Insights into bacterial colony morphology evolution and diversification during infection development in cystic fibrosis

Tese de Doutoramento em Engenharia Biomédica.

Division of labour and worker size polymorphism in ant colonies: the impact of social and genetic factors

Division of labour among workers is central to the organisation and ecological success of insect societies. If there is a genetic component to worker size, morphology or task preference, an increase in colony genetic diversity arising from the presence of multiple breeders per colony might improve division of labour. We studied the genetic basis of worker size and task preference in Formica selysi, an ant species that shows natural variation in the number of mates per queen and the number of queens per colony. Worker size had a heritable component in colonies headed by a doubly mated queen (h(2)=0.26) and differed significantly among matrilines in multiple-queen colonies. However, higher levels of genetic diversity did not result in more polymorphic workers across single- or multiple-queen colonies. In addition, workers from multiple-queen colonies were consistently smaller and less polymorphic than workers from single-queen colonies. The relationship between task, body size and genetic lineage appeared to be complex. Foragers were significantly larger than brood-tenders, which may provide energetic or ergonomic advantages to the colony. Task specialisation was also often associated with genetic lineage. However, genetic lineage and body size were often correlated with task independently of each other, suggesting that the allocation of workers to tasks is modulated by multiple factors. Overall, these results indicate that an increase in colony genetic diversity does not increase worker size polymorphism but might improve colony homeostasis.

The determinants of queen size in a socially polymorphic ant.

In social animals, body size can be shaped by multiple factors, such as direct genetic effects, maternal effects, or the social environment. In ants, the body size of queens correlates with the social structure of the colony: colonies headed by a single queen (monogyne) generally produce larger queens that are able to found colonies independently, whereas colonies headed by multiple queens (polygyne) tend to produce smaller queens that stay in their natal colony or disperse with workers. We performed a cross-fostering experiment to investigate the proximate causes of queen size variation in the socially polymorphic ant Formica selysi. As expected if genetic or maternal effects influence queen size, eggs originating from monogyne colonies developed into larger queens than eggs collected from polygyne colonies, be they raised by monogyne or polygyne workers. In contrast, eggs sampled in monogyne colonies were smaller than eggs sampled in polygyne colonies. Hence, eggs from monogyne colonies are smaller but develop into larger queens than eggs from polygyne colonies, independently of the social structure of the workers caring for the brood. These results demonstrate that a genetic polymorphism or maternal effect transmitted to the eggs influences queen size, which probably affects the social structure of new colonies.

Flexible colony-founding strategies in a socially polymorphic ant

In social insects the number of queens per nest varies greatly. One of the proximate causes of this variation may be that queens produced by multiple-queen colonies are generally smaller, and might thus be unable to found new colonies independently. We examined whether the social origin of queens and males influenced the colony-founding success of queens in the socially polymorphic ant Formica selysi. Queens originating from single-queen and multiple-queen colonies had similar survival rates and colony-founding success, be they alone or in two-queen associations. During the first 5 months, queens originating from single-queen colonies gave rise to more workers than queens originating from multiple-queen colonies. Pairs of queens were also more productive than single queens. However, these differences in productivity were transient, as all types of colonies had reached a similar size after 15 months. Mating between social forms was possible and did not decrease queen survival or colony productivity, compared to mating within social forms. Overall, these results indicate that queens from each social form are able to found colonies independently, at least under laboratory conditions. Moreover, gene flow between social forms is not restricted by mating or genetic incompatibilities. This flexibility in mating and colony founding helps to explain the maintenance of alternative social structures in sympatry and the absence of genetic differentiation between social forms.

Population genomics of eusocial insects: the costs of a vertebrate-like effective population size

The evolution of reproductive division of labour and social life in social insects has lead to the emergence of several life-history traits and adaptations typical of larger organisms: social insect colonies can reach masses of several kilograms, they start reproducing only when they are several years old, and can live for decades. These features and the monopolization of reproduction by only one or few individuals in a colony should affect molecular evolution by reducing the effective population size. We tested this prediction by analysing genome-wide patterns of coding sequence polymorphism and divergence in eusocial vs. noneusocial insects based on newly generated RNA-seq data. We report very low amounts of genetic polymorphism and an elevated ratio of nonsynonymous to synonymous changes - a marker of the effective population size - in four distinct species of eusocial insects, which were more similar to vertebrates than to solitary insects regarding molecular evolutionary processes. Moreover, the ratio of nonsynonymous to synonymous substitutions was positively correlated with the level of social complexity across ant species. These results are fully consistent with the hypothesis of a reduced effective population size and an increased genetic load in eusocial insects, indicating that the evolution of social life has important consequences at both the genomic and population levels.

