Keywords DONEPlease state 4 mandatory keywords
Host Description DONE
Please briefly describe how the research team (supervisor and/or co-supervisors) and the
conditions offered by the host institution will ensure the full implementation of the work
plan (300 words).
The Instituto Gulbenkian de Cie?ncia (IGC) is a leading international institute in research and
graduate training in biological sciences. It received maximum score in the recent international
peer-review evaluation organized by FCT, being classifying as one of the top Research Units in
Portugal. The reasons why IGC is well suited for hosting my PhD project include: 1) logistics
and services, including Drosophila rearing, and support in genomics and bioinformatics
analysis, 2) strong scientific community, with ample opportunities for interactions between
local, visiting scientist and graduate students, and 3) excellent research groups, including in the
areas of evolutionary biology and Drosophila genetics.
Patri?cia Beldade, the project’s principal investigator, has proven expertise in the genetic
dissection of inter-individual variation, including recent work on transposable element
dynamics in D. melanogaster. Her lab is fully equipped for all proposed manipulations and
analyses, and her track-record provides a solid framework for my project, both in terms of
available preliminary data and expertise, and also in terms of supervision in all relevant
concepts and approaches. She has a strong track record of publication, supervision and
funding, and is well integrated in the international community of evolutionary biologists.
The project also involves researchers at the University of Toulouse, nicely complementing the
expertise 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 also
for the student’s experience and growth.
Both host labs have funding.
Abstract (max: 150 words): DONE
Heritable phenotypic variation is an universal property of biological systems and the raw
material for evolution by natural selection. The impact of the environment on inter-individual
variation includes effects on mutation. It is well established that some environmental factors
can act as mutagens and that, even though most mutations are deleterious, high mutation
rates might help populations cope with environmental challenge. My project will analyze the
effect of environmental perturbation on an important driver of novel genetic variation:
mobilization of transposable elements (TEs). TEs are repetitive DNA sequences capable of
changing position and replicating independently in host genomes. Their mobilization in the
germline leads to the production of novel genetic variants, and has been shown to be affected
by environmental factors. This project will use Drosophila melanogaster to scrutinize the
prevalence, mechanism, and evolutionary significance of the regulation of TE dynamics by
biotic and abiotic perturbations.
Originality 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 ENVIRONMENT
Phenotypic variation is a universal property of biological systems, including disease traits, and
heritable phenotypic variation fuels evolutionary change 1. The environment contributes to
shaping phenotypic variation by affecting mutation that produces novel genetic variants,
development that translate genotype into phenotype 2 and selection that eliminates
unsuitable phenotypes. This project focuses on environmental effects on the generation of
It is well established that environmental stressors can act as mutagens and that higher
mutation rates might help cope with environmental challenge 3. Increased genetic variation
in the progeny increases chances of having variants capable of thriving under new conditions.
This can explain the advantage of mutator alleles in experimental populations evolving under
unstable conditions 4 and the recent finding that exposure to parasites/parasitoids increases
rates of recombination in gamete production 5. This project will investigate the effect of
environmental perturbation on another source of genetic variation, transposable elements
TRANSPOSABLE ELEMENTS AND GENETIC VARIATION
TEs are common DNA sequences capable of changing position and replicating independently
within genomes, in a mechanism called transposition. Transposition causes mutations and can
add genetic variation when occurring in the germline. TEs have been implicated in adaptation
in experimental 7 and natural populations 8, and in phenotypic diversification 9. They
have also been shown to play important roles in the evolution of gene regulation 10 and the
origin of novelty 11-12. Despite these examples, TE mobilization is generally harmful to host
genomes (e.g. when insertions disrupt protein-coding genes 13), and organisms have evolved
mechanisms 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 the
factors controlling TE mobilization. Several studies have shown that environmental factors
including 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 effect
including multiple TEs, genetic backgrounds and environmental factors. This project fills that
gap to provide new insight into the prevalence, mechanism and evolutionary significance of
environmentally-induced TE mobilization.
PROJECT OVERVIEW & IMPACT
I propose to study the effect of environmental perturbation, including biotic (symbionts and
parasites) and abiotic (temperature) factors, on TE mobilization in the Drosophila
melanogaster germline. This follows on preliminary findings from my MSc project in the lab of
Patri?cia Beldade at the IGC, dedicated to studying the genetic and environmental basis of
variation and diversity 19-20. The project will also involve researchers from Toulouse
interested in the ecological impact of environmental change and in TEs as source of novel
adaptive variants 6. In particular, I will work with David Duneau, an expert in host-parasite
interactions using invertebrate hosts 21-22.
Our work will impact the fields of evolutionary biology, ecology and genetics, as well as studies
of TE dynamics. The focus on environmental perturbation is especially timely, given the
unprecedented rate of global environmental change and the need for the scientific community
to understand how species will cope with it.
Objectives (max: 300 words) DONE
The project’s MAIN OBJECTIVE is to characterize the effect of environmental perturbation on
the generation of genetic variation, through mobilization of TEs in the germline. More
specifically, we will investigate environmental effects on levels of TE expression (AIM 1) and
transposition (AIM 2), as well as the mechanisms (AIM 3) and adaptive significance (AIM 4) of
those effects. We will focus on different types of perturbation (biotic and abiotic stressors) on
the germline of male and female Drosophila melanogaster of different genetic backgrounds.
We planned specific experiments and have alternative paths to be considered as work
Towards meeting AIM1, we will quantify levels of TE expression in ovaries and testes of adult
flies exposed to environmental perturbation (TASK 1). Towards meeting AIM2, we will quantify
transposition events of candidate TEs selected based on the results of TASK 1 in the progeny of
adults exposed to environmental perturbation (TASK 2). Together, the work planned for AIMs
1 and 2 will test the hypothesis that environmental perturbation can contribute to the
generation of increased genetic variation through TE mobilization, and it will enable us to
dissect 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 for
TE repression in the germline, to the effects characterized in TASKS 1 and 2. We will quantify
the effect of environmental perturbation on piRNA pathway activity (TASK 3).
