Agradecemos Molly permanece para diluição e pesticidas assistência formulação. Agradecemos também Troy Anderson para orientação análise de probidade e Zachary Adelman de assistência com design experimental. Esta pesquisa foi parcialmente apoiado por uma concessão Entomological Foundation e por fundos de bolsas Virginia Controle de Pragas.

NOTA: Este protocolo inclui dois ensaios de insecticida para tratar separadamente os ovos de percevejos e primeiros estádios. Ambos os protocolos foram realizados utilizando as mesmas insecticidas, no entanto, os protocolos tiveram que ser adaptadas para garantir a exposição insecticida e para manipular facilmente as amostras. Ensaio 1. Egg Dipping Inseticida <ol><li> Puxe acasaladas, bem alimentados adultos do sexo feminino percevejos de uma colónia e coloque as fêmeas (5-10) em uma placa de Petri com papel de filtro. Ter pelo menos dez repetições para garantir que não haverá ovos suficientes para o ensaio de insecticida. Verifique se as fêmeas colocaram ovos todos os dias. <ol><li> Uma vez que eles põem ovos, tomar as fêmeas para fora da placa de Petri (6 centímetros x 5 cm) e colocá-los em novas placas de Petri com um novo papel de filtro diariamente e registrar a data em antigos e novos pratos de Petri. </li><li> Permitir que os ovos à idade 4 a 5 dias antes de iniciar o ensaio. Suscetibilidade a inseticidas mudanças durante o desenvolvimento embrionário, sO que é importante para controlar a idade de ovo. Mantenha todas as fases da vida às 12:12 L: D fotoperíodo, 60% RH e 27 ° C. </li></ol></li><li> Uma vez que os ovos têm idade apropriada, retire os ovos de percevejos suavemente de papéis de filtro usando uma pinça soft-ponta. Raspe o fundo do ovo ligado ao papel de filtro usando a pinça e em seguida, pegue delicadamente sem aplicar muita pressão sobre o ovo para limitar atirando ovos de percevejos. </li><li> Coloque os ovos de percevejos removidos em seus respectivos grupos no interior de pratos de plástico de Petri. Limitar a eletricidade estática de arremessar ovos de percevejos, esfregando a parte inferior de cada placa de Petri no exterior com uma folha de amaciante de roupas. grupos Tratar de ovos de percevejos (5-10 ovos por grupo) de uma vez. Considere um grupo idêntico e usar, pelo menos, cinco (ou mais) replica para este ensaio. </li><li> Preparar diluições em série dos insecticidas para obter uma gama de concentrações diferentes (5 mL 0,21-21 / mL para imidacloprid (0,1%) / β-ciflutrina (0,5%) e um controlesolução com o solvente (água da torneira) somente. Adicione 20 mL de cada concentração a ter volume suficiente para o ensaio de imersão. </li><li> tubos de corte de centrifugação (50 mL) na linha 35 mL medialmente usando uma ferramenta rotativa com um cut-off de acessórios de rodas e, em seguida, cortado um furo na tampa do tubo de centrífuga (diâmetro = 2,54 cm) utilizando a mesma ferramenta rotativa com um alumínio óxido de moagem acessório pedra. Deixar 5 cm na borda da tampa durante o corte, de modo que a tampa de maneira segura, o parafuso para dentro do tubo (Figura 1). </li><li> Corte de malha fina em quadrados grandes o suficiente para caber dentro da tampa (~ 5 cm2). Parafuso a malha firmemente, eliminando todas as colisões, para os tubos de centrifugação usando a tampa de tubo de centrífuga. </li><li> Coloque um grupo de ovos de percevejos (uma repetição) usando um pincel sobre a malha no interior do tubo de centrifugação. Mergulhar o grupo de percevejos no interior do tubo de centrífuga de corte para uma concentração de insecticida durante 5 segundos. </li><li> Retire o tubo de