Les bracovirus forment une symbiose avec les guêpes parasitoïdes, demeurant dans leur génome et produits uniquement dans leurs ovaires. Nous avons caractérisé comment les cercles d’ADNdb contenu dans les particules étaient amplifiés depuis leur forme provirale avant leur encapsidation.Nous avons montré que le site d’intégration du génome viral est conservé chez les bracovirus et organisé dans le génome de la guêpe en un macrolocus regroupant la majorité des segments proviraux et 7 loci isolés. Nous avons mis en évidence 12 unités de réplication (UR) et que les 9 gènes viraux du cluster nudiviral étaient amplifiés sur une UR sans être encapsidés. Nous avons identifié des concatémères tête-tête et queue-queue comme étant les intermédiaires de réplication des UR, caractéristiques d’une réplication linéaire du génome viral. Enfin, nous avons montré que l’ADN polymérase B2 appartenait à un élément Maverick. L’absence de gènes viraux de la réplication du génome viral semble indiquer que la machinerie réplicative cellulaire serait impliquée. Il reste maintenant à mettre en évidence les différents facteurs cellulaires participant à l’amplification du génome viral. / Bracovirus form a symbiosis with parasitoid wasp, remaining in their genome and products only in their ovaries. We characterized how packaged dsDNA circles were amplified from their proviral genome before packaging in viral particles.We showed that viral genome integration site is conserved in bracovirus and organized in the wasp genome in a macrolocus where the majority of proviral segments was found and 7 isolated loci. We showed 12 replication units (UR) and the 9 nudiviral genes from cluster were amplified on a UR without being packaged. We identified concatemers head-head and tail-tail as the replication intermediates of UR, indicating a linear replication of the viral genome. Finally, we showed that DNA polymerase B2 belonged to a Maverick element.The absence of viral gene involved in the genome replication suggests that the cellular replication machinery is involved. It remains to highlight the different cellular factors involved in the amplification of the viral genome.

As sociedades de vespas da tribo Epiponini caracterizam-se pela variação no número de rainhas durante o ciclo colonial, o que implica na ocorrência cíclica de muitas rainhas (poliginia), poucas (oligoginia) e até uma rainha (monoginia). Durante as primeiras fases do ciclo colonial existe um número maior de rainhas, mas conforme a colônia cresce, o número de rainhas diminui. Os Epiponini também são caracterizados pela complexidade morfológica e as síndromes de diferenciação de castas. Entretanto, para algumas espécies dentro dos Polistinae, a única forma de separar as fêmeas reprodutoras das não reprodutoras é pelo comportamento ou pelo grau de desenvolvimento dos ovários. O estudo do comportamento e as interações sociais junto com os estudos morfométricos podem ajudar no entendimento da evolução da complexidade encontrada dentro dos Epiponini. Por isso o objetivo deste trabalho é descrever o comportamento das rainhas e operárias em Metapolybia docilis, e suas interações. Foram realizadas observações individuais do comportamento das rainhas e operárias previamente marcadas de quatro colônias de M. docilis. Foi feito um catálogo dos atos comportamentais observados com maior frequência. Para as rainhas os comportamentos incluíam tanto displays como comportamentos relacionados às necessidades básicas como alimentação e higiene, nas operárias os comportamentos incluíam principalmente trabalhos de manutenção do ninho e policiamento. Para realizar as análises morfométricas todos os indivíduos das colônias C1, C2 e C3 foram coletados, foi determinada a idade dos indivíduos da colônia e oito medidas corporais externas foram tomadas de uma amostra de 50 operárias e de todas as rainhas das colônias. A colônia C1 estava em Fase Matura, a colônia C2 em Fase de Pré- Enxameagem, as colônias C3 e C4 em Fase de Estabelecimento. Os comportamentos realizados com maior frequência pelas rainhas das quatro colônias foram: curvar o abdômen (CA), dança de display (DD), inspeção de célula (IC), trofalaxia (Tx) e auto-limpeza (Limp). Os comportamentos realizados com maior frequência pelas operárias foram IC e Tx. O comportamento CA provavelmente é um comportamento display de dominância feito pelas rainhas e o comportamento DD provavelmente está ligado a interações competitivas ou de dominância entre as fêmeas das colônias. Não foi encontrada diferenciação morfológica significativa entre rainhas e operárias de M. docilis para as colônias C2 e C3, mas sim para a colônia C1. Dentro das colônias estudadas o controle na reprodução das fêmeas é feito mediante o comportamento e não por manipulação larval. As operárias e as rainhas testam a capacidade reprodutiva das outras rainhas mediante displays de dominância não agressivos para decidir que fêmeas continuaram encarregando-se de produzir novos