Towards meeting AIM 4, we will investigate fitness of individuals born from parents that were
exposed to environmental perturbation in control versus perturbed environments (TASK 4).
This will test the hypothesis that the progeny from parents exposed to perturbation, and
having increased TE-related genetic variation, will have better chances of coping with
environmental perturbation themselves.
Detailed description (max: 1000 words) DONE
MATERIAL: FLIES & ENVIRONMENTAL FACTORS
To investigate the effects of environmental perturbation on TE mobilization, we will use
Drosophila melanogaster from a panel of 147 wild-derived fully sequenced genotypes for
which there is information about TE composition (Fig.1; 23). About half of these lines carry
Wolbachia and we can remove this symbiont using antibiotics 24. We will expose adult male
and female flies to different types of environmental perturbation agents common in natural
populations and which, we can hypothesize, affect TE dynamics.
We will test two biotic factors transmitted through the germline: Wolbachia bacterial
symbiont and DCV viral parasite. Because transposons have many properties of viruses, and
because Wolbachia is known to protect flies from viral infection 24, we hypothesize that
these 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 EXTENSION
During my MSc internship, I studied the effect of temperature change and Wolbachia on
expression levels of 9 TEs in ovaries of flies from 9 genetic backgrounds. This served for
training in relevant techniques (fly handling, ovary dissections, cDNA preparation, quantitative
real-time PCR, and data analysis) and as proof-of-principle (we found effects of temperature
and Wolbachia on TEs; Fig.2).
To identify general principles about the effects of environmental perturbation on the
generation of novel genetic variants through TE mobilization, I propose to extend that analysis
to 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 I
did 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 in
the germline (AIM1), a commonly used proxy for TE mobilization 26, to checking for
transposition 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 or
TASK 1: TE EXPRESSION IN THE GERMLINE
HYPOTHESIS: Environmental perturbation leads to increased germline expression of TEs in a
manner that differs between genetic backgrounds, TEs and sexes.
BACKGROUND: My MSc work already detected differences in the levels of TE expression in
adult ovaries depending on which temperature they were kept in and on whether they had
APPROACH: Flies from different genetic backgrounds, with and without Wolbachia, will be
reared in standard conditions and the adults exposed to different environments (up to 6
different temperatures and up to 4 doses of virus). The ovaries or testes of exposed adults will
be dissected and their RNA extracted to prepare cDNA (8 biological replicates, each of pooled
gonads from 8 same-sex individuals) to be used in qPCR reactions with primers specific for
PERSPECTIVES: Because of my MSc work, I know this approach works and that the expression
levels of some TEs differ between environmental conditions. We can also choose to test other
types of environmental perturbation, including nutrition stress that affects germline
development 27 and exposure to chemicals that affect transcription 28.
TASK 2: TE INSERTIONS IN THE PROGENY
HYPOTHESIS: Perturbation-induced increase in TE expression in adult’s gonads translates into
increased TE insertions in the corresponding progeny.
BACKGROUND: While TE expression is often used as a proxy of transposition 26, the
possibility of post-transcriptional silencing of TEs implicates that expression not always reflects
transposition. A preliminary study in our lab revealed that for 2 of 4 tested TEs increased
expression in ovaries correlated with increased copy number in the progeny.
APPROACH: We will collect eggs from TASK 1 adults and extract genomic DNA to investigate
increase in number of insertions of copy-paste TEs (retrotransposons) via qPCR.
PERSPECTIVES: The qPCR-based approach to infer TE copy number has been successfully used
in the lab. This, however, can only detect transposition for retrotransposons. Detection of
transposition for cut-paste TEs requires other approaches, which we will consider if any such
element proves especially interesting based on TASK 1. Another interesting follow-up is to
investigate whether there are preferential insertion locations for different TEs and/or in
relation to different perturbations.
TASK 3: ENVIRONMENTAL EFFECTS ON piRNA PATHWAY
HYPOTHESIS: 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 piRNA
pathway genes (qPCR) and of piRNAs themselves (RNA-seq) in gonads of individuals under
control and perturbation conditions.
PERSPECTIVES: We will also consider testing effects on post-transcriptional modifications
necessary for piRNA function (e.g. phosphorylation of Piwi 29) and on actual function of the
piRNA genes (using functional analysis tools available in Drosophila).
TASK 4: ADAPTIVE VALUE ENVIRONMENTALLY-INDUCED TRANSPOSITION
HYPOTHESIS: Increased TE mobilization in adults under environmental perturbation can be
advantageous if their progeny is exposed to perturbation.
BACKGROUND: This hypothesis is based on the “state-of-the-art” arguments accounting for
genetic mechanisms that lead to the production of genetically variable progeny under
perturbed conditions 3-5.
APPROACH: We will focus on cases where environmental perturbation resulted in increased
transposition (TASKs 1-2) and, thus, increased genetic variation in the progeny. We will expose
F1 progeny from adults from different conditions to both control and perturbation conditions
and 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 variation
under stress, we can use experimental evolution 30 to investigate how that mechanism
responds to selection under different environments.
Include a chart showing the schedule of tasks and indicating the milestone dates. To
generate the milestone chart you may use appropriate software tools for this purpose, or
even an Excel spreadsheet. To generate a pdf file with name timeline.pdf to be uploaded in
Bibliographical references (max: 30) (30 references) DONE
* : Team member on the project
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