Agradecemos ao Dr. Emir Islamovic pela assistência laboratorial e de estufa. Também agradecemos ao Dr. Sherry Flint-Garcia por fornecer as linhas de introgressão de milho x teosinte.

1. Crescimento do Material Vegetal
<ol><li>Selecione linhas de plantas para inoculação e triagem. Foram utilizadas duas linhas de milho, cinco linhas de teosinto e quarenta linhas de milho x teosintocom resistência não caracterizada aos maydis u.</li>
	<li>Sementes de plantas para experimentos experimentais (injeçãode maydis) e controle (injeção de água) inoculação de agulha. Faça isso por cada linha de plantas.</li>
	<li>Plante quatro sementes (replica) para cada linha vegetal em pequenos apartamentos empurrando as sementes cerca de 1/2 polegada para o solo com o dedo e cobrindo levemente com o solo(Figuras 1A e 1B). Não embale o solo sobre a semente. Plantar a semente mais profundamente ou embalar o solo sobre a muda pode causar problemas com o surgimento de mudas.</li>
	<li>Rege as sementes no solo. Certifique-se de que o solo está encharcado e as sementes permanecem sob o solo após a rega.</li>
	<li>Após a rega, coloque as plantas em uma câmara de crescimento com ambientes diurnos e noturnos de temperatura de 28/20 °C e 14/10 horas de fotoperíodo, respectivamente e aproximadamente 500 μmol/m2 seg   de radiaçãos fotossintéticas ativas no topo do dossel. Manter a umidade relativa durante o dia e a noite em aproximadamente 70% e 90%, respectivamente.</li>
	<li>Mantenha todas as plantas na mesma câmara de crescimento para manter um ambiente de crescimento que seja congruente durante todo o experimento.</li>
	<li>Após 10 dias, remova as plantas da câmara de crescimento e inocula as plantas com a cultura de suspensão celular dos EUA por meio de um método de inoculação de injeção de agulha. Nota: As plantas de milho podem ser vacinadas 7 dias após o plantio8-10. No entanto, as plantas de teosinte são muito pequenas depois de 7 dias. Portanto, inocular as plantas de milho e teosinto 10 dias após o plantio para consistência dentro do experimento (ver passo 2.12).</li>
</ol>2. Inoculação de injeção de agulha
<ol><li>Faça todo o trabalho em um capô de fluxo laminar. Remova os estoques de glicerol U. maydis do armazenamento do congelador. Use um laço estéril e estrias de glicerol de cepas de u. maydis tipo selvagem 1/2 (tipo acasalamento a1b1) e 2/9 (tipo acasalamento a2b2, perto de isogênico a 1/2) em placas de ágar de dextrose de batata (PDA). Mantenha as cepas separadamente.</li>
	<li>Coloque as placas PDA listradas com U. maydis em uma incubadora de 30 °C por dois dias. Se usar um patógeno biotrófico diferente, use a tensão adequada, a mídia e as condições de crescimento. Monitore o crescimento do patógeno durante o período de dois dias para garantir que a cepa de Maydis dos EUA esteja crescendo bem.</li>
	<li>Remova as placas PDA da incubadora após dois dias. As placas devem ter bom crescimento de patógenos e conter colônias únicas(Figura 2A). É importante obter colônias únicas. Se colônias únicas não estiverem presentes, recuem as placas em uma concentração menor.</li>
	<li>Faça todo o trabalho em um capô de fluxo laminar. Use um palito estéril para selecionar uma única colônia para cada cepa das placas PDA. Coloque o palito contendo uma única colônia em um caldo de dextrose de batata de 3 ml (PDB). É aconselhável ter 2-3 culturas.</li>
	<li>Coloque as culturas PDB de 3 ml em uma incubadora/agitador de 30 °C por dois dias a 200 rpm. Acompanhar o crescimento da cultura ao longo dos dois dias para garantir o crescimento da cultura. A cultura deve parecer muito nebulosa.</li>
	<li>Remova as culturas líquidas da incubadora/shaker e meça a concentração em OD600 para garantir que as células foram cultivadas a um OD de 1,0 (~1 x 107 células/ml)17.</li>
	<li>Leve as culturas de suspensão celular maydis dos EUA para uma concentração final de 1 x 106 células/ml, usando água em um volume final de cultura de 30 ml. Essa concentração resulta consistentemente em uma boa infecção das plantas com a cultura de suspensão celular patógena. 17 
</li>
