Mating behaviours and reproductive decisions are of key importance to evolutionary fitness. However, it is often difficult to measure and compare the fitness of different individuals, especially in long-lived species, and for behaviours that are subject to multiple selective forces. A striking exception though is the field of sex-allocation research, which is a triumphant success of evolutionary theory (Charnov 1982). The reason for this success is that, following Fisher (1958), the fitness consequences of different sex ratios can be readily predicted and tested analytically. These predictions are very amenable to empirical testing and provide a platform to measure the precision of adaptation, which is crucial for understanding the nature of evolutionary change.

Reproductive success is heavily influenced by life-history traits; a series of energy investment trade-offs that organisms must optimise according to their environmental conditions. These include considerations such as how many offspring and when to reproduce? The consequences of multiple trade-offs can be extremely complex, making research difficult. However, there are notable exceptions. Simple clutch size theory enabled great strides in assessing trade-offs in resource allocation, though it quickly becomes more complicated when considering investment in current versus future reproduction. Arguably, even greater success has come from consideration of investment in a particular sex. Sex allocation theory provides simple models that can be empirically tested, and has provided some of the strongest evidence for natural selection and evolution. Much of this work has focused on certain parasitoids due to their extraordinary sex ratios and the finite resources available to offspring in a host. Whilst clutch size and sex allocation theory have provided many answers, there are still questions regarding the impact of other life-history traits. In this thesis I have used the gregarious parasitoid wasp Nasonia vitripennis in laboratory experiments to assess some of these traits. I have focused on the impact of larval competition, inbreeding, host condition and host feeding on longevity, fecundity, sex allocation and mating success. By manipulating host quality through host-feeding, I was able to vary the level of resources available to offspring. Simultaneously, by manipulating the matedstatus and number of females ovipositing on a host, I was able to vary the number and sex ratio of offspring competing for resources. My research has provided an insight into how larval competition and host-feeding impact on optimal clutch size and sex allocation. Furthermore, I have attempted to assess the extent to which body size, which is commonly associated with reproductive success, can be used to predict fitness. The appendix includes work using molecular data to understand the mating behaviour and population structure of N. vitripennis in the wild, enabling models based on assumptions of laboratory-based behaviour to be applied to wild populations.

Mating in insects influences suites of behavioural and physiological changes in females. These changes can include key female traits such as dispersal, foraging, oviposition and female remating or receptivity. Whilst much is known at the phenotypic level about post-mating changes in reproductive biology across many species, much less is known at the genetic level, especially outside of established model organisms such as Drosophila melanogaster. In the parasitic wasp Nasonia vitripennis courtship behaviour, rather than copulation, is believed to be primarily responsible for driving changes in female post-mating behaviour. Here we have studied female receptivity and post-mating gene expression changes associated with courtship and copulation in Nasonia vitripennis. Firstly we considered the influence of the duration of various elements of courtship and mating on female re-mating rates. We were able to identify an association between long pre-copulatory courtship durations and females which are less likely to re-mate (after 24 hours) and suggest that this may be driven by females which are generally less receptive. We also observed that males may be capable of determining female mating state, taking longer to engage in courtship with mated females than virgin females. To further explore the influence of mating on female post-mating behavioural and physiological processes, we explored changes in gene expression occurring in response to mating. To do this we utilised two different transcriptomic sequencing approaches developed for the Illumina next-generation sequencing platform. Using a tag-seq approach we considered the differential gene expression occurring in response to mating in head and body (comprising of the thorax and abdomen) tissues across two time-points (30 minutes and four hours). We were able to identify large changes in expression in head tissues across time-points in comparison to more subtle changes in body tissues. We suggest that head tissues may be more closely associated with post-mating changes in behaviour, whilst body tissues are perhaps physiologically more associated with egg production and influenced less by mating per se. Finally, using an RNA-seq approach, we considered the gene expression changes occurring in female body tissues in response to three elements of male courtship across two time-points (30 minutes and 24 hours). We hoped to narrow down the role of male courtship and/or insemination in post-mating gene expression differences, addressing first the more limited changed in body tissues. We showed that time-point was the most important factor associated with post-mating gene expression, with the courtship components tested being associated with very little expressional change. The data presented in this thesis suggests that male courtship may not be that important for driving the post-mating behavioural and genetic changes seen in Nasonia, perhaps limiting the scope for sexual conflict over reproduction in this species.