centrífuga fora do thsolução de inseticida e. Seca-se a malha, com os ovos no interior, colocando o tubo de centrifugação directamente sobre um tecido de laboratório. </li><li> Remova o grupo de ovos para fora do tubo de centrifugação usando um pequeno, fino cerdas pincel e colocá-los em uma placa de Petri limpa com um papel de filtro limpo. </li><li> a mortalidade de ovos Record (falha para chocar) durante 14 dias para garantir que todos os ovos tiveram tempo suficiente para eclodir. </li><li> Correcta para a mortalidade no tratamento de controle usando a fórmula de Abbott (mortalidade teste% - controle% de mortalidade / 100 - mortalidade dos controlos x 100). </li><li> Inserir a mortalidade registada com as concentrações correspondentes para o software de análise de 15 para calcular os valores de LC 50. </li></ol> Ensaio 2. primeiro instar Inseticida <ol><li> Separar um grupo de percevejos bem alimentados (5 machos adultos e 5 fêmeas adultas), de preferência alimentado um dia antes, e colocá-los em uma placa de Petri (6 centímetros x 5 cm) com papel de filtro prevista mating e a postura de ovos. Permitir que os insetos adultos de cama para acasalar e pôr ovos durante cerca de 7-9 dias. Para a alimentação, use um sistema de alimentação artificial que circula água quente em tubos de vidro que cercam sangue 16. Para este estudo, utilize sangue de coelho defibronated. </li><li> Retire os adultos das placas de Petri (6 cm × 5 cm) e permita que os ovos eclodem em primeiros estádios dentro das placas de Petri. Demora cerca de 7-9 dias para os ovos de percevejos para chocar à temperatura ambiente. </li><li> Recolher os primeiros estádios em jejum da mesma idade usando um pincel. A idade pode ser determinada através da remoção dos primeiros instares diário e registar o dia em que eclodiram. Coloque-os em novos, pratos limpos Petri até o ensaio de inseticida. Primeiros ninfas não são muito resistentes dessecante, então o ensaio de inseticida deve ser concluída o mais rapidamente possível. </li><li> Para o ensaio, preparar diluições em série de imidacloprid / β-ciflutrina para obter uma gama de concentrações de 5 diferentes e um controlo Solução apenas com dissolvente. produtos formulados marcadas para o controle de percevejos nos Estados Unidos foram utilizados para estes estudos, portanto, o solvente foi água (de acordo com o rótulo). </li><li> Pipetar alíquotas de 150 ul de cada concentração de insecticida e água para o grupo de controlo para papel de filtro individuais (# 1, 4.2 cm de diâmetro). Distribuir o insecticida ao longo de todo o papel de filtro utilizando a pipeta através da aplicação de gotas ao redor de toda a borda e do centro do papel de filtro. </li><li> Coloque os papéis de filtro tratados individualmente em cima de painéis de chapas de fibra individuais (7 cm 2). </li><li> grupos de libertação de primeiros estádios (5 a 10 por grupo) para a papéis de filtro individuais enquanto ainda molhado com inseticida e cobrir os primeiros estádios com um fundo invertido de uma placa de Petri. </li><li> Coloque um peso no topo da placa de Petri para evitar que os primeiros percevejos instar de escapar da superfície tratada. </li><li> mortalidade recorde de primeiros percevejos instar após 24 h de exposição a todosconcentrações inseticida. </li><li> Correcta para a mortalidade no tratamento de controle usando a fórmula de Abbott (mortalidade teste% - controle% de mortalidade / 100 - mortalidade dos controlos x 100). </li><li> Insira a mortalidade e as concentrações registradas no software de análise de 15 para calcular LC 50 valores. </li></ol>

