Large volumes of antisera were generated against Apis melli/era venom with which to develop a novel, platform technology for the inexpensive production of anti venoms. The ovine sera contained high levels of specific antibodies which neutralised the myotoxic, phospholipase A2 and in vivo activities of the venom. Methods of processing the antisera to provide Fab or F(ab')2 were investigated. F(ab')2 was thought to be clinically advantageous and, by determining the conditions necessary for the preferential breakdown of Fc and serum components other than F(ab')2' it was possible to avoid salt precipitation. Diafiltration was then used to remove most of the unwanted small fragments and anion-exchange chromatography to remove any remaining acidic impurities such as pepsin and large aggregates. The F(ab')2 was -97% pure and the yield - 199 per L of serum. This is the first specific therapy for mass envenoming by European or Africanised bees. Spiders of the genus Latrodectus (black widows) are distributed widely and about 2,500 bites are reported annually in the USA. The neurotoxic effects of the venom were studied on the isolated phrenic nerve diaphragm preparation. Low venom concentrations (ImgIL) were stimulatory while high concentrations (IOmg/L) caused nerve blockade which was potentiated by increased calcium levels. Although effective, the Merck antivenom, which is unprocessed horse serum, causes unacceptable risks. The second purpose of this project was to prepare an improved Latrodectus spider antivenom using the new platform technology. Different immunisation schedules were studied to optimise the humoral immune response. Sheep immunised with 2mg La. hesperus venom produced the highest levels of specific antibodies as assessed by ELISA, using the isolated nerve diaphragm preparation or in vivo in mice. The new process provided a pure F(ab')2 antivenom retaining 78% of the original antisera ED so neutralising power and was - twice as effective as the Merck antivenom.

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-04-20Bitstream added on 2014-06-13T19:01:03Z : No. of bitstreams: 1 lisboa_lco_dr_rcla.pdf: 650724 bytes, checksum: 95fb2a1a2dad2bd0de9a5e34f0e6d402 (MD5) / Entre as abelhas altamente eussociais, o estudo de aspectos da morfologia e da ultra-estrutura tem ajudado a esclarecer uma série de questões sobre a sua biologia. A divisão de trabalho distingue as fêmeas em duas castas, sendo uma com função reprodutiva (rainha) e outra que desempenha as mais diversas tarefas dentro da colônia (operária). Espécies do gênero Frieseomelitta mostram esta divisão de forma mais clara, uma vez que suas operárias nunca põem ovos; começando um processo degenerativo nos ovários ainda no final de sua diferenciação. Porém, nas diferentes espécies e em diferentes condições, as operárias podem desenvolver seus ovários e realizar postura de ovos tróficos que são comidos pelas rainhas, ou dar origem a machos. O primeiro caso mostra que além da contribuição dada pelas operárias na produção de alimento para a cria com suas secreções glandulares, elas contribuem também com seus ovos para produzir alimento. Já o segundo caso pode ser um indicativo de que a divisão do trabalho reprodutivo ainda não está totalmente definida. A fertilidade das rainhas também parece se manifestar de diferentes formas. Em Apis mellifera, é evidenciada pela enorme diferença no número de ovaríolos, bem maior nas rainhas do que nas operárias. Esta diferença é devido a uma redução sofrida pelas operárias durante o desenvolvimento pós-embrionário. Já nos 9 meliponíneos o número de ovaríolos de rainhas e operárias é, para a maioria das espécies, igual e em número de quatro por ovário. Em Trigona spinipes, o número de ovaríolos dos ovários das operárias é sempre igual a quatro, enquanto que nas rainhas pode variar entre oito e quinze, entre as rainhas e entre os ovários da mesma rainha... . / The survey of morphological and ultrastructural aspects of highly eusocial bees, have cleared and supported many questions about their biology. The division of labour discern the females in to two castes, being one with reproductive function (queen) and other that perform differents tasks within the colony (worker). Frieseomelitta shows this division clearly. The workers never lay eggs; starting a degenerative process in the ovaries yet in the ending your differentiation. However, in different species and in different conditions, the workers can develop their ovaries and lay eggs that can be trophic and be eaten by the queens, or give rise males. The first condition show that further of the contribution given by the workers in the food production for the brood with yours glandular secretions, they contribute also with yours eggs for to produce food. The second condition can be an evidence that division of reproductive labour is not entirely solved yet. Queen's fertility also seems to be expressed in different ways. In honey bees Apis mellifera, there is a large difference in ovarioles number, which are bigger in queens than workers. This difference is due to the decrease in workers ovarioles number during the post-embryonic development. In Meliponini the ovarioles number of queens and workers is, for most of the species, the same; four per ovary. 11 In Trigona spinipes, the ovarioles number in workers is always four, while in queens can vary from eight to fifteen among queens and among the ovaries of the same queen. Anyway, the ovariole number present in the ovary of queens in Meliponini is very small if compared to Apis mellifera; that suggest that their efficience in egg production can be higher and attached to the ovarioles lengh... (Complete abstract, click electronic address below).

