Primary characterization of genes involved in the colony development of the insect pathogenic fungus Metarhizium anisopliae /

Strain improvement of the insect pathogenic fungus Metarhizium anisopUae is necessary to increase its virulence towards agricultural pests and thus improve its commercial efficacy. Nevertheless, the release of genetically modified conidia in crop fields may negatively affect the ecosystem. Controlling conidiation is a potential means of limiting the release of engineered strains since conidia are the infective propagules and the means of dispersal. The purpose of this study was to research the colony development of M. anisopUae to identify potential targets for genetic manipulation to control conidiation. Following Agrobacterium tumefaciem insertional mutagenesis, phenotypic mutants were characterized using Y-shaped adaptor dependent extension PCR. Four of 1 8 colony development recombinants had T-DNA flanking sequences with high homology to genes encoding known signaling pathway proteins that regulate pathogenesis and/or asexual development in filamentous fungi. Conidial density counts and insect bioassays suggested that a Serine/Threonine protein kinase COTl homolog is not essential for conidiation or virulence. Furthermore, a choline kinase homolog is important for conidiation, but not virulence. Finally, the regulator of G protein signaling CAG8 and a NADPH oxidase NoxA homolog are necessary for conidiation and virulence. These genes are candidates for further investigation into the regulatory pathways controlling conidiation to yield insight into promising gene targets for biocontrol strain improvement.

Confocal imaging of pseudomonas syringae pv. phaseolicola colony development in bean reveals reduced multiplication of strains containing the genomic island PPHGI-1.

Pseudomonas syringae pv. phaseolicola is the seed borne causative agent of halo blight in the common bean Phaseolus vulgaris. Pseudomonas syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene hopAR1 (located on a 106-kb genomic island, PPHGI-1, and earlier named avrPphB), which matches resistance gene R3 in P. vulgaris cultivar Tendergreen (TG) and causes a rapid hypersensitive reaction (HR). Here, we have fluorescently labeled selected Pseudomonas syringae pv. phaseolicola 1302A and 1448A strains (with and without PPHGI-1) to enable confocal imaging of in-planta colony formation within the apoplast of resistant (TG) and susceptible (Canadian Wonder [CW]) P. vulgaris leaves. Temporal quantification of fluorescent Pseudomonas syringae pv. phaseolicola colony development correlated with in-planta bacterial multiplication (measured as CFU/ml) and is, therefore, an effective means of monitoring Pseudomonas syringae pv. phaseolicola endophytic colonization and survival in P. vulgaris. We present advances in the application of confocal microscopy for in-planta visualization of Pseudomonas syringae pv. phaseolicola colony development in the leaf mesophyll to show how the HR defense response greatly affects colony morphology and bacterial survival. Unexpectedly, the presence of PPHGI-1 was found to cause a reduction of colony development in susceptible P. vulgaris CW leaf tissue. We discuss the evolutionary consequences that the acquisition and retention of PPHGI-1 brings to Pseudomonas syringae pv. phaseolicola in planta.

Worker demography in a large-colony, swarm-founding wasp

Neotropical swarm-founding wasps build nests enclosed in a covering envelope, which makes it difficult to count individual births and deaths. Thus, knowledge of worker demography is very limited for swarm-founding species compared with that for independent-founding species. In this study, we explored the worker demography of the swarm-founding wasp Polybia paulista, the colony size of which usually exceeds several thousand adults. We considered each wasp colony as an open-population and estimated the survival probability, recruitment rate, and population size of workers using the developments of the Cormack-Jolly-Seber model. We found that capture probability varied considerably among the workers, probably due to age polyethism and/or task specialization. The daily survival rate of workers was high (around 0.97) throughout the season and was not related to the phase of colony development. On the other hand, the recruitment rate ranged from 0 to 0.37, suggesting that worker production was substantially less important than worker survival in determining worker population fluctuations. When we compared survival rates among worker groups of one colony, the mean daily survival rate was lower for founding workers than for progeny workers and tended to be higher in progeny workers that emerged in winter. These differences in survivorship patterns among worker cohorts would be related to worker foraging activity and/or level of parasitism.

The transport of carboxylic acids and important role of the Jen1p transporter during the development of yeast colonies

On solid substrates, yeast colonies pass through distinct developmental phases characterized by the changes in pH of their surroundings from acidic to nearly alkaline and vice versa. At the beginning of the alkali phase colonies start to produce ammonia, which functions as a quorum-sensing molecule inducing the reprogramming of cell metabolism. Such reprogramming includes, among others, the activation of several plasma membrane transporters and is connected with colony differentiation. In the present study, we show that colony cells can use two transport mechanisms to import lactic acid: a ‘saturable’ component of the transport, which requires the presence of a functional Jen1p transporter, and a ‘non-saturable’ component (diffusion) that is independent of Jen1p. During colony development, the efficiency of both transport components changes similarly in central and outer colonial cells. Although the lactate uptake capacity of central cells gradually decreases during colony development, the lactate uptake capacity of outer cells peaks during the alkali phase and is also kept relatively high in the second acidic phase. This lactate uptake profile correlates with the localization of the Jen1p transporter to the plasma membrane of colony cells. Both lactic acid uptake mechanisms are diminished in sok2 colonies where JEN1 expression is decreased. The Sok2p transcription factor may therefore be involved in the regulation of non-saturable lactic acid uptake in yeast colonies.