<ol>
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Entomol. 23, 163-166 (2009).</li><li>Leverkus, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bullous+allergic+hypersensitivity+to+bed+bug+bites+mediated+by+IgE+against+salivary+nitrophorin.">Bullous allergic hypersensitivity to bed bug bites mediated by IgE against salivary nitrophorin.</a> J. invest. entomol. 126, 91-96 (2006).</li><li>Fletcher, C. L., Ardern-Jones, M. R., Hay, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+bullous+eruption+due+to+multiple+bed+bug+bites.">Widespread bullous eruption due to multiple bed bug bites.</a> Clin. Exp. Dermatol. 27, 74-75 (2002).</li><li>Pritchard, M. J., Hwang, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+anemia+from+bedbugs.">Severe anemia from bedbugs.</a> Can. Med. Assoc. J. 181, 287-288 (2009).</li><li>Goddard, J., de Shazo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+effects+of+bed+bug+attacks+(Cimex+lectularius+L).">Psychological effects of bed bug attacks (Cimex lectularius L).</a> Am. J. Med. 125, 101-103 (2012).</li><li>Harlan, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bed+bugs+101:+the+basics+of+Cimex+lectularius.">Bed bugs 101: the basics of Cimex lectularius.</a> Am. Entomol. 52, 99-101 (2006).</li><li>Pinto, L. J., Cooper, R., Kraft, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bed Bug Handbook: The Complete Guide to Bed Bugs and Their Control. , (2007).</li><li>Toloza, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+patterns+of+insecticide+resistance+in+eggs+and+first+instars+of+Triatoma+infestans+(Hemiptera:+Reduviidae)+from+Argentina+and+Bolivia.">Differential patterns of insecticide resistance in eggs and first instars of Triatoma infestans (Hemiptera: Reduviidae) from Argentina and Bolivia.</a> J. Med. Entomol. 45, 421-426 (2008).</li><li>Cueto, G. M., Zerba, E. N., Picollo, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+pyrethroid+resistance+in+eggs+of+Pediculus+humanus+capitis+(Phthiraptera:+Pediculidae)+from+Argentina.">Evidence of pyrethroid resistance in eggs of Pediculus humanus capitis (Phthiraptera: Pediculidae) from Argentina.</a> J. Med. Entomol. 45, 693-697 (2008).</li><li>Ho, S. H., Goh, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deltamethrin+as+a+potential+ovicidal+pyrethroid+against+Plutella+xylostella+L.">Deltamethrin as a potential ovicidal pyrethroid against Plutella xylostella L.</a> Toxicol. let. 22, 161-164 (1984).</li><li>Bell, C. H., Hole, B. D., Evans, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+occurrence+of+resistance+to+phosphine+in+adult+and+egg+stages+of+strains+of+Rhyzopertha+dominica+(F.)(Coleoptera:+Bostrichidae).">The occurrence of resistance to phosphine in adult and egg stages of strains of Rhyzopertha dominica (F.)(Coleoptera: Bostrichidae).</a> J. Stored Prod. Res. 13, 91-94 (1977).</li><li>Campbell, B. E., Miller, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insecticide+Resistance+in+Eggs+and+First+Instars+of+the+Bed+Bug,+Cimex+lectularius+(Hemiptera:+Cimicidae).">Insecticide Resistance in Eggs and First Instars of the Bed Bug, Cimex lectularius (Hemiptera: Cimicidae).</a> Insects. 6, 122-132 (2015).</li><li>Price, L. A., Mills, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+toxicity+of+phosphine+to+the+immature+stages+of+resistant+and+susceptible+strains+of+some+common+stored+product+beetles,+and+implications+for+their+control.">The toxicity of phosphine to the immature stages of resistant and susceptible strains of some common stored product beetles, and implications for their control.</a> J. Stored Prod. Res. 24, 51-59 (1988).</li><li>Robertson, J. L., Preisler, H. K., Russel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PoloPlus: Probit and Logit Analysis User's Guide. , (2003).</li><li>Montes, C., Cuadrillero, C., Vilella, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+a+laboratory+colony+of+Cimex+lectularius+(Hemiptera:+Cimicidae)+using+an+artificial+feeding+technique.">Maintenance of a laboratory colony of Cimex lectularius (Hemiptera: Cimicidae) using an artificial feeding technique.</a> J. Med. Entomol. 39, 675-679 (2002).</li><li>Brogdon, W. G., McAllister, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplification+of+adult+mosquito+bioassays+through+use+of+time-mortality+determinations+in+glass+bottles.">Simplification of adult mosquito bioassays through use of time-mortality determinations in glass bottles.</a> J. Am. Mosq. Control. Assoc. 14, 159-164 (1998).</li><li>Katarzyna, K., Saddler, A., Koella, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+age+and+larval+nutrition+on+phenotypic+expression+of+insecticide-resistance+in+Anopheles+mosquitoes.">Effects of age and larval nutrition on phenotypic expression of insecticide-resistance in Anopheles mosquitoes.</a> PLoS One. 8 (3), (2013).</li><li>Outram, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+resistance+of+insect+eggs+to+sulphuryl+fluoride-II:+the+distribution+of+sulphuryl-35+S+fluoride+in+insect+eggs+after+fumigation.">Factors affecting the resistance of insect eggs to sulphuryl fluoride-II: the distribution of sulphuryl-35 S fluoride in insect eggs after fumigation.</a> J. Stored. Prod. Res. 3, 353-358 (1967).</li></ol>

