PhD (Zoology) / Department of Zoology / Ecosystem services and disservices are important features in agro-ecosystems and both can have relevant economic impacts on farmers. While there has been much research on the value of ecosystem services, especially that of pest control by bats and birds, ecosystem disservices are often overlooked or estimated separately. Both, services and disservices, however, change with landscape and may be supported by natural vegetation. It is hence inevitable to assess them together to uncover their relative value and importance. Habitat loss and fragmentation are major threats to biodiversity and ecosystem services. Agricultural intensification can cause habitat loss and can negatively affect natural ecosystems and their services. However, the removal of natural vegetation can also reduce ecosystem disservices that origin from natural areas. This study focused on the economic trade-off between biocontrol by bats and birds and crop raiding by vervet monkeys in South African macadamia orchards, using vertebrate exclusion experiments. Crop quality and yield were assessed and extrapolated to income values. The study furthermore focused in more detail on the impact of orchard edge habitat and farm management on bats as ecosystem service providers as well as the arthropod community, which can provide both services (pollination, biocontrol) as well as disservices (crop damage). The macadamia industry is highly profitable and plantings are expanding worldwide. South Africa is currently the largest macadamia producer in the world and the study area, Levubu, holds some of the oldest macadamia plantings in the country. The subtropical climate and high annual rainfall make this area an intensively farmed landscape. Levubu lies at the foothills of the Soutpansberg, a centre of biodiversity and patches of natural vegetation wind through orchards of macadamia, avocado, banana and timber. Insect pest pressure is high in the macadamia industry, with several stink bug species (Hemiptera) and two moth species (Lepidoptera) accounting for most of the damage to the crop. Chapter One provides a detailed overlook over the South African macadamia industry, describes the major pest insect species and their impact on the crop and highlights the role of ecosystem services and disservices and the potential importance of remaining natural areas. Over three consecutive seasons, between September 2015 and May 2018, bats, birds and monkeys were excluded, using cages with nets, from a total of 96 macadamia trees. Four treatments were applied (Full, Day, Night, Control) to distinguish between effects of the different functional groups. Exclusions took place at orchard edges with natural or human-modified edge habitat to assess the impact of vicinity to natural vegetation on pest control and crop raiding. Results presented in Chapter Two showed that biocontrol by bats and birds was stronger near natural vegetation and significantly impacted crop quality as well as yield. Effects of bats and birds were still significant at human-modified edges, whereas crop raiding by monkeys is limited to the natural orchard edges. The economic impact analysis shows that the benefits of biocontrol through bats and birds outweighed the income losses due to crop-raiding monkeys. The value of bats and birds was as high as USD 5000 per hectare per year through prevented insect damage and a 60% crop increase. Crop-raiding affected the yield, which dropped by about 26% and resulted in losses of about USD 1500 per hectare. These results highlight the importance of integrating natural areas into agricultural landscapes, even if they incur the risk of being disadvantageous. Farmers need to be made aware of the enormous economic benefits. Effective crop raiding mitigation strategies still need to be researched to avoid negative association with natural areas. These can, however, possibly also limit access for biocntrol agents, like bats and birds and thereby reduce their economic impact. Especially the removal of natural areas or establishment of buffer zones of unpalatable crops between agricultural land and forests is not advisable. Integrative research in agro-ecosystems on trade-offs between a variety of ecosystem services and disservices is necessary in future, rather than assessing them separately. The bat community on macadamia farms was monitored monthly and compared between orchard edges. Stationary bat detectors were placed at each set of exclosures for two consecutive nights a month, automatically recording from sunset to sunrise. Recorded bat calls were identified manually to species level and activity converted to Miller’s activity index, which counts the number of active minutes per species per night. We analysed the activity per feeding guild over season and landscape setting, estimated species richness and diversity (Hill’s numbers) and species turnover between farms, season and landscape setting, using diversity partitioning. The bat community was mostly influenced by seasonality with season turn-over accounting for 21% of total bat diversity (21.25 species). Edge habitat on the other hand only accounted for 5%. We found higher species richness and activity in the high season compared to the low. While there was no difference in diversity in the high season between the two orchard edge types, species diversity at the human-modified edge was lower than in the natural during the low season. Natural habitat might therefore be more important in the low season, while its effect is overwritten by high food availability during the high season. Clutter-edge species furthermore were more active at the natural orchard edges than open-air feeders, which were in turn more active at human-modified edges. Both activities dropped significantly during the low season. Chapter Three concludes that the macadamia landscape is able to support a high bat species diversity, which is affected by seasonal differences, probably due to food availability. Natural areas are important to be maintained to support sensitive species, relying on clutter habitat and natural roosts. Habitat features as well as farm management can also influence arthropod communities. Visual observations took place each month for 20 minutes per