Considering the significant impact of school-based HIV/AIDS education, in 2007, a curriculum on HIV/AIDS was incorporated in the national curriculum for high school students of Bangladesh through the Government’s HIV-prevention program. Based on the curriculum, an intervention was designed to train teachers responsible for teaching HIV/AIDS in classes.In-depth interviews were conducted with teachers to understand their ability, skills, and confidence in conducting HIV/AIDS classes. Focus-group discussions (FGDs) were conducted with students who participated in HIV/AIDS classes. HIV/AIDS classes were also observed in randomly-selected schools. Thematic assessment was made to analyze data.The findings showed that the trained teachers were more comfortable in using interactive teaching methods and in explaining sensitive issues to their students in HIV/AIDS classes. They were also competent in using interactive teaching methods and could ensure the participation of students in HIV/AIDS classes.The findings suggest that cascading training may be scaled up as it helped increase ability, skills, and confidence of teachers to successfully conduct HIV/AIDS classes. School-based programs offer opportunities to encourage adolescents and young people to delay the onset of sexual activity, promote safe sex behavior using a condom, and increase contraceptive-use after sexual initiation . ResultsResults of studies also suggest that, to have a substantial impact of HIV/AIDS-education programs, teachers or educators should be adequately trained before involving them in any HIV/AIDS-prevention program in a school setting -12. A quImplementing HIV/AIDS education through the national curriculum is a challenging task, especially in developing cultural-sensitive curriculum and readiness of teachers in disseminating HIV/AIDS information to their pupils. In Bangladesh, HIV/AIDS is a sensitive issue in the sociocultural-religious context due to its transmission primarily through sexual contact. It may, thus, be considered too controversial to be taught in a school setting . BesidesIn 2004, the Government of Bangladesh undertook a project titled “Prevention of HIV and AIDS among young people in Bangladesh”. One of its components of the project implemented in two phases was “Integrating HIV and AIDS information into the secondary school (high school) and college curriculum as well as dissemination of developed materials”. This component had two major segments: (a) Development of curriculum and texts for class Six-Twelve and (b) Institutional capacity-building through a nationwide teachers’ training program. PIACT Bangladesh, a non-profit, non-governmental organization, is responsible for implementing the training program. A curriculum on HIV/AIDS and texts for class Six-Twelve were developed following a participatory, bottom-up approach based on public-private partnership.For institutional capacity-building, PIACT Bangladesh developed a cascade training program in which master trainers were trained first to imparting training to core trainers, who, in turn, trained subject teachers  at the institution level. For three-day master trainers’ training held at the district level, two teachers from a college, one teacher from an alim madrasha, and the Upazila Secondary Education Officer from each upazila (subdistrict) were selected. Besides, four master trainers from each upazila in a district were also selected. These master trainers trained core trainers drawn from secondary and higher secondary schools, including dakhil and alim madrashas. Two teachers, including the head teacher/principal, attended a two-day core training program held at the upazila level. Trained in batches, each batch consisted generally of 30 teachers for the core trainers’ training program. All the core trainers were responsible for training subject teachers in their respective schools/colleges and madrashas. On average, five subject teachers from each school received training for one and a half days. The core trainers also organized a half-day orientation session with the School/Madrasha Managing Committee to create a supportive environment for teaching the subject in and around the institution.The broad objective of training was to develop skills and knowledge of teachers so that they can appropriately communicate HIV/AIDS-related messages in the classroom setting. Training sessions were focused on providing appropriate information on HIV/AIDS and on the evolving ways for using interactive methods to teach the subject; for example, how to organize group discussions and puzzle games and how to encourage active participation of students using role-play method.The present study was conducted to understand the effects of teachers’ training, covering a curriculum-based HIV/AIDS-education program, on ability, skills, and confidence of teachers to teach HIV/AIDS in classes.The study was a follow-up assessment of a previously-conducted rapid assessment of a teachers’ training program . The preThe present study used qualitative techniques for data-collection. To triangulate findings, the study used multiple participant-groups as sources of information and also used various qualitative techniques. The study compared findings from teachers who received training on HIV/AIDS with findings from teachers who did not receive such training.The study was conducted in two randomly-selected districts from a list of districts in Chittagong division of Bangladesh. One of the two districts was selected from the list of districts where subject teachers of those districts received training through the PIACT’s cascading training program at least a year before data-collection, and the other one was selected from the list of districts where no such training was held. From each district, three upazilas  were randomly selected. While selecting an upazila, we considered similar upazilas from each district; however, we did exclude all Sadar (urban) upazilas due to sociocultural and economic variations between Sadar upazilas and other upazilas. Similar upazilas were identified by analyzing secondary data of each upazila, such as socioeconomic status, literacy rate, number of schools/colleges/madrashas, and the geographical distance from the district headquarter. The three upazilas were randomly selected from the list of similar upazilas.Data were collected during November 2010–June 2011 by conducting in-depth interviews with subject teachers, observing HIV/AIDS classes, and conducting focus-group discussions (FGDs) with students who participated in HIV/AIDS classes. A skilled and experienced four-member field research team was formed. The team comprised a Research Officer (RO), two Senior Field Research Assistants (SFRAs), and a Field Research Assistant (FRA) of icddr,b  for collecting data in the field. The research team was further trained on the study contexts, qualitative data-collection techniques, and non-random sampling procedures. The Principal Investigator (the first author) monitored the overall data-collection process.To conduct in-depth interviews, a flexible semi-structured interview guideline was developed. The interview guideline covered issues, such as background of participants; their experiences in training on curriculum-based HIV/AIDS education; participation in teaching of HIV/AIDS in classes; contents of text relating to HIV/AIDS taught by teachers; mode of teaching method used while educating students about HIV/AIDS; ability, skills, confidence, and experience of teachers in using interactive teaching method; how comfortable are they in discussing sensitive issues with students; their basic understanding of HIV/AIDS; and factors that hindered or facilitated during HIV/AIDS sessions. These were the prime topics of discussion; however, new issues were added based on new findings during initial interviews and analysis. On average, each interview lasted for 40-60 minutes.As per the purposive criteria, subject teachers were selected from both study districts. In the non-intervention district, teachers who were conducting HIV/AIDS classes and did not receive any training on HIV/AIDS neither from the PIACT nor from any other institution were selected. In the intervention district, teachers who were trained under cascading training on the HIV/AIDS curriculum and who were conducting HIV/AIDS classes were selected.The interviewers maintained a diary and took detailed field-notes to make results of interviews richer. A digital recorder was used for recording interviews. Immediately after interviews, the interviewers completed field-notes and transcribed the recording. After transcribing the previous interview, they proceeded for the next interview so that the experiences gathered during the previous interview help fill the gaps in the next one.The research team observed HIV/AIDS classes to understand ability of teachers in using interactive teaching method in the classroom setting and participation of students in classroom discussions. Since our intention was to observe natural methods of teaching  in HIV/AIDS classes and to minimize bias, the respective teacher of the selected class was not shared beforehand about observation, except the headmaster of the respective school. For documenting information, an observation checklist (including closed- and open-ended items) was used. The observation checklist covered the teacher's clarity and understanding of the unit objectives; ability to encourage participation of students in the learning process; receptiveness to feedback from students; ability to simplify and explain subject matter; and ability to create a comfortable classroom environment. Besides completing the observation checklist, the observers also maintained notes and a diary; after each observation, a detailed report was prepared.The research team conducted FGDs with students who participated in HIV/AIDS classes to understand the level of their participation in a class and to assess ability of teachers to use interactive method in teaching in HIV/AIDS classes. A semi-structured guideline was used to