Hymenopteran insects, which include all ants, bees and wasps, reproduce through a poorly understood form of reproduction known as haplodiploidy. A promising experimental system for understanding this developmental process is the jewel wasp, Nasonia vitripennis. A critical aspect of using Nasonia as a model is establishing an effective means for editing specific genes of interest so that their functions can be studied through genetic means. For my thesis research, I performed a pilot study of the gene editing method known as CRISPR in Nasonia. I targeted the single heterochromatin protein 1 (HP1) gene present in the Nasonia genome in order to assess the feasibility of this gene editing approach. Targeting HP1 would provide a clear phenotype when this gene is mutated due to its essential functions in early development known from studies in other eukaryotes. Additionally, creating a mutant of this gene will provide a means for studying the role of HP1 in wasp spermatogenesis, an aim that interlinks with the broader chromatin-based goals of our laboratory. Through this study I worked out a streamlined procedure for injecting CRISPR molecules into young wasp embryos, conducting genetic crosses with injected wasps, and screening through their progeny for potential mutants. I observed no mutant phenotypes in injected wasps, but instead, I isolated four potential mutants in F1 progeny. My work has helped to create a solid framework for improving this procedure in Nasonia, and they allow for a better overall understanding of the limitations of producing mutants through CRISPR gene editing in non-model organisms such as Nasonia.

Genomes usually work together to promote the fitness of the organism, but sometimes parts of the genome cause intragenomic conflict, and act selfishly in order to promote their transmission. An example of this conflict is a selfish B chromosome known as paternal sex ratio (PSR) in the jewel wasp Nasonia vitripennis. Transmitted solely to new progeny with the sperms hereditary material, PSR completely destroys the paternal genome during the first mitotic division of the newly fertilized embryo. This effect enhances transmission of the PSR chromosome because of the unique haplodiploid reproductive mode of Nasonia and other members of the hymenopteran insect group. Through transcriptomic analyses, our group recently discovered that the PSR chromosome expresses eleven transcripts in the wasp testis. A plausible hypothesis is that one or more of these transcripts play some role in paternal genome elimination. In this study I have begun to test this hypothesis by screening through a set of previously produced truncated versions of the PSR chromosome. Specifically, I used PCR in order to screen these truncated chromosomes for the presence of each of these PSR-specific transcripts. I could then correlate the level of genome elimination induced by each truncated PSR chromosome with the presence or absence of the expressed transcripts. My work has established that (i) three of the eleven transcripts are likely not involved in genome elimination; (ii) no single transcript alone causes genome elimination; (iii) the remaining eight of eleven transcripts are viable candidates for causing genome elimination; and (iv) it is likely that a sub-group of these transcripts may operate together to induce this effect. I discuss several models in which PSR-expressed RNA molecules could operate to cause genome elimination.

La plupart des études sur l’influence de la température sur la production des spermatozoïdes portent en grande partie sur les vertébrés. Néanmoins, les invertébrés constituent la majorité de la biodiversité terrestre animale, sont également plus sensibles à ces variations de température. Les travaux réalisés au cours de cette thèse ont porté sur la compréhension des capacités de reproduction, et sur l’influence d’une augmentation de température sur les fonctions mâles chez Nasonia vitripennis, un hyménoptère parasitoïde. Ce travail de thèse m’a permis de confirmer que chez cette espèce, la spermatogenèse était synchronisée et de décrire pour la première fois, un système de stockage intermédiaire des spermatozoïdes entre les testicules et les vésicules séminales. De plus, j’ai pu mettre en évidence que la spermatogénèse était sensible à une augmentation de température pendant le développement, altérant le sexe ratio de la descendance par des modifications d’allocations des sexes par les femelles. Ces résultats apportent de nouvelles connaissances sur la fertilité mâle chez cette espèce et démontrent la sensibilité de la spermatogenèse à la température. / Many studies on the impact of the temperature on spermatogenesis have largely focused on vertebrates. Nevertheless, invertebrates constitute the vast majority of terrestrial biodiversity, and are especially vulnerable to variations of temperatures. The studies realized during my thesis are focused on understanding male reproductive capacities and effects of heat stress on male fertility in a parasitic wasp Nasonia vitripennis. This work allowed to confirm that spermatogenesis was synchronized, and to describe for the first time a system of spermatozoa regulation between testis and seminal vesicles. Moreover, I was able to demonstrate that spermatogenesis was sensible to temperature variation during male development, and the offspring sex ratio was altered by modifications of sex allocation. These findings open the way to the description of male fertility in this species, and show the sensitivity of spermatogenesis to temperature variation.