tree. Observations were counted and identified to at least order level. Chapter Four concentrates on the impact of orchard edge habitat and insecticide treatment on honey bees (83% of Hymenoptera observations) abundance. Honey bees were the only taxon significantly responding to both variables, with higher abundance close to natural areas and increasing abundance with increasing time since pesticide application. Honey bees furthermore seemed to recover slightly quicker from population crashes after insecticide treatments at natural edges than they did at human-modified edges. Hymenoptera can be highly beneficial to macadamia farmers, as farmers are heavily relying on pollination by honey bees and parasitoids are known to feed on major macadamia pest insect species. These ecosystem service providers were mostly affected by habitat and management practices, which may compromise their ecosystem service provision. Although commercial bee hives are exposed throughout macadamia orchards, they do not appear to fully replace the pollination and biocontrol services provided by feral species, which can be enhanced through resource supplementation by patches of nearby natural vegetation. Chapter Five concludes with emphasizing the general importance of natural vegetation in landscape planning of agricultural areas. This study proved confidently that benefits through ecosystem services largely outweigh negative impacts of ecosystem disservices, both stemming from these natural areas. Through the inclusion, maintenance and restoration of such patches of natural vegetation, farmers can potentially increase the value and effectiveness of biocontrol by bats and birds or other services. Seasonality largely determined bat species diversity, and a general high species diversity was observed in the macadamia landscape, possibly due to a certain degree of landscape heterogeneity and high food availability. Activity patterns of clutter-edge and open-air feeding bats varied with edge habitat, while clutter-dependent bat species / NRF

Le nombre de virus détectés chez les chauves-souris est en augmentation, la plupart étant des virus à ARN. L'identification chez différentes espèces de chauves-souris, de virus ayant été responsables d'épidémies voire de pandémies chez l'homme (coronavirus agent du SRAS, virus Nipah et Hendra, filovirus Ebola et Marburg) a fait prendre conscience du risque que peuvent présenter ces animaux pour la santé humaine, ainsi que des possibilités réelles d'émergence de nouvelles pathologies dans les années futures. Ce travail avait donc pour objectifs: (i) d'identifier et caractériser les virus circulant au sein des populations de chauves-souris d'Afrique Centrale et (ii) d'explorer et d'identifier des déterminants bioécologiques, qui pourraient expliquer la richesse virale observée chez certaines espèces de chauves-souris rencontrées en Afrique tropicale forestière. A partir d'un total de 3472 individus testés, représentant 16 espèces provenant du Gabon, de la République du Congo et de la République Centrafricaine, nous avons confirmé la présence du virus Marburg chez les roussettes d'Egypte (Rousettus aegyptiacus) au Gabon, et mis en évidence des séquences virales de paramyxovirus très proches de virus zoonotiques émergents (les virus Nipah et Hendra) et réémergents (virus des oreillons) chez des chauves-souris frugivores. Des séquences de nouveaux coronavirus, flavivirus et paramyxovirus ont été également identifiées. Par ailleurs, la fragmentation de l'aire de distribution et le type de gîte ont été identifiés comme des déterminants de la richesse virale chez 15 espèces de chauves-souris d'Afrique Centrale. Les chauves-souris en Afrique Centrale seraient donc des réservoirs de virus apparentés à des virus pathogènes pour l'homme. Ces animaux pourraient donc être à l'origine de l'émergence des encéphalites à hénipavirus en Afrique et de la réémergence de certaines maladies humaines comme les oreillons, la rougeole. Des recherches futures s'orienteront vers la poursuite de la caracterisation génétique des virus détectés chez les chauves-souris d'Afrique Centrale et la détermination du risque zoonotique associé à ces virus. Des études écologiques seront également réalisées pour identifier les facteurs de risque d'émeregence des virus de chauves-souris potentiellement pathogènes pour l'homme. / The number of viruses détected in bats is growing, the most common are RNA viruses. The identification in different bat species of viruses that cause major epidemics or pandemics in human such as SARS coronavirus, Nipah and Henda viruses, the filoviruses Ebola and Marburg has raised awareness of potential risk that these animals may present to human health, as well as real possibilities of development of new diseases in future years. This work had two objectives: (i) to identify and characterize the viruses circulating in populations of bats in Central Africa and (ii) to explore and identify bioecological factors that could explain the viral richness observed in some bats species seen in tropical Africa forest. From 3472 individuals tested accounting for 16 species from Gabon, Congo and the Central African Republic, we established the presence of Marburg virus in Egyptian fruit bats (Rousettus aegyptiacus) in Gabon and identified viral sequences of paramyxoviruses close related to emerging and re-emerging zoonotic paramyxoviruses (Nipah virus, Hendra viruses and mumps virus) in fruit bats. Sequences of novel coronaviruses, paramyxoviruses and flaviviruses have also beenidentified. Moreover, the fragmentation of the range and roost type have been identified as determinants of viral richness in 15 bats species of Central Africa. Bats in Central Africa thus would be reservoirs of viruses related to viruses pathogenic for humans. These animals would lead to the emergence of encephalitis Henipavirus in Africa and the reemergence of certain human diseases such as mumps, measles. Further research will be conducted to continue the genetic characterization of viruses detected from bats in Central Africa and to determine the zoonotic risk associated with these viruses. Ecological studies will also be performed to identify the risk factors for the emergence of bats viruses potentially pathogenic for humans.