conduct FGDs, which included practical issues relating to HIV/AIDS rather than textual information (e.g. knowledge); for example, students were asked to describe details of the HIV/AIDS class, including total class time, issues/content discussed, mode of teaching method used, and their participation in discussions, and finally, they were asked to comment on ability and comfortableness of teachers in using teaching methods. Since the FGDs took place immediately after an HIV/AIDS class was over, the research team reached the respective school immediately before the class time and took permission from the headmaster of the school for conducting FGDs. The RO of each team worked as a moderator/facilitator, and the SFRA assisted him and took detailed notes on discussions and observations (e.g. body language of participants). A voice recorder was used for recoding the whole discussion. After each FGD, the research team transcribed the recorded data, and then a detailed report was prepared incorporating all notes and transcripts for each FGD. On average, each FGD lasted for 60-90 minutes.et al. that included reading, coding, displaying, reducing, and interpreting [Analysis of qualitative data was begun with first field activities . This analysis guided the improvement of quality of data as the study proceeded. The investigators regularly reviewed  the field-notes and the diary with the research team. The interviewers met the Principal Investigator at least once a week to review results of their activity sessions/interviews and to determine the best practices for further activity sessions/interviews, note-taking, and areas for questioning that should be pursued or which are not effective. From the beginning, thematic analysis was done. Themes were identified based on the existing themes and new information from the findings. The processes followed a sequence of inter-related steps recommended by Ulin rpreting . At firsThe Institutional Review Board of icddr,b, comprising a Research Review Committee and an Ethical Review Committee, approved the study. The interviewers took written consent from the respondents before conducting in-depth interviews. For observation of HIV/AIDS classes, a written institutional consent was taken from the headmaster of the school. However, before starting observation, the observer took verbal consent from the class teacher and students. The research team took written permission from the headmaster of the school for conducting FGDs. Before initiating any discussion, the team also took written assent from all FGD participants.In total, 36 in-depth interviews, 12 FGDs, and 12 observations were conducted in the two study sites: Lakshmipur district and Feni district. Fifty percent of the interviews, FGDs, and observations were conducted in each study district.The teachers perceived that the training helped them prepare class-wise lesson plans for their students. The meaning of class-wise lesson plan was that, since the students of Class Six-Eight are not matured, they would be given only a basic idea about HIV/AIDS. However, details about HIV/AIDS can be elaborated for the students of Class Nine-Ten as they are quite matured. The teachers also stated that, since the students of Class Six-Eight are in their tender age, they can become derailed or pervasive if they are given details about HIV/AIDS. They argued that, since the textbooks of Class Six-Eight contain a very basic idea about HIV/AIDS, they need not to learn more. However, when they will study in Class Nine-Ten, they can learn more details about HIV/AIDS. A subject teacher at Chagolnaiya, Feni during in-depth interview said:“To speak the truth, if we did not get the training, it would not have been possible for us to teach HIV/AIDS in the class. After training, we have been conscious about the disease, can prepare lectures according to their needs, and can teach students easily and comfortably.”The teachers further stated that training helped remove many of their misconceptions relating to HIV/AIDS; for example, many teachers believed that HIV/AIDS is a contagious disease; however, after training, they understood the real facts. A subject teacher at Chagolnaiya, Feni during in-depth interview said:“A few days ago, I heard that a person named Ali… would be tightened with a tree by confining him in a box. He would be given food using a stick.... The meaning of this is that, if he is touched, HIV can be transmitted …. After training, my misconception was removed, and I realized that it was a bitter example of stigma and discrimination!”The teachers mentioned that the training helped them gather skills to explain sensitive issues relating to HIV/AIDS to their students. Before training, they felt uneasiness to explain sensitive issues but after training, their uneasiness considerably declined. They have now learnt how to explain sensitive words using synonyms, or by story-telling, or the other way. A teacher said:Before training, the teachers would avoid to mention sensitive issues tactfully like me, saying that, since a questionnaire contains a number of questions, it is not compulsory to answer a specific question relating to HIV/AIDS …. After the training, I never avoided such topics, and now I understood how I could explain sensitive issues to students’ The non-trained teachers used lecture method while conducting HIV/AIDS classes. When the teachers were asked as to why they followed the method, they mentioned difficulties in giving a practical example concerning HIV/AIDS due to lack of knowledge on this issue. Students in FGDs stated that teachers used both lecture and question-answer methods in classes. They also mentioned that the teachers first delivered a lecture and then asked questions to them. Teachers just read out a topic that could not be heard from behind.In classroom observations, the research team found that teachers who did not have any training on HIV/AIDS used traditional lecture method only. In lecture method, the teachers delivered lectures, and students would listen. While observing classes, it also appeared that the teacher himself was not knowledgeable about HIV/AIDS. It even appeared that the teacher was not comfortable to teach the chapter on HIV/AIDS because his confidence level was so weak that he felt nervous. Chalk and duster used by him fell down from his hand due to his excessive nervousness.On the other hand, the trained teachers created some innovation in teaching methods. They mostly used discussion method and group-work with the help of a flip-chart, posters, and other visual aids. The teachers thought that, through group-work, students could easily discuss sensitive issues among themselves, which enable them to get an equal lesson. A teacher stated:Students could not remember contents of a lecture if they were taught using lecture method …. In group-work, they could take notes in the notebook by discussing with their co-students … because it is equal to write once than to read three times …. In the in-depth interview, another teacher stated:When students were taught with the help of a flip-chart, they became fascinated because they found some newness in the chart …. In the question-answer sessions, the teachers informed the students before-hand about the topic that will be discussed in the next-day class so that they can understand the issue easily and can participate in the discussion session. The teachers thought that, through this method, teacher-student interactions became closer. The student-participants in FGDs in the intervention area also stated that, in group-work, a teacher first delivered a lecture and then formed groups by giving several topics to write some points relating to the particular topic. Following this, the team leader presented some points, and other groups provided their feedback..During observation, it was noticed that a trained teacher used hand-notes (prepared by him) and a flip-chart in the class. While observing classes, it appeared that concept of a trained teacher about HIV/AIDS was clear, and he tried to explain the concept easily and comfortably. In group-work, students were divided into groups first, and they were then told to give a name of the group by the name of a river, such as Padma, Megna, or Jamuna. Each team was then given a small chit (where the topic was written), a sign pen, and a large-size paper. Ten minutes were allocated for group-work. After completing the group-work, the team leader presented findings. Then the teacher told students to ask questions. Since the students did not ask any question, the teacher discussed issues not included in group-workThe trained teachers were conscious in explaining sensitive issues in HIV/AIDS classes. They stated that, as they learnt from training, they could prepare class-specific lessons. Only basic information about HIV/AIDS was provided to students of Class Six-Eight, and some detailed information was provided to students of Class Nine and Ten. The teachers opined that students of lower classes might have been derailed if they were given details about sensitive information relating to HIV/AIDS. Synonyms were, therefore, used for explaining sensitive issues where the religious rules were emphasized. A teacher stated:We applied a technique to explain sensitive issues because students of Class Eight are in their tender age. However, since students of Class Nine-Ten are matured, they were given class-specific detailed lessons. The student-participants in FGDs stated that their teachers laid emphasis on the religious rules to explain socially-accepted sensitive issues. When the FGD participants were asked to explain as to how their teachers discussed sensitive words in the class, one FGD participant replied:The religious rule means not to involve in any physical relationship irrespective of marriage. If the religious rules would have been maintained, they would not go for any illegal physical relationship. 