<p>Докторска дисертација представља таксономску студију тестатних амеба Источне&nbsp; Херцеговине&nbsp; базирану&nbsp; искључиво&nbsp; на&nbsp; резултатима&nbsp; сопствених истраживања с обзиром да на овом подручју тестатне амебе до сада нису проучаване,&nbsp; те&nbsp; не&nbsp; постоје&nbsp; историјске&nbsp; музејске&nbsp; колекције.&nbsp; На&nbsp; подручју Источне Херцеговине регистровано је 40 врста тестатних амеба које су сврстане у 10 фамилија и један род без јасног места у класификационом систему.&nbsp; Сви&nbsp; регистровани&nbsp; таксони&nbsp; тестатних&nbsp; амеба&nbsp; су&nbsp; нови&nbsp; за&nbsp; фауну Босне и Херцеговине. Укупно је анализирано 24.549 јединки, од чега су 23.242&nbsp; јединке&nbsp; припадале&nbsp; групи&nbsp; тестатних&nbsp; амеба&nbsp; са&nbsp; лобоподијама (супергрупа&nbsp; Amoebozoa),&nbsp; а&nbsp; 1307&nbsp; јединки&nbsp; је&nbsp; припадало&nbsp; групи&nbsp; тестатних амеба са филоподијама (супергрупа Cercozoa). Најзначајнији резултат ове дисертације&nbsp; је&nbsp; опис&nbsp; пет&nbsp; нових&nbsp; врста&nbsp; за&nbsp; науку&nbsp; које&nbsp; припадају&nbsp; родовима<br />Centropyxis, Heleopera и Nebela. Морфотип означен као C.&nbsp; cf.&nbsp; aerophila&nbsp; се од врсте&nbsp; C. aerophila&nbsp; разликује по томе што се на крају љуштурице не налази пар крупних честица кварца,а и љуштурица је нешто дужа (46‒81 &mu;m код врсте C. aerophila према 67‒ 88 &mu;m код врсте C. cf. aerophila). Морфотип означен као C. cf. platystoma значајно се пре свега морфолошки разликује од врсте&nbsp; C. platystoma, те је закључено да се ради о неописаној врсти.У оквиру рода Heleopera описан је нови морфотип сличан врсти H. rosea који&nbsp; представља&nbsp; нову&nbsp; врсту&nbsp; за&nbsp; науку.&nbsp; Морфометријске&nbsp; разлике&nbsp; су релативне, тј. нису строго дискриминаторне, те се морају комбиновати са морфолошким&nbsp; разликама&nbsp; које&nbsp; су&nbsp; такође&nbsp; тешко&nbsp; yочљиве.&nbsp; Наиме,&nbsp; поред разлике&nbsp; у&nbsp; боји&nbsp; љуштурице,&nbsp; најбољи&nbsp; дискриминаторни&nbsp; морфолошки карактер&nbsp; је&nbsp; општи&nbsp; облик&nbsp; љуштурице.&nbsp; Љуштурице&nbsp; врсте&nbsp; H.&nbsp; rosea&nbsp; су робусног&nbsp; облика,&nbsp; док&nbsp; су&nbsp; љуштурице&nbsp; врсте&nbsp; Heleopera&nbsp; cf.&nbsp; rosea&nbsp; знатно елегантније&nbsp; ‒&nbsp; уже&nbsp; су&nbsp; и&nbsp; имају&nbsp; облије&nbsp; ивице.&nbsp; Највеће&nbsp; морфометријске разлике&nbsp; у&nbsp; индексним&nbsp; карактерима&nbsp; су&nbsp; забележене&nbsp; за&nbsp; однос&nbsp; ширине&nbsp; и дужине љуштурице и однос ширине апертуре и ширине љуштурице.Морфотип&nbsp; Nebela&nbsp; cf.&nbsp; collaris&nbsp; се од&nbsp; врсте&nbsp; N. collaris&nbsp; јасно разликује&nbsp; пре свега&nbsp; морфолошки&nbsp; и&nbsp; еколошки,&nbsp; а&nbsp; морфометријски&nbsp; веома&nbsp; мало.&nbsp; Наиме, најважнија морфолошка одлика која морфотип&nbsp; N.&nbsp; cf.&nbsp; collaris&nbsp; раздваја од врсте&nbsp; N.&nbsp; collaris&nbsp; су&nbsp; таласасте&nbsp; ивице&nbsp; љуштурице,&nbsp; а&nbsp; еколошка&nbsp; разлика&nbsp; се јавља&nbsp; у&nbsp; смислу&nbsp; да&nbsp; врста&nbsp; N.&nbsp; collaris&nbsp; насељава&nbsp; зелене&nbsp; маховине&nbsp; док морфотип N. cf. collaris&nbsp; насељава сфагнумске маховине. Морфотип N. cf. tincta&nbsp; var. major&nbsp; се од морфотипа N.&nbsp; cf. collaris&nbsp; разликује пре свега по јасно израженом сужењу у делу близу апертуре, тј. израженом врату. Такође, морфотип N. cf. tincta var. major никада нема таласасте ивице љуштурице,док&nbsp; се&nbsp; код&nbsp; јединки&nbsp; морфотипа&nbsp; N.&nbsp; cf.&nbsp; collaris&nbsp; ова&nbsp; карактеристика&nbsp; често јасно уочава.</p> / <p>Doktorska disertacija predstavlja taksonomsku studiju testatnih ameba Istočne&nbsp; Hercegovine&nbsp; baziranu&nbsp; isključivo&nbsp; na&nbsp; rezultatima&nbsp; sopstvenih istraživanja s obzirom da na ovom području testatne amebe do sada nisu proučavane,&nbsp; te&nbsp; ne&nbsp; postoje&nbsp; istorijske&nbsp; muzejske&nbsp; kolekcije.