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A critical step in this assay is to ensure that no eggs that are removed from a surface are damaged prior to the assay. Many insects cement their eggs to a substrate, therefore, a preliminary test may be needed to ensure that removal does not cause mortality. This test can be conducted with several replications of a treated group (bed bugs removed from filter paper) compared to a control group (bed bugs not removed). Similar low rates of mortality (or no mortality) between the treated and control group will ensure that ...

Percevejos são uma praga urbana significativo que pode causar perda de sangue, irritações da pele, insônia, depressão e ansiedade em seus hospedeiros humanos 1, 2, 3, 4, 5, 6. Os custos de eliminação e controle de percevejos são altos e muitas vezes requerem múltiplas visitas a uma casa por uma empresa de controle de pragas 7. Várias visitas são geralmente necessários por causa do comportamento críptico de percevejos e as dificuldades associadas a matá-los. Em particular, os ovos de percevejos são difíceis de controlar por causa de seu pequeno tamanho e à protecção do embrião com a casca de ovo. Atualmente, uma prática comum para muitas empresas de controle de pragas em todo os Estados Unidos é tratar uma casa para percevejos usando um inseticida químico. É bem conhecidoque os ovos são difíceis de controlar, por isso muitas empresas têm implementado um prazo de duas semanas para re-tratamentos 8. Isso permite que os ovos de percevejos tempo suficiente para chocar, de modo que o primeiro instar surgirão e supostamente mais fácil de matar com inseticidas. No entanto, há uma escassez de estudos que avaliam a eficácia de inseticidas líquidos contra ovos de percevejos e primeiros estádios. ovos de insectos têm sido documentada para ser o estágio de vida mais difícil de controlar pragas de insectos em urbanos, com excepção dos percevejos, para além de muitas pragas agrícolas. A maioria destas dificuldades têm sido atribuídas a casca do ovo, enquanto alguns estudos têm relatado resistência a inseticidas. A resistência na fase de ovo tem sido documentada em Triatoma infestans 9, Pediculus humanus capitis 10, Plutella xylostella 11, Rhyzopertha dominica 12, <em&gt; Cimex lectularius 13, e vários besouros produtos armazenados (ou seja Oryzaephilus surinamensis, Tribolium castaneum, Cryptolestes ferrugineus e Rhyzopertha dominica). 14 O desenvolvimento de resistência na fase de ovo e estágios imaturos, bem como complicações associadas com insecticida penetração através da casca do ovo, exige a necessidade de bioensaios de eficácia insecticida contra estas fases da vida. Este protocolo prevê um procedimento passo-a-passo para determinar a eficiência de inseticidas no ovo e primeiro instar fase de percevejos comuns, Cimex lectularius. Ambos estes protocolos são ensaios de resposta à concentração para permitir a quantificação dos valores de LC 50. ensaios de concentração-resposta são comumente usados ​​para estudos toxicológicos, no entanto, este protocolo foi adaptado para facilmente tratar grupos de ovos de percevejos e primeiros estádios. Estes ensaios podem ser adaptadospara os ovos de várias das espécies de insetos e fases da vida imaturas.

<table border="0" cellpadding="0" cellspacing="0" width="1067">
	<tbody>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific Inc.</td>
			<td>08-757-105</td>
			<td>Plastic</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1001-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Featherweight forceps</td>
			<td>Fisher Scientific Inc.</td>
			<td align="right">4750</td>
			<td></td>
		</tr>
		<tr>
			<td>Temprid SC (imidacloprid/bifenthrin)</td>
			<td>Bayer CropScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transport GHP (acetamiprid/bifenthrin)</td>
			<td>FMC Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td></td>
			<td>06-443-18</td>
			<td>50 mL; flat-top threaded</td>
		</tr>
		<tr>
			<td>Fine mesh</td>
			<td></td>
			<td>7250C</td>
			<td></td>
		</tr>
		<tr>
			<td>KimWipe</td>
			<td></td>
			<td>06-666A</td>
			<td>11 cm X 21 cm</td>
		</tr>
		<tr>
			<td>Small paint brush</td>
			<td></td>
			<td></td>
			<td>Any small paint brush</td>
		</tr>
		<tr>
			<td>Hardboard panels</td>
			<td></td>
			<td></td>
			<td>Composite wood or tile would be sufficient from a home improvement store</td>
		</tr>
		<tr>
			<td>Metal weights</td>
			<td></td>
			<td></td>
			<td>Any type of metal screw that will hold the weight of the Petri dish down onto a surface</td>
		</tr>
	</tbody>
</table>

a method for evaluating insecticide efficacy against bed bug, cimex lectularius, eggs and first instars

avaliações de toxicidade padrão de inseticidas contra pragas de insetos são realizados principalmente em insetos adultos. As avaliações baseiam-se em uma dose-resposta ou a curva de resposta à concentração, em que a mortalidade aumenta à medida que a dose ou concentração de um insecticida é aumentada. Concentração padrão letal (LC 50) e dose letal (LD 50) testes que resultam em mortalidade de 50% de uma população de teste pode ser um desafio para avaliação da toxicidade de inseticidas contra não-adultos fases da vida de insetos, como ovos e instar cedo ou estágios de ninfa . No entanto, esta informação é essencial para compreender a eficácia inseticida em todas as fases da vida de percevejos, o que afeta os esforços de controle e tratamento. Este protocolo usa um bioensaio de imersão padrão modificado para ovos de percevejos e um ensaio de insecticida de contacto para o tratamento de ninfa primeiros estádios. Estes ensaios de produzir uma curva de concentração-resposta para a quantificação de LC 50 valores para as avaliações insecticidas.