indivíduos. As operárias possivelmente controlam a reprodução (mediante o policiamento de ovos), seleção das rainhas, e todos os outros aspectos dentro da colônia explorando o ambiente e as necessidades do ninho. / Epiponini wasps societies are characterized by the alternation in the number of queens from many (polygyny) to few queens (olygyny) or even one (monogyny) during the colony cycle. When the colony is in the growing phase there are many queens, but as colony grows queen number decreases and new queens will be produced only when queen number is close to one (monygyny). Epiponines wasps are also characterized by the complexity of the morphological caste syndromes. However, in some Epiponini species the only way to separate reproductives from not reproductives is by behavioral acts or by the degree of ovarian development. The study of behavior and social interactions, associated with morphometric studies, could help to better understand the evolution and complexity found within the Epiponini. We studied and described behavioral acts and interactions between queens and workers in Metapolybia docilis. We performed direct and indirect (video) observations of individual behavior of previously marked queens and workers from four M. docilis colonies. We catalogued the most frequently observed behavioral acts: for queens these behaviors included displays and basic need behaviors such as feeding and hygiene; for workers they included nest maintenance and policing behaviors. All the individuals from colonies C1, C2 and C3 were collected to perform a morphometric analysis and age determination by taking eight external body measurements from a 50 workers sample and from all the queens of the colonies. Colony C1 was in an Emergence phase, colony C2 in a pre-swarming phase, colonies C3 and C4 in a pre-emergence phase. Most frequently behaviors made by queens were bending abdomen (BA), dance display (DD), cell inspection (CI), trofalaxis (Tx) and grooming (G). Most frequently behaviors made by workers were IC and Tx. Bending abdomen (BA) is probably a dominance display made by queens, and dance display (DD) was probably a test behavior made by workers and queens to test another queens. For colonies C2 and C3 there is no morphological differences between queens and workers, but there is a slight difference for colony C1. Within the colonies, the reproduction control is performed by behavior and not by larval manipulation because we did not found morphological differences between castes. Workers and queens tested the reproductive capacity of the queens by non aggressive dominance displays in order to select which females perform the reproduction work in the colony. Workers probably control the reproduction (by worker policing on the eggs laid), queen selection and all the others tasks on the colony by scanning the environment and the needs of the colony.

La consanguinité est connue par les biologistes pour diminuer la valeur sélective des individus en diminuant par exemple leur survie ou leur fertilité. De ce fait, la sélection naturelle devrait favoriser l'apparition de comportements permettant l'évitement des accouplements entre apparentes pour limiter les conséquences néfastes dues à la dépression de consanguinité. Cette dépression de consanguinité est particulièrement visible chez les Hyménoptères avec un système de détermination du sexe appelé single-locus Complementary Sex Determination (sl-CSD), où elle amène à la production de males diploïdes non viables ou stériles. Mon travail de thèse a ainsi consiste à étudier le phénomène d'évitement d'accouplements entre apparentes dans des populations naturelles d'un hyménoptère parasitoïde avec sl-CSD, Venturia canescens, ainsi que des signaux utilisés par les femelles pour déterminer l'apparentement qu'elles ont avec les individus qu'elles rencontrent. Nous avons d'abord étudié le lien unissant type d'habitat (continental, iles ou laboratoire), diversité génétique et production de males diploïdes dans 11 populations de V. canescens. En effet, un cadre théorique nomme "Vortex d'extinction du aux males diploïdes" prédit une corrélation négative entre isolations des populations, diversité génétique et production de males diploïdes pouvant amener à l'extinction de populations d'Hyménoptères. Nous avons ainsi démontré une corrélation négative entre diversité génétique et production de males diploïdes dans les populations isolées de V. canescens. Ensuite, il a été montré précédemment que les femelles de cette espèce étaient capables de discriminer les males qui leur étaient apparentes et d'éviter les accouplements entre apparentes en laboratoire. Nous nous sommes ainsi intéressés à ce phénomène d'évitement d'accouplement entre apparentes dans des populations naturelles grâce au génotypage de 450 individus du terrain et leur descendants. Nous avons montré que les femelles toléraient les accouplements entre apparentes sur le terrain ainsi qu'en laboratoire en présence