Agradecemos a Colby Carpenter e Spencer Mathias por sua ajuda no design de impressão 3D. Agradecemos a Ellen Klinger pela ajuda na produção das figuras fotográficas e jonathan B. Koch por prestar assistência com as revisões. O financiamento foi fornecido pela Unidade de Pesquisa de Biologia, Manejo e Sistemática de Insetos polinizador do USDA-ARS.

1. Imprimir estruturas de armadilhas de pólen
<ol><li>Baixe o arquivo STL apropriado para a caixa de ninho em que as abelhas estão aninhando (por exemplo, colmeias estilo Biobest ou Koppert, https://www.ars.usda.gov/pacific-west-area/logan-ut/pollinating-insect-biology-management-systematics-research/docs/pollen-traps/). Os arquivos estão disponíveis ao público, gratuitos para download e modificação pelo usuário final.</li>
	<li>Abra o arquivo STL no programa de impressora. Siga as instruções do fabricante da impressora para construir os quatro componentes da armadilha. NOTA: Deixe aproximadamente 3h para o corpo da armadilha imprimir, 2h para a bacia de captura imprimir e 30 min cada para a inserção de fechamento do filtro e armadilha para imprimir. O tamanho do corpo da armadilha é de 6 cm x 3,8 cm x 7 cm.</li>
</ol>2. Montagem da armadilha de pólen
<ol><li>Remova as estruturas de suporte impressas com o corpo da armadilha e bacia de captura, incluindo as da estrutura de peneira do corpo da armadilha(Figura 1).</li>
	<li>Use uma broca de 0,476 cm montada em uma broca de mão para limpar quaisquer fios de plástico que cruzem as bordas elevadas do filtro de pólen que possam impedir uma abelha de se mover pelos orifícios do filtro. Use uma lâmina de barbear e lixa de corte de caixa para equilibrar quaisquer solavancos ou bordas levantadas no lado plano do filtro de pólen.</li>
	<li>Coloque o filtro de pólen no corpo da armadilha empurrando suavemente o filtro plástico através de um lado do corpo da armadilha. O filtro só caberá de uma forma, pois o lado esquerdo da armadilha tem uma abertura maior para acomodar a passagem dos cones do filtro levantado.<ol>
<li>Se as fendas laterais forem muito pequenas para o filtro de pólen deslizar suavemente, raspe o plástico suficiente da fenda no corpo da armadilha com uma navalha ou outra ferramenta que pode remover pequenas porções de plástico de cada vez. Certifique-se de que o filtro de pólen se encaixa firmemente no lugar com não mais do que uma lacuna de 2 mm entre o filtro de pólen e o corpo da armadilha.</li>
	</ol>
</li>
	<li>Conecte a bacia de captura ao corpo da armadilha deslizando as bordas levantadas na parte inferior do corpo da armadilha para a ranhura no topo da bacia de captura. A bacia de captura deve ser posicionada diretamente sob a região da peneira do corpo da armadilha (Figura 1A\u2012E).</li>
	<li>Faça as modificações apropriadas cortando ou lixando o plástico para permitir a colocação suave e a remoção da bacia de captura do corpo da armadilha. A colocação da bacia de captura irá fixar o filtro de pólen no lugar e não pode ser removido até que a bacia de captura seja removida, nem o filtro de pólen possa ser inserido enquanto a bacia de captura estiver no local. NOTA: Se várias colmeias forem colocadas próximas umas das outras, fornecer a cada colmeia uma combinação de cores única do corpo da armadilha, bacia de captura e estruturas de filtro de pólen, além de implantar colmeias com orientação variada, ajudará os trabalhadores que retornam a encontrar seus ninhos.</li>
</ol>3. Preparação da colônia de abelhas bumble
<ol><li>Pare a alimentação por pólen 24\u201248 h antes de implantar colônias. Isso fará com que os trabalhadores usem qualquer pólen armazenado e os estimulem a deixar o ninho em busca de pólen.</li>
	<li>Prepare o corpo da armadilha da colmeia para instalação inserindo uma inserção de fechamento de armadilha no slot do filtro para evitar que as abelhas escapem durante a instalação da armadilha.</li>
	<li>Trabalhando sob luz vermelha para evitar que as abelhas voem, levante a caixa do ninho de plástico para fora da caixa externa de papelão.</li>
