Cochineal, a precious source of red: cochineal dyes characterization by high performance liquid chromatography with diode array detection and principal component analysis

Presented at Faculdade de Ciências e Tecnologias, Universidade de Lisboa, to obtain the Master Degree in Conservation and Restoration of Textiles

Modeling and Control of a Dragonfly-Like Robot

Dragonflies demonstrate unique and superior flight performances than most of the other insect species and birds. They are equipped with two pairs of independently controlled wings granting an unmatchable flying performance and robustness. In this paper it is studied the dynamics of a dragonfly-inspired robot. The system performance is analyzed in terms of time response and robustness. The development of computational simulation based on the dynamics of the robotic dragonfly allows the test of different control algorithms. We study different movement, the dynamics and the level of dexterity in wing motion of the dragonfly. The results are positive for the construction of flying platforms that effectively mimic the kinematics and dynamics of dragonflies and potentially exhibit superior flight performance than existing flying platforms.

A Saint Louis encephalitis and Rocio virus serosurvey in Brazilian horses

Introduction Arboviruses are an important public health problem in Brazil, in especially flaviviruses, including the Saint Louis encephalitis virus (SLEV) and the Rocio virus (ROCV), are especially problematic. These viruses are transmitted to humans or other vertebrates through arthropod bites and may cause diseases with clinical manifestations that range from asymptomatic infection, viral hemorrhagic fever to encephalitis. Methods A serological survey of horses from various regions of Brazil using an enzyme-linked immunosorbent assay (ELISA) with recombinant SLEV domain III peptides and ROCV E protein as antigens. Results Overall, 415 (55.1%) of the 753 horses that were screened were seropositive for flavivirus and, among them, monotypic reactions were observed to SLEV in 93 (12.3%) and to ROCV in 46 (6.1%). These results suggested that these viruses, or other closely related viruses, are infecting horses in Brazil. However, none of the studied horses presented central nervous system infection symptoms. Conclusions Our results suggest that SLEV and ROCV previously circulated among horses in northeast, west-central and southeast Brazil.

The Azadirachtins: potent insect growth inhibitors

In the course of their coevolution with insects, plants have learnt to protect themselves by chemical means. Semiochemical act as antifeedants or deterrents, others by disrupting growth and development. By use of the Epilachna varivestis bioassay we isolated from Azadirachta indica seed a group of triterpenoids which interfee with larval growth and development in ppm range. Main components are the azadirachtins A and B with identical biological activity. Various other azadirachtins were obtained, either as minor seed components or by chemical modification of the naturally occuring compounds. Structure vs. activity relation studies enabled us to postulate a basic structural element that should still be biologically active and with much simpler chemical structure than natural compounds. What underlies the biological activity of these insect growth inhibitors? Their interference with the hormonal regulation of development and reproduction has been studied in Locusta migratoria and Rhodnius prolixus. In addition, tritiated dihydroazadirachtin A was used. With this approach, a precise correlation between administered dose, resulting effects, and retention of the compound was established. The azadirachtins either interrupt, delay, or deviate whole developmental programs. Results from these studies provide another chemical probe for studies in insect endocrinology and physiology.

Oral insecticidal activity of root colonizing Pseudomonas fluorescens CHA0: a biocontrol agent with potential to control plant pests and diseases

The application of microbial biocontrol agents for the control of fungal plant diseases and plant insect pests is a promising approach in the development of environmentally benign pest management strategies. The ideal biocontrol organism would be a bacterium or a fungus with activity against both, insect pests and fungal pathogens. Here we demonstrate the oral insecticidal activity of the root colonizing Pseudomonas fluorescens CHA0, which is so far known for its capacity to efficiently suppress fungal plant pathogens. Feeding assays with CHA0-sprayed leaves showed that this strain displays oral insecticidal activity and is able to efficiently kill larvae of three important insect pests. We further show data indicating that the Fit insect toxin produced by CHA0 and also metabolites controlled by the global regulator GacA contribute to oral insect toxicity.

