Impacts of nymph/worker genotypes on termite incipient colony fitness

Dissertação de mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.A antraciclina Doxorrubicina (DOX) é um dos mais usados agentes antineoplásicos. No entanto, o tratamento com este composto está associado com cardiotoxicidade, que é dependente da dose e da sua acumulação. A mitocondrial Sirtuína 3 (Sirt3) é a maior deacetilase mitocondrial, modulando diversas vias, tal como a apoptose e o metabolismo celular. Assim a nossa hipótese é que a actividade da Sirt3 diminui a cardiotoxicidade induzida pela DOX. Os cardiomioblastos H9c2 foram transfectados com siRNA e plasmídeos para produzir células com Sirt3 silenciada e sobreexpressa, respectivamente. A toxicidade da DOX (0.5 e 1 µM) foi avaliada pelo ensaio da Sulforodamina B e por citometria de fluxo. A despolarização mitocondrial e a produção do anião superóxido foi determinada por microscopia de fluorescência e o conteúdo de proteínas específicas por western blot. A sobre e sub-expressão da Sirt3 foi confirmada por western blot e RT-PCR. A toxicidade da DOX envolveu a indução de morte celular em todos os grupos. O aumento do conteúdo de Sirt3 mediado pela sobreexpressão parece diminuir a toxicidade da DOX, maioritariamente pela manutenção da integridade da rede mitocondrial e redução do stress oxidativo. Por outro lado, a p53 parece ser um alvo directo da Sirt3 e a protecção conferida contra a morte celular pela Sirt3 pode ser relacionada com esta proteína.The anthracycline Doxorubicin (DOX) is one of the most widely used anti-neoplastic agents. However, treatment with this drug is associated with a cumulative and dose dependent cardiotoxicity. Mitochondrial Sirtuin 3 (Sirt3) is the major mitochondrial deacetylase, modulating several pathways, such as apoptosis and metabolism. Thus, our hypothesis is that mitochondrial Sirt3 activity decreases DOX-induced cardiotoxicity. H9c2 cardiomyoblasts were transfected with siRNA and a plasmid construct to produce Sirt3 knock-down and Sirt3 overexpressing cells, respectively. DOX (0.5µM and 1µM) toxicity was evaluated by the Sulforhodamine B assay and by flow cytometry using the Life/Death assay. Mitochondrial depolarization and superoxide production was determined by fluorescence microscopy and content in specific proteins by western blot. Sirt3 overexpression or knock-down was confirmed by Western Blot and qRT-PCR. In all experimental groups, DOX induced cell death. Increase in Sirt3 content by transfection-mediated overexpression appeared to decrease DOX toxicity, most by maintaining the integrity of mitochondrial network and reducing oxidative stress. On the other hand, p53 seems to be a direct target of Sirt3 and the protection against cell death conferred by Sirt3 could be related to this protein

Neotenic reproductives influence worker caste differentiation in the termite Reticulitermes speratus (Isoptera; Rhinotermitidae)

Division of labor among castes in social insect colonies increases ergonomic efficiency and colony-level fitness, and has played a key role in the ecological success of social insects. Knowledge of the factors that regulate castes is important for understanding adaptive social organization. Our previous study on the termite Reticulitermes speratus demonstrated that the presence of a pair of nymphoid reproductives during development affected offspring caste ratios. In the present study, we investigated further the influence of individual neotenics on offspring caste ratios. Parthenogenetically-produced offspring were reared in worker-tended experimental colonies with the addition of different forms (nymphoid or ergatoid) and numbers of neotenics, and compared the caste ratios of the offspring between the different experimental treatments. We found that all offspring in worker-only tended colonies became nymphs, while a proportion of offspring in colonies with a single neotenic (with the exception of male ergatoids) differentiated into workers. These results show offspring caste ratios are influenced by the presence of single female ergatoids, single female and male nymphoids, while they remain unaffected by the presence of male ergatoids

Molecular phylogeny and evolution of <i>Parabasalia</i> with improved taxon sampling and new protein markers of actin and elongation factor-1α

Background: Inferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa—in particular determining the root—is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences. Principal Findings: Actin and elongation factor-1a genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported. Conclusions/Significance: We conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in these structures. Therefore, systematics based solely on morphology does not reflect the evolutionary history of parabasalids