'Safe sex’ was explained as having marriage by maintaining the religious rules. According to the non-trained teachers, explaining sensitive issues to students was embarrassing and sometimes difficult as they asked many questions that were impossible to answer. They were sometimes unaware of the appropriate answer or the way of explaining the issue. Moreover, when sensitive words were discussed in the class, students laughed and did not feel interest to learn about HIV/AIDS. The teachers just read out sensitive words without explaining these to students. They stated that some words, e.g. sperm and vaginal fluids, were difficult to explain. A teacher stated:I neither uttered any sensitive word loudly nor did I put less emphasis on those words because students might feel shy or might put their heads down. So, I just read sensitive words just like a person reading a book …. Almost similar findings were revealed from FGDs with students in the area where teachers did not receive training. They stated that their teachers did not even look at them, and they just read out the text. If any student would try to ask any question, the teacher showed disinterest to respond. The teacher even sometimes acted like as if he could not hear the student’s question. During observations, the study team noticed that the teacher just read out text relating to modes of transmission and prevention while students were just listeners. When the teacher said that HIV/AIDS could be transmitted through an illegal relationship, students appeared to feel shy but the teacher did not notice it.The trained teachers mostly avoided the words 'condom-use’. Students stated that, while talking to them in FGDs, their teachers avoided the words 'sexual intercourse’. While observing the class, the research team noticed the same.However, non-trained teachers found many words sensitive to them in classroom discussions. They usually avoided the words, such as sexual intercourse, condom-use, sperm, safe sex, and vaginal fluid. In FGDs, the student-participants reported that their teachers avoided to mention the words—sexual intercourse and condom-use. While observing the class, the research team as well noticed that teachers were trying to avoid these words.The findings of FGDs in schools where teachers did not receive any training revealed that students did not participate spontaneously in HIV/AIDS classes. In most cases, the teachers did not encourage them to participate in the class, and they seemed to be annoyed when students asked questions. The research team observed that students were laughing in the HIV/AIDS class. It was also observed that teachers were delivering lectures in their own way but students had no concentration. Some teachers in the control area sometimes encouraged students to ask questions. Some teachers asked questions to students as a technique to keep the environment calm and quiet or to stop laughing. Students who participated in FGDs reported that their teachers did not take any initiative to encourage them to participate in discussions. During observations, the research team also noticed that the teachers taught their students in their own way. However, the trained teachers encouraged their students to participate in group-work and provided feedback during feedback sessions. Similar findings were observed during observations that teachers conducting a class through group-work and question-answer sessions. Students echoed same finding in FGDs.During in-depth interviews, teachers were asked to identify practical issues they faced while conducting HIV/AIDS classes. Teachers, irrespective of their training status, identified issues that were confounding their HIV teaching, such as class time, lack of support from the headmaster, teaching materials, and preference of teachers either for co-education or for separating boys and girls in the class, i.e. a separate class arrangement.The allocated time for HIV/AIDS class was 35 minutes in both study areas. The control-area teachers reported that inadequate time was allocated to teach new and sensitive issues such as HIV/AIDS. They recommended increasing class time from 35 minutes to one hour. When we interviewed the head teachers, they argued that class time is a technical issue and was determined taking into consideration all possible issues, including the importance of different class-contents. They considered lesson on HIV/AIDS less important in terms of the examination context, and a class was allocated after the lunch period.The trained teachers confirmed that allocated time for teaching HIV/AIDS was adequate. During training, the teachers learnt how to accommodate required issues within a speculated class time. They argued that class time could not be increased due to the schedule problem as it will hamper the usual schedule of other classes. They suggested that a teacher could divide the HIV/AIDS curriculum into units to teach it in one or more classes while making the schedule for term examination.The trained teachers also reported that the head teacher instructed them to teach the chapter in allocated class time. They also added that the Managing Committee of the school, local people, and other teachers were informed about the issue, which helped them teach such sensitive topics comfortably and easily. All intervention-area teachers also reported that training material helped them teach the HIV/AIDS subject in the class. Besides, the material helped enhance their mental courage because they thought that whenever they would forget any issue, they can get help from the material. Although they preferred a separate class for girls and boys, they thought that co-education is okay.Training on HIV and AIDS helped the study teachers to increase their ability and skills to prepare class-specific lessons. The trained teachers followed these curricula considering the adaptability of young students to explain sensitive issues and words to students and started the class by giving an introduction about the importance of the topic in their personal life. Lack of training for teachers was also identified as a concern in teaching sexual health programs in the school context ,22,23 beStudies have shown that training helps improve knowledge, attitudes, and readiness of teachers to impart HIV/AIDS-prevention information to their students ,26,27. TIn terms of teaching methods used in conducting HIV/AIDS classed, training had a greater impact which was reflected among teachers who had prior training. They used group-work and discussion method in classes in accordance with the situation or environment of the classroom setting. The non-trained teachers mostly used lecture method, which was also used in other classes. They, however, had no idea about any other methods to teach the topic perfectly. While previous studies also identified these as the key barriers to implementing HIV education, such as teachers’ knowledge gap, lack of guidelines and resources, and discomfort in using interactive teaching methods ,30.The findings of the present study demonstrated that the training enhanced skills and ability of teachers ensuring participation of students in classroom discussions. The trained teachers encouraged their students to participate in group-work and provided feedback to them to ensure their participation in discussions. While participation of students in HIV/AIDS classes was not spontaneous in the control area, in most cases, teachers did not encourage their students to participate in discussions.Teaching school students about HIV/AIDS may be especially problematic in Bangladesh and other South Asian countries where HIV is mostly transmitted through heterosexual contact but where discussion of sexual issues among adults and students has been culturally restricted. Studies in other countries have identified that cultural prohibition created barriers to discussing these sensitive terms with students ,31. The Overall, the results of the assessment revealed that the teachers’ training program had a positive impact on ability, skills, and self-confidence of teachers in conducting HIV/AIDS classes, who received training on HIV/AIDS through the cascading training process compared to teachers who did not receive it. The positive effects of the cascading training were also visible in this study when experiences of teachers who did not receive any training were considered. HIV/AIDS being a new subject contains some sensitive terms or words; therefore, the teachers should be trained on teaching methods, and at the same time, they need to prepare their students to be ready to learn such sensitive words or terms. Results of a study also suggest that teachers should be well-trained to be able to teach children and adolescents about human sexuality and prevention of HIV/AIDS comfortably and competently; otherwise, they will be in a disadvantageous position in dealing with populations at risk of HIV infection .The present study had some limitations. The findings may not be generalized in the wider context due to its qualitative nature and approaches that applied to select teachers from different schools. The non-random recruitment and sampling of participants through a purposive sampling technique is prone to selection bias. Another potential limitation was that the study used direct observation; it is susceptible to observer-bias and observer-effect, which refer to the way in which presence of an observer in some way influences behavior of teachers and students being observed. However, to reduce such biases, we used trained and skilled research staff to conduct observations. The results at best give a comprehensive and qualitative understanding of the effects of teachers’ training on curriculum-based HIV/AIDS-education program on ability, skills, and confidence of teachers to teach HIV/AIDS in the classroom setting.Overall, the findings suggest that cascading training should be scaled up as it demonstrated to have its impact on teachers. This suggestion is supported because the training program particularly helped increase ability, skills, and confidence of teachers to conduct HIV/AIDS classes, increase their knowledge and skills to implement interactive teaching methods, and ensure participation of students in HIV/AIDS clasess.The authors declare that they have no competing interests.All authors of this manuscript were the investigators of the research and participated in preparing the manuscript, from conception to finalization. HS designed the study, supervised and monitored data collection, analysis and drafted the manuscript. MAI participated in study design, data-collection and write-up. RG participated in study design, data analysis and write-up. All authors read, critically revised, and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/990/prepub