abstract: During the 1960s, the long-standing idea that traits or behaviors could be explained by natural selection acting on traits that persisted "for the good of the group" prompted a series of debates about group-level selection and the effectiveness with which natural selection could act at or across multiple levels of biological organization. For some this topic remains contentious, while others consider the debate settled, even while disagreeing about when and how resolution occurred, raising the question: "Why have these debates continued?" Here I explore the biology, history, and philosophy of the possibility of natural selection operating at levels of biological organization other than the organism by focusing on debates about group-level selection that have occurred since the 1960s. In particular, I use experimental, historical, and synthetic methods to review how the debates have changed, and whether different uses of the same words and concepts can lead to different interpretations of the same experimental data. I begin with the results of a group-selection experiment I conducted using the parasitoid wasp Nasonia, and discuss how the interpretation depends on how one conceives of and defines a "group." Then I review the history of the group selection controversy and argue that this history is best interpreted as multiple, interrelated debates rather than a single continuous debate. Furthermore, I show how the aspects of these debates that have changed the most are related to theoretical content and empirical data, while disputes related to methods remain largely unchanged. Synthesizing this material, I distinguish four different "approaches" to the study of multilevel selection based on the questions and methods used by researchers, and I use the results of the Nasonia experiment to discuss how each approach can lead to different interpretations of the same experimental data. I argue that this realization can help to explain why debates about group and multilevel selection have persisted for nearly sixty years. Finally, the conclusions of this dissertation apply beyond evolutionary biology by providing an illustration of how key concepts can change over time, and how failing to appreciate this fact can lead to ongoing controversy within a scientific field. / Dissertation/Thesis / Doctoral Dissertation Biology 2014

The gammaproteobacterium Arsenophonus nasoniae infects Nasonia vitripennis (Hymenoptera: Pteromalidae), a parasitic wasp that attacks filth flies. This bacterium kills virtually all male offspring of infected females. Female wasps transmit A. nasoniae both vertically (from mother to offspring) and horizontally (to unrelated Nasonia developing in the same fly). This latter mode may enable the bacterium to colonize novel species and spread throughout a filth fly-parasitoid guild. This spread may be important for maintenance of the bacterium. The ecology of novel hosts may be significantly impacted by infection. The actual and potential host range of A. nasoniae was assessed. I used Arsenophonus-specific primers to screen a large sample of filth flies and their parasitoids. The bacterium infects a wide range of wasp species in the environment. The potential host range was determined by inoculating three wasp and one fly species with an isolate of A. nasoniae from Lethbridge, AB. The bacterium successfully infected all insects and was transmitted by two wasp species. It reduced host longevity, but did not kill males, in Trichomalopsis sarcophagae. It also caused pupal mortality in Musca domestica.