	<li>Localize a entrada do ninho de plástico na frente do ninho. Existem dois estilos de entrada dependendo do fornecedor do ninho: caixas estilo Koppert (etapa 3.5) e caixas estilo Biobest (passo 3.6).</li>
	<li>Para as entradas de colmeias estilo Koppert, monte a armadilha de pólen na entrada do ninho, puxando para cima na guia de entrada até que ambos os buracos de entrada estejam abertos.<ol>
<li>Insira os dois tubos da armadilha de pólen nos orifícios de entrada, garantindo que a peneira da armadilha do pólen esteja no fundo. Empurre suavemente para baixo na guia de entrada de plástico para fixar a armadilha de pólen no lugar.</li>
	</ol>
</li>
	<li>Para instalar a armadilha de pólen nas entradas da colmeia estilo Biobest, use uma chave de fenda de cabeça plana para retirar suavemente o dispositivo de entrada de plástico da caixa do ninho. Insira a armadilha de pólen nos buracos de entrada do ninho até que a armadilha do pólen esteja firmemente contra o ninho(Figura 1E).<ol>
<li>Fixar a armadilha no ninho usando fita adesiva ou cola seca rápida onde a armadilha entra em contato com a caixa do ninho, se necessário.</li>
	</ol>
</li>
	<li>Devolva a caixa do ninho de plástico para a caixa de papelão. O papelão pode precisar ser cortado para acomodar a armadilha do pólen.</li>
</ol>4. Implantação de ninhos
<ol><li>Coloque caixas de ninho na área de estudo. Fornecer cobertura para proteger contra precipitação e ancoragem para vento, pois estes podem afetar negativamente a qualidade e a quantidade de pólen que é coletado. Forneça colmeias colocadas em estufas com cobertura solar adequada para reduzir o superaquecimento.</li>
	<li>Remova a inserção de fechamento da armadilha do corpo da armadilha para permitir que as abelhas forrageiam livremente para que os invasores possam se orientar com a área circundante e a localização de seu ninho. O tempo de voo de orientação deve ser completo em 24 horas em condições normais.</li>
</ol>5. Coleta de pólen
<ol><li>Para engatar a armadilha, deslize o filtro de pólen para dentro da ranhura do filtro, garantindo que ela esteja bem no lugar.</li>
	<li>Instale a bacia de captura deslizando-a para o corpo da armadilha da frente até que esteja totalmente fechada. Se a bacia de captura estiver excessivamente solta ou cair do corpo da armadilha, use um elástico para fixá-la no corpo da armadilha.</li>
	<li>Observe as abelhas entrando e saindo da armadilha de pólen na primeira implantação para garantir que os orifícios do filtro de pólen sejam grandes o suficiente para acomodar as abelhas.<ol>
<li>Se os trabalhadores não conseguirem passar pelo filtro de pólen, remova o filtro e use brocas maiores que 0,476 cm para aumentar o tamanho dos furos. Faça isso de forma sequencial, aumentando o diâmetro do orifício 1/32 in. (0,079 cm) cada vez, pois os orifícios muito grandes não coletarão pólen.</li>
		<li>Uma vez que as abelhas são capazes de passar pelo filtro, continue a observar a entrada para garantir que o pólen esteja sendo removido após a reentrada. NOTA: As abelhas devem ter alguma dificuldade em se mover pelos orifícios do filtro de pólen, especialmente nas primeiras vezes que passam. Se eles passarem muito facilmente, o pólen pode não ser desalojado do corbiculado.</li>
	</ol>
</li>
	<li>Após o período designado de coleta de pólen, deslize e remova a bacia de captura do corpo da armadilha.</li>
	<li>Processe as cargas de pólen de acordo com seu projeto experimental.</li>
	<li>Remova o filtro de pólen para permitir que os trabalhadores forragem livremente até o próximo período de coleta de pólen. O corpo da armadilha pode permanecer preso à colmeia durante a duração do experimento. NOTA: Armadilhas de pólen podem ser engajadas enquanto o pesquisador desejar coletar pólen de uma colônia. No entanto, a implantação de armadilhas de pólen por mais de 24 h em uma semana pode resultar em fome de ninhada em um desenvolvimento de uma colmeia e colônia retardada.</li>
</ol>