</ol>Nota: Várias concentrações de suspensão celular devem ser testadas ao usar diferentes cepas de patógenos para determinar o título celular adequado necessário para a inoculação18,19. A concentração final dada para a cultura de suspensão celular pode ser usada como ponto de partida para titulação. A concentração adequada da cultura de suspensão celular patógena deve ser verificada visualizando os fenótipos vegetais com boa infecção (Figuras 3A-E).
<ol start="8"><li>Misture volumes iguais das duas cepas U. maydis antes da inoculação. Se o uso de uma cepa de patógeno proceder à etapa 2.9. Prepare culturas frescas de suspensão celular U. maydis para cada experimento de inoculação e descarte culturas de suspensão celular após dois dias.</li>
	<li>Para a inoculação experimental de injeção de agulha, encha uma seringa de 3 ml com a cultura de suspensão celular dos eua, puxando a cultura de suspensão celular para a seringa.</li>
	<li>Para a inoculação de injeção de agulha de controle, encha uma seringa de 3 ml com água17. Use o mesmo procedimento para a inoculação experimental da injeção de agulha.</li>
	<li>Conecte uma agulha hipodérmica de 0,457 mm x 1,3 cm ao final de cada seringa de 3 ml. O tamanho da agulha selecionada fornecerá a cultura de suspensão celular entre as folhas da planta com danos mínimos ao tecido vegetal.</li>
	<li>Remova as plantas experimentais e de controle da câmara de crescimento 10 dias após o plantio em preparação para inoculações de injeção de agulha(Figura 2B) (ver passo 1.7).</li>
	<li>Insira cuidadosamente a agulha hipodérmica contendo a cultura de suspensão celular U. maydis no caule de uma planta experimental em um ângulo de 90° logo acima da linha do solo. Insira a agulha até que esteja no meio da haste. Não empurre a agulha através da haste(Figura 2C).</li>
	<li>Injete a planta experimental com cerca de 100 μl da cultura de suspensão celular maydis 18,19. Isso vai variar ligeiramente dependendo da altura da muda. A cultura de suspensão celular vai empurrar através do caule e mover-se para o turbilhão da planta. A cultura de suspensão celular será visível no turbilhão da planta. Continue injetando 100 μl da cultura de suspensão celular em cada planta individual até que a seringa de 3 ml esteja vazia.</li>
	<li>Após a injeção, remova cuidadosamente a agulha da haste da planta. Retire a agulha da seringa agora vazia de 3 ml e encha com água. Coloque a agulha de volta na seringa e empurre a água através da agulha para remover qualquer tecido vegetal que possa ser pego na ponta da agulha.</li>
	<li>Repita as etapas 2.9-2.15 para cada planta experimental. Siga o mesmo protocolo para as plantas de controle injetando água.</li>
	<li>Coloque as plantas experimentais e de controle inoculadas de volta na câmara de crescimento. Rega as plantas diariamente molhando o solo, não o tecido vegetal.</li>
	<li>Verifique as plantas diariamente para detectar o desenvolvimento de patógenos e reações de resistência vegetal.</li>
</ol>3. Triagem de reação de resistência
<ol><li>Escore e regise as reações de resistência para cada planta 7, 10, 14 e 21 dias pós-inoculação (dpi) usando uma escala de classificação de reação de resistência de 1 a 5. A gravidade da doença aumenta à medida que os valores numéricos na escala de classificação aumentam(Tabela 2). Uma reação de resistência de 1C (clorose da folha), 1A (produção de antocianina de folha) ou 2 (pequenas galos de folhas) indicam resistência. Uma reação de resistência de 3 (galos de haste), 4 (gail basal) ou 5 (morte vegetal) indica suscetibilidade(Figuras 3A-E e Tabela 2)18,19.</li>
	<li>Escore tanto plantas experimentais quanto de controle e classificações de reação de resistência recorde.</li>
	<li>Compare as reações de resistência das plantas experimentais e de controle. Selecione plantas experimentais com uma classificação de reação de resistência de 1C, 1A ou 2. Essas plantas são consideradas resistentes aos U. maydis18,19.</li>
	<li>Repita todo o experimento para verificar os fenótipos da planta.</li>
</ol>

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Os autores não têm nada a revelar.