La leptospirose est une zoonose d’importance médicale majeure dans les îles du Sud-Ouest de l’Océan Indien (SOOI) dont certaines enregistrent des incidences parmi les plus élevées au monde. Durant la dernière décennie, les données épidémiologiques moléculaires obtenues avec une approche « One Health » ont mis en évidence une grande diversité de lignées de leptospires ainsi que différentes chaines de transmission sur les différentes îles de la région. Les données moléculaires montrent la présence de leptospires pathogènes et de réservoirs animaux introduits ou endémiques de cette région. La distribution de ces différentes lignées de leptospires est associée à (i) un contraste épidémiologique incluant des différences dans la sévérité des cas humains et (ii) des niveaux de spécificité d’hôtes différents selon les leptospires considérés. Plus particulièrement, les leptospires endémiques du SOOI semblent être moins pathogènes chez les humains et montrent une plus forte affinité pour leur réservoir que les leptospires cosmopolites. Pour compléter nos connaissances sur l’histoire évolutive des leptospires du SOOI, nous avons produit des données provenant de chauves-souris de l’Afrique de l’Est. Ces données confirment la spécificité de certaines lignées de leptospires envers leurs hôtes chiroptères et suggèrent que les chauves-souris d’Afrique ont colonisé Madagascar tout en étant infectées par leurs leptospires. Afin de mieux comprendre le rôle des différents leptospires dans l’épidémiologie régionale de la leptospirose, nous avons mesuré la pathogénicité de trois souches de leptospires retrouvées dans cette région à l’aide d’un modèle hamster. Des souches de Leptospira mayottensis et Leptospira borgpetersenii ont été isolés respectivement de Tenrec ecaudatus (tenrec) et Triaenops menamena (chauve-souris), deux mammifères endémiques du SOOI. Une souche de Leptospira interrogans, dont le génotype est retrouvé dans la majorité des cas humains graves à la Réunion, a été isolée de Rattus rattus (rat). En cohérence avec les données épidémiologiques humaines de Mayotte et de La Réunion, les leptospires endémiques se sont révélées être significativement moins pathogènes que la souche L. interrogans. La spécificité d’hôte des deux souches isolées de mammifères endémiques a été mise à l’épreuve par des infections expérimentales de Rattus norvegicus, connu comme un réservoir important de leptospires. Les rats ont été infectés avec les trois isolats précédemment utilisés. Les rats infectés par les souches endémiques n’ont pas développé d’infection rénale chronique contrairement à la souche cosmopolite. Ces résultats montrent que la spécificité d’hôte des leptospires endémiques observée in natura est probablement due à des facteurs génétiques plutôt qu’à des facteurs écologiques, comme un manque de contacts physiques entre les réservoirs animaux endémiques et introduits. Enfin, le séquençage complet de souches de leptospires du SOOI a été réalisé afin d’identifier des caractéristiques génétiques pouvant être associées à la pathogénicité et la spécificité d’hôte des leptospires pathogènes. Une classification précise de souches de leptospires du SOOI a pu être réalisée sur la base des génomes complets. La comparaison de ces génomes a permis d’identifier des gènes spécifiques à un groupe ou une espèce de leptospires. Cependant des modifications génomiques complexes rendent difficiles l’identification de caractéristiques génomiques responsables d’un phénotype particulier tel que la virulence ou la spécificité d’hôte. / Leptospirosis is a zoonosis of main medical concern on several islands of southwestern Indian Ocean (SWIO), some of which recording among the highest human incidence worldwide. Over the last decade, molecular epidemiology investigations carried out under a One Health framework have revealed a wide variety of Leptospira lineages and distinct transmission chains throughout the islands of the region. These islands are home to pathogenic Leptospira lineages and animal reservoirs that are either introduced or endemic to the SWIO region. Interestingly, the regional distribution of Leptospira diversity is associated with (i) a contrasted severity of human cases and (ii) distinct levels of specificity of Leptospira towards their mammalian hosts. Specifically, endemic Leptospira appear less pathogenic in humans and display higher specificity towards their animal reservoirs than their cosmopolitan counterparts. To complete the dataset of Leptospira diversity in the SWIO region, we produced data from bats of eastern Africa. Results support the previously observed pattern of host specificity of Leptospira towards their bats hosts and, overlaid upon the biogeographic history of Malagasy bats, suggest that these volant mammals have colonized Madagascar from continental Africa while hosting pathogenic Leptospira. To better understand the role of distinct Leptospira lineages in the contrasted epidemiology observed in the SWIO, we investigated the pathogenicity of three Leptospira isolates from this region using a hamster model. Leptospira mayottensis and Leptospira borgpetersenii isolates were obtained from Tenrec ecaudatus (tenrec) on Mayotte and Triaenops menamena (bat) in Madagascar, respectively, both mammals endemic to the SWIO region. A Leptospira interrogans strain, which genotype has been reported in the majority of human acute cases on La Réunion, was isolated from the introduced Rattus rattus (rat). In keeping with a distinct severity of the disease on Mayotte and La Réunion, endemic bat-borne and tenrec-borne Leptospira were significantly less pathogenic than the control cosmopolitan rat-borne isolate. The host specificity of the isolates obtained from endemic hosts was addressed using experimental infection of Rattus norvegicus, a known reservoir of pathogenic Leptospira. This animal model was challenged with all three isolates and mostly failed in supporting chronic infection with bat-borne and tenrec-borne Leptospira. Hence, the strong host-specificity of endemic Leptospira toward their hosts observed in the wild likely results from genetic determinants shaped by long-term co-evolutionary processes rather than from ecological constraints such as a lack of physical contact between introduced and endemic animal reservoirs. Finally, we undertook full genome sequencing of regional strains in order to highlight genomic features that may be associated with virulence and host specificity. Whole genome sequencing allowed the accurate classification of Leptospira isolates obtained on SWIO islands. Comparative genomics allowed to identify genes specific to a group or species of Leptospira but complex changes in Leptospira genome make difficult the identification of genomic elements responsible for specific traits such as virulence and host specificity.