L’intensification des pratiques agricoles a été identifiée comme cause majeure du déclin de la biodiversité. Plusieurs études ont documenté l’impact de la fragmentation du paysage naturel et de l’agriculture intensive sur la diversité des espèces, mais très peu ont quantifié le lien entre la structure du paysage et les interactions trophiques, ainsi que les mécanismes d’adaptation des organismes. J’ai étudié un modèle biologique à trois niveaux trophiques composé d’un oiseau hôte, l’hirondelle bicolore Tachycineta bicolor, de mouches ectoparasites du genre Protocalliphora et de guêpes parasitoïdes du genre Nasonia, au travers d’un gradient d’intensification agricole dans le sud du Québec. Le premier objectif était de déterminer l’abondance des espèces de mouches ectoparasites et de leurs guêpes parasitoïdes qui colonisent les nids d’hirondelles dans la zone d’étude. La prévalence de nids infectés par Protocalliphora spp. était de 70,8% en 2008 et 34,6% en 2009. Le pourcentage de nids comprenant des pupes de Protocalliphora parasitées par Nasonia spp. était de 85,3% en 2008 et 67,2% en 2009. Trois espèces de Protocalliphora ont été observées (P. sialia, P. bennetti et P. metallica) ainsi que deux espèces de Nasonia (N. vitripennis et N. giraulti). Il s’agit d’une première mention de P. bennetti et de N. giraulti dans la province de Québec. Mon deuxième objectif était d’évaluer l’impact de l’intensification agricole et de la structure du paysage sur les relations tri-trophiques entre les organismes à l’étude. Les résultats révèlent que les réponses à la structure du paysage de l’hirondelle, de l’ectoparasite et de l’hyperparasite dépendantent de l’échelle spatiale. L’échelle spatiale fonctionnelle à laquelle les espèces répondent le plus varie selon le paramètre du paysage modélisé. Les analyses démontrent que l’intensification des pratiques agricoles entraîne une diminution des populations d’oiseaux, d’ectoparasites et d’hyperparasites. De plus, les populations de Protocalliphora et de Nasonia sont menacées en paysage intensif puisque la dégradation du paysage associée à l’intensification des pratiques agricoles agit directement sur leurs populations et indirectement sur les populations de leurs hôtes. Mon troisième objectif était de caractériser les mécanismes comportementaux permettant aux guêpes de composer avec la variabilité de la structure du paysage et de la qualité des hôtes. Nos résultats révèlent que les femelles Nasonia ajustent la taille de leur ponte en fonction de la taille de la pupe hôte et de l’incidence d’hyperparasitisme. Le seul facteur ayant une influence déterminante sur le ratio sexuel est la proportion de paysage dédié à l’agriculture intensive. Aucune relation n’a été observée entre la structure du paysage et la taille des filles et des fils produits par les femelles Nasonia fondatrices. Ce phénomène est attribué aux comportements d’ajustement de la taille de la ponte et du ratio sexuel. En ajustant ces derniers, minimisant ainsi la compétition entre les membres de leur progéniture, les femelles fondatrices sont capables de maximiser la relation entre la disponibilité des ressources et la valeur sélective de leur progéniture. En conclusion, ce travail souligne l’importance de considérer le contexte spatial des interactions trophiques, puisqu’elles influencent la biodiversité locale et le fonctionnement de l’écosystème. / Landscape fragmentation and homogenization are considered to be the main causes of the worldwide decline in biological diversity. The degradation of habitat quality is mainly caused by the expansion and intensification of human land-use activities, primarily for agricultural purposes. Many studies documented the impact of landscape fragmentation and agricultural intensification on the overall fitness of animals across various taxa, but few works have studied this phenomenon in relation to trophic interactions. Here, we investigated the effects of landscape structure on the tri-trophic interactions between a bird host (the Tree Swallow Tachycineta bicolor (Vieillot)), its blowfly ectoparasites (Protocalliphora Hough), and their parasitoid wasps (Nasonia Walker) along a gradient of agricultural intensification covering 10, 200 km2 in southern Québec, Canada. The first objective was to describe the assemblages of Protocalliphora and Nasonia species found in Tree Swallow nests within our system. The prevalence of nest infestation by Protocalliphora was of 70.8% in 2008 and 34.6% in 2009. The percentage of nests containing Protocalliphora pupae parasitized by Nasonia spp. was of 85.3 % in 2008 and 67.2% in 2009. Three species of Protocalliphora were collected (P. sialia, P. bennetti and P. metallica) and two species of Nasonia (N. vitripennis and N. giraulti). Secondly, I evaluated the impact of landscape structure and agricultural intensification on the number of Tree Swallow fledglings, number of Protocalliphora per nest and the level of hyperparasitism by Nasonia. Our results revealed that organisms from different trophic levels perceived the landscape at distinctive spatial extents. This perception, however, differed based on whether the proportions of intensive or extensive culture in the landscape were considered. Furthermore, the number of Tree Swallow fledglings, the abundance of P. sialia and the level of hyperparasitism by N. vitripennis all decreased with an increase in the proportion of intensive culture in the landscape. Protocalliphora and Nasonia were more susceptible to extinction within highly intensive landscapes as they are confronted with both the direct effect of habitat degradation on their populations and the indirect effect of habitat degradation on their host populations. The last objective was to investigate how parasitoid wasps respond to environmental variability. We showed that the size of the P. sialia pupae hosts decreased in more intensive landscapes. Wasps clutch size was shown to increase within increasing host size and the sex ratio of offspring produced by N. vitripennis became more male biased as the proportion of intensive culture increased in the landscape. In addition, both female and male size was influenced by resource availability (size of host and number of competitors). Our data indicate that by producing smaller male biased clutches in the smaller hosts within intensive landscapes, females were able to respond to poor environments and maximize the size of their offspring. To conclude, this work highlights the importance of considering the landscape context of trophic interactions, as these interactions dictate local biodiversity and ecosystem function.

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