Neste estudo, o método de inoculação de injeção de agulha usado para fornecer uma cepa de U. maydis na haste de 700 plantas de milho e teosinto foi bem sucedido. Além disso, uma escala revisada de classificação de resistência à doença foi usada para rastrear as plantas e detectar o desenvolvimento de patógenos. Como resultado do uso de ambos os métodos, foram identificadas linhas vegetais resistentes aos maydis u. foram identificadas entre 700 plantas de milho e teosinto que agora podem ser...

As doenças fúngicas das plantas representam uma das ameaças mais eminentes à agricultura. A necessidade de desenvolver culturas com maior resistência à doença está aumentando devido às necessidades alimentares de uma população mundial em crescimento. Patógenos vegetais infectam naturalmente plantas agrícolas no campo causando doenças que impactam negativamente o rendimento da cultura6. Foi demonstrado que identificar e utilizar plantas resistentes pode melhorar a resistência e diminuir a perda de rendimento. Cultivares resistentes foram identificadas em muitas espécies vegetais, incluindo milho, trigo, arroz e sorgo, inoculando as plantas com um patógeno vegetal e selecionando para linhas resistentes7. Portanto, o desenvolvimento e o uso de um método de inoculação eficiente permitiriam que muitas plantas fossem inoculadas e rastreadas para resistência. Vários métodos de inoculação têm sido utilizados, incluindo a inoculação de mergulho, a pipetação da cultura de suspensão celular do patógeno no giro da planta e a inoculação da injeção de agulha8-11. A cada método, o patógeno deve ser introduzido de forma confiável entre as folhas vegetais onde o patógeno entra na planta através da formação de appresoria para garantir o desenvolvimento do patógeno e a infecção vegetal12,13.
O método de inoculação de mergulho envolve submergir uma muda de planta em uma cultura de suspensão de células patógenas, enquanto o método de pipetação requer colocar a cultura de suspensão de células patógenas no turbilhão da muda vegetal. No entanto, há problemas com ambos os métodos. Em primeiro lugar, ambos os métodos dependem do movimento natural do patógeno da superfície da folha para o tecido vegetal, que é altamente variável. A maioria dos patógenos entra naturalmente na planta através de aberturas estomatais ou feridas na superfície da folha vegetal. No entanto, há uma variabilidade significativa na capacidade dos patógenos de penetrar a superfície da folha vegetal através da estomata e/ou feridas na superfície da folha. Portanto, a penetração do patógeno não pode ser controlada com qualquer método de inoculação potencialmente resultando em dados inconsistentes. Em segundo lugar, ao selecionar um grande número de plantas, submergir as mudas em uma cultura de suspensão celular patógena pode ser demorado e pode limitar o número de plantas que podem ser rastreadas. Por outro lado, o protocolo de inoculação de injeção de agulha descrito aqui fornece a cultura de suspensão de células patógenas entre as folhas da planta facilitando a formação de appressoria14. O patógeno então utiliza a appressoria recém-desenvolvida para entrar na planta eliminando o problema de penetração do patógeno. Além disso, o protocolo de inoculação de injeção de agulha fornece uma gama de fenótipos para plantas de milho e teosinto que foram inoculadas com U. maydis e demonstram boa infecção. Os fenótipos podem ser usados como marcador para determinar a melhor concentração para a cultura de suspensão celular patógena, resultando em fenótipos vegetais consistentes dentro e entre diferentes experimentos.