Evolution, maintenance and ecological consequences of genetic caste determination in " Pogonomyrmex " harvester ants

Summary Division of labor between reproducers (queens) and helpers (workers) is the main characteristic of social insect societies and at the root of their ecological success. Kin selection models predict that phenotypic differences between queens and workers should result from environmental rather than from genetic differences. However, genetic effects on queen and worker differentiation were found in two populations-of Pogonomyrmex harvester ants. Each of the two populations is composed of two genetically distinct lineages. Queens (which can be of either lineage) generally mate with males of their own and of the alternate lineage and produce two types of female offspring, those fertilized by males of the queens' lineage which develop into queens and those fertilized by males of the alternate lineage which develop into workers. All four lineages were further suggested to be themselves of hybrid origin between-the species P: barbatus and P. rugosus, in which queens and workers do not differ genetically. In a first set of experiments, we tested if female caste determination (the differentiation into queens and workers) in the lineages was genetically hardwired and if it was associated with costs in terms of the ability to optimally allocate resources to the production of queens and workers. To this end we first mated queens of-two lineages to a single male. Queens mated to a male of the alternate lineage successfully raised worker offspring whereas queens mated to a male of their own lineage almost always failed to produce workers. This reveals that pure-lineage individuals have lost the ability to develop into workers. Second, we analyzed offspring produced by naturally mated queens. During the stage of colony founding when only workers are produced, naturally mated queens laid a high proportion of pure-lineage eggs but the large majority of these eggs failed to develop. As a consequence, the number of offspring produced by incipient colonies decreased linearly with the proportion of pure-lineage eggs laid by queens. Moreover, queens of the lineage most commonly represented in a given population produced more pure-lineage eggs, in line with the view that they mate randomly with the two types of males and indiscriminately use their sperm. Altogether these results predict frequency-dependent founding success for pairs of lineages because queens of the more common lineage will produce more pure-lineage eggs and their colonies be less successful during the stage of colony founding. To describe the distribution of populations characterized with genetic caste determination relative to the populations with environmental caste determination we genotyped queens and workers collected during a large survey of -additional populations. Genetic caste determination associated with pairs of interbreeding lineages was frequent and widespread in the studied range and we identified four additional lineages displaying genetic caste determination. Overall, there were thus eight highly differentiated lineages with genetic caste determination. These lineages always co-occurred in the same complementary lineage pairs. Three of the four lineage pairs appeared to have a common origin, while their relationship with the forth could not be resolved. The genetic survey also revealed that, in addition to being genetically isolated from one another, all eight lineages were genetically distinct from P. rugosus and P. barbatus, even when colonies of interbreeding lineages co-occurred with colonies of either putative parent at the same site. This raised the question of the mechanisms involved in the reproductive isolation between the lineages and the parental species and between the two lineages of a lineage pair. At a site where one lineage pair co-occurred with P. rugosus, we identified two pre-zygotic mechanisms (differences in timing for mating flights between P. rugosus and the lineage pair and assortative mating) and one post-zygotic mechanism (high levels of hybrid unviablility) which in combination may largely account for the reproductive isolation between the lineages and their parental species. The mechanisms accounting for the reproductive isolation between the two lineages of a lineage pair varied across lineage pairs. In one lineage pair, inter-lineage individuals exclusively occurred in the sterile worker caste, raising the possibility that inter-lineage eggs have completely lost the ability to develop into queens in this lineage pair and that there is thus no opportunity for gene flow. In each of the three remaining lineage pairs, inter-lineage queens were produced by a minority of colonies. In these lineage pairs, colonies headed by inter-lineage queens failed to grow sufficiently to produce reproductive individuals which may account for the reproductive isolation between co-occurring lineages in three lineage pairs. In conclusion, the results of this thesis show that genetic caste determination is costly but widespread in Pogonomyrmex harvester ants. Reproductive isolation among the lineages and between the lineages and the parental species as well as frequency-dependent founding success for co-occurring lineages may contribute to the persistence of this extraordinary system. Résumé La division du travail entre individus reproducteurs (les reines) et individus non-reproducteurs (ouvrières) représente la caractéristique principale des sociétés d'insectes et est à la base de leur succès