Colony cycle of the social wasp Mischocyttarus consimilis Zikán (Hymenoptera, Vespidae)

Colony cycle of the social wasp Mischocyttarus consimilis Zikán (Hymenoptera, Vespidae). This study describes some aspects of the colony cycle of the Neotropical social wasp Mischocyttarus consimilis, from data obtained under field conditions. Our results showed that the colony cycle in M. consimilis is annual and asynchronous in relation to the months of the year. The colonies remained active for approximately eight months. Most of the abandonments were associated with natural causes, and were most frequent in the pre-emergence stage. The nests were constructed preferentially in man-made structures, especially in sites protected from direct sunlight and rain. Colony foundation was either by haplometrosis or pleometrosis, being the first form predominant.

Resources Taken to the Nest by Protopolybia exigua (Hymenoptera, Vespidae) in Different Phases of the Colony Cycle, in a Region of the Medio Sao Francisco River, Bahia, Brazil

The present study aimed to identify what resources were taken to the nest by workers of Protopolybia exigua (de Saussure), to verify if there is a relationship between physical factors of weather and phases of colony development and the collection of different resources and also to observe if the wasps carry macerated prey in the crop. The species collected nectar (62.8%), wood pulp for nest building (6.1%), water(2.9%), prey (0.8%) and resin (0.2%). Moreover, workers of P exigua do carry macerated prey in the crop. There was a positive correlation (r = 0.85, p = 0.0004) between the presence of larvae in the nest and the carrying of solid contents in the crop, which provides strong evidence that this species macerates its prey while still in the field, before taking it to the nest. There was positive influence of the temperature on the collection of nectar (r = 0.89, p = 0.0001) and water (r = 0.46, p = 0.0066) and negative influence of relative air humidity on the collection of the same resources (r = - 0.88, p = 0.0001 and r = - 0.37, p = 0.0160, respectively). Luminosity influenced the gathering of water (r = 0.43, p = 0.0089) and Pulp for nest building (r = 0.31, p = 0.0274) and the wind speed did nor influence the collection of any resources. Nectar is the main item taken to the nest during all phases of the colony.

Caste flexibility and variation according to the colony cycle in the swarm-founding wasp, Parachartergus fraternus (Gribodo) (Hymenoptera : Vespidae : Epiponini)

Differences between castes and related aspects in Parachartegus fraternus (Gribodo) have been studied in various stages of the colony cycle. Five colonies from three colony development stages (namely, pre-emergence, worker-producing and male-producing) were analyzed. The main results were: irrespective of colony age, castes were always morphologically indistinguishable; non-inseminated ovary-developed females were found in four colonies; because differences of castes were physiological rather than morphological, castes are evidently post-imaginally determined and this is probably taking place according to the colony conditions. These findings support the occurrence of caste totipotency in Parachartergus fraternus.

Foraging activity of Protopolybia exigua (Hymenoptera, Vespidae) in different phases of the colony cycle, at an area in the region of the Médio São Francisco River, Bahia, Brazil

In this work, the foraging activity of Protopolybia exigua (de Saussure) was studied, aiming to verify the amplitude of the foraging time, the relation between the physical factors of weather (temperature, relative air humidity, luminosity and wind speed) and the daily activity of searching for resources, as well as to analyze the influence of the colony development phases and the number of individuals (adults and immatures) engaged in such activity. The study was carried out in two areas of the city of Bom Jesus da Lapa, Bahia, Brazil, during the period from January to June 2006, when the observation of 12 colonies of P. exigua was performed. The results revealed this species presented an amplitude of activity of almost 13 hours. There was an average of 44.4 nest exits and 37.6 returns a day by the nest workers and the Returns with Resources Index for this species was 93.5%. The foraging activity increased as of 10:01, and became more intense, with the peak from 1:01 p.m. to 4:00 p.m., when the highest temperatures (°C) and the lowest values of relative air humidity (%) were observed. The colonies that had a larger number of larvae presented a higher average number of trips to the field per hour (10.94) and the number of females in the colony tends to induce foraging activity. There were no differences between the numbers of nest exits by the workers regarding the three phases of colony development.