Population Trends of a Mixed-Species Colony of Humboldt and Magellanic Penguins in Southern Chile after Establishing a Protected Area

Worldwide marine protected areas (MPAs) have been designated to protect marine resources, including top predators such as seabirds. There is no conclusive information on whether protected areas can improve population trends of seabirds when these are further exploited as tourist attractions, an activity that has increased in past decades. Humboldt Penguins (Spheniscus humboldti) and Magellanic Penguins (S. magellanicus) breed sympatrically on Puñihuil Islets, two small coastal islands off the west coast of Chiloé Island (41° S) in southern Chile that are subject to exploitation for tourism. Our goal was to compare the population size of the mixed colony of Humboldt and Magellanic Penguins before and after protection from unregulated tourism and freely roaming goats in 1997. For this purpose, two censuses were conducted in 2004 and 2008, and the numbers compared with those obtained in 1997 by other authors. The proportion of occupied, unoccupied, and collapsed/flooded burrows changed between years; there were 68% and 34% fewer collapsed burrows in 2004 and 2008, respectively, than in 1997. For the total number of burrows of both species, we counted 48% and 63% more burrows in 2004 and 2008, respectively, than in 1997. We counted 13% more burrows of Humboldt Penguins in 2008 than in 1997, and for Magellanic Penguins, we estimated a 64% increase in burrows in 2008. Presumably, this was as a result of habitat improvement attributable to the exclusion of tourists and the removal of goats from the islets. Although tourist visits to the islets are prohibited, tourism activities around the colonies are prevalent and need to be taken into account to promote appropriate management.

Colony dehydration and water collection by specialized caste in the leaf-cutting ant Atta sexdens rubropilosa

Social organization enables leaf-cutting ants to keep appropriate micro-ecological nest conditions for the fungus garden (their main food), eggs, larvae and adults. To maintain stability while facing changing conditions, individual ants must perceive destabilising factors and produce a proper behavioral response. We investigated behavioral responses to experimental dehydration in leaf-cutting ants to verify if task specialization exists, and to quantify the ability of ant sub-colonies for water management. Our setup consisted of fourteen sub-colonies, ten of which were randomly assigned to different levels of experimental dehydration with silica gel, whereas the remaining four were controls. The ten experimental sub-colonies were split into two groups, so that five of them had access to water. Diverse ant morphs searched for water in dehydrated colonies, but mainly a caste of small ants collected water after sources had been discovered. Size specialization for water collection was replicable in shorter experiments with three additional colonies. Ants of dehydrated colonies accumulated leaf-fragments on the nest entrance, and covering the fungus garden. Behaviors that may enhance humidity within the nests were common to all dehydration treatments. Water availability increased the life span of dehydrated colonies.

Granulocyte Colony-stimulating Factor Reduces Mortality by Suppressing Ventricular Arrhythmias in Acute Phase of Myocardial Infarction in Rats

Our aim was to evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on early cardiac arrhythmias after myocardial infarction (MI) and the impact on survival. Male Wistar rats received repeated doses of 50 mu g/kg G-CSF (MI-GCSF group) or vehicle (MI group) at 7, 3, and 1 days before surgery. MI was induced by permanent occlusion of left corollary artery. The electrocardiogram was obtained before occlusion and then for 30 minutes after surgery. Events and duration of ventricular arrhythmias were analyzed. The levels of connexin43 (Cx43) were measured by Western blot immediately before MI production. Survival was significantly increased in MI-GCSF pretreated group (74% versus 52.0% MI. P < 0.05). G-CSF pretreatment also significantly reduced the ventricular premature beats when compared with the untreated-MI group (201 +/- 47 versus 679 +/- 117, P < 0.05). The number and the duration of ventricular tachycardia were smaller in the MI-G-CSF group, as well as the number of ventricular fibrillation episodes (10% versus 69% in NIL P < 0.05). Cx43 levels were significantly increased by G-CSF treatment (1.27 +/- 0.13 versus 0.86 +/- 0.11; P < 0.05). The MI size 24 hours after occlusion was reduced by G-CSF pretreatment (36 +/- 3% versus 44 +/- 2% of left ventricle in MI group; P < 0.05). The increase of Cx43 expression in the heart may explain the reduced incidence in ventricular arrhythmias in the early phases after coronary artery occlusion in rats, thus increasing survival after MI.

Reproductive Patterns and Birth Seasonality in a South-American Breeding Colony of Common Marmosets, Callithrix jacchus

SOUSA,M.B.C. et al. Reproductive Patterns and Birth Seasonality in a South-American Breeding Colony of Common Marmosets, Callithrix jacchus. Primates, v.40, n.2, p. 327-336, Apr. 1999.

Relationship Between Population Size and Symbiont Fungus Culture Volume in Colonies of Acromyrmex balzani (Hymenoptera: Formicidae)

In order to quantify the population of Acromyrmex balzani Emery, 1890 nests and to verify whether the population dynamic of the colony is correlated with the symbiont fungus volume, the principal energy source of the colony, five nests located in grassland areas were evaluated. The nests were sprayed with neutral talcum powder to improve visualization and digging. The symbiont fungus and the entire population existent in the chambers were collected. The mean fungus volume and total nest population in excavated nests were, respectively, 74.76 ml and 1,095 individuals. Simple linear correlation analysis verified that the fungus volume grew proportionally to the number of individuals. Despite the correlation between fungus volume and population dynamic of colonies, the factors that determine this relationship are little known since other microorganisms live in association with the colony.

Internal Architecture and Population Size of Acromyrmex subterraneus molestans (Hymenoptera, Formicidae) Nests: Comparison Between a Rural and an Urban Area

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Digging effort in leaf-cutting ant queens (Atta sexdens rubropilosa) and its effects on survival and colony growth during the claustral phase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The size of excavators within a polymorphic termite species governs tunnel topology

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

Background: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli ( Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.Results: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside ( IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.Conclusion: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Discrete dimorphism among castes of the bald-faced hornet Dolichovespula maculata (Hymenoptera: Vespidae) in different phases of the colony cycle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)