écologique. Des modèles de sélection de parentèle prédisent que les différences phénotypiques entre reines et ouvrières devraient provenir d'effets environnementaux plutôt que de différences génétiques. Malgré ce fait, des effets génétiques sur la différentiation entre reines et ouvrières ont été montrés dans deux populations de fourmis moissonneuses du genre Pogonomyrmex. Chacune des deux populations est composée de deux lignées génétiquement distinctes. Les reines de chaque lignée s'accouplent en général avec des mâles de leur propre lignée ainsi qu'avec des mâles de l'autre lignée et produisent deux types d'oeufs, ceux qui sont fécondés par les mâles de leur propre lignée qui se développent en nouvelles reines et ceux qui sont fécondés par les mâles de l'autre lignée qui se développent en ouvrières. Il a été suggéré que les lignées sont elles-mêmes des hybrides entre les deux espèces P. barbatus et P. rugosus. Dans ces deux espèces, les reines et ouvrières ne sont pas génétiquement distinctes. Dans une première série d'expériences, nous avons testé si la détermination de la caste femelle (le développement en reine ou en ouvrière) est génétiquement rigide et si elle est associée à des coûts en terme de capacité à allouer de façon optimale les ressources pour la production de reines et d'ouvrières. Pour cela nous avons accouplé des reines de deux lignées avec un seul mâle. Les reines accouplées avec un mâle de l'autre lignée ont élevé de nouvelles ouvrières avec succès alors que les reines accouplées avec un mâle de leur propre lignée ont presque toujours échoué à produire des ouvrières. Ceci montre que les individus de lignée pure ont perdu la capacité de se développer en ouvrière. Deuxièmement, nous avons analysé la descendance de reines qui se sont accouplées naturellement. Durant le stade de fondation de la colonie, où seules des ouvrières sont élevées, les reines accouplées naturellement ont pondu une grande proportion d'oeufs de lignée pure mais la majorité de ces derniers ne se sont pas développés. En conséquence, le nombre de descendants produits par des colonies fondatrices diminuait linéairement avec la proportion des oeufs de lignée pure pondus par la reine en accord avec l'hypothèse que les reines s'accouplent au hasard avec les deux types de mâles et utilisent leur sperme aléatoirement. Dans l'ensemble; ces résultats prédisent un succès de fondation fréquence-dépendant pour les deux lignées, car les reines de la lignée la plus fréquente produiront .plus d'oeufs de lignée pure et leurs colonies auront moins de succès lors de la fondation de colonies par rapport aux colonies de la lignée la moins fréquente. Pour décrire la distribution des-populations caractérisées par une détermination génétique des castes par rapport aux populations caractérisées par une détermination environnementale des castes, nous avons génotypé des reines et des ouvrières qui ont été collectées lors d'une analyse de populations supplémentaires. La détermination génétique des castes associée à des croisements entre lignées est fréquente et largement répartie dans l'aire étudiée. Nous avons identifié quatre lignées supplémentaires, ayant une détermination génétique des castes, pour un total de huit lignées. Ces huit lignées forment quatre paires de lignées et on ne trouve jamais deux lignées de paires différentes, dans une population. Trois des quatre paires de lignées s'avèrent avoir une origine commune alors que leur relation avec la quatrième paire de lignées n'a pas pu être résolue. L'analyse génétique de populations supplémentaires a également révélé qu'en plus d'être génétiquement isolées les unes des autres, les huit lignées sont génétiquement distinctes de P. rugosus et P. barbatus même si les colonies d'une paire de lignées se trouvent en sympatrie avec l'une ou l'autre des espèces parentales. Ceci relève la question des mécanismes impliqués dans l'isolation reproductive entre les lignées et les espèces parentales ainsi qu'entre les deux lignées d'une paire. En étudiant un site où une paire de lignées se trouve en sympatrie avec P. rugosus, nous avons identifié deux mécanismes pré-zygotiques (des différences dans le timing du vol nuptial entre P. rugosus et les lignées et des accouplements assortis) ainsi qu'un mécanisme post-zygotique (un niveau élevé de non-viabilité des hybrides). En combinaison, ces mécanismes peuvent largement expliquer l'isolement reproductif entre les lignées et leurs espèces parentales. Les mécanismes contribuant à l'isolement reproductif entre les deux lignées d'une paire variaient entre paires de lignées. Dans une paire, les individus de génotype inter-lignée se trouvent uniquement dans la caste stérile des ouvrières, suggérant qu'il n'y a pas d'opportunité pour avoir du flux de gènes entre les deux lignées ce cette paire. Dans chacune des trois autres paires de lignées des nouvelles reines de génotype inter-lignée sont produites par une minorité de colonies. Par contre, les colonies avec une reine mère de génotype inter-lignée ne se développent pas suffisamment pour produire des individus reproducteurs. Ceci peut donc expliquer pourquoi il n'y a pas de flux de gènes entre les deux lignées de trois paires. En conclusion, les résultats de cette thèse montrent que la détermination génétique de la caste est coûteuse mais très répandue chez les fourmis. moissonneuses du genre Pogonomyrmex. L'isolement reproductif des lignées entre elles et avec les espèces parentales, ainsi qu'un succès de fondation fréquence-dépendant contribuent à la persistance de ce système extraordinaire.