O estudo de prevalência do vírus rábico foi realizado em uma amostra de morcegos capturados na Mata Atlântica da região sudeste do Brasil. Os morcegos são um dos principais reservatórios silvestres do vírus rábico. No Brasil existem aproximadamente 144 espécies de morcegos, e pouco se sabe sobre a circulação do vírus rábicos nessas espécies. Foram realizadas estimativas – com duas metodologias de isolamento - para detectar a presença do vírus rábico na população estudada. Os resultados foram obtidos pelo cruzamento entre a variável infectividade (presença de vírus rábico) e as variáveis epidemiológicas (espécies de morcegos, sexo, idade, local de captura ). Observou-se que o método de isolamento que utiliza as células McCoy isolou com maior facilidade vírus de morcegos insetívoros, além de apresentar maior capacidade de detectar a infecção na fase latente (subclínica). Já as células N2A foram mais eficientes na detecção do vírus rábico em morcegos hematófagos D. rotundus. As duas metodologias utilizadas apresentaram maior proporção de isolamento do vírus rábico em morcegos insetívoros, nectarívoros e fitófagos. Tais resultados sugerem que os morcegos insetívoros desempenham importante papel na manutenção do vírus no reservatório cuja população foi estudada. Também foi possível constatar que a circulação do vírus ocorre inter e intra-espécies, mas estudos especificamente desenhados para avaliar esse aspecto devem ser implementados. / The prevalence study of the rabies virus was carried out in a sample of bats captured in the Brazilian southeastern São Paulo. Bats are one of the main wild reservoirs of the rabies virus. Brazil holds 144 species of bats and little is know about the circulation of such virus in these species. Two metodologies were used for the estimates of the presence of the rabies virus in the captured in the Parque Estadual Intervales. The results were obtained crossing the variable (presence of rabies virus) with epidemiological variables (bat species, sex, age, site of capture). The McCoy cell line method proved isolating more easily the virus of insectivorous bats besides presenting more capability of detection of infection in the latent phase (sub-clinic phase). On the other hand the N2A cell line were more efficient in detecting the rabies virus in D. rotundus hematophagous bats. It was also observed that for both cells the insectivorous, nectarivorous and phytophagous bats presented higher rabies virus isolation proportion. These results suggest that insectivorous bats play in important role in the maintence of the virus in this reservoir. Although could also be observed that the circulation of the virus occurs intra and inter species, but studies specially designed to asses this issue must be re-evaluated.

O estudo de prevalência do vírus rábico foi realizado em uma amostra de morcegos capturados na Mata Atlântica da região sudeste do Brasil. Os morcegos são um dos principais reservatórios silvestres do vírus rábico. No Brasil existem aproximadamente 144 espécies de morcegos, e pouco se sabe sobre a circulação do vírus rábicos nessas espécies. Foram realizadas estimativas – com duas metodologias de isolamento - para detectar a presença do vírus rábico na população estudada. Os resultados foram obtidos pelo cruzamento entre a variável infectividade (presença de vírus rábico) e as variáveis epidemiológicas (espécies de morcegos, sexo, idade, local de captura ). Observou-se que o método de isolamento que utiliza as células McCoy isolou com maior facilidade vírus de morcegos insetívoros, além de apresentar maior capacidade de detectar a infecção na fase latente (subclínica). Já as células N2A foram mais eficientes na detecção do vírus rábico em morcegos hematófagos D. rotundus. As duas metodologias utilizadas apresentaram maior proporção de isolamento do vírus rábico em morcegos insetívoros, nectarívoros e fitófagos. Tais resultados sugerem que os morcegos insetívoros desempenham importante papel na manutenção do vírus no reservatório cuja população foi estudada. Também foi possível constatar que a circulação do vírus ocorre inter e intra-espécies, mas estudos especificamente desenhados para avaliar esse aspecto devem ser implementados. / The prevalence study of the rabies virus was carried out in a sample of bats captured in the Brazilian southeastern São Paulo. Bats are one of the main wild reservoirs of the rabies virus. Brazil holds 144 species of bats and little is know about the circulation of such virus in these species. Two metodologies were used for the estimates of the presence of the rabies virus in the captured in the Parque Estadual Intervales. The results were obtained crossing the variable (presence of rabies virus) with epidemiological variables (bat species, sex, age, site of capture). The McCoy cell line method proved isolating more easily the virus of insectivorous bats besides presenting more capability of detection of infection in the latent phase (sub-clinic phase). On the other hand the N2A cell line were more efficient in detecting the rabies virus in D. rotundus hematophagous bats. It was also observed that for both cells the insectivorous, nectarivorous and phytophagous bats presented higher rabies virus isolation proportion. These results suggest that insectivorous bats play in important role in the maintence of the virus in this reservoir. Although could also be observed that the circulation of the virus occurs intra and inter species, but studies specially designed to asses this issue must be re-evaluated.