Orientador: Ricardo de Oliveira Orsi / Banca: Ligia Maria Ruv Carelli Barreto / Banca: Simone Fernandes / Resumo: Os objetivos do presente trabalho foram avaliar a presença dos minerais cálcio (Ca), cobre (Cu), enxofre (S), ferro (Fe), fósforo (P), magnésio (Mg), potássio (K) , sódio (Na) e zinco (Zn) em amostras de méis de eucalipto (Eucalyptus sp.), laranjeira (Citrus sinensis L. Osbeck) e silvestre de abelhas Apis mellifera L. da região do Pólo Cuesta, estado de São Paulo, Brasil. As análises dos minerais foram realizadas por espectrofotometria de absorção atômica e os resultados avaliados por meio da Análise de Variância (ANOVA), seguido pelo teste de Tukey-Kramer para comparação das médias (P<0,05). Houve diferenças para os minerais Ca, S, P, Mg, K e Na entre as diferentes origens botânicas. Pode-se concluir que os méis da região do Pólo Cuesta apresentam minerais essenciais ao organismo humano, sendo que o mel de eucalipto apresentou os maiores teores para a maioria dos minerais analisados / Abstract: The aim of this study were to evaluate the presence of minerals calcium (Ca), copper (Cu), sulfur (S), iron (Fe), phosphorus (P) , magnesium (Mg), potassium (K), sodium (Na) and zinc (Zn) in eucalyptus (Eucalyptus sp.), orange (Citrus sinensis L. Osbeck) and wild honeys samples of Apis mellifera L. bees from Pólo Cuesta region, São Paulo state, Brazil. Analyses of minerals were carried out by atomic absorption spectrophotometry and the results evaluated by analysis of variance (ANOVA) followed by Tukey-Kramer test for comparison of means (P<0.05). There were differences for Ca, S, P, Mg, K and Na minerals between different botanical origins. It was concluded that the honey's Pólo Cuesta region shows minerals essentials to human organism, and the eucalyptus honey had the majority minerals analysed / Mestre

Classical experiments on honey bee time-memory showed that foragers trained to collect food at a fixed time of day return the following day with remarkable time-accuracy. Previous field experiments revealed that not all foragers return to a food source on unrewarded test days. Rather, there exist 2 subgroups: “persistent” foragers reconnoiter the source; “reticent” foragers wait in the hive for confirmation of source availability. To examine how these foragers contribute to a colony’s ability to reallocate foragers across sources with rapidly changing availabilities, foragers were trained to collect sucrose during a restricted window for several days and observed over 3 days throughout which the feeder was empty. In 2 separate trials, activity monitoring revealed a high level of activity apparently directed at other food sources. This “extracurricular” activity showed extensive temporal overlap with visits to the feeder, indicating that honey bees can manage at least 2 different overlapping time memories.

It has been observed that returning honey bee foragers congregate with unemployed foragers and food receiver bees in a localized region of the hive known as the dance floor. Here, the returning foragers advertise food sources via the waggle dance. It was hypothesized that the close proximity of the dance floor to the hive entrance was related to foragers minimizing time and travel inside the hive. The hive entrance is conventionally located at the bottom of the hive. It was suggested that this location was ideal for easy removal of debris. This correlation between dance floor location and hive entrance location invokes further examination of the relationship. Is the hive entrance location used to establish dance floor location? Using scan sampling- the hive was visually scanned along rows in a descending fashion from the top right corner to hive entrance. The location of each observed waggle dance was recorded for 30 minutes. Observations were conducted for three consecutive days, then the hive entrance location was displaced. The observation hive was altered to contain three hive entrances located adjacent to the bottom first frame, adjacent to the center of the second frame, and adjacent to the middle of the third frame. Only one hive entrance was open at a given time. For the last three days of the experiment, the bottom hive entrance was made accessible again. Regardless of entrance position, the dance floor was seen to be established adjacent to the hive entrance.

