Le sujet de thèse « Hormones and female aggressiveness in Eupelmus vuilleti (Hymenoptera: Eupelmidae) » (2016-2019), dont le financement est assuré est proposé à l’IRBI (UMR CNRS 7261 – Université François-Rabelais de Tours) sous la direction de Marlène Goubault
et en co-tutelle avec Ryan Earley (University of Alabama, USA).

Le projet et les conditions de travail à Tours et à Tuscaloosa (Alabama) sont détaillées ci-dessous..

Nous cherchons un(e) étudiant(e) ayant de solides connaissances en écologie comportementale, avec si possible des compétences en analyses d’hormones et/ou manipulation d’insectes.

Pour candidater, merci de m’adresser pour le 25 mai au plus tard:
– Un CV (en anglais)
– une lettre de motivation (en anglais)
– les relevés de notes de licence et master.

Marlène Goubault
Tel: +33 (0)2 47 36 70 87
Email: [email protected]

Hormones and female aggressiveness in Eupelmus vuilleti

Directeurs de thèse (co-tutelle): Dr Marlène Goubault (Université François-Rabelais de Tours) & Dr Ryan Earley (University of Alabama, USA, http://rlearley.people.ua.edu)

GENERAL CONTEXT: Hormones are chemical messengers that translate information about the biotic or abiotic environments into adaptive changes in behavior and physiology (Adkins-Regan 2005, Neat 2008). For instance, in vertebrate males, fighting often alters circulating concentrations of androgenic hormones such as testosterone, which in turn moderate social behaviors such as aggression. It is well established that androgens have diverse effects on individuals’ behavior and physiology, with high levels being beneficial in some contexts but costly in others. High titers of testosterone generally increase male reproductive success by enhancing their competitive and sexual behaviors, while reducing their survival and immune function (Adkins-Regan 2005). Due to the cost associated with high androgen levels, individuals should maintain relatively low androgen titres most of the time and upregulate them only when beneficial (e.g. during the breeding season, in the presence of a competitor). The ‘Challenge hypothesis’ offers a framework for understanding when such variation should be observed (Wingfield et al. 1990). It proposes that during challenges, such as an intrusion within a reproductive territory, levels of testosterone should increase, enabling the owner to successfully meet the challenge and win the contest.
Initially developed for birds, then extended to vertebrates, the ‘challenge hypothesis’ is currently being considered by entomologists (Scott 2006, Trumbo 2007, Tibbetts & Huang 2010, Tibbetts & Crocker 2014). Insects do not have testosterone. Instead, juvenile hormone (JH) affects insect physiology and behavior like androgens do in vertebrates. JH mediates fecundity-survival trade-offs in many insects (Flatt et al. 2005): it generally correlates positively with fertility and negatively with immune responses and life expectancy (see Tibbetts & Crocker 2014). High levels of JH are also often associated with high dominance and aggression (Scott 2006, Kou et al. 2008, 2009, Tibbetts & Huang 2010, Tibbetts & Crocker 2014). Despite the major differences in structure and evolutionary origin of hormones in vertebrates and insects, considering the challenge hypothesis in such diverse taxa offers the opportunity to identify convergent evolution of hormone function. It will permit us to disentangle effects resulting from evolutionary adaptations from those resulting from hormone-specific constraints (Tibbetts & Crocker 2014).
To date, rapid endocrine responses to social challenges, like intrusion, have been poorly investigated in insects, and the rare studies have focused on JH (Scott 2006, Kou et al. 2008). However, other hormones, like the ecdysteroids, can also be involved because they are known to mediate insect growth and reproduction and could potentially modulate responses to social and non-social environments (see Raikhel et al 2005 for a review). In Eupelmus vuilleti, the 20-hydroxy-ecdysone (20-HE) is a good candidate mediator of social responses. In this species, females aggressively defend their host, on which they lay their eggs, against competitive intruders (Fig.1; Mohamad et al. 2010, 2011, 2012) and they show a winner effect but no loser effect (Goubault & Decuignière 2012). Additionally, their level of 20-HE increases almost immediately after they detect a host (Casas et al. 2009). Higher ovarian concentrations of ecdysone is further associated with the maturation of new eggs (Casas et al. 2009). As winning a contest means exploiting a host, the maturation of new eggs by winners could enhance the value that they place in the host (subjective Resource Value), making them more willing to fight (Mohamad et al. 2010). In contrast, losers’ estimation of the host would remain unchanged, thereby favoring the evolution of winner effects but not of loser effects. Although the winner effect observed shortly after the first fight could simply be a by-product of host exploitation, the fact that it persists for at least 4 hours suggests that other mechanisms are involved (Goubault & Decuignière 2012). We suspect a stronger or longer-lasting upregulation of hormone titers in winners, due to the encounter with a competitor, which would then mediate their fighting behaviors in a following contest.
The main aim of this PhD project is therefore to investigate the role of the two hormones, JH and 20-HE, on female aggressiveness and contest outcome in E. vuilleti. This work will involve a series of correlative and manipulative experiments that explore feedbacks between hormone levels and fighting behaviors in this species.