de plusieurs males, nous permettant de mettre en lumière l'importance des conditions environnementales sur le choix du partenaire sexuel. Nous nous sommes enfin concentrés sur le système de reconnaissance des apparentes au niveau mécanistique en étudiant les signaux chimiques utilisés par les femelles pour reconnaitre leurs apparentes dans deux contextes écologiques différents, le choix du partenaire sexuel et l'évitement du superparasitisme lors de la ponte. Nous avons ainsi montré des similitudes entre les compositions chimiques de ces deux signaux mais aussi qu'ils n'étaient pas interchangeables entre les deux contextes écologiques étudiés. Au final, les résultats obtenus apportent un nouvel éclairage sur les conditions nécessaires à l'apparition d'un évitement d'accouplements entre apparentes dans des populations naturelles ainsi que sur les signaux utilisés lors de la reconnaissance de parentèle chez un hyménoptère parasitoïde / Inbreeding is well known by biologists to lower the fitness of individuals by or example decreasing survival or fertility. Therefore, natural selection should favour behaviours preventing the reproduction of genetically-related individuals or mitigating harmful consequences, called inbreeding depression. Inbreeding depression is particularly visible in Hymenoptera with a sex-determination system called single-locus Complementary Sex Determination (sl-CSD), where it leads to the production of diploid males that are either unviable or sterile. My PhD work has thus been devoted to the study of sib-mating avoidance in natural populations of a parasitoid with sl-CSD, Venturia canescens, and to understand the cues used by females recognize their kin. We first studied the link between habitat type (continental, island or captive), genetic diversity and diploid male production in 11 V. canescens populations. Indeed, a theoretical framework called "Diploid male extinction vortex" predict a negative correlation between populations’ isolation, genetic diversity and diploid male production that could lead to the extinction of hymenopteran populations.We actually showed a negative correlation between genetic diversity and diploid male production in isolated populations. Previous studies have furthermore demonstrated kin discrimination and sib-mating avoidance by V. canescens females in the laboratory. We therefore studied the sibmating avoidance behaviour in natural populations of this species by genotyping more than 450 wild individuals and their offsprings. We demonstrated that females tolerated inbreeding in the wild as well as in the laboratory when several males were present. We highlighted the importance of environmental conditions on mate choice. At last, we were interested in the kin recognition system and researched the chemical cues used by females in two ecological contexts, mate choice and superparasitism avoidance. This allowed us to identify similarities in the composition of the two chemical signals and that they were not interchangeable between the two studied ecological contexts. In the end, the results we obtained shed new light on the necessary conditions for the apparition of sib-mating avoidance in natural populations, as well as on the cues used for kin recognition in a parasitoid

The nature of aerosols in hot exoplanet atmospheres is one of the primary vexing questions facing the exoplanet field. The complex chemistry, multiple formation pathways, and lack of easily identifiable spectral features associated with aerosols make it especially challenging to constrain their key properties. We propose a transmission spectroscopy technique to identify the primary aerosol formation mechanism for the most highly irradiated hot Jupiters (HIHJs). The technique is based on the expectation that the two key types of aerosols-photochemically generated hazes and equilibrium condensate clouds-are expected to form and persist in different regions of a highly irradiated planet's atmosphere. Haze can only be produced on the permanent daysides of tidally locked hot Jupiters, and will be carried downwind by atmospheric dynamics to the evening terminator (seen as the trailing limb during transit). Clouds can only form in cooler regions on the nightside and morning terminator of HIHJs (seen as the leading limb during transit). Because opposite limbs are expected to be impacted by different types of aerosols, ingress and egress spectra, which primarily probe opposing sides of the planet, will reveal the dominant aerosol formation mechanism. We show that the benchmark HIHJ, WASP-121b, has a transmission spectrum consistent with partial aerosol coverage and that ingress-egress spectroscopy would constrain the location and formation mechanism of those aerosols. In general, using this diagnostic we find that observations with the James Webb Space Telescope and potentially with the Hubble Space Telescope should be able to distinguish between clouds and haze for currently known HIHJs.