&nbsp; Na&nbsp; području Istočne Hercegovine registrovano je 40 vrsta testatnih ameba koje su svrstane u 10 familija i jedan rod bez jasnog mesta u klasifikacionom sistemu.&nbsp; Svi&nbsp; registrovani&nbsp; taksoni&nbsp; testatnih&nbsp; ameba&nbsp; su&nbsp; novi&nbsp; za&nbsp; faunu Bosne i Hercegovine. Ukupno je analizirano 24.549 jedinki, od čega su 23.242&nbsp; jedinke&nbsp; pripadale&nbsp; grupi&nbsp; testatnih&nbsp; ameba&nbsp; sa&nbsp; lobopodijama (supergrupa&nbsp; Amoebozoa),&nbsp; a&nbsp; 1307&nbsp; jedinki&nbsp; je&nbsp; pripadalo&nbsp; grupi&nbsp; testatnih ameba sa filopodijama (supergrupa Cercozoa). Najznačajniji rezultat ove disertacije&nbsp; je&nbsp; opis&nbsp; pet&nbsp; novih&nbsp; vrsta&nbsp; za&nbsp; nauku&nbsp; koje&nbsp; pripadaju&nbsp; rodovima<br />Centropyxis, Heleopera i Nebela. Morfotip označen kao C.&nbsp; cf.&nbsp; aerophila&nbsp; se od vrste&nbsp; C. aerophila&nbsp; razlikuje po tome što se na kraju ljušturice ne nalazi par krupnih čestica kvarca,a i ljušturica je nešto duža (46‒81 &mu;m kod vrste C. aerophila prema 67‒ 88 &mu;m kod vrste C. cf. aerophila). Morfotip označen kao C. cf. platystoma značajno se pre svega morfološki razlikuje od vrste&nbsp; C. platystoma, te je zaključeno da se radi o neopisanoj vrsti.U okviru roda Heleopera opisan je novi morfotip sličan vrsti H. rosea koji&nbsp; predstavlja&nbsp; novu&nbsp; vrstu&nbsp; za&nbsp; nauku.&nbsp; Morfometrijske&nbsp; razlike&nbsp; su relativne, tj. nisu strogo diskriminatorne, te se moraju kombinovati sa morfološkim&nbsp; razlikama&nbsp; koje&nbsp; su&nbsp; takođe&nbsp; teško&nbsp; yočljive.&nbsp; Naime,&nbsp; pored razlike&nbsp; u&nbsp; boji&nbsp; ljušturice,&nbsp; najbolji&nbsp; diskriminatorni&nbsp; morfološki karakter&nbsp; je&nbsp; opšti&nbsp; oblik&nbsp; ljušturice.&nbsp; LJušturice&nbsp; vrste&nbsp; H.&nbsp; rosea&nbsp; su robusnog&nbsp; oblika,&nbsp; dok&nbsp; su&nbsp; ljušturice&nbsp; vrste&nbsp; Heleopera&nbsp; cf.&nbsp; rosea&nbsp; znatno elegantnije&nbsp; ‒&nbsp; uže&nbsp; su&nbsp; i&nbsp; imaju&nbsp; oblije&nbsp; ivice.&nbsp; Najveće&nbsp; morfometrijske razlike&nbsp; u&nbsp; indeksnim&nbsp; karakterima&nbsp; su&nbsp; zabeležene&nbsp; za&nbsp; odnos&nbsp; širine&nbsp; i dužine ljušturice i odnos širine aperture i širine ljušturice.Morfotip&nbsp; Nebela&nbsp; cf.&nbsp; collaris&nbsp; se od&nbsp; vrste&nbsp; N. collaris&nbsp; jasno razlikuje&nbsp; pre svega&nbsp; morfološki&nbsp; i&nbsp; ekološki,&nbsp; a&nbsp; morfometrijski&nbsp; veoma&nbsp; malo.&nbsp; Naime, najvažnija morfološka odlika koja morfotip&nbsp; N.&nbsp; cf.&nbsp; collaris&nbsp; razdvaja od vrste&nbsp; N.&nbsp; collaris&nbsp; su&nbsp; talasaste&nbsp; ivice&nbsp; ljušturice,&nbsp; a&nbsp; ekološka&nbsp; razlika&nbsp; se javlja&nbsp; u&nbsp; smislu&nbsp; da&nbsp; vrsta&nbsp; N.&nbsp; collaris&nbsp; naseljava&nbsp; zelene&nbsp; mahovine&nbsp; dok morfotip N. cf. collaris&nbsp; naseljava sfagnumske mahovine. Morfotip N. cf. tincta&nbsp; var. major&nbsp; se od morfotipa N.&nbsp; cf. collaris&nbsp; razlikuje pre svega po jasno izraženom suženju u delu blizu aperture, tj. izraženom vratu. Takođe, morfotip N. cf. tincta var. major nikada nema talasaste ivice ljušturice,dok&nbsp; se&nbsp; kod&nbsp; jedinki&nbsp; morfotipa&nbsp; N.&nbsp; cf.&nbsp; collaris&nbsp; ova&nbsp; karakteristika&nbsp; često jasno uočava.</p> / <p>The&nbsp; PhD&nbsp; thesis&nbsp; is&nbsp; a&nbsp; taxonomic&nbsp; study&nbsp; of&nbsp; testate&nbsp; amoebae&nbsp; from&nbsp; East Herzegovina based exclusively on the results of our own research, given that testate amoebae have not been studied in this region so far, and there are no historical museum collections. In the region&nbsp; of East Herzegovina,40 testate amoeba species have been registered, which are classified into 10&nbsp; families&nbsp; and&nbsp; one&nbsp; genus&nbsp; without&nbsp; a&nbsp; clear&nbsp; place&nbsp; in&nbsp; the&nbsp; classification system. All registered testate amoeba taxa are new to the fauna of Bosnia and Herzegovina.