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A coleta de pólen das entradas de colônias de abelhas podem permitir uma variedade de estudos ecológicos e agrícolas. Identificar as fontes florais das quais as abelhas bumble coletam pólen fornece informações valiosas e insights sobre a diversidade de plantas que contribuem para a dieta geral de uma colônia19. Identificar a fonte de pólen tem implicações tanto para a produção agrícola quanto para estudos de serviços ecossistêmicos em terras silvestres12<sup...

As abelhas bumble(Bombus spp.) são grandes insetos robustos que são encontrados nas regiões temperadas, alpinas e árticas do mundo1. São importantes para plantar comunidades e fornecer importantes serviços de polinização para as culturas agrícolas que visitam2. Declínios recentes na abundância e distribuição de várias espécies trouxeram sua importância como polinizadores à vanguarda da conscientização pública3. Pesquisadores identificaram vários estressores que provavelmente estão contribuindo para o declínio populacional, incluindo a falta de recursos florais diversos e abundantes nos quais as abelhas se forragem4. Identificar de quais espécies de plantas as abelhas desossa permitem que pesquisadores e gestores de terras entendam como as abelhas podem estar respondendo a mudanças na disponibilidade de recursos, competição e distúrbios antropogênicos5,6.
Estudos que investigam as preferências de forrageamento de pólen de abelhas bumble são frequentemente conduzidos por pesquisadores capturando abelhas individuais forrageando flores e, em seguida, removendo as cargas de pólen corbicular de espécimes para posterior processamento e identificação7,8,9,10. Embora este método forneça uma visão de como uma espécie ou um conjunto de espécies de abelhas bumble utiliza os recursos em uma área7, é tempo de que as diferenças intensivas e potenciais nas preferências entre as colmeias não podem ser discernidas sem análises moleculares adicionais para identificar colônia de origem da abelha forrageiras11.
Para alguns estudos de dinâmica de forrageamento, deseja-se realizar os estudos em colônias individuais; no entanto, os ninhos de abelhas selvagens estão geralmente localizados no subsolo ou no nível do solo, tornando-os difíceis de localizar12. As colmeias de abelhas bumble produzidas comercialmente fornecem aos pesquisadores maior acesso e melhor controle experimental e a remoção do pólen dos trabalhadores ainda é conduzida principalmente pela captura de forasteiros à medida que retornam à colmeia e removem manualmente suas cargas de pólen corbicular13,14. A remoção de pólen à mão do corbicula de uma abelha é intensiva em tempo com baixo rendimento por hora de pólen especialmente nas entradas da colmeia onde a taxa de indagas de pólen que retornam pode ser baixa. Além disso, a remoção manual do pólen das abelhas pode resultar em picadas de trabalhadores perturbados.
Armadilhas de pólen têm sido usadas para remoção experimental de pólen de abelhas de mel por décadas15; ainda assim, um método passivo para remover pólen de abelhas bumble não foi desenvolvido. O principal obstáculo no desenvolvimento de um mecanismo para remover o pólen das abelhas inquecíveis que retornam é a grande variação do tamanho do trabalhador que existe em uma colônia de abelhasbumble 16. As armadilhas de pólen de abelhas são eficazes em grande parte porque o tamanho do trabalhador das abelhas não varia muito. Além disso, essas armadilhas requerem apenas pequenas manipulações após a instalação e não exigem que as abelhas sejam sacrificadas17. Isso é conseguido usando telas ou superfícies plásticas que desalojam o pólen das patas traseiras dos trabalhadores à medida que retornam à colmeia. Essas armadilhas removem apenas uma parte das cargas de pólen dos forasteiros que retornam e os vários desenhos desses resultam em eficiências variadas na coleta de pólen. À medida que o pólen é removido das pernas das abelhas, ele cai através de uma tela e em uma bacia de coleta à qual as abelhas não têm acesso, para que o pesquisador possa removê-lo com apenas pequenos distúrbios para a colmeia.
O objetivo do presente estudo é adaptar as técnicas utilizadas para a coleta de pólen das colmeias de abelhas de mel e aplicá-los em ninhos de abelhas bumble usando estruturas impressas em 3D e testar os desenhos de armadilhas em colônias de Bombus huntii. O processo de projeto seguiu as premissas de que as armadilhas deveriam ser baratas de produzir, adaptáveis a uma variedade de espécies de abelhas bumble, causar danos mínimos ou perturbação às abelhas, e que a taxa de remoção de pólen deve exceder a coleta manual de pólen. A tecnologia de impressão tridimensional é versátil, facilmente acessível e uma ferramenta econômica que permite aos pesquisadores replicar e modificar objetos para fins específicos18. A técnica aqui apresentada instrui o usuário a construir armadilhas de pólen e anexá-las a colônias de abelhas disponíveis comercialmente. As armadilhas não foram projetadas para serem usada com colônias selvagens. Essas armadilhas removem passivamente as cargas de pólen corbicular das patas traseiras do pólen carregando abelhas enquanto retornam às suas caixas de ninho.