Survival and Molting Incidence after Heat and Cold Shocks in Panstrongylus megistus Burmeister

Survival and molting incidence were studied after heat (40°C) and cold (0°C) shocks in specimens of Panstrongylus megistus with the aim of establishing its response to temperature stress under laboratory rearing conditions and to understand occasional changes in the biological characteristics of specimens captured in nature. The response to the thermal shocks was found to vary as a function of the temperature and duration of the shock, developmental phase and sex of the specimens, and in certain cases, the insect habit and nourishment conditions. P. megistus specimens were found to be less resistant to the heat shock assay than Triatoma infestans, another reduviid species. The short cold shock affected survival of P. megistus more than did the heat shock, survival of fully-nourished specimens being preferential. The response of adults to the short cold shock was affected by sex, males being generally less resistant. The insect sylvatic habit was found to seldom affect the thermal shock response established for specimens with domestic habit. A decrease in molting frequency and sometimes a slowdown of the molting rate were found after the short heat and cold shocks, possibly promoted by change in hormonal balance, and differing from patterns reported for T. infestans. The results indicate that no generalization should be made for different reduviid species in terms of the effects of temperature shocks.

Comparison of some behavioral and physiological feeding parameters of Triatoma infestans klug, 1834 and Mepraia spinolai porter, 1934, vectors of chagas disease in Chile

There are two vectors of Chagas disease in Chile: Triatoma infestans and Mepraia spinolai. We studied the feeding behavior of these species, looking for differences which could possibly explain the low impact of the latter species on Chagas disease. Both species used thermal cues to locate their feeding source and consumed a similar volume of blood which was inversely related to the body weight before the meal and directly related to the time between meals. The average time between bites were 6.24 and 10.74 days. The average bite of M. spinolai lasted 9.68 min, significantly shorter than the 19.46 min for T. infestans. Furthermore, while T. infestans always defecated on the host, this behavior was observed in M. spinolai in only one case of 27 (3.7%). The delay between the bites and defecation was very long in M. spinolai and short in T. infestans. These differences may affect the reduced efficiency of transmission of Chagas infection by M. spinolai.

Development of Hepatozoon caimani (Carini, 1909) Pessôa, De Biasi & De Souza, 1972 in the Caiman Caiman c. crocodilus, the frog Rana catesbeiana and the mosquito Culex fatigans