Data collecting and preparing is generally considered a crucial process in data science projects. Especially for image data, adding semantic attributes when preparing image data provides much more insights for data scientists. In this project, we aim to implement a general-purpose central image data repository that allows image researchers to collect data with semantic properties as well as data query. One of our researchers has come up with the specific challenge of collecting images with weight data of infants in least developed countries with limited internet access. The rationale is to predict infant weights based on image data by applying Machine Learning techniques. To address the data collecting issue, I implemented a mobile application which features online and offline image and annotation upload and a web application which features image query functionality. This work is derived and partly decoupled from the previous project – ImageSfERe (Image Sharing for Epilepsy Research), which is a web-based platform to collect and share epilepsy patient imaging.

1. Today honey bee colonies face a wide range of challenges in modern agricultural landscapes which entails the need for a comprehensive investigation of honey bees in a landscape context and the assessment of environmental risks. Within this dissertation the pollen foraging of honey bee colonies is studied in different agricultural landscapes to gain insight into the use of pollen resources and the influence of landscape structure across the season. General suggestions for landscape management to support honey bees and other pollinators are derived. 2. Decoding of waggle dances and a subsequent spatial foraging analysis are used as methods in Chapters 4 and 5 to study honey bee colonies in agricultural landscapes. The recently developed metabarcoding of mixed pollen samples was applied for the first time in honey bee foraging ecology and allowed for a detailed analysis of pollen, that was trapped from honey bees in front hive entrances (Chapter 6). 3. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach to light microscopy, which still is a tedious and error-prone task. In this study we assessed mixed pollen probes through next-generation sequencing and developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialized palynological expert knowledge. 4. During maize flowering, four observation hives were placed in and rotated between 11 landscapes covering a gradient in maize acreage. A higher foraging frequency on maize fields compared to other landuse types showed that maize is an intensively used pollen resource for honey bee colonies. Mean foraging distances were significantly shorter for maize pollen than for other pollen origins, indicating that effort is put into collecting a diverse pollen diet. The percentage of maize pollen foragers did not increase with maize acreage in the landscape and was not reduced by grassland area as an alternative pollen resource. Our findings allow estimating the distance-related exposure risk of honey bee colonies to pollen from surrounding maize fields treated with systemic insecticides. 5. It is unknown how an increasing area of mass-flowering crops like oilseed rape (OSR) or a decrease of semi-natural habitats (SNH) change the temporal and spatial availability of pollen resources for honey bee colonies, and thus foraging distances and frequency in different habitat types. Sixteen observation hives were placed in and rotated between 16 agricultural landscapes with independent gradients of OSR and SNH area within 2 km to analyze foraging distances and frequencies. SNH and OSR reduced foraging distance at different spatial scales and depending on season, with possible benefits for the performance of honey bee colonies. Frequency of pollen foragers per habitat type was equally high for SNH, grassland and OSR fields, but lower for other crops and forest. In landscapes with a small proportion of SNH a significantly higher density of pollen foragers on SNH was observed, indicating the limitation of pollen resources in simple agricultural landscapes and the importance of SNH. 6. Quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment we rotated 16 honey bee colonies across 16 agricultural landscapes (see also Chapter 5), used traps to get samples of collected pollen and observed the intra-colonial dance communication to gain information about foraging distances. Neither the amount of collected pollen nor pollen diversity were related to landscape diversity. The revealed increase of foraging distances with decreasing landscape diversity suggests that honey bees compensate for a lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. 7. Our results show the importance of diverse pollen resources for honey bee colonies in agricultural landscapes. Beside the risk of exposure to pesticides honey bees face the risk of nutritional deficiency with implications for their health. By modifying landscape composition and therefore availability of resources we are able to contribute to the wellbeing of honey bees. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes. / 1. Honigbienen stehen heutzutage vor einer Vielzahl von Herausforderungen in der modernen Agrarlandschaft, was umfassende Untersuchungen von Honigbienen im Landschafskontext erforderlich macht. Im Rahmen dieser Arbeit wurde das Pollensammeln von Honigbienenvölkern in verschiedenen Agrarlandschaften studiert, um Einblick in die Nutzung von Pollenressourcen und auf den Einfluss der Landschaftsstruktur zu gewinnen. 2. Die Dekodierung von Schwänzeltänzen und eine anschließende räumliche Analyse des Sammelverhaltens werden als Methoden in den Kapiteln 4 und 5 eingesetzt, um Bienenvölker in Agrarlandschaften zu untersuchen. Das kürzlich entwickelte Metabarcoding von gemischten Pollenproben wurde zum ersten Mal in der Honigbienenökologie angewandt und ermöglichte eine detaillierte Analyse von Pollenproben, die per Pollenfallen vor den Stockeingängen gesammelt wurden (Kapitel 6). 3. Pollenbestimmung durch molekulare Sequenzierung und DNA Barcoding wurde als Alternative zur Lichtmikroskopie vorgeschlagen, die immer noch sehr mühsam und fehlerbehaftet ist. In dieser Studie bestimmten wir gemischte Pollenproben durch Next-Generation-Sequenzierung und entwickelten einen bioinformatischen Arbeitsablauf um diese Hochdurchsatz-Daten mit einer neu kreierten Referenzdatanbank zu analysieren. Um die Durchführbarkeit zu evaluieren verglichen wir Ergebnisse aus der klassischen Identifizierung via Lichtmikroskopie derselben Proben mit unseren Sequenzier-Ergebnissen. Häufigkeitsschätzungen auf Basis der Sequenzierdaten waren signifikant mit den gezählten Häufigkeiten via Lichtmikroskopie korreliert. Next-Generation-Sequenzierung stellt daher einen nützlichen und effizienten Arbeitsablauf dar, um Pollen auf dem Gattungs- und Artniveau zu bestimmen ohne spezielles palynologisches Expertenwissen zu benötigen. 4. Während der Maisblüte wurden vier Beobachtungsstöcke in 11 Landschaften mit einem Maisflächengradienten platziert und zwischen diesen rotiert. Maisfelder wurden intensiver genutzt als Flächen anderer Landnutzungstypen. Die mittleren Sammeldistanzen waren signifikant niedriger für Maispollen als Pollen anderer Herkunft, was darauf hinweist, dass Aufwand in das Sammeln einer diversen Pollendiät gesetzt wird. Der Anteil an Maispollensammlerinnen stieg nicht mit der Maisanbaufläche in der Landschaft und wurde nicht durch Grünlandfläche als alternative Pollenressource reduziert. Unsere Ergebnisse ermöglichen die Schätzung des entfernungsbezogenen Expositionsrisikos von Honigbienenvölker auf Pollen aus den umliegenden Maisfeldern, die mit systemischen Insektiziden behandelt werden. 5. Es ist nicht bekannt, wie eine Zunahme von Massentrachten wie Raps (OSR) oder eine Abnahme von halbnatürlichen Habitaten (SNH) die zeitliche und räumliche Verfügbarkeit von Pollenressourcen für die Honigbienen, und damit Sammeldistanzen und -frequenzen in verschiedenen Lebensraumtypen verändert. Sechzehn Beobachtungsstöcke wurden in 16 Agrarlandschaften mit unabhängigen Gradienten an OSR- und SNH-Fläche innerhalb von 2 km platziert und regelmäßig rotiert, um Sammeldistanzen und -frequenzen zu analysieren. SNH und OSR reduzierten die Sammeldistanzen auf verschiedenen räumlichen Skalen und je nach Saison, mit möglichen Vorteilen für die Leistungsfähigkeit von Bienenvölkern. Die Häufigkeit der Pollensammler pro Habitattyp war gleich hoch für SNH, Grünland und OSR, aber niedriger für andere Kulturen und Wald. In Landschaften mit einem kleinen Anteil von SNH wurde eine deutlich höhere Dichte von Pollensammlerinnen auf SNH beobachtet, was auf die Begrenzung der Pollenressourcen in einfachen Agrarlandschaften und die Bedeutung von SNH hinweist. 6. Menge und Diversität des gesammelten Pollens können das Wachstum und die Gesundheit von Honigbienenvölkern beeinflussen, aber es ist wenig über den Einfluss der Landschaftsstruktur auf die Pollendiät bekannt. In einem Feldexperiment rotierten wir 16 Honigbienenkolonien über 16 Agrarlandschaften (siehe auch Kapitel 5), nutzten Pollenfallen um Proben des gesammelten Pollens zu nehmen und beobachteten die intrakoloniale Tanzkommunikation, um Informationen über die Sammeldistanzen zu erhalten. Weder Pollenmenge noch -diversität waren von der Landschaftsdiversität abhängig. Der offenbarte Anstieg von Sammeldistanzen mit abnehmender Landschaftsdiversität legt nahe, dass Honigbienen durch die Erweiterung des Pollensammelbereichs eine niedrigere Landschaftsdiversität kompensieren, um Pollenmenge und -diversität zu erhalten. 7. Unsere Ergebnisse zeigen die Bedeutung eines diversen Pollenangebots für Bienenvölker in der Agrarlandschaft. Neben dem Risiko einer Exposition gegenüber Pestiziden, stehen Bienenvölker vor der Gefahr von Mangelernährung mit Auswirkungen auf ihre Gesundheit. Durch eine Änderung der Landschaftzusammensetzung und damit der Verfügbarkeit von Ressourcen können wir zum Wohlergehen der Honigbienen beitragen. Agrarumweltmaßnahmen mit dem Ziel Bestäuber zu unterstützen, sollten sich auf mögliche räumliche und zeitliche Lücken in der Pollenverfügbarkeit und Vielfalt in der Agrarlandschaft konzentrieren.