Molecular Phylogeny and Evolution of Parabasalia with Improved Taxon Sampling and New Protein Markers of Actin and Elongation Factor-1α

BACKGROUND: Inferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa-in particular determining the root-is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences. PRINCIPAL FINDINGS: Actin and elongation factor-1α genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported. CONCLUSIONS/SIGNIFICANCE: We conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in these structures. Therefore, systematics based solely on morphology does not reflect the evolutionary history of parabasalids

Phylogenetic Comparison of Endosymbionts with Their Host Ants Based on Molecular Evidence

Some Formicid ants have symbiotic intracellular bacteria in the epithelial cells of their midgut. These endosymbionts are believed to be derived from a common ancestor. A recent study revealed that endosymbionts of the ant genus Camponotus are closely related to Enterobacteriaceae, but their relationship to endosymbionts of other genera of ants is unknown. In this study, the nucleotide sequences of 16S ribosomal RNA (rRNA) of endosymbionts and mitochondrial cytochrome oxidase subunit I (COI) of their host were determined in five genera of the subfamily Formicinae (Hymenoptera: Formicidae). Based on these molecular data, we constructed phylogenetic trees in order to characterize the systematic position of the symbionts and to estimate the relationship of symbionts and hosts. The analysis showed that the endosymbionts were all connected with the Enterobacteriaceae but did not constitute a monophylitic group, while the three genera belonging to the tribe Camponotini, the endosymbionts and their hosts made a clade. The topologies of these trees were identical for the most part. These results suggest that the endosymbionts of ants have plural origins, and that in the Camponotini, ancestral symbionts have coevolved with their host ants, which are so divergent to several genera as to construct one tribe

Neotenic reproductives influence worker caste differentiation in the termite Reticulitermes speratus (Isoptera; Rhinotermitidae)

Division of labor among castes in social insect colonies increases ergonomic efficiency and colony-level fitness, and has played a key role in the ecological success of social insects. Knowledge of the factors that regulate castes is important for understanding adaptive social organization. Our previous study on the termite Reticulitermes speratus demonstrated that the presence of a pair of nymphoid reproductives during development affected offspring caste ratios. In the present study, we investigated further the influence of individual neotenics on offspring caste ratios. Parthenogenetically-produced offspring were reared in worker-tended experimental colonies with the addition of different forms (nymphoid or ergatoid) and numbers of neotenics, and compared the caste ratios of the offspring between the different experimental treatments. We found that all offspring in worker-only tended colonies became nymphs, while a proportion of offspring in colonies with a single neotenic (with the exception of male ergatoids) differentiated into workers. These results show offspring caste ratios are influenced by the presence of single female ergatoids, single female and male nymphoids, while they remain unaffected by the presence of male ergatoids.</p

Studies of the social evolution of termites using introduction of selfish-genotype individuals into incipient colonies.

研究成果の概要（和文）：本研究では、初期コロニーを祖先シロアリのコロニーのモデルとして用い、シロアリの社会性進化を促した要因を解析した。初期コロニーのカスト構成やコロニーサイズ、利己的遺伝子型個体の比率を操作し、その挙動を調べた。単為生殖コロニーの単独および競争環境下での適応度成分との調査から、生殖虫に分化しやすい利己的個体は、王や女王と闘争しコロニーの適応度を下げることが示された。遺伝的カスト決定システムが利己的個体の生産を抑制していると考えられる。競争環境下でコロニーサイズとソルジャーの存在はともに初期コロニーの適応度を大きく上げた。また、コロニー融合の頻度に血縁関係が影響する事が示された。研究成果の概要（英文）：In this study, factors that promoted social evolution of termites were analyzed, using incipient colonies as model of ancestral termite colonies. Investigation of fitness　components of parthenogenetic colonies under solitary and competitive environments revealed that individuals of selfish-genotype easily differentiated into supplementary reproductives and fight against original reproductives, which drastically decrease fitness of the colonies. Genetic caste determination system probably works to suppress the production of selfish individuals. Under competitive environments, both colony size increase and presence of soldiers greatly increase the fitness of colonies. Frequency of incipient colony fusion was affected by the　relatedness between the reproductives