Blattella germanica as the model and RNAi for transcript depletion, we have confirmed that Met transduces the antimetamorphic signal of JH in young nymphs and plays a role in the last nymphal instar moult in this species. Previously, the function of Met as the JH receptor had been demonstrated in the Eumetabola clade, with experiments in Holometabola (in the beetle Tribolium castaneum) and in their sister group Paraneoptera (in the bug Pyrrhocoris apterus). Our result shows that the function of Met as JH receptor is also conserved in the more basal Polyneoptera. The function of Met as JH transducer might thus predate the evolutionary innovation of metamorphosis. Moreover, expression of Met was also found in last nymphal instar of B. germanica, when JH is absent. Depletion of Met in this stage provoked deficiencies in wing growth and ecdysis problems in the imaginal moult. Down-regulation of the ecdysone-inducible gene E75A and Insulin-Like-Peptide 1 in these Met-depleted specimens suggest that Met is involved in the ecdysone and insulin signalling pathways in last nymphal instar, when JH is virtually absent.Juvenile Hormone (JH) represses metamorphosis of young instars in insects. One of the main players in hormonal signalling is Methoprene-tolerant (Met), which plays the role of JH receptor. Using the Polyneopteran insect  In essence, insect metamorphosis is regulated by the interplay of two hormones, 20-hydroxyecdysone (20E), which triggers the successive moults throughout the life cycle, and juvenile hormone (JH), which represses the transition to the adult stage Drosophila melanogaster strains D. melanogaster Met protein could bind JH with a very high affinity (Kd of 5.3 nM), providing the first clear indication that Met might play the role of JH receptor. More recently, it has been observed that depletion of Met mRNA levels with RNAi in early larval stages of the holometabolan species T. castaneum triggers precocious pupal morphogenesis Pyrrhocoris apterusT. castaneum, Charles et al. While molecular mechanisms regulating the action of 20E have largely been unveiled, those underlying the action of JH have remained almost a complete mystery until recent years, when the transcription factor Methoprene-tolerant (Met) has been proclaimed as the JH receptor D. melanogasterT. castaneumBlattella germanicaP. apterus and Rhodnius prolixusIn the meantime, it was found that the JH signal downstream of Met is transduced by Krüppel-homolog 1 (Kr-h1), a transcription factor with a DNA binding domain consisting of eight zinc fingers. The function of Kr-h1 as a transducer of the antimetamorphic action of JH was demonstrated through RNAi experiments in holometabolan species, such as Bombyx morikJHRE, in the promoter region of B. mori Kr-h1. Interestingly, kJHRE contains an E-box to which bHLH-PAS proteins may bind, and reporter assays in mammalian HEK293 cells  showed that while kJHRE-specific reporter activity was triggered in the presence of JH when the complete open reading (ORF) frame of B. mori Met was expressed, this activation was higher when Taiman ORF was coexpressed with Met. Therefore, these results suggest that Met and Tai jointly interact with the kJHRE located in the Kr-h1 promoter region and that this interaction is JH-dependent T. castaneumkJHRE, this time located in both the promoter region and in the first intron of T. castaneum Kr-h1. Moreover, reporter assays performed with HEK293 cells also showed that kJHRE reporter activity was triggered when Met was expressed in the presence of JH and the activation was enhanced when Taiman was coexpressed with Met P. apterusT. castaneumFrom a mechanistic point of view, results obtained in NIAS-Bm-aff3 cells from the silkworm B. germanica as model. Moreover, our preliminary experiments showed that Met is also expressed in last nymphal instar of this cockroach, intriguingly when JH is absent. A second purpose of the present work was, then, to study the function of Met in this stage.The present work aims to extend the investigation into the JH receptor role of Met in metamorphosis to Polyneopteran insects, taking the cockroach B. germanica specimens used in the experiments were from a colony reared in the dark at 30±1°C and 60–70% relative humidity. Freshly ecdysed female nymphs were selected and used at the chosen ages. Prior to injection treatments, dissections and tissue sampling, the specimens were anaesthetized with carbon dioxide.The We carried out total RNA extraction from the whole body (excluding the digestive tube to avoid intestine parasites) or specific tissues using the miRNeasy extraction kit (QIAGEN). A sample of 500-ng from each RNA extraction was treated with DNase (Promega) and reverse transcribed with first Strand cDNA Synthesis Kit (Roche) and random hexamers primers (Roche). RNA quantity and quality was estimated by spectrophotometric absorption at 260 nm using a Nanodrop Spectrophotometer ND-1000 (NanoDrop Technologies).B. germanica transcriptome obtained with RNA extracts from whole body of penultimate instar female nymphs and sequenced with a 454 Junior sequencer  at the Technical and Scientific Services of the Biomedical Research Park of Barcelona (PRBB). Transcriptome sequences were validated with RT-PCR using specific primers and cDNA from penultimate instar female nymphs of B. germanica as a template. Further 3′ and 5′ rapid amplification of cDNA ends  allowed us to obtain a practically complete full length ORF of BgMet. All PCR products were subcloned into the pSTBlue-1 vector (Novagen) and sequenced.BgMet mRNA was obtained from a Quantitative real time PCR (qRT-PCR) reactions were carried out in triplicate in an iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories), using SYBR Green . A template-free control was included in all batches. The primers used to detect mRNA levels are detailed in B. germanica were topically applied in dorsal abdomen with JH III (Sigma-Aldrich), which is the native JH of B. germanicaca. 50% of the biologically active (10R)-JH III, thus the active dose applied was around 10 µg per specimen.Freshly emerged last instar female nymphs of Autographa californica nucleopoyhedrovirus was used as control dsRNA (dsMock). The dsRNAs were prepared as reported elsewhere The detailed procedures for RNAi experiments were as described previously B. germanica transcriptomes available in our laboratory and PCR strategies, we obtained a cDNA of 1783 bp (GenBank accession number HG965209) whose conceptual translation rendered a 594 amino acid protein with sequence similarity to Met proteins that we called BgMet. The BgMet sequence contains the bHLH, PAS A, PAS B and PAC  motifs that are typical of Met proteins  was with Met1 of B. mori  or last (N6) nymphal instars . ExpressB. germanica metamorphosis was studied by RNAi approaches using a double-stranded RNA (dsRNA) which targets BgMet (dsMet-a) (ca. 30%) in dsMet-a-treated specimens (ca. 50%) , whereas Tai mRNA levels did tend to be lower, the differences with respect to dsMock-treated specimens were not statistically significant (Involvement of Met in dsMet-a) . In a fipecimens . At the pecimens . In a seca. 50%) . We alsonificant .At the phenotypic level, we first analysed the specimens that received a double treatment of dsMet-a or dsMock in N4. All dsMock-treated specimens (n = 27) moulted to N5, then to N6 and to adult, all stages showing normal morphological features . As regaThen, we analysed the specimens that received a double treatment of dsMet-a or dsMock in N5. All dsMock-treated specimens (n = 19) moulted to normal N6 and then to normal adults. None of the dsMet-a-treated specimens (n = 35) died during N5 or N6. After the moult from N5, 5 out of the 35 specimens (14%) were nymphoids with adult features , 1 speciTo assess the specificity of dsMet-a, we tested an alternative Met-targeting dsRNA (dsMet-b) designed in another region of the BgMet sequence . We carrca. 65%) in dsMet-a-treated specimens  than controls, whereas those of RXR tended to be lower but differences with respect to controls were not statistically significant. The ecdysone inducible gene E75A was significantly down-regulated (ca. 65%) (B. germanica orthologue of Insulin-Like-Peptide 1 (ILP-1), which is the most highly expressed ILP in B. germanica brain . ILP-1 mRNA levels in dsMet-a-treated specimens were significantly, and dramatically, lower (ca. 90%) than in control specimens  . As moulpecimens .B. germanica Met is the first Met sequence reported in Dictyoptera, a Polyneopteran within the Pterygota subclass of hemimetabolan insects. In terms of overall percentage of identity, BgMet is closer to T. domestica (an Apterygota ametabolan species) than to the phylogenetically closer Pterygota hemimetabolan L. migratoria. As expected, the homologous sequences of Diptera and Lepidoptera, the most modified orders of Pterygota Holometabola, are the most divergent with respect to BgMet.P. apterus) and holometabolan (T. castaneum) species B. germanica N4 did not attain such a size and therefore moulted to N5 despite dsMet treatment. Moreover, adult features triggered by Met depletion were more apparent and with higher penetrance when the specimens were treated in earlier stages, N4, compared to N5, possibly because the experimental design allowed more time for tissue growth and development and for more effective Met depletion. These observations are in line with the recently proposed concept that insects acquire competence to