The inadequacy of arbitrarily classifying interactions between species as antagonistic, neutral or mutualistic has become clear in recent years. Both direct and indirect interactions between species can vary between mutualism and antagonism, depending on the intrinsic and extrinsic contexts of the interaction. This study investigated the characteristics of an ant-plant-homopteran interaction in southern Africa. The polyphagous homopteran Hilda patruelis (Tettigometridae) feeds primarily on the trunk-borne fruiting branches and figs of the Cape fig tree, Ficus sur, and produces honeydew which attracts tending ants. Ten of the sixteen ant species/species groups present on F. sur tended H. patruelis, with Pheidole megacephala the most frequent attendant. Ants attracted to F. sur by H. patruelis honeydew or other liquid food sources also preyed on insects on the tree, including adults of the small agaonid fig wasps whose larvae feed on the ovules in the developing figs. One fig wasp species (Ceratosolen capensis) is also the tree's only pollinator. No benefits to H. patruelis from being tended by ants were detected, either in terms of reduced parasitism, or predation by a lycaenid caterpillar. A P. megacephala colony foraging on a F. sur tree was found to receive a high proportion of its likely energy requirements from the tree, mainly in the form of H. patruelis honeydew, during periods when it was bearing fruit. It is probable that the H. patruelis-P. megacephala interaction constitutes a direct mutualism at times, but that benefits to the homopteran are intermittent or weak. Both H. patruelis and ants benefitted from F. sur, directly or indirectly, through the provision of food (and for some ants, nesting sites). The removal of phloem sap by H. patruelis did not detectably reduce the trees' reproductive output, either in terms of pollinator or viable seed production. The indirect effects of ant and H. patruelis presence on the F. sur trees were on average positive, as ants preyed disproportionately heavily on fig wasp species parasitic on or competing with the pollinator, thus increasing pollinator production. Effects of ant presence on seed production were not investigated, but have been demonstrated as beneficial elsewhere. However, there is great varatlon both in the composition of the wasp fauna arriving to oviposit at different crops, and in ant densities per fig, on several temporal and spatial scales. This results in high variability in the effects of ants on the pollinator and, through it, the tree, from positive to zero and potentially even negative. Despite this conditionality of beneficial outcomes for the tree, the mean effect of ants on the F. sur population studied was to increase pollinator production by up to nearly 20%. This study is among the few to have demonstrated an overall benefit to a plant of having homopteran-tending ants present on it.

Fig trees (Ficus spp., Moraceae) and fig wasps (Chalcidoidea, Agaonidae) are uniquely associated. In one fig wasp group, the pollinators (Agaoninae), each species is generally host species-specific. The relationship is one of obligate mutualism where the wasps provide pollination services and in return utilises some of the ovules for larval development. Non-pollinating fig wasps (generally belonging to subfamilies other than the Agaoninae) may be gallers or parasitoids, and can also be host species-specific. In the accompanying studies we examined the factors governing the interactions between fig wasps and their host trees. Surveys of fig trees and their associated pollinating fig wasps conducted in southern Africa, Madagascar and The Comores generally confirmed their specific relationships. An examination of F. sycomorlls in Madagascar resulted in the reclassification of F. sakalavarum as a distinct species with its own specific pollinator species. Biological and chemical evidence is presented demonstrating that the pollinators were able to distinguish their hosts through volatiles which emanated from the figs when they were ready to be pollinated. Environmental factors were found to influence wasp behaviour. Ambient temperature governed the timing of wasp emergence from their natal figs. When dispersing from their natal figs, the fig wasps flew upwards and then were blown downwind. Once nearing trees bearing figs ready to be pollinated, the wasps lost height and flew upwind towards the trees. E. baijnathi females apparently avoided figs which already contained a conspecific foundress. Scanning electron microscope studies of pollinating female fig wasp antennae showed that while all the species possessed multiporous plate sensilla, in only a few species were these sensilla elongated. Multiporous plate sensilla elongation is rare or absent among other female chalcids and may have evolved within the Agaoninae in order to facilitate their location on receptive host figs. Pollinator choice specificity appears to break down in a number of cases. In the first case examined, two pollinator species were recorded from the figs of African F. sycomorus. One. C. arabicus, pollinates the figs while the other, C. galili, acts as a 'cuckoo' by utilising some of ovules for oviposition without providing pollen. In the second case three pollinating fig wasp species were recorded from the rigs of F. lutea. Two were found to be incidental visitors and were not specifically attracted to the tree. The hybrid seeds from these crosses were successfully germinated but the seedlings did not grow passed the cotyledon stage of their development. In the concluding study the consequences of Ficus phenology and the structure of the fig's unusual inflorescence on the nonpollinating fig wasp community were examined. Various factors affecting the population levels and species richness were also examined. Future possible research directions were discussed.