Seguindo a inoculação vegetal com uma cultura de suspensão de células patógenas, as plantas são tipicamente rastreadas para detectar um fenótipo resistente ou suscetível8-11,15. Embora as escalas de classificação da doença tenham sido usadas extensivamente para triagem e classificação de fenótipos vegetais, as escalas de classificação diferem dependendo do patógeno que está sendo analisado. Portanto, um estabelecimento de protocolo de escala de classificação de doenças para as interações de u. maydis e milho pode ser utilizado para patógenos fúngicos semelhantes16.
A presente série de protocolos detalha a inoculação da injeção de agulha com uma cultura de suspensão celular dos EUA e triagem de reação de resistência à doença de linhas de introgressão de milho, teosinte e milho x teosinto. Os protocolos atuais não se limitam à inoculação de injeção de agulha de U. maydis em plantas de milho, mas podem ser utilizados para relativamente qualquer patógeno fúngico e espécies vegetais. Portanto, incluir os detalhes de ambos os métodos no mesmo protocolo permitirá que os pesquisadores utilizem diretamente os protocolos de inoculação e triagem ou manipulem os protocolos originais para melhor se adequarem ao patógeno e às espécies de interesse vegetal.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Seed for plants</td>
			<td></td>
			<td></td>
			<td>Collected from original crosses</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Conviron</td>
			<td>PGR14 REACH-IN</td>
			<td></td>
		</tr>
		<tr>
			<td>Planting flats</td>
			<td>Hummert International</td>
			<td>14-3385-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Soil (3 parts pine bark; 1 part peat moss with perlite)</td>
			<td>Hummert International</td>
			<td>10-1059-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Lab Conoco</td>
			<td>70875372</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol stock of pathogen (U. maydis) or fungal pathogen of interest</td>
			<td></td>
			<td></td>
			<td>Stocks were grown from original culture</td>
		</tr>
		<tr>
			<td>Sterile loop</td>
			<td>Fisher Scientific</td>
			<td>S17356A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potato dextrose agar (PDA) plates</td>
			<td>Fisher Scientific</td>
			<td>R454311</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator set to 30 &#176;C</td>
			<td>Fisher Scientific</td>
			<td>11-690-650F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile toothpicks</td>
			<td>Walmart</td>
			<td></td>
			<td>Purchased from Walmart and sterilized by autoclave</td>
		</tr>
		<tr>
			<td>Potato dextrose broth (PDB)</td>
			<td>Fisher Scientific</td>
			<td>ICN1008617</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator-shaker set to 30 &#176;C</td>
			<td>New Brunswick</td>
			<td>14-278-179</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Fisher Scientific</td>
			<td>4001000</td>
			<td></td>
		</tr>
		<tr>
			<td>U. maydis&#160;cell suspension culture (1 x 106 cells/ml)</td>
			<td></td>
			<td></td>
			<td>Grown from glycerol stock as described in the methods</td>
		</tr>
		<tr>
			<td>3 ml Syringes</td>
			<td>Becton Dickinson</td>
			<td>309606</td>
			<td></td>
		</tr>
		<tr>
			<td>.457 mm x 1.3 cm Hypodermic needles</td>
			<td>Kendall Brands</td>
			<td>8881250321</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and efficient method for assessing pathogenicity of ustilago maydis on maize and teosinte lines