Bumblebees in a Changing Climate: Evaluating the Effects of Temperature on Queen Performance

Bumblebees are a very essential group of pollinating insects, but their populations have declined drastically during the past decades. We need to understand why their numbers are decreasing and what can be done to reverse this trend. Climate change-related phenomena, such as changes in the overwintering temperatures and spring conditions, are among the most prominent threats to bumblebees. Queens have a special role in the lifecycle of bumblebees because they overwinter and start new colonies the next year. Their successful performance: survival, overwintering ability, longevity, immune competence, and nest establishing capability in spring, is highly important for bumblebee populations. However, the effects of climate change on bumblebee queen performance remain unknown. The main objective of this thesis was to assess how temperature affects the performance of bumblebee queens during and after overwintering. The effects of warm temperature predicted by climate change scenarios on queen survival and stress-tolerance were studied by a four-month artificial diapause of bumblebee queens at two temperatures (9°C and 1.8°C). Bumblebee colonies were also reared in a laboratory and factors affecting colony characteristics were examined. In addition, queen performance during spring was studied in a starvation experiment using two temperatures (15°C as normal; 24°C as warmer than average) and queens collected from nature right after their emergence. My research revealed how temperature affects queen performance, and queen size was found to be an important factor determining the direction of some of these effects. We found a 0.4g weight threshold for bumblebee queens to be able to survive overwintering. In addition, during mild winters, larger queens have a higher chance than smaller ones to survive through winter and also to cope with immunological stresses after overwintering. During cold conditions, which are normal in the current climatic situation, this advantage disappears. In the spring starvation experiment, the starved queens survived approximately eight days longer in 15°C than in 24°C, which means that starvation risk rises significantly with increasing spring temperature, in a situation where food is scarce due to for example frost damage or asynchrony between bumblebees and their important food plants. These results could mean that in the future climate, larger queens are better able to survive the winter, initiate their nests and start rearing their offspring. This may be problematic, because I also detected two alternative strategies of colony development that differ between large and small queens; larger queens start to lay eggs earlier at nest initiation, their colonies mature later, they produce more workers, and they have a more strongly male biased sex allocation compared with smaller queens. If larger queens have a greater change of producing offspring after a mild winter, this could lead to a significant decline in the total production of new queens at a population level. Thus, it seems that queen size could act as one mechanism regulating the population level outcomes in different temperatures. The new information presented in my thesis reinforces that basic research, monitoring, and local species conservation of bumblebees both in Finland and globally must be increased to ensure that this highly important pollinator group survives in the face of climate change.

Susceptibility of the ant-cultivated fungus Leucoagaricus gongylophorus (Agaricales : Basidiomycota) towards microfungi

The aim of this study was to select virulent strains of microfungi against Leucoagaricus gongylophorus, a symbiotic fungus cultivated by leaf-cutting ants. The results from in vitro assays showed that microfungal strains had a variable and significant impact on the colony development of L. gongylophorus. Specifically, Trichoderma harzianum, Escovopsis weberi CBS 810.71 and E. weberi A088 were more effective, inhibiting the L. gongylophorus colonies by 75, 68 and 67%, respectively (P < 0.05) after 15 days. Strain E. weberi A086 and Acremonium kiliense were less effective: 43 and 26%, respectively (P < 0.05). In spite of the current negative perspective of a microbiological control approach for these ants, the present work discusses the possibility of using mycopathogenic fungi for the control of these insects, and points out the importance of encouraging more studies in this area.

Queen lipid content and nest growth in the leaf cutting ant (Atta sexdens rubropilosa) (Hymenoptera: Formicidae)

The relationship between the queens' lipid content and nest growth (population size, biomass and nest architecture) was studied from founding up to 1 year. Nests aged 3, 4, 5, 6, 9 and 12 months were dug in the field, and their dimensions were measured. The ant nest population and fungus garden was also collected. The sample was taken to the laboratory where we counted the worker population and weighed the biomass (fungus plus offspring) and queens. Queens were separated for the determination of lipids. The lipid content in the bodies of queens decreased in the first months, then stabilized (at 4-6 months) before increasing in months 9 and 12. Nest biomass (symbiotic fungus and offspring) and worker population increased over time. The structural growth of the nests was observed by excavating around them. Initially nests (3 months old) had one chamber at an average depth of 15 cm. By 1 year, the nests had three or four deep chambers, and were about 3-4 m deep. Our study contributes to knowledge of the dynamics of the energy-reserve expenditure by queens during colony founding and colony development for up to 1 year. © 2013 Copyright Taylor and Francis Group, LLC.

Horários de atividade forrageadora e itens coletados por Protopolybia exigua (de Saussure) (Hymenoptera, Vespidae) na região do médio São Francisco, Bahia

Pós-graduação em Ciências Biológicas (Zoologia) - IBRC

Desenvolvimento e produção de mel de colônias de abelhas tiúba, Melipona fasciculata Smith, 1854 (Apidae: Meliponina), em diferentes modelos de colmeias e localidades do Maranhão

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Glândula ectomandibular e comportamento de Polistes versicolor (Olivier) (Hymenoptera, vespidae)

Pós-graduação em Ciências Biológicas (Zoologia) - IBRC

First-year nest growth in the leaf-cutting ants Atta bisphaerica and Atta sexdens rubropilosa

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