemicals, including 35 with few modifications. Briefly, silk sericin solutions  were added drop-wise (10 µl/5 s) into an organic phase of a non-solvent (acetone) under forceful stirring (800 rpm) at room temperature  was directly dispersed in UP-Water at various concentrations ) under moderate stirring at room temperature. Then, a protein-based nanocapsule was prepared via a flash-nanoprecipitation system adapted from Nelemans et al.ure Fig. . At thatThe Resveratrol (RES) and Melatonin (MEL) solved in absolute dimethyl sulfoxide (DMSO) were loaded within sericin based nanocapsules via the direct system of dissolution into the aqueous phase (protein phase) at the final concentration of 0.6 mg/mL. The next step involved the addition of the dissolved RES + MEL aqueous phase into the acetone organic phase under forceful stirring (800 rpm) and pH 7.45 (3 wt% RES + MEL in blank-sericin based nanocapsules). This addition was done without the need for any tools or devices and only with an insulin syringe, 1 drop per 5 s, under intense stirring. Sericin-based nanocapsules (SNC) containing the drug were also recovered by completely evaporating water and acetone. Formerly, Intracellular Co-delivery of Resveratrol-melatonin utilizing a pH-responsive Sericin-based nanocarriers for anticancer therapeutics the antioxidants were loaded in optimal sericin-based-nanocapsule prepared formulations (0.1% w/v). The SNC was prepared in triplicate, freeze-dried, stored at − 20 °C, and protected from light. In addition to physical appearance, ease of reconstitution, and storage stability, the MR-SNC (Melatonin and Resveratrol-loaded sericin-based nanocapsule) was tested for variations in quality features, when added to cell culture media.Particle size, poly dispersity index (PDI), and particle surface charge  of SNC and MR-SNC were determined by Dynamic Light Scattering technique via Zetasizer instrument  at 25 °C. At that time, other main characteristics of SNC and MR-SNC, including aggregation, size, morphology and shape were assessed with Scanning Electron Microscope (SEM) , at an operating voltage of 25 kV.-1 with a spectral resolution of 4 cm-1 via FT-IR spectrophotometer .FT-IR spectroscopy was used to determine the chemical composition, molecular properties, and surface adsorption of nano-particles functional groups. Briefly, 1–2 mg of the pure antioxidants (RES/MEL), lyophilized blank SNC (B-SNC), and MR-SNC were mixed and then triturated with 100 mg potassium bromide . The mixture was then placed in the FT-IR sample holder and pressed. Spectra were recorded over the scanning range of 200–4000 cmThe structures of sericin, B-SNC, MEL, RES, MEL + RES, and MR-SNC dispersal were evaluated by circular dichroism spectrophotometer  via a 1 mm path length-quartz cell. Through this examination, the samples were scanned three times with a scan rate of 100 nm/min using ultraviolet light within 180–250 nm.36. At first, MR-SNC solution was ultracentrifuged  for 15 min at 18,200 rpm to separate the free drug from MR-SNCs. Then 1 mL of supernatant was assessed via UV–vis spectrophotometry  for evaluating of the unentrapped RES or MEL at 297 and 277 nm, respectively37. Next, a standard calibration curve was prepared using various concentraions of RES and MEL . The free drugs amounts were obtained using these curves. Finally, the entrapment efficiency (EE) and drug loading (DL) of RES and MEL on MR-SNC were calculated using the following formula:Entrapment efficiency and drug loading were assessed by ultracentrifugation methodAll the experiments were performed in triplicate, results are presented as mean ± SD.39. Briefly, 5 mL of MR-SNC was placed in a dialysis bag, followed by immersion in a fixed volume of release medium . Then, the bottels were incubated in an orbital mixer (Benchmark Scientific) for 55 h at 37 ± 0.5 °C at 300 rpm. At planned time intervals , 1 mL of PBS dialysate was removed and replaced with the same volume of fresh media and then analyzed by UV–vis spectrophotometry , at two different maximum absorption wavelengths (λmax), corresponding to 297 nm for RES and 277 nm for MEL. At each pH, the amount of MEL and RES released from MR-SNC was calculated as the drug release percentage at planned time intervals relating to the quantity of the entrapped drug.In vitro profiles of MEL and RES released from MR-SNC were studied at different pH values using a dialysis bag  as described elsewhere® Pasteur institute of Iran, Karaj, Iran) was chosen as an in vitro model because it preserves several features of differentiated mammary epithelium including the expression of functional estrogen receptor, responsiveness to estradiol and the ability to invade and metastasize. Furthermore, MCF-7 