Keywords DONEPlease state 4 mandatory keywordsEnvironmental perturbationTransposable elementGenetic variationAdaptive evolution Host Description DONE Please briefly describe how the research team (supervisor and/or co-supervisors) and theconditions offered by the host institution will ensure the full implementation of the workplan (300 words). The Instituto Gulbenkian de Cie?ncia (IGC) is a leading international institute in research andgraduate training in biological sciences. It received maximum score in the recent internationalpeer-review evaluation organized by FCT, being classifying as one of the top Research Units inPortugal.

The reasons why IGC is well suited for hosting my PhD project include: 1) logisticsand services, including Drosophila rearing, and support in genomics and bioinformaticsanalysis, 2) strong scientific community, with ample opportunities for interactions betweenlocal, visiting scientist and graduate students, and 3) excellent research groups, including in theareas of evolutionary biology and Drosophila genetics. Patri?cia Beldade, the project’s principal investigator, has proven expertise in the geneticdissection of inter-individual variation, including recent work on transposable elementdynamics in D. melanogaster. Her lab is fully equipped for all proposed manipulations andanalyses, and her track-record provides a solid framework for my project, both in terms ofavailable preliminary data and expertise, and also in terms of supervision in all relevantconcepts and approaches. She has a strong track record of publication, supervision andfunding, and is well integrated in the international community of evolutionary biologists. The project also involves researchers at the University of Toulouse, nicely complementing theexpertise of the host lab in different aspects: 1) focus on organisms’ interaction with(changing) environments, 2) recent work on TEs as source of novel adaptive variants, and 3)experience with experimental work on interaction between Drosophila and its parasites,notably, the project’s co-supervisor David Duneau, who is already a collaborator of the IGC group. The opportunity to learn from him and to interact with the Toulouse community,through various visits and extended stays, will be invaluable not only for the project, but alsofor the student’s experience and growth. Both host labs have funding.

299 words Abstract (max: 150 words): DONE Heritable phenotypic variation is an universal property of biological systems and the rawmaterial for evolution by natural selection. The impact of the environment on inter-individualvariation includes effects on mutation. It is well established that some environmental factorscan act as mutagens and that, even though most mutations are deleterious, high mutationrates might help populations cope with environmental challenge. My project will analyze theeffect of environmental perturbation on an important driver of novel genetic variation:mobilization of transposable elements (TEs). TEs are repetitive DNA sequences capable ofchanging position and replicating independently in host genomes.

Their mobilization in thegermline leads to the production of novel genetic variants, and has been shown to be affectedby environmental factors. This project will use Drosophila melanogaster to scrutinize theprevalence, mechanism, and evolutionary significance of the regulation of TE dynamics bybiotic and abiotic perturbations. 146 words Stat-of-the-art. DONEOriginality and innovative nature of the project, including the relevance of the expected results in the broad scientific area of the project (max: 500 words) PHENOTYPIC VARIATION AND THE ENVIRONMENTPhenotypic variation is a universal property of biological systems, including disease traits, andheritable phenotypic variation fuels evolutionary change 1. The environment contributes toshaping phenotypic variation by affecting mutation that produces novel genetic variants,development that translate genotype into phenotype 2 and selection that eliminatesunsuitable phenotypes. This project focuses on environmental effects on the generation ofgenetic variation. It is well established that environmental stressors can act as mutagens and that highermutation rates might help cope with environmental challenge 3. Increased genetic variationin the progeny increases chances of having variants capable of thriving under new conditions.

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This can explain the advantage of mutator alleles in experimental populations evolving underunstable conditions 4 and the recent finding that exposure to parasites/parasitoids increasesrates of recombination in gamete production 5. This project will investigate the effect ofenvironmental perturbation on another source of genetic variation, transposable elements(TEs) 6. TRANSPOSABLE ELEMENTS AND GENETIC VARIATIONTEs are common DNA sequences capable of changing position and replicating independentlywithin genomes, in a mechanism called transposition. Transposition causes mutations and canadd genetic variation when occurring in the germline. TEs have been implicated in adaptationin experimental 7 and natural populations 8, and in phenotypic diversification 9.

Theyhave also been shown to play important roles in the evolution of gene regulation 10 and theorigin of novelty 11-12. Despite these examples, TE mobilization is generally harmful to hostgenomes (e.g.

when insertions disrupt protein-coding genes 13), and organisms have evolvedmechanisms to repress TEs, such as the germline-specific piRNA pathway 14. Despite their prevalence in genomes 15 and impact on fitness, little is known about thefactors controlling TE mobilization. Several studies have shown that environmental factorsincluding temperature, salinity and UV exposure can affect transposition 16-18. However,published data is controversial and there is no systematic and broad analysis of this effectincluding multiple TEs, genetic backgrounds and environmental factors. This project fills thatgap to provide new insight into the prevalence, mechanism and evolutionary significance ofenvironmentally-induced TE mobilization.