1- Development of methods for hormone dosage and micro-injection: Benefiting from Ryan Earley’s experience of dosing vertebrate steroids, 20-HE and JH will be extracted from E. vuilleti individuals then their titers will be measured using a Liquid Chromatography-Mass Spectrometer (LC-MS). This technique has already been successfully used to simultaneously measure both hormones in insects (Westerlund, PhD dissertation 2004). In the proposed project, each sample will have to be calibrated so that variation between samples can be accurately measured, as routinely done for vertebrate hormone measurements in the Earley lab.

To determine the impact of hormone levels on fighting behaviors, injection of hormones will be realized. In order to mimic realistic increase in hormone titers, JH and 20-HE (which can be commercially bought) will be injected in small amount into the haemolymph of the wasps. Such microinjections will be adapted from previous those successfully developed by Giron et al. 2002, when manipulating E. vuilleti circulating concentration of sugar to test its impact on female survival.
Estimated duration: 4-5 months. Feasibility and collaboration: Hormone quantification will be done in the Earley lab. The student will benefit from Ryan Earley’s long expertise in measuring hormones in vertebrates, using different techniques including LC-MS. In France, the PhD student will also benefit from the collaboration already established by Marlène Goubault with Charlotte Lécureuil (IRBI, Tours) and Nathalie Mondy (Univ. Lyon), both experts in reproductive physiology (incl. endocrinology) of insects, as well as David Siaussat (Univ. Paris 6) studying the cellular action of JH and 20-HE in insects. The student will also benefit from the long-standing expertise of David Giron (IRBI, Tours) on manipulating and measuring circulating nutrients in E. vuilleti and Jerôme Casas (IRBI, Tours) on studying the role of 20-HE on egg maturation in E. vuilleti.

2- Correlative study: is female aggressiveness and probability of winning correlated to JH and ecdysteroid titers? Is the winner effect associated with hormones titers?

To date, social dominance and aggressiveness in insects have been associated with high levels of JH (see Tibbetts & Crocker 2014 for a review). Nevertheless, 20-HE is also known to affect insect development and reproduction and could therefore also be associated with agonistic behaviors. In E. vuilleti, because 20-HE is known to affect egg maturation (Casas et al. 2009) and egg load, which in turn influences female aggressiveness and success rate during contests (Mohamad et al. 2010), this hormone is also likely to be associated with aggressive behaviors and probability of winning. To measure this likely correlation between hormones and contest behaviors, a series of detailed behavioral observations of contest between two females will be carried out and followed by quantification of both hormones. To test whether the winner effect, in particular, is associated with elevated titers of 20-HE as suggested by Goubault & Decuignière (2012), hormones levels will be measured in winners and losers obtained as described in this study.
Estimated duration: 1yr. Feasibility: Behavioral studies will be carried out at the IRBI where the PhD student will benefit from the long experience of Marlène Goubault on studying behavioral contest in parasitoid wasps, especially in E. vuilleti. Then the quantification of hormone concentrations will be done in the Earley lab, as previously mentioned.

3- Manipulative study: do JH or 20-HE levels influence aggressive behaviors and female probability to win contests?