is a HER2-negative, and progesterone recepto- positive cell line41.In the present study, the human breast adenocarcinoma cell line  supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution  and was incubated at 5% CO43. Briefly, cells were plated in 96-well plates at a density of 3 × 103 cells/well and, following overnight incubation, were treated with RES + MEL, B-SNC, and MR-SNC at the concentrations indicated above and using the media at different pHs for 24 and 48 h , B-SNC  and MR-SNC  at different pHs  on MCF-7 cells was measured using a colorimetric MTT assay kit (Sigma Aldrich) as decribed previously8 h Fig. . After t41. First, a suspension of MR-SNC with a concentration of 1 mg/mL was mixed with the same volume of FITC (5 µg/mL) and stirred for 10 h in the dark at room temperature. Then the suspension was centrifuged at 13,000 rpm for 10 min and washed with PBS three times. Next, MCF-7 cells were seeded at a density of 2 × 105 cells per well in 6-well culture plates. After discarding the medium, the cells were washed twice with PBS. Next, cells were treated with 1000 µg/mL of stained MR-SNC at pH 6 (obtained from the MTT assay) for 8 h. Then the cells were washed three times with PBS, and 4% paraformaldehyde (100 µL/well) was added to fix the MCF-7 monolayers for 15 min.To study the cellular uptake of the fabricated nanoparticle, the cells were stained with FITC as describedNuclear evaluation was also studied by DAPI  staining. After 24 h incubation, the cells exposed to the MR-SCN at IC50 concentrations were washed with PBS, fixed with 4% (v/v) paraformaldehyde for 30 min, and rinsed twice with PBS. Next, DAPI was added for 20 min in the dark to stain the nucleus. Finally, cells were rinsed with PBS 3 times, and the well (n = 3) was evaluated by cytation cell imaging reader .The obtained data were analyzed using SPSS for Windows, version 23.0, and GraphPad Prism 8 software. The one-way analysis of variance (ANOVA) was used to determine the statistically significant differences between the groups. All the experiments were performed in triplicate, and mean values ± standard deviation (SD) were used to present the data. A p-value less than 0.05 was considered to be statistically significant.The current study was approved by the Ethics Committee of the Kermanshah university of medical science (IR.KUMS.REC.1400.331).35. Physico-chemical characteristics of all sericin nanoparticles were evaluated in different sericin concentrations  in a single MR-SNC was 98%. The antioxidants were loaded in the SNC, with a DL (%) of approximately 27  stretching vibration. The band at 1606 cm−1 is related to C = O stretching vibration, and the peaks at 1587 and 1382 cm−1 correspond to the C = C and C-N stretching, respectively. The RES spectrum , 1527 cm−1 , and 1244 cm−1 , which suggest the presence of a random coil structure. Another band at 1384 cm−1 was credited to C–H and O–H stretching and C–OH banding of hydroxyl amino acid side chains, like serine.Since the Blank-SNC is comprised of Sericin (S) alone; and after the freeze-dry proceedure, the FTIR peaks for both SNC and sericin powder (S) were quite similar, only one of them is displayed in the FTIR diagram Figs. , S3c. Th−1 were observed in MR-SNC FTIR spectrum. In this spectrum prominent shifting of the peak was observed from 1527 to 1537, 1462 to 1465, 1244 to 1267, 1159 to 1188, and 877 to 875 cm-1 in comparison to those of SNC. Additionally, comparative peaks with SNC in this spectrum were of higher intensity as opposed to the SNC, which was due to the formation of intermolecular hydrogen-bonding between NH and OH group of SNC with MEL and RES. Moreover, the new peaks appeared at 1587, 1516, 1323, 1188, 968, 713, and 677 cm−1 at the MR-SNC FTIR spectrum, indicating a shoulder peak in the spectra of MR-SNC, signifying MEL and RES were joined into SNC. Simultaneously, the expanded intensity of amide I, II, and III demonstrated the construction of a new amide linkage in MR-SNC. As opposed to SNC, a shoulder peak at 677–1500 cm−1 was seen in the spectra of MR-SNC, which could be to the C=O vibration of the C–H bunch from MEL and RES. These results confirmed the presence of MEL and RES in the spectrum of MR-SNC.MR-SNC was synthesized by three monomers; MEL, RES, and Sericin. In the spectrum of MR-SNC, all the bands relating to MEL, RES, and SNC were identified. As shown in Figs. Circular dichroism (CD) is a sensitive technique to monitor the conformational changes in a protein. CD, as a biophysical method, was used to probe sericin structure following its formation of nanocapsules by the nanoprecipitation method. It measures a difference in the absorption of right and left circularly polarized light to