The sporogony of Hepatozoon caimani has been studied, by light microscopy, in the mosquito Culex fatigans fed on specimens of the caiman Caiman c. crocodilus showing gametocytes in their peripheral blood. Sporonts iniciate development in the space between the epithelium of the insect gut and the elastic membrane covering the haemocoele surface of the stomach. Sporulating oocysts are clustered on the gut, still invested by the gut surface membrane. Fully mature oocysts were first seen 21 days after the blood-meal. No sporogonic stages were found in some unidentified leeches fed on an infected caiman, up to 30 days following the blood-meal. When mosquitoes containing mature oocysts were fed to frogs (Leptodactylus fuscus and Rana catesbeiana), cysts containing cystozoites developed in the internal organs, principally the liver. Feeding these frogs to farm-bred caimans resulted in the appearance of gametocytes in their peripheral blood at some time between 59 and 79 days later, and the development of tissue cysts in the liver, spleen, lungs and kidneys. Transmission of the parasite was also obtained by feeding young caimans with infected mosquitoes and it is suggested that both methods occur in nature. The finding of similar cysts containing cystozoites in the semi-aquatic lizard Neusticurus bicarinatus, experimentally fed with infected C. fatigans, suggests that other secondary hosts may be involved.

Cloning and characterization of a V-ATPase subunit C from the American visceral leishmaniasis vector Lutzomyia longipalpis modulated during development and blood ingestion

Visceral leishmaniasis (VL) is a serious tropical disease that affects approximately 500 thousand people worldwide every year. In the Americas, VL is caused by the parasite Leishmania (Leishmania) infantum chagasi mainly transmitted by the bite of the sand fly vector Lutzomyia longipalpis. Despite recent advances in the study of interaction between Leishmania and sand flies, very little is known about sand fly protein expression profiles. Understanding how the expression of proteins may be affected by blood feeding and/or presence of parasite in the vector's midgut might allow us to devise new strategies for controlling the spread of leishmaniasis. In this work, we report the characterization of a vacuolar ATPase subunit C from L. longipalpis by screening of a midgut cDNA library with a 220 bp fragment identified by means of differential display reverse transcriptase-polymerase chain reaction analysis. The expression of the gene varies along insect development and is upregulated in males and bloodfed L. longipalpis, compared to unfed flies.

Insecticide treated mosquito nets for malaria control in India-experience from a tribal area on operational feasibility and uptake

The study assessed the operational feasibility and acceptability of insecticide-treated mosquito nets (ITNs) in one Primary Health Centre (PHC) in a falciparum malaria endemic district in the state of Orissa, India, where 74% of the people are tribes and DDT indoor residual spraying had been withdrawn and ITNs introduced by the National Vector Borne Disease Control Programme. To a population of 63,920, 24,442 ITNs were distributed free of charge through 101 treatment centers during July-August 2002. Interview of 1,130, 1,012 and 126 respondents showed that the net use rates were 80%, 74% and 55% in the cold, rainy and summer seasons, respectively. Since using ITNs, 74.5-76.6% of the respondents observed reduction of mosquito bites and 7.2-32.1% reduction of malaria incidence; 37% expressed willingness to buy ITNs if the cost was lower and they were affordable. Up to ten months post-treatment, almost 100% mortality of vector mosquitoes was recorded on unwashed and washed nets (once or twice). Health workers re-treated the nets at the treatment centers eight months after distribution on a cost-recovery basis. The coverage reported by the PHC was only 4.2%, mainly because of unwillingness of the people to pay for re-treatment and to go to the treatment centers from their villages. When the re-treatment was continued at the villages involving personnel from several departments, the coverage improved to about 90%.Interview of 126 respondents showed that among those who got their nets re-treated, 81.4% paid cash for the re-treatment and the remainder were reluctant to pay. Majority of those who paid said that they did so due to the fear that if they did not do so they would lose benefits from other government welfare schemes. The 2nd re-treatment was therefore carried out free of charge nine months after the 1st re-treatment and thus achieved coverage of 70.4%. The study showed community acceptance to use ITNs as they perceived the benefit. Distribution and re-treatment of nets was thus possible through the PHC system, if done free of charge and when personnel from different departments, especially those at village level, were involved.

Thermal preferences and limits of Triatoma brasiliensis in its natural environment - Field observations while host searching

The goal of this work was to explore the thermal relationship between foraging Triatoma brasiliensis and its natural habitat during the hottest season in the state of Ceará, Brazil. The thermal profiles were determined using infrared analysis. Although the daily temperature of rock surfaces varied in a wide range, T. brasiliensisselected to walk through areas with temperatures between 31.7-40.5ºC. The temperature of T. brasiliensisbody surface ranged from 32.8-34.4ºC, being higher in legs than the abdomen. A strong relationship was found between the temperature of the insect and the temperature of rock crevices where they were hidden (r: 0.96, p < 0.05). The species was active at full sunlight being a clear example of how the light-dark rhythm may be altered, even under predation risk. Our results strongly suggest a thermal borderline for T. brasiliensisforaging activity near 40ºC. The simultaneous determination of insect body and rock temperatures here presented are the only obtained in natural habitats for this or other triatomines.