metamorphose only in late nymphal or larval instars Expression of BgMet in the penultimate (N5) nymphal instar, when JH is present, is not surprising. In this instar, we propose that Met transduces the JH signal that represses adult morphogenesis, as Met depletion during this stage triggers precocious metamorphosis at the next moult. When dsMet was administered in the antepenultimate (N4) nymphal instar the adult characters appeared after two moults. Similar findings relating to the precocious apparition of adult characters after depleting Met in pre-final juvenile stages have been reported in both hemimetabolan  nymphal instar of B. germanica ILP-1 was dramatically reduced in Met-depleted specimens, which suggests that Met enhances ILP-1 transcription without the contribution of JH. JH and Met involvement in the IIS pathway has already been reported in T. castaneum adults T. castaneum ILPs  in brain and fat body. In the fat body, vitellogenin transcription, which is JH-dependent, was also reduced in Met-depleted specimens and the authors showed that JH-induced vitellogenin expression in the fat body is mediated by ILP signalling T. castaneum decreased lipid and carbohydrate metabolism and extended their survival B. germanica, when JH is absent, would merit further study to elucidate the underlying mechanisms.The delayed onset of moulting and ecdysis plus wing size complications, might also be caused by metabolic and growth deficiencies related to the Insulin/IGF-1 signalling (IIS) pathway. Our experiments showed that expression of B. germanica, a representative of Polyneoptera. This function of Met had previously been demonstrated in Holometabola (T. castaneum) and Paraneoptera (P. apterus), thus meaning that it is present across Eumetabola, according the phylogeny of the extant hexapod orders reported by Wheeler et al. T. domesticaT. domestica is ametabolan, Met function in this species cannot be related to metamorphosis, but it could, however, be related to JH transduction for vitellogenin production and oocyte growth, as occurs in the hemipteran P. apterus, according to the study of Smykal et al. As a main conclusion, our results show that Met transduces the JH signal and, therefore, represses metamorphosis in the cockroach Figure S1Comparison of BgMet with other insect Met protein sequences. In addition to the percentage of overall identity, we indicate the percentage of identity for each of the characteristic domains of the protein. The species included (and the respective code and accession number in GenBank) are: Thermobia domestica , Rhodnius prolixus , Locusta migratoria , Tribolium castaneum , Acyrtosiphon pisum , Aedes aegypti , Pyrrhocoris apterus , Drosophila melanogaster Germ cell-expressed , Bombyx mori , Drosophila melanogaster Methoprene-tolerant , Bombyx mori . * indicate that the sequence lacks the region between the initial Met and the bHLH domain and a part of the latter, and ** that the sequence lacks the region between the initial Met and the bHLH domain, but the latter is complete.(TIF)Click here for additional data file.Figure S2Mild phenotypes obtained after treating Blattella germanica N4 with dsMet-a. (A) Dorsal and ventral view of a specimen that died during the ecdysis to N6; the detail shows that the lateral expansions in the mesonotum and metanotum are somewhat enlarged and apparently flexible (indicated with an arrow in the detail). (B) Dorsal and ventral view of a specimen that moulted from N6 to adult with the tegmina and wings correctly patterned but not well extended, and with the hind tibiae deformed.(TIF)Click here for additional data file.Figure S3Effects of Met depletion with dsMet-b in Blattella germanica. (A) Dorsal and ventral view of a precocious adult obtained after treating with dsMet-b in N5. (B) Dorsal and ventral view of a male precocious adult obtained after treating with dsMet-b in N4. (C) Effects, at transcript level, of dsMet-b treatment in N5; insects received a 3-µg dose in N5D0 and another on N5D3; transcript levels  were measured on N5D6. Each point represents 4 biological replicates and results are expressed as the mean ± SEM; data are normalized against the dsMock-treated samples , and the asterisk indicates statistically significant differences with respect to controls (p<0.05), according to the REST software tool (TIF)Click here for additional data file.Table S1Primers used to detect transcript levels by qPCR and those used to prepare the dsRNAs for RNAi experiments.(PDF)Click here for additional data file.Table S2Summary of the effects of Met depletion at phenotypic level in the experiments treating with dsMet-a or dsMet-b and at the stages N4, N5 or N6. In all cases the corresponding dsRNA was administered in two 3 µg-doses, one on day 0 and the other on day 3 of the given instar. Controls were equivalently treated with dsMock. Methodological details are described in the main text.(PDF)Click here for additional data file.


N=142; sampled at two time points) is associated with a long-lasting shift in microbiota composition and metabolism. The shift includes depletion of Actinobacteria, increase in Bacteroidetes and Proteobacteria, decrease in bile-salt hydrolase and increase in macrolide resistance. Furthermore, macrolide use in early life is associated with increased risk of asthma and predisposes to antibiotic-associated weight gain. Overweight and asthmatic children have distinct microbiota compositions. Penicillins leave a weaker mark on the microbiota than macrolides. Our results support the idea that, without compromising clinical practice, the impact on the intestinal microbiota should be considered when prescribing antibiotics.Early-life antibiotic use is associated with increased risk for metabolic and immunological diseases, and mouse studies indicate a causal role of the disrupted microbiome. However, little is known about the impacts of antibiotics on the developing microbiome of children. Here we use phylogenetics, metagenomics and individual antibiotic purchase records to show that macrolide use in 2–7 year-old Finnish children ( The impact of antibiotics on the microbiome and health of children is poorly understood. Here, Korpela et al. study the gut microbiome of 142 children and show that the use of macrolides, but not penicillins, is associated with long-lasting shifts in microbiota composition and increased risk of asthma and overweight. Antibiotics are the most commonly used drugs in the pediatric populations of western countries14589101315We show that antibiotic use in childhood is associated with marked changes in the intestinal microbiota composition, which persist for over 6 months. Macrolides, particularly, appear to modify the microbiota and their functions, being the strongest driver of inter-individual differences in microbiota composition in our cohort. Among the children who received macrolides in early life, we find a positive correlation between overall lifetime antibiotic use and body mass index (BMI), as well as an increased risk of asthma, suggesting that macrolide use may alter the microbiota in infants in a way that predisposes to antibiotic-associated weight gain and asthma in later childhood.Our cohort consists of 236 Finnish children aged 2–7 years (median age 5 years), attending day care at the time of the study. A total of 257 fecal samples were obtained from 142 children  who donaBifidobacterium as the dominant genus, was reduced in this group, in many cases to below detectability, and the abundances of the Gram-negative phyla Bacteroidetes and Proteobacteria were increased. These changes were largely resolved in the M12 and M24 groups, indicating that the phylum-level balance of the microbiota recovered within 1 year after a macrolide course. The penicillin-user groups  did not have a distinctly different phyla composition. The genus-level principal coordinates analysis (PCoA) indicated that the main driver determining the overall microbiota composition was macrolide use: most of the M6 samples clustered together with low scores on component 1, whereas the rest of the samples showed less clear patterns , indicating that the beta diversity among these samples was associated with recovery from the most recent macrolide course. Penicillin use was not strongly associated with the principal components. The same patterns were evident in both the 2–4- and 5–7-year-old children, indicating that the dominant effect of macrolide exposure on the microbiota was not age-dependent. Furthermore, the pattern was evident regardless of previous antibiotic exposure background. We thus conclude that macrolides appear to set the microbiota to a certain state, regardless of the baseline composition.Although the indications for penicillin and macrolide courses were the same—both were prescribed mainly for respiratory infections—macrolide use specifically was associated with a clear shift in the microbiota composition . The gropatterns . The timAntibiotic use, and macrolide use in particular, was associated with a long-term reduction in microbial richness, which did not reach the level of the control samples even 12–24 months after the course . The agez-score and health , 18 of the 48 genera and 7 of the 14 orders that were detected in >30% of the samples were significantly associated with recent antibiotic use, that is, were significantly different in the groups M6 or P6 as compared with the control group (Eggerthella) and three decreased 4–10-fold  in the group M6. Of these, Collinsella was significantly reduced in all groups compared with the control group, suggesting that this genus is strongly affected by antibiotics. Among the Bacteroidetes, Bacteroides was twofold elevated in the group M6 and Parabacteroides in the groups M6, M12, P6 and P24. Among the other Gram-negative bacteria, a twofold increase in Proteobacteria in the group M6 was notable, but apparently distributed among several genera with a member of Enterobacteriaceae as the most significantly affected . Among the Firmicutes, bacteria from the order Gemellales were significantly and