O milho é uma grande cultura de cereais em todo o mundo. No entanto, a suscetibilidade aos pat pato patógenos biotróficos é a principal restrição ao aumento da produtividade. U. maydis é um patógeno fúngico biotrófico e o agente causal do milho smut no milho. Esta doença é responsável por perdas significativas de rendimento de aproximadamente US $ 1,0 bilhão anualmente nos EUA1 Vários métodos, incluindo rotação de culturas, aplicação de fungicida e tratamentos de sementes são atualmente usados para controlar o smut de milho2. No entanto, a resistência do hospedeiro é o único método prático para gerenciar o smut do milho. A identificação de plantas agrícolas, incluindo milho, trigo e arroz resistentes a vários patógenos biotróficos, diminuiu significativamente as perdas de rendimento anualmente3-5. Portanto, o uso de um método de inoculação de patógenos que fornece de forma eficiente e reprodutivelmente o patógeno entre as folhas da planta, facilitaria a identificação rápida de linhas de milho resistentes aos U. maydis. Como, um primeiro passo para a indentificação das linhas de milho resistentes aos U. maydis,um método de inoculação de injeção de agulha e um método de triagem de reação de resistência foi utilizado para inocular as linhas de introgressão de milho, teosinte e milho x teosinto com uma cepa de maydis U. maydis e para selecionar plantas resistentes.
Foram plantadas linhas de introgressão de milho, teosinte e de milho x teosinto, compostas por cerca de 700 plantas, inoculadas com uma cepa de U. maydis,e rastreadas para resistência. Os métodos de inoculação e triagem identificaram com sucesso três linhas de teosinto resistentes aos u. maydis. Aqui é apresentado um protocolo detalhado de inoculação de injeção de agulha e retração de resistência para linhas de introgressão de milho, teosinto e milho x teosinto. Este estudo demonstra que a inoculação de injeção de agulha é uma ferramenta inestimável na agricultura que pode fornecer maydis entre as folhas da planta e forneceu linhas vegetais resistentes aos maydis u. que agora podem ser combinados e testados em programas de reprodução para melhor resistência à doença.

Uma inoculação de injeção de agulha bem sucedida pode ser determinada visualizando o fenótipo das plantas inoculadas com U. maydis (experimental). A maioria das plantas experimentais eram suscetíveis à infecção por U. maydis. As plantas suscetíveis apresentaram desenvolvimento de doenças muito graves demonstradas pela formação de caule e gail basal com teliosporos pretos (Figuras 3D e 3E, Tabela 2). Várias plantas morreram após a inocula?...

Watch this Scientific Journal Video about A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines at JoVE.com

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Descreve-se o uso de um método de injeção de agulha para inocular plantas de milho e teosinto com o pato patogênico biotrófico Ustilago maydis. O método de inoculação da injeção de agulha facilita a entrega controlada do patógeno fúngico entre as folhas da planta onde o patógeno entra na planta através da formação de appresoria. Este método é altamente eficiente, permitindo inoculações reprodutíveis com u. maydis.

Um método rápido e eficiente para avaliar a patogenicidade dos maydis ustilago nas linhas de milho e teosinte

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

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Environment

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

8.4K visualizações.  University of Georgia. Descreve-se o uso de um método de injeção de agulha para inocular plantas de milho e teosinto com o pato patogênico biotrófico Ustilago maydis. O método de inoculação da injeção de agulha facilita a entrega controlada do patógeno fúngico entre as folhas da planta onde o patógeno entra na planta através da formação de appresoria. Este método é altamente eficiente, permitindo inoculações reprodutíveis com u. maydis.

Vídeo: A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

Uso de Chironomidae (Diptera) Pupal Exuviae como um Bioavaliação Protocolo Rápido para massas de água de superfície flutuante

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Ambiente

The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6)&#160;tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal&#8217;s muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

The Use of an Automated System GreenFeed to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals

Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals.

O uso de um sistema automatizado (GreenFeed) para monitorar entérica de metano e dióxido de carbono As emissões dos ruminantes

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

Administrando e Detecção de Marcas de proteína em artrópodes para a dispersão de Pesquisa

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

Método confiável para avaliar a germinação de sementes, dormência, e Mortalidade em condições de campo

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Protocolo para avaliar os efeitos relativos do ambiente e da genética na galhada e no crescimento corporal de um cervídeo de vida longa

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

Preparação de DMMTAV DMDTAV usando DMAV para aplicações ambientais: síntese, purificação e confirmação

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

O protocolo de Havaí para acompanhamento científico da broca do café: um modelo para agroecossistemas de café no mundo

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

Um flexível baixo custo sistema hidropônico para avaliar as respostas de planta para pequenas moléculas em condições estéreis

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

Elevado-throughput, microescala protocolo para a análise de parâmetros de processamento e qualidades nutricionais em milho (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Avaliar as habilidades de remoção de partículas de árvore folhas

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...