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-26T12:52:36Z No. of bitstreams: 1 2015 - William Douglas de Carvalho.pdf: 2195014 bytes, checksum: b6deab05010b9d498b671e9f59a39141 (MD5) / Made available in DSpace on 2017-01-26T12:52:36Z (GMT). No. of bitstreams: 1 2015 - William Douglas de Carvalho.pdf: 2195014 bytes, checksum: b6deab05010b9d498b671e9f59a39141 (MD5) Previous issue date: 2015-12-09 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Many world hotspots for conservation are located in mountains, which are considered priority areas for preservation of plants and vertebrates species. Species that occur in these locations have functional traits that favor the colonization of forests in high areas. Furthermore, we know little about the biology of these species along elevational gradients, for example, reproductive rate. Thus, the objectives of this thesis were divided into two sections, namely: (1) verify how the species and the different Phyllostomidae guilds vary in richness along an altitudinal gradient and identify the functional traits that facilitate the colonization of forests in high areas, and (2) determine if the reproductive rate of fruit bats Phyllostomidae varies locally and if this variation can be explained by local responses to variations in the abundance of resources. For this, we compiled studies in the Atlantic Forest of southern and southeastern Brazil through database (Capes bank of thesis; Scielo; Web of Science), journals and the database of Laborat?rio de Diversidade de Morcegos (LADIM) of Instituto de Biologia of Universidade Federal Rural do Rio de Janeiro (UFRRJ). After the selection of 35 different species of Phyllostomidae bats of 45 different locations, where the altitude varies between 60 and 2.450 m.a.s.l., 10 functional traits for each species were selected: trophic guild (GT), trophic level (NT), relative wing load (CRA), aspect ratio (AA), degree requirement forest habitat (GEHF), using natural cavities (UCN), southern latitudinal limit (LLS), body mass (MC), forearm size (TA) and body size (TC). The selected functional traits were related to an altitudinal index, calculated for each species, and those with greater correlation were chosen to be the best model to describe and determine the main functional traits that lead the species to colonize forests in high areas. Furthermore, we considered species with over 300 captures (Carollia perspicillata, Sturnira lilium e Artibeus spp.) to verify the effect of altitude on their reproductive rate. We found that there was a decline in the abundance and richness of Phyllostomidae species in high altitudes, with a decline in the abundance of fruit bats and increase of hematophagous species. The nectarivorous bats, gleaners and omnivore bats were not affected by altitude. The results also indicated that the forest habitat requirements degree was the main functional trait that leads the species to colonize forests in high areas, and the use of natural cavities and southern latitudinal limit could be relevant to colonize. All bat species had a higher reproductive rate in months of major rainfall (October to March). Sturnira lilium was the species that has the highest reproductive rate in high altitudes, which coincides with the area where there is a greater wealth and abundance of Solanum sp. Artibeus sp. had a higher reproductive rate in medium and low altitudes, where there is a greater richness of Ficus species and Carollia perspicillata showed no altitudinal preference for reproduction. At the areas with larger bat abundance, more reproducing females were observed, as these sites appear to have a higher amount of resources. The conservation of the whole altitudinal gradient with the possible implementation of conservation units is necessary, because bat species occur in greater abundance at different altitudes and reproduce at different altitudes. / Em ?reas montanhosas localizam-se muitos hotspots mundiais para conserva??o, sendo que esses locais s?o considerados ?reas priorit?rias para preserva??o de esp?cies de plantas e vertebrados. Esp?cies que ocorrem nesses locais possuem tra?os funcionais que propiciam a coloniza??o de florestas em ?reas elevadas. Al?m disso, pouco ? conhecido sobre a biologia dessas esp?cies em gradientes altitudinais, como por exemplo sua taxa reprodutiva. Sendo assim, os objetivos da presente tese foram divididos em dois cap?tulos, sendo eles: (1) verificar como as esp?cies e as diferentes guildas de Phyllostomidae variam ao longo de um gradiente altitudinal e identificar que tra?os funcionais facilitam a coloniza??o de florestas em ?reas elevadas e (2) determinar se a taxa reprodutiva de morcegos Phyllostomidae frug?voros varia localmente e se esta varia??o pode ser explicada pelas respostas locais ? varia??o na abund?ncia de recursos. Para isto, compilamos estudos realizados na Floresta Atl?ntica do sul e sudeste do Brasil atrav?s de banco de dados (banco de teses da CAPES; Scielo; Web of Science), revistas