consistently reduced in the penicillin-user groups. The order Lactobacillales was increased in all macrolide user groups. This was largely owing to an increase in Streptococcus, whereas Lactobacillus was decreased. Lactobacillus was also decreased in the groups P6 and P12. Erysipelotrichales as an order, and its member genus Eubacterium, were elevated in the group M6. An unassigned member of Clostridiales, a member related to Christensenella, and Anaerostipes were reduced in the group M6, whereas the genera Clostridium and Dorea were increased.After controlling for age, BMI ol group . Four ofP<0.001 in a generalized linear model). Hence, after controlling for recent antibiotic use, the cumulative lifetime use had a minimal association with the microbiota composition. Four genera, all in the order Clostridiales, were significantly positively associated with total lifetime macrolide use . Megamonas was also positively associated with lifetime penicillin use (P=0.003). Microbial richness was not associated with lifetime antibiotic use after controlling for the most recent antibiotic course.In addition to stratifying the children by the most recent antibiotic course, we investigated the possibility of cumulative effects of lifetime antibiotic exposure (number of antibiotic courses per year of life). Recent antibiotic use correlated with lifetime use: all antibiotic user groups had higher lifetime use than the control group, as the control group was selected partly based on low lifetime antibiotic use . There was a clear positive correlation between qPCR-measured abundance of bsh genes and time since the last macrolide course  compared with the non-exposed (N=116) was 6.11 . We also observed a strong correlation between total lifetime antibiotic use and the BMI z-score in the children with >2 macrolide courses before the age of 2 years  and overweight (N=9) children with those of matched healthy and normal-weight controls. The asthmatic cases were distinguished from the healthy controls by a set of three bacterial genera with significantly different abundances: Blautia, Rothia and Coprobacillus (Clostridium (Erysipelotrichaceae), Clostridium (Clostridiaceae), Akkermansia and Enterococcus . When antibiotics are used annually or more frequently, the microbiota may not have time to recover and the antibiotic-associated microbiota composition may persist.We observed clear differences in the microbiota composition in the antibiotic-treated children as compared with those who were not exposed to antibiotics for >2 years. Some aspects of the microbiota, such as the abundance of The children receiving antibiotics had infections, whereas those not receiving antibiotics did not have bacterial infections, requiring antibiotic treatment—although they may have had viral infections— and hence we considered the possibility that the microbiota changes could be attributed to the infections. However, both macrolide and penicillin antibiotics were prescribed mostly for respiratory infections, but were associated with clearly different, even opposite, changes in the microbiota. Furthermore, it has previously been shown that fever does not alter the microbiota composition of children8913In an earlier mouse study, it was shown that although the microbiota recover when antibiotic administration is ceased, the metabolic changes persistz-scores, specifically in a group of children that were exposed to macrolides in early life. The growth-promoting effect of antibiotics in animals224z-scores. Furthermore, it does not explain why priming specifically with macrolides predisposes to the antibiotic-associated weight gain. Rather, the weight gain is likely to occur via macrolide-associated changes in the microbiota, which promote excessive weight gain, as demonstrated experimentally with miceBifidobacteriumChristensenellaWe found a strong positive association between recorded antibiotic use and BMI Bile acid metabolism is one of the key functions performed by the intestinal bacteria, with strong effects on host energy metabolism29Lactobacillus and/or Bifidobacterium3738Bacteroides fragilisEggerthella spp. observed after macrolide exposure. Most Eggerthella spp. are pathogens9Previous birth-cohort studies and large international studies have found an association between asthma and early-life antibiotic useTo conclude, macrolide use was associated with microbiota characteristics that have previously been associated with the risk of immunological and metabolic diseases, as well as obesity. Furthermore, macrolide use promoted a marked increase in macrolide resistance of the microbiota. Our results confirm and extend previous results from mouse experiments10z-scores were calculated according to the LMS parameters obtained from the Centers of Disease Control and Prevention. Data on allergies were obtained from questionnaires filled out by the parents. Nearly all (>95%) of the children had received breastfeeding (>70% for at least 6 months), as this is highly promoted in Finland where mothers obtain leave of absence for up to 3 years with job guarantees. All children that developed asthma had received breastfeeding, and all but one received it for 6 months or more. The children received three daily meals at the day-care centre; the majority of their diet was thus largely similar. Furthermore, income inequality in Finland is low compared with other western countriesOur cohort consists of 236 Finnish children aged 2–7 years (median age 5 years), attending the same day-care centres at the time of the study. Register-based information and full background information was available for all children; 142 children donated fecal samples. The children are part of a larger cohort originally recruited for a probiotic trialIn Finland, antibiotics are only available by prescription. Information on antibiotic purchases was obtained from the records of the Finnish Social Insurance Institution, which subsidizes health-care costs. We collected data on all antibiotics purchased for the study children from the date of birth to the date of donation of the last fecal sample. Individuals with chronic illnesses are eligible for special reimbursement of their drug purchases, and the eligibility information is stored in the national database. We collected data on diagnosed asthma and allergic dermatitis (until the end of 2012), the most common developing chronic illnesses in the study cohort. The antibiotics were grouped into penicillins (amoxicillin with or without clavulanic acid and penicillin V), macrolides (azithromycin and clarithromycin), first-generation cephalosporins and sulphonamide–trimethoprim. The latter two groups of antibiotics were very seldom used, and therefore we focused on the penicillin and macrolide use. Azithromycin and clarithromycin became available in Finnish ambulatory care in 1994 and 1996, respectively, that is, before the birth of the study children. In our cohort, 50% of all antibiotic purchases were penicillins, 24% macrolides and the rest cephalosporins and sulfonamide–trimethoprim. These figures correspond to the population level data from FinlandBased on the parent-reported symptoms during the 7-month interval, we were able to assign potential indications for the use of antibiotics during the interval period. Respiratory infections were the most common apparent indication, occurring in association with 88% of the courses; other infections (mainly of the urinary tract) were associated with 5% and no concurrent reported symptoms with 7% of the courses. The antibiotic courses were not prescribed for gastrointestinal infections; pediatric gastrointestinal infections in Finland are nearly always viral and hence not treated with antibiotics. According to the physician in charge of the study, concurrent symptoms, indications for the antibiotics, symptom severity or disease history of the children receiving macrolides during the interval period did not differ from those receiving penicillins. Macrolides were typically prescribed due to presumed penicillin allergy or parents' request owing to easy administration or previous experience.z-score were assessed using Pearson correlations. Associations between antibiotic use during the first 2 years of life and asthma (N=15), and allergic dermatitis (N=5) were assessed using the Fisher's test.Associations between health and antibiotic use were analysed using the full cohort of 236 children. Associations between antibiotic use and BMI −1.The fecal samples were collected at home and transported immediately to the study centre for storage in −70 °C. DNA was extracted from the fecal samples using the Promega Wizard Genomic DNA Purification Kit as describedDe novo operational taxonomic unit (OTU) picking was done using Qiime version 1.8.0 . Colonies were counted on each plate after 2-day incubation at 37 °C, and the fraction of resistant colonies was calculated as the number of colony-forming units (c.f.u.'s) per 1 g of sample on the antibiotic plate divided by the c.f.u. on the control plate.Phenotypic antibiotic-resistant analysis was conducted by cultivating bacteria anaerobically from 80 fecal samples as mixed cultures. Bacteria were grown as dilution series on Brain-Heart Infusion agar (Oxoid) with or without 10 mg mlbsh, ermB and ermF were performed with primers specified in ermB and ermF genes have been previously publishedbsh primers to target this gene in the dominant phyla Firmicutes and Bacteroidetes. We focused on species that have confirmed bile-salt hydrolase activity and no penicillin V amidase activitybsh genes encoded by Bacteroides ovatus, Ruminococcus obeum and Eubacterium ventriosum based on sequence data obtained from GenBank using BLAST. The optimal primer concentrations and annealing temperatures were determined for each assay, and the specificity of the primer pairs was verified by melting curve analysis as well as agarose gel electrophoresis to confirm the correct product size. Reactions were run in triplicate on iCycler iQ Real-Time PCR Detection System  in a volume of 25 μl. Each reaction contained 1 × HOT FIREPol EvaGreen qPCR Mix Plus , 0.5 μM of each primer and 25 ng of fecal DNA. The amplification protocol involved one cycle at 95 °C for 5 min for initial denaturation followed by 40 cycles of denaturation at 95 °C for 20 s, primer annealing for 20 s, extension at 72 °C for 30 s and an additional incubation step at 80–82 °C for 30 s to collect the fluorescence data. The mean Ct value per sample  was used as a proxy for relative abundance of the target gene.The metagenomic analysis indicated that apart from an increase in macrolide resistance, macrolide use was associated with a decrease in bile-salt hydrolase genes. We therefore conducted qPCR analyses to quantify the abundances of these genes in a total of 130 DNA samples from 117 children. Quantification of bacterial genes The fecal samples were grouped based on antibiotic use . The conMicrobiota richness was calculated as the number of species-level OTUs (97% similarity cutoff) detected. Microbiota diversity was calculated as the inverse Simpson diversity index based on the species-level data. PCoAs were conducted using the genus-level data and Bray–Curtis dissimilarities. The associations of the first principal components with background variables such as antibiotic use were visualized by inverse distance-weighted interpolation of the variable values over the component space. Microbiota maturity index, which has previously been shown to differentiate between healthy children and those with health issues58P value testing and unreliable results. The detected abundance of rare genera is likely to be influenced considerably by chance and therefore less reliable than the abundance of common genera. The main focus of interest was to assess how the different groups (z-score of the child (which is likely to reflect lifestyle and diet at home), health status  and lifetime antibiotic use (when comparing antibiotic user groups) or antibiotic user group (when assessing the cumulative effect of lifetime antibiotic use). False disovery rate (FDR)-corrected P valuesP value <0.15) from the control group after controlling for all the above-mentioned variables, were considered potentially influenced by antibiotic use.Generalized linear models with negative binomial distribution (function glm.nb in the library MASS in R) were used to identify individual bacterial taxa that were associated with antibiotic use, either as being significantly different in the antibiotic user groups compared with the control group or significantly linearly associated with the total lifetime number of macrolide or penicillin courses (per years of life). We focused only on the 48 genera that we detected in >30% of the samples, in order to avoid unnecessary t groups  differ iN=8), and two age- and antibiotic use history-matched healthy controls for each (N=16). For analysis of the microbiota–overweight association, we selected the samples from all cases with BMI z-score >2 (N=9) and two (if available) age and antibiotic use history-matched normal-weight controls for each (N=16). For two overweight individuals, only one matching control case was available. For the asthma association analysis, we calculated the average microbiota composition between the first and second time point samples, if two samples were available. For the overweight analysis, we used only the first time point sample, as the weight and height were only recorded once at the beginning of the study. To find the bacterial groups that best distinguished asthma or overweight cases from healthy or normal-weight cases, we first fitted binomial models with asthma or overweight as the response variable, and each genus-level bacterial group separately as an explanatory variable. We used P values from these models as indicators of discriminating potential, and selected all bacterial groups with P<0.2 for further analysis. These groups were combined as explanatory variables in one model. AIC-based stepwise model selection was then used to find the set of bacterial groups that best discriminated between the asthma/overweight and healthy/normal-weight cases.To determine whether the asthmatic cases had distinct features in their microbiota composition, we selected the samples from all cases with current or developing asthma  under the study name PRJEB11685.How to cite this article: Korpela, K. et al. Intestinal microbiome is related to lifetime antibiotic use in Finnish pre-school children. Nat. Commun. 7:10410 doi: 10.1038/ncomms10410 (2016).Supplementary Figures 1-5, Supplementary Tables 1-4 and Supplementary References


Leishmania research is why most species cause cutaneous infections but others cause fatal visceral disease. Interestingly, L. donovani causes both visceral and cutaneous leishmaniasis in Sri Lanka. L. donovani clinical isolates were therefore obtained from cutaneous leishmaniasis (CL-SL) and visceral leishmaniasis (VL-SL) patients from Sri Lanka. The CL-SL isolate was severely attenuated compared to the VL-SL isolate for survival in visceral organs in BALB/c mice. Genomic and transcriptomic analysis argue that gene deletions or pseudogenes specific to CL-SL are not responsible for the difference in disease tropism and that single nucleotide polymorphisms (SNPs) and/or gene copy number variations play a major role in altered pathology. This is illustrated through the observations within showing that a decreased copy number of the A2 gene family and a mutation in the ras-like RagC GTPase enzyme in the mTOR pathway contribute to the attenuation of the CL-SL strain in visceral infection. Overall, this research provides a unique perspective on genetic differences associated with diverse pathologies caused by Leishmania infection.A central question in  Leishmania donovani clinical isolate from Sri Lanka and compared it to a typical visceral disease causing clinical isolate. Through whole genome sequencing, bioinformatics analysis, experimental infection in mice and functional genomic analysis, this study provides novel information on what differentiates a deadly visceral strain from a benign cutaneous strain. Results indicate that the ability of Leishmania parasites to cause visceral or cutaneous leishmaniasis may be determined by mutations or amplification of a few genes, or combinations of these factors. Overall, this work contributes to the understanding of parasite virulence and may help guide disease control efforts.Visceral leishmaniasis is one of the most lethal parasitic diseases, and the mechanisms that govern its survival in visceral organs are not understood. Here, we obtained an atypical cutaneous  Leishmania protozoan parasites transmitted by infected sand flies Leishmania species can infect humans; however only the Leishmania donovani complex including L. infantum cause the vast majority of visceral leishmaniasis cases worldwide Leishmaniasis is a neglected tropical disease present in 98 countries, with over 350 million people at risk of infection and is caused by L. donovani (strain MON-37) has been responsible for thousands of cutaneous leishmaniasis cases in the past decade L. donovani typically causes visceral leishmaniasis in Asia and Africa. Visceral leishmaniasis is rare in Sri Lanka, with the first recorded case only in 2007 L. donovani MON-37 L. donovani MON-37 are responsible for visceral and cutaneous leishmaniasis in Sri Lanka.In Sri Lanka, an atypical Leishmania research is why some species remain at the site of the sandfly bite and cause cutaneous infections and others metastasize to the internal organs where they cause visceral disease. By comparing genomes of Leishmania species which cause different pathology, we previously identified several L. donovani genes including A2 and Ldbpk_280340 which are required for visceral organ tropism Leishmania species is insufficient to fully define determinants of disease tropism and pathology because of their evolutionary distance, introducing genetic changes unrelated to human pathology Leishmania isolates of the same species that cause different human pathologies. We therefore undertook a phenotypic and genotypic analysis of L. donovani clinical isolates derived from cutaneous and visceral leishmaniasis patients in Sri Lanka. Characterization of these L. donovani clinical isolates provides invaluable insight into the etiology of visceral and cutaneous leishmaniasis in Sri Lanka and provides unique insight into the genetic basis of visceral leishmaniasis.Among the most important questions in L. donovani isolate (CL-SL) from Sri Lanka was derived from a skin lesion as detailed in methods and the visceral leishmaniasis L. donovani strain (VL-SL) has recently been reported L. donovani MON-37  isolates were obtained directly from cutaneous or visceral sites of human infection; 2) isolates could be cultured and identified by isoenzyme gene sequencing; 3) isolates induced similar pathologies in experimental animals as in humans; and 4) the same strains could be re-isolated from the experimental infection site. Since the mouse infection phenotypes were similar to the human infections, this would argue that these isolates are representative of the cutaneous and visceral disease causing strains circulating in Sri Lanka.Overall, these results conform to Koch's postulates demonstrating that cutaneous and visceral leishmaniasis are caused by distinct but closely related strains of L. donovani isolates were phenotypically distinct and therefore provided strong justification for comparing their genomes. Whole genome sequencing was performed using the Illumina GAIIx next generation sequencer >200× coverage as detailed in supplementary methods  genome are summarized in L. donovani strain from Nepal. Furthermore, although the majority of variants are homozygous, about 20% of variants are heterozygous. As expected, SNPs accounts for the vast majority of variants which occur mostly in the intergenic regions, while about 20% of the SNPs are located in coding regions. Interestingly, there are over 70 pseudogenes resulting from frame shifts or stop codon variations which are in common to the Sri Lanka