In July 2001 in the region of Kastri Platanistou in Evia, Greece a wind power park of 5 MWinstalled capacity, began its commercial power production under the supervision of the companyMELTEMI-KASTRI.During these 6 years of its operation significantly reduced incomes were recorded compared to theinitial study financial analysis. Apparently loss of income occurred due to technical failures;nevertheless more costly might have been a suboptimal primary energy analysis.Herein, the collected data from the nacelle of the wind turbines and a net installed in the site wereanalyzed in order to estimate the site’s wind energy potential and gain knowledge of the parametersthat so far affected the overall production. The analysis was performed through Windrose andWAsP software.Aiming to maximize the exploitation of the site’s wind energy potential, several wind turbinemodels in different installation points were examined. Further analysis of the exported resultsindicated the optimum scenario, finally suggested to the owners of the wind farm.

Cells use energy to maintain order, as living systems are inherently non-equilibrium. Or- der in the cytoplasm is achieved by compartmentalization. One type of compartment that gained interest in recent years is membraneless organelles (MLOs). Observations of the liquid-like properties of MLOs led to their interpretation in analogy to Liquid-Liquid Phase Separation (LLPS). However, LLPS alone implies a passive closed system that tends towards equilibrium, which is incompatible with the physical nature of the cell. It is unclear then what non-equilibrium interactions give rise to the dynamics of MLOs in the cell. We sought to decipher the regulatory interactions that give rise to active condensation in the actomyosin cortex of C. elegans. The components of the actomyosin cortex, F- actin and its branching nucleation module Arp2/3 and N-WASP (WSP-1 in C. elegans) have been described as a phase separated system in previous reports. In vitro, phase separated N-WASP compartments do not have the non-equilibrium growth and disas- sembly dynamics observed in the multicomponent clusters in vivo. Therefore, our goal is to examine WSP-1, Arp2/3 and F-actin interactions in the endogenous context. We chose the stage in which the quiescent oocyte cortex becomes actively contractile. During the transition out of quiescence, we observed transient WSP-1 Arp2/3 F-actin puncta that assemble and disassemble. To capture growth dynamics for all puncta, we devel- oped a novel phase portrait analysis tool. The phase portrait allows us to simultaneously study puncta growth and disassembly rates as a function of internal composition. The growth rate dependence on internal composition reflects the non-trivial changes to nu- cleation profiles that accompany condensation in active, open, multi-component systems. We observed superlinear WSP-1 growth rates consistent with condensation. Further, we identified the in vivo equivalent of a nucleation barrier for WSP-1 condensation. The in vivo nucleation barrier increases with branching F-actin reaction, which tunes con- densation. Correspondingly, the reactive components WSP-1 and Arp2/3 are important for condensate dynamics. Combining condensation and the branching reaction, we for- mulated a coarse-grained model which captures non-equilibrium condensate dynamics. Altogether, our results showed that WSP-1 grows like condensation, and its growth is steered away from equilibrium by Arp2/3 mediated branching reaction. In summary, combining high-resolution imaging, quantitative analysis and theory, we identified the interactions that could explain non-equilibrium condensation in the acto- myosin cortex. The living dynamics that arise from the interplay between condensation and reaction. The interplay between physical processes (like condensation) and biological regulation (such as reactions) may be a common organizing principle behind MLO for- mation, as well as other non-equilibrium processes in the cell. The methods and concepts developed in this work hold the promise to deepen our understanding of how living cells regulate their dynamic organization, in order to maintain themselves in a non-equilibrium ordered state.:1 Introduction 1 1.1 Evolving concepts of cellular organization 1 1.2 