determine a protein’s secondary structure. Sericin has a molecular weight of 198.6 kDa with 1758 amino acid residues. As its spectra reveal Figs. , sericinThe in vitro release of MEL and RES from MR-SNC was examained by the dialysis bag method, under different pH values , up to 55 h  of MEL occurred in pH 6 at 55 h.To assess viability of the MCF-7 cells after treatment with RES + MEL , B-SNC  and MR-SNC  at different pHs , the MTT assay was done. There were no significant differences between controls and treated groups after 24 h (data not shown).Exposure to RES + MEL (1–100 µg/mL) for 48 h also showed similar cell viability to the control group. However, at 200 µg/mL, the cell viability significantly decreased at all pH values   after 4 h exposure at pH 6 is shown in Fig. 45. According to the captured images  in nature are its other advantages51. We tested five different sericin concentrations ) for protein-based nanocapsules preparation to find out the optimum concentration  in SNC, releasing MEL and RES24. Furthermore, MEL and RES are more stable at low pH values57. This stability in the acidic environment can potentially help with SNC diffusion.Sericin is a hydrophilic protein, that is composed of 40% serine and aspartic acid with a high potential for self-assembling. The carboxyl groups of aspartic acids render sericin a net negative charge at neutral pH. As can be seen in Fig. 58. At low doses it has antioxidant and protective effects on normal and tumor cells. However, the high doses it shows toxicity towards cancer cells46. Our results are consistent with these observations. Also, the cytotoxic effects of MR-SNC on the viability of the MCF-7 cells was observed at 100, 250, 500 and 1000 µg/mL at all pH values and was significantly higher than that exposed to SNC after 48 h. This finding strongly suggests potential synergy between the three antioxidants .MEL and RES had a significant toxicity on MCF-7 cell viability at 48 h, especially at 200 µg/mL and all pH values. SNC also reduced cell viability at 250, 500 and 1000 µg/mL at pH 6.5. Sericin has a remarkable antioxidant activity via preventing lipidic peroxidation and oxidative stressOur result with MR-SNC after 48 h showed high cell toxicity at pH 6 accross the concentrations tested. Thses results are consistant with the charge-reversal property of MR-SNC at low pH values.59. In addition, only positively-charged nanoparicles can attach to cencer cells61. Our uptake studies at pH 6 altered the nanocarrier surface charge from negative to positive via increasing the amino/carboxyl ratio in MR-SNC through a charge reversal process24. So, FITC-labeled MR-SNC attached to the MCF-7 membrane quite easily and was picked up by these cells via transcytosis.Our results with FITC showed high fluorescent signal. Cancer cells have a high negatively-charged surface due to high concentration of glycoproteins on their membrane. Many of these glycoproteins contain sialic acid, which in part contributes to the negative charge cell membrane55. At the time of conducting this research, it was not possible for us to investigate intracellular and extracellular ROS, due to the deficit and high cost of the required materials and equipment, in our country due to the sanctions condition that it was a limitation for present work.Our DAPI results illustrated chromatin condensation and DNA fragmentation in MCF-7 cells. DNA damage is an apoptosis hallmark, so our results suggest that the MEL and RES encapsulation in SNC leads to apoptosis of MCF-7 cells. Sericin based nanocarriers can induce cell apoptosis via caspase-3 activation, Bax proteins upregulation and Bcl-2 downregulationIn summary, we created a pH-sensitive nanocarrier with a low sericin concentration by nanoprecipitation as a simple and reproducible method. MEL and RES were loaded in this nanocarrier with charge-reversal property, that turned its surface charge from negative to positive in a mildly acidic environment. Our morphological and analytical investigations indicated the intact sericin, MEL, and RES structures after nanocarrier preparation and the desirable size, shape and size distribution of nanoparticle. The pH-dependent drug release behavior of MR-SNC, with the maximum release in pH 6, showed this smart strategy's potential efficacy in releasing antioxidants in the acidic tumor microenvironment. Moreover, MR-SNC significantly decreased MCF-7 cell viability in acidic pH. MR-SNC was picked up by MCF-7 cells in pH 6, accumulated inside them and caused DNA damage. In conclusion, the charge-reversal protein-based nanocarrier will be an excellent strategy to maximize the efficiency of anti-cancer agents and minimize the drug’s unspecific toxicity.Supplementary Figures.