especializadas, e do banco de dados do Laborat?rio de Diversidade de Morcegos (LADIM) do Instituto de Biologia da Universidade Federal Rural do Rio de Janeiro (UFRRJ). Ap?s a sele??o de 35 diferentes esp?cies de morcegos Phyllostomidae de 45 diferentes localidades, onde a altitude variou entre 60 e 2.450 m de altitude, selecionamos 10 tra?os funcionais para cada esp?cie: guilda tr?fica (GT), n?vel tr?fico (NT), carga relativa da asa (CRA), aspecto da asa (AA), grau de exig?ncia de habitat florestal (GEHF), utiliza??o de cavidades naturais (UCN), limite latitudinal sul (LLS), massa corporal (MC), tamanho do antebra?o (TA) e tamanho corporal (TC). Os tra?os funcionais selecionados foram relacionados com um ?ndice altitudinal, representando a altitude em que a esp?cie ? mais abundante, e aqueles tra?os funcionais que apresentaram maior correla??o foram submetidos a escolha de melhor modelo para descrever quais foram os principais tra?os funcionais que levam as esp?cies a colonizarem florestas em ?reas elevadas. Al?m disso, consideramos as esp?cies com mais de 300 capturas (Carollia perspicillata, Sturnira lilium e Artibeus spp.) para verificar o efeito da altitude sobre sua taxa reprodutiva, relacionando a propor??o de animais em reprodu??o com a altitude. Encontramos uma queda na abund?ncia e riqueza de esp?cies de Phyllostomidae em altitudes elevadas, com queda na abund?ncia de frug?voros e aumento de esp?cies de h?bitos hemat?fagos. As guildas de nectar?voros, catadores e on?voros n?o foram influenciadas pela altitude. Nossos resultados tamb?m indicaram que o grau de exig?ncia de habitat florestal foi o principal tra?o funcional que leva as esp?cies a colonizarem florestas em ?reas elevadas, sendo que a utiliza??o de cavidades naturais e o limite latitudinal sul tamb?m podem ser relevantes para a coloniza??o. Todas as esp?cies de morcegos apresentaram maior taxa reprodutiva nos meses em que historicamente ocorrem maiores precipita??es (de outubro a mar?o). Sturnira lilium foi a esp?cie que apresentou maior taxa reprodutiva em altitudes elevadas, sendo esta taxa coincidente com a ?rea onde h? maior riqueza e abund?ncia de Solanum sp. Artibeus sp. apresentou maior taxa reprodutiva em m?dias e baixas altitudes, locais onde h? maior riqueza de esp?cies de Ficus e Carollia perspicillata n?o apresentou prefer?ncia altitudinal para a reprodu??o. Naquelas localidades onde a abund?ncia de morcegos foi maior, mais f?meas estavam reproduzindo-se, sendo que estes locais parecem ter maiores quantidades de recursos. Mostra-se necess?rio a conserva??o de todo o gradiente altitudinal com a poss?vel implanta??o de unidades de conserva??o, pois determinadas esp?cies de morcegos ocorrem em maior abund?ncia em altitudes diferentes, al?m de se reproduzirem em altitudes diferentes

Este estudo teve como objetivo detectar a presença do vírus da raiva em diferentes órgãos de morcegos do gênero Artibeus empregando as técnicas moleculares como RT-PCR, hnRT-PCR e Real Time RT-PCR. De aproximadamente 4000 espécimes de morcegos recebidas no Instituto Pasteur para o diagnóstico da raiva, foram selecionados 30 morcegos do gênero Artibeus, com resultados positivos para raiva pelas técnicas tradicionais de IFD e inoculação em células N2A utilizando suspensões feitas a partir do SNC. Para as técnicas moleculares, foram retirados glândulas salivares, bexigas urinárias, rins, pulmões e conteúdos fecais e ainda foram lavadas as calotas cranianas dos espécimes. Os órgãos e conteúdos fecais foram diluídos a 1:10 (P/V) e as bexigas urinárias a 1:20 (P/V). As suspensões foram inoculadas em células N2A para o isolamento viral. Foi realizada a extração do RNA total usando o TRIzol®, foram realizadas a transcrição reversa seguida da PCR e hnRT-PCR com utilização de primers específicos para o gene codificante da proteína N. A partir do produto da transcrição reversa foi realizada a técnica de Real Time RT-PCR, utilizando primers e sonda específicos para variante antigênica 3. Das 30 suspensões de lavado cerebral, 28 (93,33%) resultaram positivos, na inoculação em cultura de células, seguido de glândulas salivares (36,67%), bexigas (16,67%) e conteúdos fecais (3,33%). Os resultados encontrados da sensibilidade nas técnicas de RT-PCR, hnRT-PCR e Real Time RT-PCR foram 56,25%, 82,57% e 82,19% quando avaliadas as 180 amostras analisadas. A comparação das técnicas de hnRT-PCR e Real Time RT-PCR feita pelo teste exato de Fisher quanto a proporção de positivos detectados mostrou que para o lavado cerebral, órgãos e conteúdos fecais a proporção foi igual (P>0,05). Em relação à positividade os resultados encontrados nas técnicas de hnRT-PCR e Real Time RT-PCR foram 100% em lavado cerebral; 90% e 93,33% em glândulas salivares; 83,33% e 90% em bexigas; 80% e 93,33% em rins; 76,67% e 50% em pulmões e 43,33% em ambas as técnicas em conteúdos fecais. Esses resultados sugerem que tanto as técnicas de hnRT-PCR como Real Time RT-PCR podem ser utilizadas como métodos complementares para o diagnóstico da raiva e são sensíveis o bastante para o uso em estudos de patogênese. A técnica de Real Time RT-PCR realizada neste estudo se mostrou eficiente em detectar o RABV em diferentes órgãos e tecidos extraneurais com a vantagem de ser uma técnica mais rápida