Condensation of biomolecules 3 1.2.1 Terminology for biomolecular condensates 5 1.2.2 Technical considerations for identifying liquid-like properties and LLPS 7 1.2.3 Thermodynamics of condensation 10 1.2.4 The problem of an equilibrium description of living systems 13 1.2.5 Towards active condensation 14 1.3 Actomyosin cortex self-organization 16 1.3.1 F-actin treadmilling and nucleation 17 1.3.2 N-WASP and Arp2/3 regulation 18 1.3.3 Multivalent interactions in condensation of transmembrane receptors and actin regulators 22 1.3.4 Cortex activation in C. elegans 23 2 Aims 25 3 Results 26 3.1 C. elegans cortical activation begins at fertilization 26 3.1.1 C. elegans oocytes as an ex utero model for cortex self-organization 27 3.2 WSP-1, Arp2/3 and F-actin form dynamic multicomponent phases 32 3.2.1 Capping proteins outcompete Formin in WSP-1 Arp2/3 puncta preventing F-actin elongation 32 3.2.2 WSP-1 and Arp2/3 are required for punctate F-actin formation and dynamics 34 3.2.3 Summary of the characterization of cortical activation 34 3.3 Establishment of systematic phase portrait analysis for multicomponent clusters 36 3.3.1 Non-equilibrium features of the multicomponent puncta 36 3.3.2 Recording intensity traces of multicomponent cluster over time 37 3.3.3 Probability flux of composition in the phase portrait show a closed cycle 38 3.3.4 WSP-1 F-actin puncta have a preferred joint concentration 38 3.3.5 The phase portrait is robust to cell-to-cell noise 41 3.3.6 Choosing the appropriate bin size 41 3.4 Existence of a tuned critical size and signatures of active condensation 45 3.4.1 Growth rate dependence on internal composition 45 3.4.2 Stoichiometric growth laws of WSP-1 F-actin clusters 47 3.4.3 Estimation of WSP-1 cluster critical size in vivo 47 3.4.4 Theoretical description of WSP-1 and F-actin interactions in regulating puncta dynamics 48 3.4.5 Summary of 2D phase portrait findings 52 3.5 Towards three dimensional phase portrait analysis of the reaction network 54 3.6 Initial assessment of the compartment’s external environment 54 3.7 Identification of modulators of puncta dynamics 56 3.7.1 CDC-42 controls cortical levels of WSP-1 56 3.7.2 RHO-1 and Formin CYK-1 are not involved in WSP-1 F-actin condensate dynamics 58 3.7.3 WSP-1 and Arp2/3 dynamics are independent of NCK-1 and VAB-1 58 3.7.4 Arp2/3 regulates condensate dynamics 60 3.8 Summary of perturbations 63 4 Conclusions and outlook 64 4.1 Concluding remarks 64 4.2 Discussion 66 4.3 Future directions 67 4.3.1 Realizing the full potential of the phase portraits in identifying biochemical interactions 67 4.3.2 Resolving the ultrastructure of condensates . 70 4.3.3 Further investigation of the biological function 71 4.3.4 Applying full-dynamic data acquisition to other membraneless organelles 71 5 Materials and Methods 72 5.1 C.elegans maintenance and strains 72 5.2 Sample preparation 72 5.2.1 In utero imaging 72 5.2.2 Oocyte imaging 73 5.2.3 C.elegans HaloTag staining 73 5.2.4 Oocyte chemical inhibitor treatments 73 5.3 RNAi Feeding 73 5.4 Microscopy 73 5.4.1 Spinning disk microscopy 73 5.4.2 SIM-TIRF microscopy 74 5.5 TIRF microscopy 74 5.6 Phase portrait analysis pipeline 74 5.7 Kymographs 76 / Zellen verbrauchen Energie, um Ordnung aufrechtzuerhalten, da lebende Systeme von Natur aus ungleichgewichtig sind. Ordnung im Zytoplasma wird durch Kompartimen- tierung erreicht. Eine Art von Kompartiment, das in den letzten Jahren an Interesse gewonnen hat, sind membranlose Organellen (engl.: membraneless organelles, MLOs). Beobachtungen der flu ̈ssigkeits ̈ahnlichen Eigenschaften dieser MLOs fu ̈hrten zu ihrer In- terpretation in Analogie zur Flu ̈ssig-Flu ̈ssig-Phasentrennung (engl.: liquid-liquid phase separation, LLPS). Die LLPS allein impliziert jedoch ein passives geschlossenes System, das zum Gleichgewicht neigt und mit der physikalischen Natur der Zelle nicht kompatibel ist. Es war bisher nicht bekannt, welche Ungleichgewichtswechselwirkungen die Dynamik von MLOs in der Zelle hervorrufen. Wir wollten die regulatorischen Wechselwirkungen entschlu ̈sseln, die zu aktiver Konden- sation im Aktomyosin-Kortex von C. elegans fu ̈hren. Die Komponenten des Aktomyosin- Kortex, F-Aktin und seines verzweigten Nukleationsmoduls Arp2/3 und N-WASP (WSP- 1 in C. elegans) wurden in fru ̈heren Studien als phasengetrenntes System beschrieben. In vitro weisen phasengetrennte N-WASP-Kompartimente allerdings nicht dieselben un- gleichgewichtigen Wachstums- und Zerlegungsdynamiken auf, die in kultivierten Zellen beobachtet werden. Daher wollten wir die Wechselwirkungen zwischen WSP-1, Arp2/3 und F-Aktin im Kontext des Fadenwurms C. elegans untersuchen. Wir haben das C.elegans Lebenstadium gew ̈ahlt, in dem die ruhende Eizellenrinde aktiv kontraktil wird. Wa ̈hrend des U ̈bergangs aus der ruhigen in die aktive Periode konnten wir voru ̈bergehende WSP- 1 Arp2/3 F-Aktin-Puncta beobachten, die sich zusammensetzen und zerlegen. Um die Wachstumsdynamik fu ̈r alle Puncta zu erfassen, haben wir ein neuartiges Tool zur Anal- yse von Phasenportr ̈ats entwickelt. Das Phasenportr ̈at ermo ̈glicht es uns, gleichzeitig die Wachstums- und die Zerlegungsraten von Puncta in Abha ̈ngigkeit der inneren Zusam- mensetzung zu messen. Die Abha ̈ngigkeit der Wachstumsrate von der inneren Zusam- mensetzung spiegelt die nicht trivialen A ̈nderungen der Nukleationsprofile wider, die mit der Kondensation in aktiven, offenen Mehrkomponentensystemen einhergehen. Wir kon- nten superlineare WSP-1-Wachstumsraten beobachten, die mit der Kondensation u ̈bere- instimmen. Ferner konnten wir das In-vivo-A ̈quivalent einer Nukleationsbarriere fu ̈r die WSP-1-Kondensation identifizieren. Die In-vivo-Nukleationsbarriere nimmt mit der verzweigten F-Actin-Reaktion zu, die die Kondensation reguliert. Dementsprechend sind die reaktiven Komponenten WSP-1 und Arp2/3 wichtig fu ̈r die Dynamik des Konden- sats. Wir haben die Kondensations- und Verzweigungsreaktionen kombiniert, um damit ein grobko ̈rniges Modell zu formulieren, das die Ungleichgewichtskondensationsdynamik erfasst. Insgesamt haben unsere Ergebnisse gezeigt, dass WSP-1 kondensiert und diese Kondensation durch Arp2/3-vermittelte Verzweigungsreaktionen aus dem Gleichgewicht gebracht wird. Zusammenfassend konnten wir durch Kombination von hochauflo ̈sender Bildgebung, quan- titativer Analyse und Theorie die Wechselwirkungen identifizieren, die die Ungleichgewicht- skondensation im Aktomyosin-Kortex erkla ̈ren ko ̈nnten. Die Dynamik im lebendem Sys- tem ergibt sich aus dem Zusammenspiel von Kondensation und Reaktion. Die Interaktion zwischen physikalischen Prozessen (wie Kondensation) und biologischen Regulationen (wie Reaktionen) kann ein gemeinsames Organisationsprinzip hinter der MLO-Bildung sowie anderen Ungleichgewichtsprozessen in der Zelle sein. Die in dieser Arbeit entwickel- ten Methoden und Konzepte k ̈onnen daher helfen, unser Versta ̈ndnis daru ̈ber zu vertiefen, wie lebende Zellen ihre r ̈aumlich-zeitlichen Proteinverteilungen dynamisch regulieren, um sich in einem ungleichgewichtigen, geordneten Zustand zu halten.:1 Introduction 1 1.1 Evolving concepts of cellular organization 1 1.2 Condensation of biomolecules 3 1.2.1 Terminology for biomolecular condensates 5 1.2.2 Technical considerations for identifying liquid-like properties and LLPS 7 1.2.3 Thermodynamics of condensation 10 1.2.4 The problem of an equilibrium description of living systems 13 1.2.5 Towards active condensation 14 1.3 Actomyosin cortex self-organization 16 1.3.1 F-actin treadmilling and nucleation 17 1.3.2 N-WASP and Arp2/3 regulation 18 1.3.3 Multivalent interactions in condensation of transmembrane receptors and actin regulators 22 1.3.4 Cortex activation in C. elegans 23 2 Aims 25 3 Results 26 3.1 C. elegans cortical activation begins at fertilization 26 3.1.1 C. elegans oocytes as an ex utero model for cortex self-organization 27 3.2 WSP-1, Arp2/3 and F-actin form dynamic multicomponent phases 32 3.2.1 Capping proteins outcompete Formin in WSP-1 Arp2/3 puncta preventing F-actin elongation 32 3.2.2 WSP-1 and Arp2/3 are required for punctate F-actin formation and dynamics 34 3.2.3 Summary of the characterization of cortical activation 34 3.3 Establishment of systematic phase portrait analysis for multicomponent clusters 36 3.3.1 Non-equilibrium features of the multicomponent puncta 36 3.3.2 Recording intensity traces of multicomponent cluster over time 37 3.3.3 Probability flux of composition in the phase portrait show a closed cycle 38 3.3.4 WSP-1 F-actin puncta have a preferred joint concentration 38 3.3.5 The phase portrait is robust to cell-to-cell noise 41 3.3.6 Choosing the appropriate bin size 41 3.4 Existence of a tuned critical size and signatures of active condensation 45 3.4.1 Growth rate dependence on internal composition 45 3.4.2 Stoichiometric growth laws of WSP-1 F-actin clusters 47 3.4.3 Estimation of WSP-1 cluster critical size in vivo 47 3.4.4 Theoretical description of WSP-1 and F-actin interactions in regulating puncta dynamics 48 3.4.5 Summary of 2D phase portrait findings 52 3.5 Towards three dimensional phase portrait analysis of the reaction network 54 3.6 Initial assessment of the compartment’s external environment 54 3.7 Identification of modulators of puncta dynamics 56 3.7.1 CDC-42 controls cortical levels of WSP-1 56 3.7.2 RHO-1 and Formin CYK-1 are not involved in WSP-1 F-actin condensate dynamics 58 3.7.3 WSP-1 and Arp2/3 dynamics are independent of NCK-1 and VAB-1 58 3.7.4 Arp2/3 regulates condensate dynamics 60 3.8 Summary of perturbations 63 4 Conclusions and outlook 64 4.1 Concluding remarks 64 4.2 Discussion 66 4.3 Future directions 67 4.3.1 Realizing the full potential of the phase portraits in identifying biochemical interactions 67 4.3.2 Resolving the ultrastructure of condensates . 70 4.3.3 Further investigation of the biological function 71 4.3.4 Applying full-dynamic data acquisition to other membraneless organelles 71 5 Materials and Methods 72 5.1 C.elegans maintenance and strains 72 5.2 Sample preparation 72 5.2.1 In utero imaging 72 5.2.2 Oocyte imaging 73 5.2.3 C.elegans HaloTag staining 73 5.2.4 Oocyte chemical inhibitor treatments 73 5.3 RNAi Feeding 73 5.4 Microscopy 73 5.4.1 Spinning disk microscopy 73 5.4.2 SIM-TIRF microscopy 74 5.5 TIRF microscopy 74 5.6 Phase portrait analysis pipeline 74 5.7 Kymographs 76

European Paper Wasps (Polistes dominula) are social insects that build round, symmetrical nests. Current models indicate that these wasps develop colonies by following simple heuristics based on nest stimuli. Computer simulations can model wasp behavior to imitate natural nest building. This research investigated various building heuristics through a novel Markov-based simulation. The simulation used a hexagonal grid to build cells based on the building rule supplied to the agent. Nest data was compared with natural data and through visual inspection. Larger nests were found to be less compact for the rules simulated.

Metapolybia wasps live in small societies (around one hundred adults) and rear their young in nests they construct on flat surfaces from plant materials. For processing nest paper, they must gather plant materials and process it into pulp with water. The water is collected by water foragers and is transferred to pulp foragers indirectly via a "common stomach." The common stomach, or social crop, is formed by generalist wasps called laborers. These wasps can engage in water exchange, store water in their crops, and may become specialist foragers or builders. We provide an alternative model for regulating task partitioning in construction behavior by using an agent based modeling framework parameterized by our field observations. Our model predicts that assessing colony needs via individual interactions with the common stomach leads to a robust regulation of task partitioning in construction behavior. By using perturbation experiments in our simulations, we show that this emergent task allocation is able to dynamically adapt to perturbations of the environment and to changes in colony-level demands or population structure. The robustness of our model stems from the fact that the common stomach is both a strong buffer and a source of several feedback mechanisms that affect the individual wasps. We show that both the efficiency and the task fidelity of these colonies are dependent upon colony size. We also demonstrate that the emergence of specialist wasps (individuals with high task fidelity) does not require any special initial conditions or reinforcement at the individual level, but it is rather a consequence of colony-level workflow stability. Our model closely mimics the behavior of Metapolybia wasps, demonstrating that a regulation mechanism based on simple pair-wise interactions through a common stomach is a plausible hypothesis for the organization of collective behavior.