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		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:71px;">MakerBot Replicator+</td>
			<td style="width:87px;">MakerBot</td>
			<td style="width:87px;">Model PABH65</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:71px;">MakerBot Tough Material</td>
			<td style="width:87px;">PLA Filament</td>
			<td style="width:87px;"></td>
			<td>various colors</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:71px;">Nest Box</td>
			<td style="width:87px;">Biobest</td>
			<td style="width:87px;"></td>
			<td>Not sold publicly without bee purchase</td>
		</tr>
	</tbody>
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a 3d printed pollen trap for bumble bee (bombus) hive entrances

Para verificar as fontes vegetais das quais as abelhas se forragem para pólen, os indivíduos devem ser coletados para remover suas cargas de pólen corbicular para análise. Isso tem sido tradicionalmente feito por invasores de rede nas entradas do ninho ou nas flores, resfriando as abelhas no gelo, e depois removendo as cargas de pólen do corbiculado com fórceps ou um pincel. Este método é demorado e trabalhoso, pode alterar o comportamento normal de forragem, e pode resultar em incidentes de picada para o trabalhador que realiza a tarefa. Armadilhas de pólen, como as usadas em colmeias de abelhas, coletam pólen desalojando cargas de pólen corbicular das pernas dos trabalhadores enquanto passam pelas telas na entrada do ninho. As armadilhas podem remover uma grande quantidade de pólen de abelhas invasoras que retornam com mão-de-obra mínima, mas até o momento nenhuma armadilha está disponível para uso com colônias de abelhas. Os trabalhadores dentro de uma colônia de abelhas podem variar em tamanho tornando difícil adaptar esse mecanismo a colmeias de abelhas desarmadas comercialmente. Usando programas de design de impressão 3D, criamos uma armadilha de pólen que remove com sucesso as cargas de pólen corbicular das pernas de intrusos de abelhas bumble retornando. Este método reduz significativamente o tempo exigido pelos pesquisadores para coletar pólen de intrusos de abelhas que retornam à colônia. Apresentamos o projeto, resultados de testes de eficiência de remoção de pólen, e sugerimos áreas de modificações para os pesquisadores adaptarem armadilhas a uma variedade de espécies de abelhas ou desenhos de caixas de ninhos.

Oito diferentes desenhos de filtros de pólen foram testados para determinar sua eficácia e eficiência na remoção de cargas de pólen corbicular dos trabalhadores de abelhas bumble retornando. Todos os projetos foram bem sucedidos em remover pelo menos uma carga de pólen corbicular de um forasteiro retornando. No entanto, alguns foram encontrados para retardar os trabalhadores de sair ou entrar na colmeia ou não remover cargas de pólen(Tabela 1). Armadilhas de pólen com vários filtros foram tes...

Watch this Scientific Journal Video about A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances at JoVE.com

A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

Apresentamos um mecanismo não letal e automatizado para coletar pólen de trabalhadores de abelhas bumble(Bombus)que retornam a uma colmeia. Instruções para produzir, preparar, instalar e usar os dispositivos estão incluídas. Ao usar objetos impressos em 3D, a modificação no design foi oportuna, eficiente e permitiu uma rápida reviravolta para testes.

Uma armadilha de pólen impresso em 3D para entradas de colmeias de abelhas bumble(Bombus)

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis.  ...

a-3d-printed-pollen-trap-for-bumble-bee-bombus-hive-entrances

Research

JoVE Journal

Biology

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances

5.1K visualizações.  Utah State University. Apresentamos um mecanismo não letal e automatizado para coletar pólen de trabalhadores de abelhas bumble(Bombus)que retornam a uma colmeia. Instruções para produzir, preparar, instalar e usar os dispositivos estão incluídas. Ao usar objetos impressos em 3D, a modificação no design foi oportuna, eficiente e permitiu uma rápida reviravolta para testes.

Vídeo: A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

Medir a rigidez à flexão de células bacterianas Usando uma armadilha óptica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Biologia

Associated Chromosome Trap for Identifying Long-range DNA Interactions

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.

The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

Armadilha cromossomo associados para Identificar Interações de longo alcance DNA

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Um Sistema de 3D para condrócitos Cultivar Humanos Articular no líquido sinovial

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Análise Quantitativa de Autophagy usando microscopia de fluorescência avançada 3D

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Imunofluorescência combinados e FISH DNA em 3D preservado núcleos interfásicos para estudar mudanças na organização nuclear 3D

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

Obtenção de amostras com retardado, acelerado e invertida Envelhecimento no Modelo Bee Mel

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Métodos para comparar Nutrientes na beebread Feito por africanizadas e mel europeu abelhas e os efeitos sobre Títulos Hemolinfa Proteína

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

Dissecção e Observação de Honey Bee Dorsal navio de Estudos da função cardíaca

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

Monitoramento de nível de colônia os efeitos da exposição de pesticidas subletais em mel de abelhas

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

We describe methods for collecting corbicular pollen from honey bees as well as protocols for color sorting, acetolysis, and microscope slide preparation of pollen for taxonomic identification. In addition, we present pellet color and taxonomic diversity of corbicular pollen collected from five cropping systems using pollen traps.

Coleta e Identificação de Pólen das Colônias de Abelhas

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...