PROJECT OVERVIEW & IMPACTI propose to study the effect of environmental perturbation, including biotic (symbionts andparasites) and abiotic (temperature) factors, on TE mobilization in the Drosophilamelanogaster germline. This follows on preliminary findings from my MSc project in the lab ofPatri?cia Beldade at the IGC, dedicated to studying the genetic and environmental basis ofvariation and diversity 19-20. The project will also involve researchers from Toulouseinterested in the ecological impact of environmental change and in TEs as source of noveladaptive variants 6.

In particular, I will work with David Duneau, an expert in host-parasiteinteractions using invertebrate hosts 21-22. Our work will impact the fields of evolutionary biology, ecology and genetics, as well as studiesof TE dynamics. The focus on environmental perturbation is especially timely, given the unprecedented rate of global environmental change and the need for the scientific communityto understand how species will cope with it. 500 words Objectives (max: 300 words) DONE The project’s MAIN OBJECTIVE is to characterize the effect of environmental perturbation onthe generation of genetic variation, through mobilization of TEs in the germline.

Morespecifically, we will investigate environmental effects on levels of TE expression (AIM 1) andtransposition (AIM 2), as well as the mechanisms (AIM 3) and adaptive significance (AIM 4) ofthose effects. We will focus on different types of perturbation (biotic and abiotic stressors) onthe germline of male and female Drosophila melanogaster of different genetic backgrounds.We planned specific experiments and have alternative paths to be considered as workprogresses. Towards meeting AIM1, we will quantify levels of TE expression in ovaries and testes of adultflies exposed to environmental perturbation (TASK 1). Towards meeting AIM2, we will quantifytransposition events of candidate TEs selected based on the results of TASK 1 in the progeny ofadults exposed to environmental perturbation (TASK 2). Together, the work planned for AIMs1 and 2 will test the hypothesis that environmental perturbation can contribute to thegeneration of increased genetic variation through TE mobilization, and it will enable us todissect the effects of host genetic backgrounds and sex to this.

For AIM 3, we will focus on the contribution of the piRNA pathway, the specific mechanism forTE repression in the germline, to the effects characterized in TASKS 1 and 2. We will quantifythe effect of environmental perturbation on piRNA pathway activity (TASK 3). Towards meeting AIM 4, we will investigate fitness of individuals born from parents that wereexposed to environmental perturbation in control versus perturbed environments (TASK 4).This will test the hypothesis that the progeny from parents exposed to perturbation, andhaving increased TE-related genetic variation, will have better chances of coping withenvironmental perturbation themselves. 295 words Detailed description (max: 1000 words) DONE MATERIAL: FLIES & ENVIRONMENTAL FACTORSTo investigate the effects of environmental perturbation on TE mobilization, we will useDrosophila melanogaster from a panel of 147 wild-derived fully sequenced genotypes forwhich there is information about TE composition (Fig.

1; 23). About half of these lines carryWolbachia and we can remove this symbiont using antibiotics 24. We will expose adult maleand female flies to different types of environmental perturbation agents common in naturalpopulations and which, we can hypothesize, affect TE dynamics. We will test two biotic factors transmitted through the germline: Wolbachia bacterialsymbiont and DCV viral parasite. Because transposons have many properties of viruses, andbecause Wolbachia is known to protect flies from viral infection 24, we hypothesize thatthese two biotic factors can affect TE mobilization.

We will also test one abiotic factor:temperature, which has been shown to affect TEs in different species 16, 25. PRELIMINARY STUDY & PROPOSED EXTENSIONDuring my MSc internship, I studied the effect of temperature change and Wolbachia onexpression levels of 9 TEs in ovaries of flies from 9 genetic backgrounds. This served fortraining in relevant techniques (fly handling, ovary dissections, cDNA preparation, quantitativereal-time PCR, and data analysis) and as proof-of-principle (we found effects of temperatureand Wolbachia on TEs; Fig.2). To identify general principles about the effects of environmental perturbation on thegeneration of novel genetic variants through TE mobilization, I propose to extend that analysisto include: 1) more TEs (up to 20, with different properties) and more genotypes (up to 20); 2)a wider range of perturbations: more than 3 temperatures (including low temperature stress Idid not investigate in my MSc), controlling for Wolbachia titers (assessment by qPCR; 24) ,and adding viral infection (the co-supervisor has experience and will assist in this); and 3)testing also the male germline. The work proposed will go beyond quantifying TE expression inthe germline (AIM1), a commonly used proxy for TE mobilization 26, to checking fortransposition in the next generation genomes (AIM2), as well as to exploring the mechanism(AIM3) and adaptive significance (AIM4) of environmentally-regulated transposition. We propose 4 tasks (Timeline; Fig.3) for which we present the specific hypothesis addressed,relevant background information, proposed approach and perspectives for follow-up orcontingency work.