e sensível. / This study was aimed to detect the presence of rabies virus in different organs of the genus Artibeus bats using molecular techniques such as RT-PCR, hnRT-PCR, and the Real Time RT-PCR. From about 4,000 specimens of bats received for rabies diagnosis at the Pasteur Institute, 30 bats of the genus Artibeus were then selected. The selected bats presented positive results by the traditional DFA and N2A-cells inoculation test using brain tissue suspensions. Samples of salivary glands, urinary bladders, kidneys, lungs, and fecal contents and washings of the skulls were collected for the molecular techniques testing. The organs and the fecal contents were diluted at 1:10 (w/v) and the urinary bladder, at 1:20 (w/v) and these suspensions were inoculated into N2A cells for viral isolation. The extraction of the total RNA was performed by using TRIzol® and followed by the reverse transcription and the PCR and the hnRT-PCR were performed by using specific primers for the gene encoding the protein N. The product obtained by the reverse transcription technique was submitted to the Real Time RT-PCR technique, using primers and probe specific for antigenic variant 3 of the rabies virus. Of the 30 suspensions of the brain washings, 28 (93.33%) were positive in N2A cell culture inoculation, followed by the suspensions of the salivary glands (36.67%), bladders (16.67%) and fecal contents (3.33%). For the 180 samples evaluated, the results of sensitivity found for the RT-PCR, hnRT-PCR and Real Time RT-PCR techniques were 56.25%, 82.57%, and 82.19%, respectively. A comparison of hnRT-PCR and Real Time RT-PCR techniques performed by Fisher\'s exact test showed that the proportion of positives detected by the brain washings, organs and of the fecal content was non-significant (P> 0.05). Regarding the results found in hnRT-PCR and Real Time RT-PCR techniques, 100% positives were in brain washing, 90% and 93.33% in salivary glands, 83.33% and 90% in bladders, 80% and 93.33% in kidneys, 76.67% and 50% in lungs and 43.33% for both techniques on fecal contents. These results suggest that both hnRT-PCR and Real-Time PCR techniques can be used as complementary methods for the diagnosis of rabies and are sensitive enough for use in pathogenesis studies. The Real Time RT-PCR technique performed in this study proved to be faster and more sensitive and effective in detecting RABV in different organs and extra neural tissues of bats.

Water is a vital resource for species inhabiting arid and semi-arid regions and can shape the biotic communities that we observe. Because water is considered a limiting resource for many species in desert environments, there is the potential for competitive interactions between species to occur at or around water sources. For this dissertation I tested hypotheses related to resource competition among different species of wildlife in the Great Basin and Mojave Deserts of western Utah. Chapter one evaluated the influence of feral horses (Equus caballus) on patterns of water use by communities of native birds and mammals. Chapter two determined if feral horses competed with pronghorn (Antilocapra americana) and mule deer (Odocoileus hemionus) for access to water. In chapters one and two, we found evidence that horses compete with native wildlife for water. In chapter one, horses were associated with decreased richness and diversity of native species at water sources. Native species also had fewer visits and spent less time at water sources frequented by horses. In chapter two, we found that pronghorn and mule deer used water sources less often where horse activity was high. There were also significant differences in temporal activity for pronghorn, but not mule deer, at horse-occupied sites versus sites where horses were absent or uncommon. Our results indicated that horses spatially and temporally displaced other species at water sources providing evidence of a negative influence on how communities of native wildlife access a limited resource in an arid environment. Chapter three assessed whether dominant carnivores (coyote (Canis latrans) and bobcat (Lynx rufus)) negatively influenced the spatial use of water sources by the subordinate kit fox (Vulpes macrotis). Our results did not reveal strong negative associations between kit fox visits to water sources and visits by dominant carnivores; in fact, dominant carnivores contributed very little to the use of water by kit foxes. Instead, kit fox visits were more closely associated with habitat features at water sources. Our findings indicate that dominant carnivores are not the primary driver of use of water sources by subordinate carnivores. Chapter four evaluated whether a simulated loss of water due to climate change/increased human use would differentially affect desert bats based on flight morphology and maneuverability. When we experimentally reduced surface area of water sources, larger, less-maneuverable bats experienced a 69% decrease in drinking success and increased competition with smaller, maneuverable bats. Anticipated reductions in the sizes of water sources due to climate change may lead to species with less maneuverability being unable to access water efficiently and facing increased competition from more agile bats.