CL-SL and VL-SL isolates yet are functional in the reference L. donovani strain  non-synonymous and 92 (60 homo+32 hetero) synonymous changes specific to the CL-SL isolate , S1. AltL. donovani strain and if the SNP appeared to be in a functionally important part of the gene as described above. To determine the potential impact of CL-SL specific SNPs on visceral infection and to complement potential functional defects, we cloned 7 corresponding genes from the VL-SL isolate described above, transfected them into the CL-SL isolate and measured the survival of the transgenic parasites in the spleen 4 weeks after infection. All of the CL-SL specific SNPs were verified by Sanger sequencing prior to transfection and their expression in the transfected CL-SL isolate verified by RT-PCR  in the CL-SL isolate has a homozygous non-conservative amino acid substitution (R231C). As shown in TOR genes (TOR 1–3) identified in the Leishmania genome and TOR 1–2 are essential whereas TOR3 is not essential for survival in the promastigote stage but is required for survival in macrophages and infected mice The Leishmania species L. donovaniChromosome somy can vary in different We next examined whether the differences in chromosome somy was reflected in differences at the transcript level. cDNA libraries were prepared using mRNA derived from axenic promastigotes (Pro), axenic amastigotes (Ax) and macrophage-derived amastigotes (Am) and entire chromosome transcript levels were compared between these two isolates . SeveralWhen comparing the individual gene transcript levels in different life cycle stages, the vast majority of genes were expressed at similar levels. A list of the most differentially expressed genes with at least a 4 fold difference in mRNA levels in the amastigote infected macrophage stage is shown in One of the genes with an increased copy number  and highL. donovani genomic library and previously used to characterize the role of the A2 locus in visceralization Because of the difference in A2 proteins between the VL-SL and CL-SL isolates, we investigated whether this was functionally relevant for the different pathologies displayed by these strains. A2 expression was therefore experimentally increased in the CL-SL strain by transfection with the A2 gene-containing pKSneo plasmid expression vector. This construct was generated from a We further confirmed the importance of A2 in these clinical isolates by downmodulation of A2 expression in the VL-SL strain and this was associated with impaired survival in the visceral organs . OverallL. donovani strains are responsible for cutaneous and visceral leishmaniasis. We provide compelling evidence here including fulfilment of Koch's postulates that different strains of L. donovani MON-37 are responsible for visceral and cutaneous disease in Sri Lanka. Most strikingly, the CL-SL clinical isolate was severely attenuated for survival in visceral organs in experimentally infected BALB/c mice, yet acquired the ability to cause cutaneous leishmaniasis in humans. Atypical cutaneous leishmaniasis in Sri Lanka caused by L. donovani is therefore most likely due to parasite-specific rather than host-specific determinants.A major question concerning leishmaniasis in Sri Lanka is whether one or different L. donovani VL-SL and CL-SL clinical isolates examined here represent unique strains to investigate genetic determinants affecting disease tropism. There were no homozygous gene deletions and only one pseudogene  specific to the CL-SL strain that were wildtype in the VL-SL strain. SNPs and protein level variations are most likely responsible for disease tropism and pathology differences, and the Rag C and A2 genes identified within are examples of genes which may contribute to the different pathologies caused by the CL-SL and VL-SL strains. It will be of considerable interest to characterize additional CL-SL isolates and determine how widespread changes in these and other genes are on a larger population of parasites from cutaneous leishmaniasis patients in Sri Lanka.The L. donovani and L. infantum but found as pseudogenes in Old World cutaneous leishmaniasis species such as L. major and L. tropicaL. mexicanaL. donovani, A2 is required for survival in visceral organs and protects L. donovani against high temperature (fever) and oxidative stress L. donovani CL-SL strain has lost the threshold level of A2 expression necessary for survival in visceral organs and that this represents a major determinant of its attenuation.Genome sequencing , Fig. 4ARag C gene which in higher eukaryotic cells is involved in the regulation of the mTOR pathway. There are 3 TOR genes in Leishmania which are all essential for Leishmania survival in macrophages or infected mice TOR genes, this pathway could be compromised by polymorphism in the Rag C gene. Expression of the VL-SL Rag C gene in the CL-SL isolate significantly increased the ability of the CL-SL isolate to survive in visceral organs . Written informed consent was obtained prior to parasite isolation from the two adult patients.Isolation of All BALB/c mouse infections (permit # 7395) were approved by the McGill University Animal Care Committee following guidelines from the Canadian Council on Animal Care (CCAC).L. donovani parasites were isolated from two Sri Lankan patients from the Vavuniya district as previously shown geographically Leishmania antibodies as previously described Leishmania promastigote culture medium Leishmania promastigotes were shifted to 37°C, pH 5.5 culture media to mimic the macrophage phagolysosome environment. Transfections were performed as previously described with the pKSneo-control plasmid, the pKSneo-A2 plasmid or the pKSneo-A2 (R) antisense plasmid Leishmania parasite number determined by limiting dilution. The number of stationary promastigotes injected is indicated for each experiment in the Figure legends. Liver parasite burdens were also measured by counting the number of amastigotes in Giemsa-stained liver imprints, expressed as Leishman-Donovan Units (LDU): number of amastigotes per 1000 cell nuclei×liver weight Female BALB/c mice weighing 17–20 g were purchased from Charles River Breeding Laboratories and maintained in the animal care facility under pathogen-free conditions. BALB/c mice were infected by tail vein injection of stationary-phase promastigotes. Four weeks post infection, amastigotes were isolated from the liver and spleen, and the L. donovani BPK282/Ocl4, cloned line from Nepal). Somy and copy number information for each chromosome were calculated independently using custom written perl script entitled “find_copy_number.pl”  and was made into a paired-end DNA sequencing (DNA-seq) library using the Genomic DNA Sample Preparation Kit . Libraries were sequenced using the Genome Analyzer IIx (Illumina) at the High Throughput Genomics Unit at the University of Washington. Reads were aligned against the reference genome  reactions were performed with QIAGEN OneStep RT-PCR Kit. One primer was specific for the transfected VL-SL gene and other primer was specific to the expression vector sequence contained in the 3′ untranslated region All information was deposited in GenBank under bioproject ID PRJNA210295. DNA sequencing data can be accessed from the SRA database using accession no. SRS484822 and SRS484824. RNA-seq data has been deposited in the GEO database using accession no SRP026479. Open source software tools we used are referenced from methods section. The locally developed scripts are publicly available to download from github project ‘bifxscripts’ under GPLicence  zymodeme (MON-2) sequence is given on top. The previously reported (5) L. donovani Sri Lanka (Ld-SrLan) zymodeme (MON-37) is shown below. CL-SL (CL) and VL-SL (VL) are the Sri Lanka L. donovani isolates. L. tro, L. tropica; L. maj, L. major; L. mex, L. mexicana; and L. bra, L. braziliensis are shown for comparison to other Leishmania species. Note: the single amino acid difference at amino acid 326 in red distinguishes the Indian strain  from the Sri Lanka strain  and the CL and VL isolates are identical with D at amino acid 326.Partial amino acid sequence alignments of the 6-phosphogluconate dehydrogenase (6PGDH) isoenzyme from different (DOCX)Click here for additional data file.Figure S26 stationary phase promastigotes and sacrificed at five weeks post-infection (at the peak of footpad swelling). Footpad parasite burden was determined by limiting dilution of footpad homogenates. Average values plus standard error for two independent experiments with at least 4 mice/group are shown.Footpad parasite burden is comparable for CL-SL (CL) and VL-SL (VL). Mice were infected subcutaneously in the hind footpad with 5×10(DOCX)Click here for additional data file.Figure S3Panel A. Western blot showing down-regulation of A2 in VL by antisense RNA. The VL isolate was transfected with the pKSneo vector encoding antisense A2 RNA that down-regulates A2 protein expression (VL - A2) or the control empty pKSneo vector (VL). A2 proteins (upper panels) and tubulin loading control (lower panels) were detected by Western blot after 4 h at 40°C to induce A2 expression. Panel B. Virulence of VL-SL with reduced A2 expression (VL - A2) was assessed four weeks following intravenous injection in the tail vein of BALB/c mice (5×107 stationary phase promastigotes) and spleen parasite burden was determined by limiting dilution of spleen homogenates. Values plus standard error are displayed.Down-regulation of A2 in the VL-SL (VL) isolate results in loss of virulence. (DOCX)Click here for additional data file.Table S1Non-synonymous changes identified in the CL-SL isolate.(DOCX)Click here for additional data file.Table S2Genes with transcript levels more than 2 fold Up-regulated in VL-SL.(DOCX)Click here for additional data file.Table S3Genes with transcript levels more than 2 fold Up-regulated in CL-SL.(DOCX)Click here for additional data file.Table S4List of primers used for RT-PCR.(DOCX)Click here for additional data file.Test S1Supporting methods.(DOCX)Click here for additional data file.