TASK 1: TE EXPRESSION IN THE GERMLINEHYPOTHESIS: Environmental perturbation leads to increased germline expression of TEs in amanner that differs between genetic backgrounds, TEs and sexes.BACKGROUND: My MSc work already detected differences in the levels of TE expression inadult ovaries depending on which temperature they were kept in and on whether they hadWolbachia.APPROACH: Flies from different genetic backgrounds, with and without Wolbachia, will bereared in standard conditions and the adults exposed to different environments (up to 6different temperatures and up to 4 doses of virus). The ovaries or testes of exposed adults willbe dissected and their RNA extracted to prepare cDNA (8 biological replicates, each of pooledgonads from 8 same-sex individuals) to be used in qPCR reactions with primers specific fordifferent TEs.PERSPECTIVES: Because of my MSc work, I know this approach works and that the expressionlevels of some TEs differ between environmental conditions. We can also choose to test other types of environmental perturbation, including nutrition stress that affects germlinedevelopment 27 and exposure to chemicals that affect transcription 28.

TASK 2: TE INSERTIONS IN THE PROGENYHYPOTHESIS: Perturbation-induced increase in TE expression in adult’s gonads translates intoincreased TE insertions in the corresponding progeny.BACKGROUND: While TE expression is often used as a proxy of transposition 26, thepossibility of post-transcriptional silencing of TEs implicates that expression not always reflectstransposition. A preliminary study in our lab revealed that for 2 of 4 tested TEs increasedexpression in ovaries correlated with increased copy number in the progeny.APPROACH: We will collect eggs from TASK 1 adults and extract genomic DNA to investigateincrease in number of insertions of copy-paste TEs (retrotransposons) via qPCR.PERSPECTIVES: The qPCR-based approach to infer TE copy number has been successfully usedin the lab. This, however, can only detect transposition for retrotransposons.

Detection oftransposition for cut-paste TEs requires other approaches, which we will consider if any suchelement proves especially interesting based on TASK 1. Another interesting follow-up is toinvestigate whether there are preferential insertion locations for different TEs and/or inrelation to different perturbations. TASK 3: ENVIRONMENTAL EFFECTS ON piRNA PATHWAYHYPOTHESIS: Increased TE expression is due to environmental effects on the piRNA pathway.BACKGROUND: piRNAs are a class of small RNAs that interact with the proteins Piwi,Aubergine, and Argonaute3 to silence TEs in the germline 14.APPROACH: To test if instances of increased TE expression (TASK 1) and transposition (TASK 2)are due to disturbed piRNA pathway activity, we will investigate expression of the main piRNApathway genes (qPCR) and of piRNAs themselves (RNA-seq) in gonads of individuals undercontrol and perturbation conditions.PERSPECTIVES: We will also consider testing effects on post-transcriptional modificationsnecessary for piRNA function (e.

g. phosphorylation of Piwi 29) and on actual function of thepiRNA genes (using functional analysis tools available in Drosophila). TASK 4: ADAPTIVE VALUE ENVIRONMENTALLY-INDUCED TRANSPOSITIONHYPOTHESIS: Increased TE mobilization in adults under environmental perturbation can beadvantageous if their progeny is exposed to perturbation.

BACKGROUND: This hypothesis is based on the “state-of-the-art” arguments accounting forgenetic mechanisms that lead to the production of genetically variable progeny underperturbed conditions 3-5.APPROACH: We will focus on cases where environmental perturbation resulted in increasedtransposition (TASKs 1-2) and, thus, increased genetic variation in the progeny. We will exposeF1 progeny from adults from different conditions to both control and perturbation conditionsand test their fitness by assessing number of F2 progeny and different life-history traits(development time, body size, longevity).PERSPECTIVES: If we find evidence of an adaptive genetic mechanism that increases variationunder stress, we can use experimental evolution 30 to investigate how that mechanismresponds to selection under different environments. 1000 words Cronograma DONE Include a chart showing the schedule of tasks and indicating the milestone dates. Togenerate the milestone chart you may use appropriate software tools for this purpose, oreven an Excel spreadsheet.

To generate a pdf file with name timeline.pdf to be uploaded inthesis webpage. Bibliographical references (max: 30) (30 references) DONE* : Team member on the project 1 Stern DL (2000). Evolutionary developmental biology and the problem of variation.Evolution 54: 1079-1091. *2 Beldade P, Mateus ARA and Keller RA (2011). Evolution and molecular mechanisms ofadaptive developmental plasticity.

Mol Ecol. 20: 1347-1363. 3 Ram Y and Hadany L (2012). The evolution of stress-induced hypermutation in asexualpopulations. Evolution 66: 2315-2328. 4 Giraud A, Matic I, Tenaillon O, Clara A, Radman M, Fons M and Taddei F (2001).

Costs andbenefits of high mutation rates: adaptive evolution of bacteria in the mouse gut. Science291:2606-2608. 5 Singh ND, Criscoe DR, Skolfield S, Kohl KP, Keebaugh ES and Schlenke TA (2015). Fruit fliesdiversify their offspring in response to parasite infection. Science 349: 747-750.

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Mutation Research 616: 46-59. 14 Aravin A, Hannon GJ and Brennecke J. (2007). The piwi-piRNA pathway provides anadaptive defense in the transposon arms race. Science 318: 761-764.

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