Step 2 (correlative approach) will give essential information about the natural ranges and peaks of the concentration of hormones circulating into E. vuilleti wasps when contesting for hosts. However, due to feedbacks between hormones and behaviors, the exact impact of hormones on agonistic behavior can only be determined by manipulating the titers of circulating hormones in the wasps. To do so, microinjections of known and realistic doses of JH and/or 20-HE (combined with the use of antagonists) in females’ hemolymph, followed by behavioral assays, will allow us to assess the amplitude of hormone action on female aggressiveness. The differential manipulation of both hormones will permit to disentangle the relative role of each hormone on agonistic behaviors.
Estimated duration: 1.5yr. Feasibility: Micro-injection and behavioral studies will be carried out at the IRBI and subsequent dosage at the Earley’s lab.

PROJECT ORIGINALITY: the originality of this project will be to finely study the relative role of the two main insect hormones, JH and 20-HE, on female aggressiveness and probability of winning a contest in E. vuilleti, by combining two different methodological approaches involving correlative and manipulative studies. To date, compared to the large and still growing body of literature on the role of hormones on aggressive behaviors in vertebrates, very little is known in insects, and the rare studies have focused on JH. This study will therefore be the first to consider simultaneously both hormones in the context of animal contest in insects. It will thus represent a milestone in the understanding of the relationship between both hormones and aggressive behaviors in insects. The comparison of the results resulting from this study on insects with those obtained on vertebrates will also provide new insight on the possible convergent evolution of the function of insect and vertebrate hormones, despite their major differences in structure and evolutionary origin. This study will thus participate in disentangling effects resulting from evolutionary adaptions from those resulting from hormone-specific constraints.

STUDENT INTEGRATION IN BOTH LABS: The PhD student will benefit from the different and complementary expertise of both advisors, M Goubault and R Earley, as well as the skills, knowledge and techniques available in their respective labs. Marlène Goubault is a specialist of behavioral ecology in insects, especially in the context of animal contest. At the IRBI, the student will find all the required support for insect rearing, behavioral studies and microinjections. As mentioned above, he/she will benefit from the long history of studies on E.
vuilleti carried at the IRBI by many researchers on different topics: nutrition physiology (David Giron, Jérome Casas), reproductive physiology (Jerome Casas, Christophe Bressac, Claude Chevrier). He/she will also have the opportunity to discuss with numerous researchers studying different aspect of insect biology, such as the neuro-ethology of insect (Claudio Lazrri) and their reproductive physiology (Charlotte Lécureuil). In Ryan Earley’s laboratory in the US, the student will have the opportunity to learn various endocrine techniques including hormone extraction and purification, sample preparation, and methods for assaying hormone concentrations (e.g., LC-MS, enzymeimmunoassay). The student will gain an understanding of the mechanisms underlying aggression and contest behavior by examining relationships between JH and 20-HE and behavioral output. The Earley lab specializes in investigations of this sort (e.g., Garcia et al. 2015; Kolluru et al. 2015; Li et al. 2014; Earley et al. 2013), and the student will interact with a group of over 70 students who conduct research related to the endocrine basis of behavior.
FINANCIAL AND SCIENTIFIC RESOURCES: The financial resources of the IRBI team will allow the student to carry out his/her research activity in France as well as attending national and international conferences where he/she will present his/her results. Quantification of hormone concentrations will be conducted in the Earley lab and will be funded by the US group. Additionally, Marlène Goubault and Ryan Earley will apply for a PICS grant (CNRS grant funding travels between collaborative groups from two different countries).
The PhD student will also have the opportunity to meet other international collaborators with whom the two advisors already work with: Ian Hardy (Univ. Nottingham, UK), expert in animal contest in insects and developing a metabolomic approach on this topic, Mark Briffa (Univ. Plymouth, UK) studying animal personalities and metabolic fighting costs in hermit crabs and Yuying Hsu (National Taiwan Normal University, Taiwan) specialist of the neuroendocrinology of the experience effect in fish contest. These different labs could potentially represent great opportunities for the student to continue his/her research career as a post-doctoral research fellow.
APPORT POUR L’ÉTUDIANT : this integrative project proposes to combine an evolutionary approach to animal behavior with endocrinology mechanisms. It will offer the student the opportunity to acquire theoretical and empirical expertise in behavioral ecology as well as state-of-the-art physiological techniques to quantify hormones titers in small animals. He/she will also benefit from working in two different countries: understanding both French and American research systems will an advantage for him/her when applying for grants in the future. By the end of his/her PhD, he/she will be fluent in both French & English, if not when starting the project.
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