Domain shuffling in a sensor protein contributed to the evolution of insect pathogenicity in plant-beneficial Pseudomonas protegens.

Pseudomonas protegens is a biocontrol rhizobacterium with a plant-beneficial and an insect pathogenic lifestyle, but it is not understood how the organism switches between the two states. Here, we focus on understanding the function and possible evolution of a molecular sensor that enables P. protegens to detect the insect environment and produce a potent insecticidal toxin specifically during insect infection but not on roots. By using quantitative single cell microscopy and mutant analysis, we provide evidence that the sensor histidine kinase FitF is a key regulator of insecticidal toxin production. Our experimental data and bioinformatic analyses indicate that FitF shares a sensing domain with DctB, a histidine kinase regulating carbon uptake in Proteobacteria. This suggested that FitF has acquired its specificity through domain shuffling from a common ancestor. We constructed a chimeric DctB-FitF protein and showed that it is indeed functional in regulating toxin expression in P. protegens. The shuffling event and subsequent adaptive modifications of the recruited sensor domain were critical for the microorganism to express its potent insect toxin in the observed host-specific manner. Inhibition of the FitF sensor during root colonization could explain the mechanism by which P. protegens differentiates between the plant and insect host. Our study establishes FitF of P. protegens as a prime model for molecular evolution of sensor proteins and bacterial pathogenicity.

Evolution at Two Levels in Fire Ants: The Relationship between Patterns of Gene Expression and Protein Sequence Evolution.

Variation in protein sequence and gene expression each contribute to phenotypic diversity, and may be subject to similar selective pressures. Eusocial insects are particularly useful for investigating the evolutionary link between protein sequence and condition-dependent patterns of gene expression because gene expression plays a central role in determining differences between eusocial insect sexes and castes. We investigated the relationship between protein coding sequence evolution and gene expression patterns in the fire ants Solenopsis invicta, S. richteri, and their hybrids to gain greater insight into how selection jointly operates on gene expression and coding sequence. We found that genes with high expression variability within castes and sexes were frequently differentially expressed between castes and sexes, as well as between species and hybrids. These results indicate that genes showing high variation in expression in one context also tend to show high variation in expression in other contexts. Our analyses further revealed that variation in both intra- and interspecific gene expression was positively associated with rate of protein sequence evolution in Solenopsis. This suggests that selective constraints on a gene operate both at the level of protein sequence and at the level of gene expression regulation. Overall, our study provides one of the strongest demonstrations that selective constraints mediate both protein sequence evolution and gene expression variability across different biological contexts and timescales.

A cnidarian homologue of an insect gustatory receptor functions in developmental body patterning.

Insect gustatory and odorant receptors (GRs and ORs) form a superfamily of novel transmembrane proteins, which are expressed in chemosensory neurons that detect environmental stimuli. Here we identify homologues of GRs (Gustatory receptor-like (Grl) genes) in genomes across Protostomia, Deuterostomia and non-Bilateria. Surprisingly, two Grls in the cnidarian Nematostella vectensis, NvecGrl1 and NvecGrl2, are expressed early in development, in the blastula and gastrula, but not at later stages when a putative chemosensory organ forms. NvecGrl1 transcripts are detected around the aboral pole, considered the equivalent to the head-forming region of Bilateria. Morpholino-mediated knockdown of NvecGrl1 causes developmental patterning defects of this region, leading to animals lacking the apical sensory organ. A deuterostome Grl from the sea urchin Strongylocentrotus purpuratus displays similar patterns of developmental expression. These results reveal an early evolutionary origin of the insect chemosensory receptor family and raise the possibility that their ancestral role was in embryonic development.