In an earlier project completed in our laboratory a seasonal fluctuation in Phosphoglucomutase (PGM) phenotype frequencies was found, so that the winter bees were almost all heterozygotes and long lived than the summer bees among which homozygotes were significantly at high frequencies at Pgm locus. Same results were obtained in populations of three subspecies, A. m. meda, A. m. caucasica, and A. m. carnica from different climatic regions. In the current study environmental cues related with seasonal change in PGM phenotype frequency was examined along with the correlation between PGM heterozygosity and overwintering success. Cessation of food influx was found to be effective by itself as an environmental cue that causes a sudden and sharp increase in PGM heterozygosity. In addition to that, PGM heterozygosity of the colonies with greater overwintering success was found to be significantly higher than the ones with intermediate or low overwintering success. Benefiting from the previous studies and the results of current study, ethyl oleate was suggested as a chemical signal that functions in the regulation of PGM heterozygosity.

The honeybee is one of the model species in insect olfaction and its sense of smell is well studied. However, knowledge about the spectrum of odorants detectable to honeybees is limited. One class of odorants that has never been tested so far are the amino acids, which are important constituents of floral nectar. The experiments reported here were conducted in order to (1) determine if the odor of amino acids is detectable to honeybees (Apis mellifera), and (2) determine olfactory detection thresholds in honeybees for detectable amino acid odors. To this end, the proboscis extension reflex, a classical conditioning paradigm that takes advantage of the honeybee’s ability to build a robust association between an odor stimulus and a nectar reward, was used. The results demonstrate that five out of 20 amino acids presented at 100 mM were detectable. The honeybees’ median olfactory detection thresholds for these five amino acids are 12 mM for L-tyrosine and L-cysteine, 50 mM for L-asparagine and L-tryptophan, and 100 mM for L-proline. These threshold values are high in comparison to naturally occurring concentrations in floral nectar, and compared to threshold values obtained in vertebrate species. One possible explanation for these findings is that the size of the olfactory receptor repertoire of honeybees limits their olfactory capabilities in terms of detectability and sensitivity for the odor of amino acids

Pollinering är avgörande för ett antal kommersiella grödor samt för olika växtarter i inhemsk vegetation. Honungsbi (Apis mellifera) är en av de viktigaste pollinerarna. I regionen Taranaki i Nya Zeeland finns två aspekter av kommersiell pollinering, dels betalar många biodlare för tillgång till manuka för de antibakteriella egenskaperna honungen får av den, dels betalar frukt- och grönsaksodlare för pollinering av sina grödor. Markägare kan ha ett intresse av att spara manuka på sina marker och få en utkomst från biodlarna genom att dessa betalar för åtkomsten. Studien syftar till att klarlägga de effekter som kommersiell pollinering har i Taranaki-regionen idag och för framtida användning och har utgått från intervjuer med biodlare, frukt- och grönsaksodlare och personer som representerar myndigheterna. Resultaten visar att de ekonomiska effekterna av kommersiell pollinering i dag i Taranaki-regionen är relativt små. Även den kommersiella pollineringens roll för skydd och förvaltning av inhemsk natur undersöks. Kommersiell pollinering kan vara en viktig del inom naturskydd men är samtidigt beroende på om interaktionen med andra bin kan vara skadlig. Pollineringstjänster kan utvecklas dels genom information till markägare och jordbrukare, dels genom marknadsföring från biodlare. / Pollination is crucial for a number of commercial crops as well as for native vegetation. The honey bee (Apis mellifera) is one of the most important pollinators. In the Taranaki region in New Zealand there are two aspects of commercial pollination; beekeepers pay for access to manuka due to the antibacterial quality it gives the honey and orchardists pay for pollination of their crops. Landowners could have an interest in managing the manuka and having beekeepers paying for the access. This study seeks to elucidate the effects that commercial pollination has in Taranaki region, today as well as for future use, through interviews with beekeepers, orchardists and authority personnel. The results show that the effects of commercial pollination in Taranaki are of minor extent. Also the role commercial pollination plays for environmental management is highlighted. Commercial pollination may be a part of nature protection and play a particular role in effort to increase biodiversity, but it is much depending on the interaction with native bees. Pollination services may be developed in different ways including informing landowners and farmers and marketing of beekeepers.