Le virus Nipah (NiV) est un virus hautement pathogène responsable d’encéphalites et de syndromes respiratoires sévères chez l’humain. Les chauves-souris appartenant au genre Pteropus sont le réservoir naturel du NiV et ne développent pas de symptômes cliniques d’infection. Comprendre les relations entre l’hôte réservoir et le pathogène requiert la disponibilité de modèles pertinents pour l’étude des interactions. Les études portent à la fois sur le virus et son hôte. Ainsi, nous avons caractérisé phylogénétiquement la souche cambodgienne du NiV isolée de chauves-souris Pteropus et nous l’avons comparée avec les souches isolées chez l’homme. De plus, en absence du génome de référence pour l’espèce de chauve-souris Pteropus giganteus, nous avons séquencé et assemblé le génome de cette espèce, hôte réservoir de la souche NiV-Bangladesh, qui est en circulation actuellement. Enfin, afin d’obtenir des phénotypes cellulaires plus pertinents que des cellules immortalisées pour l’étude des interactions entre le NiV et les chauves-souris du genre Pteropus – les seules disponibles actuellement - nous avons utilisé la reprogrammation somatique sur des cellules primaires de chauve-souris Pteropus. Cette technique permet d’obtenir des cellules souches présentant la capacité d’autorenouvellement et de différenciation. En utilisant une combinaison originale de trois facteurs de transcription, nous avons généré les premières cellules reprogrammées de chauves-souris Pteropus exprimant des caractéristiques de cellules souches. Nous avons démontré que ces cellules sont très susceptibles à l’infection par le NiV mais incapables de produire de l’interféron et d’activer les cascades de signalisations antivirales en réponse à une stimulation avec de l’ARN double brin, contrairement aux cellules primaires. Le développement de ce modèle original ouvre de nouvelles perspectives pour l’étude des interactions entre l’hôte réservoir et le pathogène et pour l’identification de facteurs contrôlant la susceptibilité à l’infection par le NiV, et potentiellement par d’autres virus hébergés par des chauves-souris. / Nipah virus (NiV) is a highly pathogenic virus that causes encephalitis and severe respiratory syndromes in humans. Pteropus bats are the reservoir of NiV and do not show any clinical symptoms. In order to understand the host reservoir - pathogen interactions, the relevant models are needed. Such studies focus on both the virus and its host. A phylogenetically characterization of the NiV Cambodian strain obtained from Pteropus bats was performed and this virus was compared with human ones. In addition, we sequenced and assembled the genome of Pteropus giganteus bat, the natural host of the NiV-Bangladesh strain, which is currently circulating. Up to date, most studies have used immortalized primary cells that are not natural target of the virus. In order to get reprogrammed stem cells, a somatic reprogramming approach was applied to various Pteropus primary cells. The reprogrammed cells are capable of self-renew and differente in different cell lineages. Using an original mix of transcription factors, we derived reprogrammed cells exhibiting stem cells features. We demonstrated the high susceptibly of these cells to henipavirus infections compared with the very low level of infection of the initial primary cells. Generated bat reprogrammed cells do not induce interferon production and signalisation in response to dsRNA. The development of this original model opens new perspectives on virus-host interaction studies, especially that of cellular anti-viral response by identifying factors controlling either susceptibility or restriction to the NiV infection, and possibly other viruses hosted by bats.

Bats and birds are known to be the reservoirs of a number of zoonotic diseases. The capacity of flight and the diversity of these animals may make them special in maintaining and disseminating diverse viruses leading to instances of emerging zoonoses. In particular bats are increasingly recognized to be reservoirs of a wide range of viruses, including Nipah, Ebola and severe acute respiratory syndrome (SARS) coronaviruses. In most instances these viruses appear to establish long-term persistence in bats. In this thesis I report the identification of novel astroviruses from different insectivorous species of apparently healthy bats sampled in Hong Kong and in 11 provinces of Mainland China with high positive rates. Astroviruses are important causes of diarrhea in many animal species, including humans. This study revealed a remarkably high genetic diversity of bat astroviruses, which form novel distinct phylogenetic groups in the genus Mamastrovirus. Evidence for varying degrees of host restriction for bats astroviruses has been found. The finding of diverse astroviruses in Miniopterus bats captured within a single cave habitat in Hong Kong illustrates a very unusual virus host relationship between astroviruses and these bats. Surveillance of astroviruses in rodents, the only mammal with species numbers surpassing that of bats, has revealed a novel astrovirus in only 1.6 % of the faecal samples of urban brown rat (Rattus norvegicus) in Hong Kong in marked contrast with the prevalence and diversity of astroviruses in bats. Rat astrovirus was phylogenetically related to human astroviruses MLB1 which was detected from clinical samples from diarrhoeal patients in Hong Kong in this study. The unusually high positive rates of astroviruses in bats have been again highlighted. Avastroviruses were detected in 7.1% of the aquatic wild bird samples. Avastrovirus have also been detected in doves in Hong Kong, pond herons and a less whistling duck in Cambodia. A phylogenetic analysis of these novel astroviruses together with other previously known astroviruses revealed that avastrovirus can be divided into 3 monophyletic groups. On the other hand, avian coronaviruses was detected in 12.5% of the aquatic wild bird samples. Phylogenetic analysis of these avian coronaviruses has led us to suggest taxonomic separation of these viruses into two groups as gammacoronaviruses and deltacoronaviruses. Frequent interspecies transmissions of gammacoronaviruses between duck species were demonstrated. Analysis of the avian viral sequences and host mitochondrial DNA sequences suggested that some coronaviruses may have coevolved with birds from the same order. With the discoveries of coronaviruses and astroviruses in mammalian and birds, we now have a better understanding on the diversity and ecology of these two virus families in wildlife. These findings provide new insights into the ecology and evolution of these viruses in nature and have revealed possible inter-species transmissions of these viruses. The role of bats as a reservoir of viruses with potential to pose zoonotic threats to human health was also reinforced. Studies of the virus ecology in wildlife as demonstrated in this thesis will help formulating better strategies for controlling emerging diseases in the future. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy