Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis--University of California. / Cover title. Extra t.p., with thesis statement, attached to cover. Bibliography: p. 180-181.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Trypanosomatid protozoa of Leishmania genus are vector-borne parasites that infect humans causing a wide range of diseases known as Leishmaniasis. Severity of clinical forms generally depends on infecting species, thus its correct identification becomes critical to diagnosis, prognosis and treatment. The current biochemical and molecular methods for typing strains of Leishmania species are mostly effective. Although these tools remain important in the diagnosis and identification of Leishmania, they are insufficient for definitive identification. After isolate 7 clinical parasites strains, some of them belonging to atypical cases of cutaneous/mucocutaneous and visceral Leishmaniasis, we aimed to identify the specie of Leishmania involved in these cases. By combining structural analysis of the body shape, such as presence of flagellum and kinetoplast, we saw that these parasites shared morphologic characteristics with others Tripanosomatidae, however, they were not conclusive for species identification. Since PCR and isozymes assays were unsuccessful to identify 6 out of 7 clinical isolates reported here, we proceeded to determine their genome sequence. Through whole-genome sequencing analysis of parasite strains, we showed that a new pathogenic Trypanosomatid was the etiological agent in clinical cases diagnosed as Leishmaniasis in Brazil. By comparing coding sequences of over orthologous genes within 36 Trypanosomatidae organisms, we found that this new parasite is closely related to Crithidia fasciculata, which parasites exclusively mosquitoes and is considered non-infective to humans. Phenotypic characterization of 2 of these isolates from the skin and bone marrow of the same patient showed that skin isolate was attenuated compared to the bone marrow isolate for survival in the spleen in BALB/c mice. However, both of them were able to infect and induce inflammation in the liver after 4 and 6 weeks post infection. Conversely, only skin isolate was able to survive in dermis, leading to ear swelling, accompanied of presence of parasites in the ear and lymph nodes. Besides that, bone marrow isolate infection in murine macrophages showed higher number of infected cells and also increased number of parasites within macrophages comparing to the skin isolate infection. Furthermore, bone marrow isolate negatively modulated the nitric oxide production in murine macrophages, corroborating with increased infection. Our findings raise concerns about an emerging infectious disease easily confused with Leishmaniasis, opening a research path towards epidemiological questions about identification of vectors, reservoirs, distribution and reassessment of Leishmaniasis cases in Brazil. Due to its proximity to the monoxenous Crithidia genus and the geographical location of atypical Leishmaniasis cases reported here, we proposed naming this new parasite species as Cridia sergipensis. Overall, this research provides a unique knowledge on phenotipic differences associated with diverse pathologies caused by Cridia infection. / Protozoários Tripanossomatídeos do gênero Leishmania são parasitos transmitidos por vetores que infectam hospedeiros vertebrados, causando um amplo espectro de doenças denominadas leishmanioses. A gravidade das formas clínicas depende, dentre outros fatores, da espécie, assim, a identificação correta se torna crítica para o diagnóstico, prognóstico e tratamento. As ferramentas bioquímicas e moleculares disponíveis para caracterizar espécies de Leishmania são, na maioria das vezes, eficientes. Embora essas metodologias sejam importantes para o diagnóstico e identificação de Leishmania, são insuficientes para identificação conclusiva. Após isolar 7 amostras clínicas de parasitos, sendo alguns deles de casos atípicos de leishmaniose cutânea/mucocutânea e visceral, o principal objetivo foi identificar a espécie de Leishmania envolvida. Através da análise estrutural do citoesqueleto, que envolve a presença de flagelo e cinetoplasto, por exemplo, foi observado que esses parasitos compartilham características morfológicas com outros Tripanossomatídeos, entretanto, não se pode afirmar qual a espécie em questão. Uma vez que os resultados da identificação por PCR e eletroforese de isoenzimas foram inconclusivos, foi realizado o sequenciamento total do genoma. O sequenciamento dos isolados clínicos dos pacientes diagnosticados com leishmanioses mostrou que se trata de uma nova espécie patogênica dentro do grupo dos Tripanossomatídeos. Ao comparar as sequências codificantes dos genes ortólogos de 36 Tripanossomatídeos, foi observado que esses parasitos são intimamente relacionados com Crithidia fasciculata, parasito exclusivo de mosquitos, e considerado não patogênico para humanos. A caracterização fenotípica de 2 desses isolados clínicos, sendo um deles isolado da pele e outro da medula óssea do mesmo paciente mostrou que o isolado da pele não foi capaz de estabelecer doença no baço de camundongos BALB/c infectados. Por outro lado, ambos os isolados infectaram e induziram reação inflamatória no fígado dos animais após 4 e 6 semanas de infecção. No entanto, apenas o isolado da pele foi capaz de sobreviver na derme dos camundongos, induzindo inflamação local com presença de parasitos na orelha e linfonodos. Além disso, a infecção com o isolado da medula óssea em macrófagos murinos exibiu um maior número de células infectadas e maior número de amastigotas dentro das células, comparada ao da pele. Adicionalmente, o isolado da medula óssea foi capaz de modular negativamente a produção de óxido nítrico em macrófagos murinos, corroborando com o aumento da infecção. Essas descobertas levantam preocupações sobre uma doença infecciosa emergente facilmente confundida com leishmaniose, abrindo caminhos para a pesquisa nos campos epidemiológicos, de vetores, reservatórios, distribuição e reavaliação de casos de leishmaniose. Devido a proximidade com os parasitos do gênero Crithidia e a localização geográfica de onde foram isolados, a nomenclatura de Cridia sergipensis foi proposta para agrupar essa nova espécie. O presente estudo contribui também para o conhecimento das diferenças fenotípicas associadas com as diversas patologias causadas pela infecção com Cridia. / Aracaju

A subfamília Leishmaniinae compreende espécies de tripanossomatídeos dos gêneros Crithidia, Leptomonas, Endotrypanum e Leishmania. O objetivo deste estudo foi caracterizar isolados da subfamília Leishmaniinae através de DNA Barcoding e inferir suas relações filogenéticas utilizando os genes SSUrDNA, gGAPDH, CATB, HSP70 e ITS1rDNA como marcadores genéticos. As espécies estudadas foram segregadas em dois clados principais, um formado por Endotrypanum-Leishmania e genótipos de L. costarricensis posicionados como clado basal. E outro clado que contém espécies monoxênicas as quais foram separados em diferentes subclados (Crithidia, Leptomonas, Lotmaria, Termófilo e três subclados homogêneos representados por C. inconstans, C. acanthocephali e isolados de Rondônia). No gênero Leishmania as análises posicionaram as espécies pouco estudadas do complexo L. enriettii como o grupo mais basal. Os isolados de lagartos Moçambique formaram um grupo monofilético com as espécies do subgênero L. (Sauroleishmania), corroborando, também, sua relação com o subgênero L. (Leishmania). / The Leishmaniinae subfamily comprises trypanosomatid species from genera Crithidia, Leptomonas, Endotrypanum and Leishmania. The aim of this study was to characterize Leishmaniinae isolates through DNA Barcoding and further infer their phylogenetic relationships using SSUrDNA, gGAPDH, CATB, HSP70 and ITS1rDNA genes as genetic markers. The Leishmaniinae species was segregated into two major clades, one formed by Endotrypanum-Leishmania with L. costarricensis genotypes resolving as a basal clade. The other clade contained the monoxenic species which were separated in different subclades (Crithidia, Leptomonas, Lotmaria, Termofilo, and three homogeneous subclades represented by C. inconstans, C. acanthocephali and Rondônia isolates). In Leishmania genus analyzes positioned the poorly investigated species of the complex L. enriettii as the most basal group. Isolates from Mozambique lizards formed a monophyletic group with the species of the subgenus L. (Sauroleishmania), was corroborated its relationship to the subgenus L. (Leishmania) too.

Plants are under selection to simultaneously attract pollinators while deterring herbivores. This dilemma can lead to tradeoffs in floral traits, which are traditionally thought to be optimized for pollinators. My dissertation addresses the ecological costs and putative functional significance of nectar secondary metabolites, a paradoxical but widespread phenomenon in the angiosperms. I investigate this issue from the pollinator’s perspective using a series of controlled laboratory investigations focused primarily on the bumble bee Bombus impatiens and the nectar alkaloid gelsemine, from Gelsemium sempervirens. I begin by demonstrating that nectar enriched with the alkaloid gelsemine significantly deters visits from bumble bees at a range of natural alkaloid concentrations. However, this aversion can be mitigated by increasing the sucrose concentration such that the alkaloid-rich nectar is more rewarding than its alkaloid-free counterpart. I then demonstrate that the consumption of gelsemine-rich nectar can inhibit oocyte development and protein utilization in bees, but that this effect is limited to bees of suboptimal condition. Continuous consumption of the nectar alkaloid gelsemine also leads to a reduction in the pathogen load of bumble bees infected with Crithidia bombi, but direct interactions between the pathogen and the alkaloid have no impact on infection intensity. Gelsemine also fails to inhibit floral yeast growth, suggesting that nectar alkaloids may not be universally antimicrobial. Finally, I demonstrate that gross nectar cardenolides from the genus Asclepias are strongly correlated with gross leaf cardenolides and that the majority of individual cardenolides found in nectar are a subset of those identified in leaves. This pattern suggests that nectar cardenolides are a consequence of defense for Asclepias; however, they may not be a costly corollary because bumble bees show an overall preference for nectar cardenolides at mean concentrations. Altogether, my dissertation provides a new perspective on the role of chemical defenses against herbivores in plant-pollinator interactions.

Plants are under selection to simultaneously attract pollinators while deterring herbivores. This dilemma can lead to tradeoffs in floral traits, which are traditionally thought to be optimized for pollinators. My dissertation addresses the ecological costs and putative functional significance of nectar secondary metabolites, a paradoxical but widespread phenomenon in the angiosperms. I investigate this issue from the pollinator’s perspective using a series of controlled laboratory investigations focused primarily on the bumble bee Bombus impatiens and the nectar alkaloid gelsemine, from Gelsemium sempervirens. I begin by demonstrating that nectar enriched with the alkaloid gelsemine significantly deters visits from bumble bees at a range of natural alkaloid concentrations. However, this aversion can be mitigated by increasing the sucrose concentration such that the alkaloid-rich nectar is more rewarding than its alkaloid-free counterpart. I then demonstrate that the consumption of gelsemine-rich nectar can inhibit oocyte development and protein utilization in bees, but that this effect is limited to bees of suboptimal condition. Continuous consumption of the nectar alkaloid gelsemine also leads to a reduction in the pathogen load of bumble bees infected with Crithidia bombi, but direct interactions between the pathogen and the alkaloid have no impact on infection intensity. Gelsemine also fails to inhibit floral yeast growth, suggesting that nectar alkaloids may not be universally antimicrobial. Finally, I demonstrate that gross nectar cardenolides from the genus Asclepias are strongly correlated with gross leaf cardenolides and that the majority of individual cardenolides found in nectar are a subset of those identified in leaves. This pattern suggests that nectar cardenolides are a consequence of defense for Asclepias; however, they may not be a costly corollary because bumble bees show an overall preference for nectar cardenolides at mean concentrations. Altogether, my dissertation provides a new perspective on the role of chemical defenses against herbivores in plant-pollinator interactions.

The increased honey bee colony losses in the last decade gain a considerable attention of scientists and public. The causes of these losses include a wide range of biotic and abiotic factors, but pathogens and parasites are probably the main ones. Monoxenous trypanosomatids in honey bee gut Lotmaria passim and Crithidia mellificae were neglected for a long time but according to recent studies they seem to participate in those colony declines. Trypanosomatids are widespread parasites, including also the causes of some human illnesses, such as sleeping sickness, Chagas disease and leishmaniasis. The mechanism of the effect on honey bee health is not well understood so far. The aim of this thesis was to verify the occurrence of the trypanosomatids in honey bee samples from two regions in Czech Republic. The methods, based on detection of specific DNA loci, confirm the high prevalence of L. passim, which was founded in majority (71 %) of the samples. On the contrary, no samples were positive to C. mellificae.

Many bumblebee species are suffering from the effects of habitat fragmentation and population isolation. In some cases, populations have lost genetic diversity due to genetic drift and it is possible they are now at heightened risk of extinction. Inbreeding may be particularly costly to bumblebees because, as Hymenoptera, their complementary sex determination system can lead to the production of sterile or inviable diploid males. However, little is known about the effect that diploid male production has on bumblebee colony fitness. Here, the consequences of brother-sister mating in the bumblebee Bombus terrestris are investigated, and the production of diploid males was found to exert considerable costs at the colony level by reducing productivity and survival. Diploid males may therefore act as indicators of the genetic health of populations, and their detection could be used as an informative tool in hymenopteran conservation. Due to the costs associated with inbreeding, selection may have favoured the evolution of kin recognition systems in bumblebees. Data are presented that suggest that B. terrestris can discriminate between kin and non-kin as gynes were less willing to mate with siblings compared to non-relatives. Theory predicts that inbreeding may impose further costs on bumblebees through increased levels of parasitism, but empirical data are scarce. The relationship between population genetic diversity and parasite prevalence is assessed using Hebridean island populations of Bombus muscorum and Bombus jonellus. In the more outbred B. jonellus, there was no relationship between parasite prevalence and population heterozygosity. But prevalence of the gut parasite Crithidia bombi and the tracheal mite Locustacarus buchneri were found to be higher in populations of B. muscorum that had lower genetic diversity. In addition to assessing infection status, the activity of the immune system was assessed in each individual bee. However, there was no relationship between population heterozygosity and these immune parameters. This suggests that, in some Hymenopteran species, as populations lose genetic diversity the impact of parasitism will increase, potentially pushing threatened populations closer to extinction. Therefore, preventing population fragmentation by the creation of suitable habitats and by ensuring connectivity between habitat patches are important aspects of hymenopteran conservation. Finally, this thesis investigates the potential threat of pathogen spread from commercially reared bumblebees used for crop pollination to wild bumblebees. Although no direct evidence for parasite spillover is found, the prevalence of C. bombi was significantly higher in B. terrestris by the end of the season on farms that used commercial bumblebees compared to farms that did not. This high prevalence does suggest that pathogen spillover is a potential threat and it would be preferable to reduce the usage of commercial bumblebees where possible. For example, sowing wild flower mixes could boost natural pollinator populations, which in turn would benefit soft fruit pollination. Overall, this thesis contributes to our knowledge of the consequences of inbreeding in bumblebees and the relationship between genetic diversity and parasite prevalence. It provides a greater understanding of the factors that might be pushing threatened pollinators towards extinction and as a whole provides important information that may inform conservation practitioners, whose aim is to protect the future of our hymenopteran pollinators.

Immunodeficient honey bee (Apis mellifera) colonies suffer from broad range of parasites including eukaryotic protozoa. Despite this fact, the eukaryotic parasites are still poorly documented in the Czech Republic. The presence of eukaryotic parasites (Nosema ceranae, Nosema apis, Crithidia mellificae and Apicystis bombi) was observed in different apiaries in the Czech Republic. The samples were taken in 9 apiaries in 53 beehives during the 2014/2015 season. From each beehive, 10 adult of honey bees were taken from the peripheral comb in triplicate. DNA was isolated from every sample of honey bees. The parasites were detected by polymerase chain reaction (PCR) with specific primers. The treatment fall of parasitic mite Varroa destructor was obtained from beekeepers for season of 2014. Crithidia mellificae was detected by 5 types of specific primers (SEF, SER; SSU, SSU rRNA, Cyt b, Tryp cyt b) and positive amplicons were cloned and sequenced. The obtained sequences were compared with GeneBank and showed similarity from 98-100% to sequences of Lotmaria passim (Trypanosomatid). Crithidia mellificae was not detected. L. passim had prevalence of 79,2% and is reported in the Czech Republic for the first time. Primer Tryp-cyt b is recommended for the routine detection of L. passim. Nosema ceranae was...