&nbsp; A total of 24,549 individuals belonged to the group of testate&nbsp; amoebae&nbsp; with&nbsp; lobopodia&nbsp; (supergroup&nbsp; Amoebozoa),&nbsp; and&nbsp; 1307 individuals&nbsp; belonged&nbsp; to&nbsp; the&nbsp; group&nbsp; of&nbsp; testate&nbsp; amoebae&nbsp; with&nbsp; filopodia (supergroup Cercozoa). The most significant results of this PhD thesis are the descriptions of five new species for science belonging to the genera Centropyxis, Heleopera, and Nebela. The morphotype&nbsp; Centropyxis&nbsp; cf.&nbsp; aerophila&nbsp; differs from&nbsp; C. aerophila&nbsp; in that&nbsp; there&nbsp; is&nbsp; no&nbsp; large&nbsp; quartz&nbsp; particles&nbsp; at&nbsp; the&nbsp; shell&nbsp; end,&nbsp; and&nbsp; the&nbsp; shell&nbsp;&nbsp; is slightly&nbsp; longer&nbsp; (46‒81&nbsp; &mu;m&nbsp; in&nbsp; C.&nbsp; aerophila&nbsp; versus&nbsp; 67‒88&nbsp; &mu;m&nbsp; in&nbsp; C.&nbsp; cf. aerophila).&nbsp; The&nbsp; morphotype&nbsp; C.&nbsp; cf.&nbsp; platystoma&nbsp; differs&nbsp; significantly morphologically&nbsp; from&nbsp; C.&nbsp; platystoma,&nbsp; so&nbsp; it&nbsp; was&nbsp; concluded&nbsp; that&nbsp; it&nbsp; is&nbsp; an undescribed species. Within the genus&nbsp; Heleopera&nbsp; a new &nbsp; morphotype similar to&nbsp; H. rosea&nbsp; has been&nbsp; described,&nbsp; representing&nbsp; a&nbsp; new&nbsp; species&nbsp; for&nbsp; science.&nbsp; Morphometric differences are relative, i.e. they are not strictly discriminatory, and must be combined with morphological differences that are difficult to detect. Namely, in addition to the difference in the color of the shell, the best discriminatory morphological character is the general shell shape. Shells of&nbsp; H. rosea&nbsp; are red and robust in shape, while shells of&nbsp; H.&nbsp; cf. rosea&nbsp; are volet and much more&nbsp; elegant ‒&nbsp; they are narrower and have rounded edges. The&nbsp; largest&nbsp; morphometric&nbsp; differences&nbsp; in&nbsp; the&nbsp; index&nbsp; characters&nbsp; were observed for shell width/shell length ratio and aperture width/shell width ratio. The&nbsp; morphotype&nbsp; Nebela&nbsp; cf.&nbsp; collaris&nbsp; clearly&nbsp; differs&nbsp; from&nbsp; N.&nbsp; collaris primarily&nbsp; morphologically&nbsp; and&nbsp; ecologically,&nbsp; but&nbsp; morphometrically&nbsp; very little.&nbsp; Namely,&nbsp; the&nbsp; most&nbsp; important&nbsp; morphological&nbsp; character&nbsp; that&nbsp; N.&nbsp; cf. collaris separates from N. collaris are the wavy edges of the shell, and the&nbsp; ecological difference occurs in the sense that&nbsp; N. collaris&nbsp; inhabits green mosses while &nbsp; N. cf. collaris inhabits Sphagnum mosses. The morphotype N.&nbsp; cf.&nbsp; tincta&nbsp; var.&nbsp; major&nbsp; from&nbsp; the&nbsp; morphotype&nbsp; N.&nbsp; cf.&nbsp; collaris&nbsp; differs primarily by a clearly pronounced narrowing in the part near the aperture, i.e. pronounced neck. Also, the morphotype N. &nbsp; cf. tincta var. major never has a wavy edge of the shell, while in&nbsp; N.&nbsp; cf.&nbsp; collaris&nbsp; this feature is often clearly observed.</p>

Les espèces bactériennes opportunistes responsables d'infections nosocomiales chez l'Homme se rencontrent dans les environnements terrestres et aquatiques. Elles sont très souvent caractérisées par une résistance naturelle aux antibiotiques leur conférant un phénotype appelé Multi-Drug Resistant (MDR). L'efflux d'antibiotiques via des pompes, est un des mécanismes à l'origine de cette multi-résistance. Alors que le rôle de ces pompes chez des bactéries isolées en milieu clinique est connu, aucune donnée n'est disponible concernant leur rôle chez les bactéries associées avec d'autres organismes eucaryotes du sol tels que les amibes. Pourtant des données de la littérature indiquent que les amibes, jusqu'alors principalement connues pour leur rôle prédateur de bactéries sont susceptibles d'héberger des bactéries " résistantes " aux amibes (ARB). Parmi ces ARB, des pathogènes opportunistes ont été identifiés dont certains sont connus pour être porteurs de pompes à efflux. Les pompes à efflux de ces bactéries pourraient donc intervenir dans l'adaptation aux amibes du sol. Afin de vérifier cette hypothèse, nous avons, dans un premier temps, isolé et identifié la flore amibienne et les ARB de différents sols. Parmi les ARB identifiées, Stenotrophomonas maltophilia, Pseudomonas aeruginosa et Burkholderia cepacia sont caractérisées par des propriétés d'antibiorésistance contrastées et de virulence élevées. Des études d'interaction ont montré que S. maltophilia se multipliait dans des amibes axéniques et que des pompes à efflux Sme étaient surexprimées. Par ailleurs des molécules sécrétées par l'amibe stimulent la croissance bactérienne et des études préliminaires de profilage métabolique ont montré la présence de différents métabolites secondaires produits par l'amibe au cours de l'interaction avec S. maltophilia pouvant jouer un rôle dans l'expression des pompes à efflux / The opportunistic bacterial species, responsible for nosocomial infections in humans, occurs in terrestrial and aquatic environments. They are often characterized by natural resistance to antibiotics giving them a phenotype called Multi-Drug Resistant (MDR). The efflux of antibiotics via pumps, is one of the mechanisms behind this multi-resistance. While the role of these pumps in bacteria isolated from hospital is known, no data are available regarding their role in bacteria associated with other soil eukaryotic organisms such as amoebae. Nevertheless, data from the literature indicate that amoebae, mainly known to be predators of bacteria, are likely to harbour "amoeba resistant bacteria” (ARB). Among these ARB, opportunistic pathogens have been identified, some of which are known to be carriers of efflux pumps. The efflux pumps of these bacteria could thus interfere in the adaptation to soil amoebae. In order to verify this hypothesis, we first isolated and identified the amoebal population and the ARB of different soils. Among the identified ARB, Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Burkholderia cepacia are characterized by high contrast antibiotic resistance and high virulence. Interaction studies showed that S. maltophilia could multiplied in axenic amoebae and Sme efflux pumps were overexpressed. Furthermore, molecules secreted by the amoeba stimulate bacterial growth and preliminary studies of metabolic profile have shown that production of various secondary metabolites by the amoeba during the interaction with S. maltophilia could play a role in the efflux pumps expression

La co-culture d’amibes est utilisée afin d’isoler des microorganismes. Ainsi le premier virus géant,fut découvert. Cependant, les méthodes de culture sur protozoaires sont délicates et fastidieuses. De ce fait, le développement de ces cultures représente une difficulté pour les microbiologistes, limitant ainsi l’analyse d’un nombre important d’échantillons et la caractérisation de nouveaux virus. De nouvelles stratégies et des améliorations des modèles actuels sont donc nécessaires. Notre travail a été de développer de nouvelles stratégies permettant l’isolement de microorganismes associés aux amibes. Dans la 1ere partie nos travaux ont permis une amélioration des cultures avec le développement de nouveaux milieux de culture et l’utilisation ciblée d’antimicrobiens.La clé de ces stratégies est l’association des techniques rapides aux étapes améliorées de culture et leur application à un large panel de protozoaires pouvant abriter des microorganismes. Les résultats ont permis le développement d’un système d’isolement à haut débit très efficace. Nous avons notamment mis au point des techniques de tri de virus géants par cytométrie.Dans la seconde partie, nos travaux ont porté sur la description et la caractérisation des nouveaux isolats.Les résultats obtenus démontrent l’importance de poursuivre l’isolement et la caractérisation de ces microorganismes afin de mieux appréhender l’évolution de ces microorganismes, leur biotope et leur pathogénicité.De nouveaux outils sont nécessaires,notre manque d’imagination et l’absence de systèmes automatisés seront les limites aux nouvelles stratégies dans le monde de la microbiologie. / Amoebae are predators without distinction and they can also act as hosts to several different microorganisms that may coexist simultaneously. Some protozoa are sources of human pathogens where they act as reservoir of any human pathogens like Legionellae, Chlamydiaceae and others. In addition, the first giant virus, Acanthamoeba Polyphaga imivirus, was discovered using Amoeba as cell host. Since then, many other giant viruses have been isolated. For decades, amoebae were used as cell hosts in the culture- based process to isolate microorganisms, and allowed to recover new giant viruses and bacterial species from human and environmental samples. In contrast the co-culture system with protozoa is tedious and fastidious. Microbiologists are limited to routine culture methods, limiting by this the speed of screening potential samples and the efficiency of yielding new isolates. Much effort and improvement were needed. Our work consisted in the development of new strategies and techniques for the isolation of new microorganisms associated to protozoa. In the first part of this work, we described, all the improvements we brought to the protists culture system for the isolation of intracellular microorganisms especially giant viruses and Chlamydiaceae. Major improvements were based on modified culture enrichment steps, adapted culture media, and targeted use of specific drugs. The key of this new strategy was the implementation of high-throughput technologies to the ameliorated culture based systems, and the application of this later to a wide panel of protozoa used as potential host cells. These presented advances and strategies demonstrated significant time saving, and higher sensitivity than older techniques, they considerably increased the potential of collecting new environmental or clinical isolates and also new undiscovered microorganisms especially new giant virus familiesand particular Chlamydiaceae associated to amoebae. We continued to ameliorate the efficiency of the flow cytometric technology by reviewing its contributions to the virology field, then by applying it to the isolation system by sorting the new isolates as a new strategy for better genomic and proteomic analysis. In the second part of this work, we focused on the characterization of new isolates at the level of developmental cycle and genomic description. We used electron microscopy, and genome sequencing as main tools to describe our newly isolated giant viruses but also report new species of Chlamydiaceae and managed to decipher Chlamydiaceae species with a host dependent replication cycle, an issue that has not, thus far, been observed in protozoa-associated Chlamydiaceae. The strategies and results described herein show the importance of pursuing the isolation of new associatedamoebal microorganisms in order to give rise to new insights into the evolution of these microorganisms, their respective biotopes, and their potential or hidden pathogenicity. The more we need to search the more tools are needed, only our lack of imagination and appropriate automated systems will put limits on any needed strategy in the field of microbiology.

By combining laboratory experiments with field work, I have looked at the following aspects of cellular slime mould (CSM) biology: (a) the genetic structure of social groups (fruiting bodies) in the wild and its relation to the role of large mammals as dispersal agents; (b) social behaviour in clonal, intra-species polyclonal and interspecies social groups and (c) fitness-related trade-offs with respect to life history traits as a possible mechanism for coexistence and cooperative behaviour in CSMs. The major findings of this study are as follows: (a) individuals belonging to different strains of a species, different species and genera occur in close proximity, even on a speck of soil (250µm–1mm) or the same dung pat; (b) social groups formed in the wild by Dictyostelium giganteum and D. purpureum are generally multiclonal; (c) genetically diverse strains can co-aggregate and form chimaeric social groups; (d) in chimaeric social groups, strains differ in their relative sporulation efficiencies; (e) the fact that strains co-exist in spite of this may be attributable in part to trade-offs between various fitness-related traits as can be demonstrated in the case of wild isolates of D. giganteum in pair wise mixes. The Dictyostelids or CSMs are haploid, eukaryotic, soil dwelling social amoebae with an unusual life cycle (Bonner, 1967; Raper, 1984). They exist as single cells in the presence of food (bacteria, yeast, fungal spores). Once the food is exhausted, they enter the social phase of their life cycle. Approximately 102 to 106 amoebae aggregate at a common collection point and form a starvation resistant structure called the fruiting body. In many species a fruiting body is made up of an aerial stalk of dead cells and a ball of viable spores on top. In other CSM species (not part of this study), all amoebae in a fruiting body differentiate into spores and the stalk is an extracellular secretion. The CSM life cycle raises fundamental questions related to the evolution of an extreme form of ‘altruism’ in the form of reproductive division of labour in social groups. The spore–stalk distinction in the CSMs is analogous to the germ–soma distinction in metazoans, although, the CSMs achieve multicellularity not by repeated divisions of a zygote but via the aggregation of many cells which may or may not be clonally related (Bonner, 1982; Kaushik and Nanjundiah, 2003). Social behaviour in the CSMs offers interesting parallels to what is seen in the social insects (Gadagkar and Bonner, 1994). The origin and maintenance of ‘altruism’ has been a long-standing issue in sociobiology. Because of their simple life cycle and experimental tractability, the CSMs are ideal for studying the evolutionary origin and maintenance of social behaviour, in particular of ‘altruistic’ behaviour. By elevating spores above soil level, stalk cells, protect them from noxious compounds and predators present in soil and also facilitate their passive dispersal. In the course of doing so they die. The death of stalk cells appears to be an extreme form of altruism. Knowledge of the genetic structure of social groups and populations including patterns of kinship is essential for modelling the evolution of ‘altruism’. Thus, it is important to understand the genetic structure of CSM social groups in the wild. For this, social groups (fruiting bodies) of CSMs were isolated from undisturbed forest soil of the Mudumalai forest reserve in South India. Soil and animal dung samples were brought to the laboratory and quasi-natural social groups were generated by inoculating the samples on non-nutrient agar. The fruiting bodies from various CSM species were formed by these isolates. Since soil and dung samples were not perturbed in any way, the fruiting bodies were formed as they would have in nature. When compared to soil, dung samples contained a higher CSM diversity and more CSM propagules. The presence of CSMs in fresh animal dung makes it likely that they were transported and dispersed over long distances through the gut of these animals. Such dispersal is likely to be preceded by a thorough mixing of spores in the gut. That increases the probability of co-occurrence of different genotypes in a social group. This possibility was confirmed by genetically characterizing spores in social groups of Dictyostelium giganteum and D. purpureum collected from the wild. Random amplification of polymorphic DNA (RAPD), a simple and reliable molecular technique, was used for genotyping spores within a fruiting body. 17 fruiting bodies (8 from animal dung and 9 from soil) were studied. 15 out of 17 (9 out of 11 of D. giganteum and 6 out of 6 D. purpureum) were polyclonal; the minimum number of distinct clones in a single fruiting body was 3 to 7 (animal dung) and 1 to 9 (soil). Therefore in D.giganteum and D. purpureum, chimaeric social groups seem to be the norm. This suggests that other species of CSMs form intra-species chimaeric social groups in wild, though clonal fruiting bodies occur too. The next objective of this thesis was to test whether genetic heterogeneity had functional consequences. That is, when different strains come together in an aggregate, do they contribute equally to the reproductive (spore) and non-reproductive (stalk) pathways? Amoebae of different clones (strains) of D. giganteum or D. purpureum were mixed and developed together and the number of spores formed by each strain was counted. These experiments confirmed that strains of D. giganteum or D. purpureum can aggregate together and form chimaeric fruiting bodies. The ability to mix (measured as the frequency of chimaerism) depended on the strains used and varied from one mix to another. One strain was often found to ‘exploit’ the other during sporulation, that is, it formed more spores than its expected share. Despite this, strains are found in very close proximity in the soil, which raises an important question: when one strain is more efficient at sporulating than other, how can the two co-exist stably? To investigate what might lie behind the stable co-existence of strains, I studied various fitness-related traits in the life cycle of D. giganteum. They included the rate of cell division, the time taken to go through multicellular development, the efficiency of slug migration through various depths of soil and the probability of differentiation into a spore. Measurements were carried out on strains taken separately and on their pair wise mixes. Five different D. giganteum wild strains (46a3, 46d2, 48.1a1, F5 and F16) were used. All were isolated from the Mudumalai forest (India). 46a3 and 46d2 came from soil within 10 cm of each other, 48.1a1 from soil about 200m away from 46a3; and F5 and F16 from the same fruiting body (Kaushik et al., 2006; Sathe et al., 2010). Members of a pair differed significantly in the measured fitness-related traits. For example, in the case of 48.1a1 and 46d2, 48.a1 grew faster than 46d2 both individually and in a mix. After starvation, 48.1a1 formed fruiting bodies faster than 46d2; a mix of the two developed at the rate of the faster member, implying that the slower one (46d2) gained from the association with 48.1a1. During slug migration, slugs formed by 48.1a1 came up through a higher depth of soil than 46d2 slugs and did so earlier. Chimaeric slugs were like the more efficient member, 48.1a1, in terms of the maximum depth of soil that was covered, but like the less efficient member, 46d2, in terms of the time taken for slugs to be seen on the soil surface. 48.1a1 seems to have an advantage over 46d2 in all these respects. However, during sporulation in chimaeras, 48.1a1 formed relatively fewer spores than 46d2. Similar trade-offs were seen in all mixes. F5 and F16 displayed an unexpected feature during sporulation; the spore-forming efficiency of either strain depended on its proportion in the initial mix in a frequency-dependent manner that was consistent with a stable equilibrium. Thus, trade-offs between different fitness-related traits contribute to the co-existence of strains. Next, I studied interactions between members of different CSM species. Several species of CSMs were isolated from the same environment (Sathe et al., 2010); a question of interest was to see if amoebae of different species came together to form a chimaeric multicellular body. Five strains (two D. purpureum and three D. giganteum) were used in this study. Amoebae of D. giganteum and D. purpureum co-aggregated. However, there were factors that caused amoebae of the two species to sort out thereafter. The extent of segregation differed between strains, a characteristic that inter-species mixes share with intra-species mixes. In conclusion, the ability of cellular slime moulds to form multiclonal social groups in the wild suggests that one should look to factors in addition to close relatedness to understand the evolution of CSM social behaviour. The existence of fitness-related trade-offs between different traits indicates that individual-level selection can also contribute to the maintenance of chimaeric social groups.

Les amibes sociales sont des organismes eucaryotes présents dans le sol de presque toutes les zones climatiques. Ils sont remarquables pour leur passage d'un état unicellulaire à un état multicellulaire en réponse à la carence en nutriments. En période de carence, des millions de cellules forment des agrégats qui constituent chacun un nouvel organisme multicellulaire, contenant des spores, cellules reproductives, et des cellules de tige, cellules mortes qui favorisent la dispersion des spores. Ce comportement, de par le coût payé par les cellules de tige, a permis d'utiliser les amibes sociales en tant que système-modèle pour aborder des questions majeures de l'évolution de la coopération et de la multicellularité. Dans cette étude, nous examinons trois aspects différents du comportement des amibes sociales; agrégation, non-agrégation et compétition, et nous analysons comment ces aspects contribuent à notre compréhension de la coopération chez les amibes et systèmes microbiens en général.Nous avons exploré le fait bien connu mais négligé qu'en phase de carence nutritive, une fraction des cellules ne participent pas à la formation des agrégats pas et ne sont pas engagées dans le développement multicellulaire. Nous décrivons les facteurs phénotypiques et génétiques qui déterminent la fraction de cellules hors-agrégats chez D. discoideum. Les deux stratégies, d'agrégation et de non-agrégation, sont coûteuses ou bénéfiques d'un point de vue évolutif selon la durée de la phase de carence. Nous avons développé un modèle pour simuler ce processus. Nous proposons que le partitionnement de la population dans des états unicellulaire et multicellulaire est adaptative dans des environnements fluctuants avec une durée imprévisible des périodes de carence nutritive. Les amibes sociales sont donc situées à l'intersection de deux thèmes émergents en évolution microbienne, la coopération et le "placement des paris".Dans la deuxième partie, nous proposons un nouveau cadre pour aborder les observations a priori contradictoires de la diversité génétique dans les populations naturelles d'amibes sociales et une faible diversité nécessaire pour la coopération. Nous proposons que le cycle de vie complexe des amibes sociales fournit plusieurs points de compétition qui peut servir à la fois comme stabilisateur de la diversité et de la coopération. Nous explorons cette hypothèse expérimentalement avec un modèle en analysant la compétition entre 6 isolats naturels de D. discoideum. Notre simulation-modèle indique que la compétition à différents stades du cycle de vie peut conduire à l'exclusion des "gagnants sociaux". Toutefois nous n'avons pas réussi à expliquer la coexistence à long terme de souches génétiquement distinctes. Bien que préliminaires, nos résultats soulignent l'importance d'intégrer l'écologie des espèces dans les études de coopération microbienne.Enfin, nous nous concentrons sur une nouvelle dynamique d'agrégation chez P. pallidum observée dans notre laboratoire. L'agrégation est un processus au niveau de la population au cours duquel la population se divise en nombreuses sous-populations (agrégats) qui font face à la sélection de manière indépendante. Un tel fractionnement de la population peut avoir de fortes conséquences évolutives du point de vue de la coopération qui n'ont pas encore été explorées expérimentalement. Nous décrivons la dynamique des populations qualitativement et proposons plusieurs mesures quantitatives de partitionnement de la population en agrégats. Nos résultats préliminaires suggèrent qu'il existe une préférence pour les agrégats d'une certaine taille, mais qu'il n'existe aucune organisation spatiale des agrégats. / Social amoebae are eukaryotic organisms that inhabit soil of almost every climate zone. They are remarkable for their switch from unicellularity to multicellularity as an adaptation to starvation. When starved, millions of single cells aggregate and form a multicellular fruiting body, which contains reproductive spore cells and dead stalk cells, which help in spore dispersion. This costly behavior made social amoebae a model system for addressing major questions of the evolution of cooperation and multicellularity. In this study we look at three different aspects of social amoebae behavior; aggregation, non-aggregation and competition, and ask how they contribute to our understanding of cooperation in social amoebae and microbial systems in general.We explored the known but neglected observation that, upon starvation, not all cells aggregate and engage in multicellular development. We describe phenotypically and genetically non-aggregating cell proportion in D. discoideum species. Both aggregating and non-aggregating strategy are costly or beneficial depending on duration of starvation. With our computational model we propose that partitioning the population into unicellular and multicellular states is adaptive in fluctuating environments with unpredicted duration of starvation periods. Social amoebae may therefore lie at the intersection of cooperation and bet-hedging. In the second part, we provide a new framework for addressing the contrasting observations of high genetic diversity in natural populations of social amoebae and experimentally suggested low diversity-high relatedness required for cooperation. We propose that complex life cycle of social amoebae provides multiple competition points that can possibly play an important role in maintaining diversity and cooperation. We explore this experimentally and computationally by looking at competition over the whole life cycle between 6 natural isolates of D. discoideum. Our simulation model indicates that competition at different stages of the life cycle can lead to exclusion of “social winners”. Though we failed to explain strain coexistence. Although preliminary, our results emphasize the importance of integrating species ecology in cooperative studies.Finally, we focus on a new aggregation dynamics in P. pallidum species observed in our lab. Aggregation is a population level process during which population gets divided into numerous subpopulations/aggregates that face selection independently. Such population partitioning can have strong evolutionary consequences on cooperation that have not yet been explored experimentally. We describe the population dynamics qualitatively and propose several quantitative measurements of population partitioning into aggregates. Our preliminary results suggest that there is a preference for aggregates of certain size, but there is no spatial organization of aggregates.

Both testate amoebae and fungi are common inhabitants of coniferous litter. Their interactions in this environment were rarely studied, although they reach high biodiversity and can play a significant role in nutrient cycling in this environment. In this study, a cultivation of litter needles in the damp chambers was used to investigate interactions between fungi and testate amoebae. Observation of spruce litter needles in environmental scanning electron microscope was used to better characterize testate amoebae communities directly on the needles. Additionally, two experiments changing the biotic conditions in the microcosm were used to follow a principle of the interactions. Three species of testate amoebae from litter needles were able to colonize the filter paper on the bottom of the damp chambers. Occurrence of Phryganella acropodia and Assulina muscorum on the filter paper was significantly fuelled by the presence of mycelium. Assulina muscorum was associated with the fungal spores and Arcella discoides was attracted by sporulating colonies of Cladosporium spp. in the damp chambers. By contrast, no association of putatively mycophagous Phryganella acropodia with fungal spores was observed. Arcella discoides was attracted both by live and death mycelium in additional experiment. Interestingly, the...