Os ovos foram imersas em 5 diferentes concentrações de imidacloprid / β-ciflutrina (uL / mL). Os primeiros instares foram colocados sobre superfícies tratadas de cinco concentrações diferentes de imidacloprid / β-ciflutrina (Figura 2). Foram utilizados três diferentes populações de C. lectularius: Harlan, Richmond, e épico Center (Tabela 1). A cepa Harlan é su...

Watch this Scientific Journal Video about A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars at JoVE.com

A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

avaliações de inseticidas são muitas vezes alvo contra insetos adultos, em vez de estágios imaturos. Aqui, apresentamos um protocolo para avaliar inseticidas contra ovos de percevejos com uma comparação à primeira fase de percevejos de ninfa. Estes protocolos pode ser modificada para outros insetos para avaliar a eficácia do inseticida nas fases da vida não-adultos.

Um método para avaliar Inseticida Eficácia contra Bed Bug,<em&gt; Cimex lectularius</em&gt;, Ovos e primeiros estádios

Standard toxicity evaluations of insecticides against insect pests are primarily conducted on adult insects. Evaluations are based on a dose-response or ...

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A Method for Evaluating Insecticide Efficacy against Bed Bug, Cimex lectularius, Eggs and First Instars

11.2K visualizações.  University of Florida.  avaliações de inseticidas são muitas vezes alvo contra insetos adultos, em vez de estágios imaturos. Aqui, apresentamos um protocolo para avaliar inseticidas contra ovos de percevejos com uma comparação à primeira fase de percevejos de ninfa. Estes protocolos pode ser modificada para outros insetos para avaliar a eficácia do inseticida nas fases da vida não-adultos. 

Vídeo: A Method for Evaluating Insecticide Efficacy against Bed Bug,  Cimex lectularius, Eggs and First Instars

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

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

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

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with 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 ...

Ambiente

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

Um método simples para a extração automatizada de fase sólida das amostras de água para análise imunológica de Pequenas Poluentes

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

O uso de uma bateria de métodos químicos e ecotoxicológicos para a avaliação da eficácia dos Processos de Tratamento de Águas Residuais remover estrogénica Potência

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

A Tabela Gusseted Thermogradient para controlar a temperatura do solo para avaliar o crescimento da planta e monitoramento de processos do solo

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

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 ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

Um Sistema de Exposição Fluxo através de sedimentação de Efeitos Avaliando suspensos sobre a vida aquática

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Um método de extração e análise de lipídios para caracterizar micróbios de solo em experimentos com muitas amostras

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Image-based Lagrangian Particle Tracking in Bed-load Experiments

Image analysis has been increasingly used for the measurement of river flows due to its capabilities to furnish detailed quantitative depictions at a relatively low cost. This manuscript describes an application of particle tracking velocimetry (PTV) to a bed-load experiment with lightweight sediment. The key characteristics of the investigated sediment transport conditions were the presence of a covered flow and of a fixed rough bed above which particles were released in limited number at the flume inlet. Under the applied flow conditions, the motion of the individual bed-load particles was intermittent, with alternating movement and stillness terms. The flow pattern was preliminarily characterized by acoustic measurements of vertical profiles of the stream-wise velocity. During process visualization, a large field of view was obtained using two action-cameras placed at different locations along the flume. The experimental protocol is described in terms of channel calibration, experiment realization, image pre-processing, automatic particle tracking, and post-processing of particle track data from the two cameras. The presented proof-of-concept results include probability distributions of the particle hop length and duration. The achievements of this work are compared to those of existing literature to demonstrate the validity of the protocol.

The manuscript presents a protocol for the conduction of bed-load sediment transport experiments where the moving particles are tracked by image analysis. The experimental facility, the procedures for run realization and data processing, and finally some proof-of-concept results are presented here.

Rastreamento de partículas Lagrangian baseado em imagem em experiências de carga de cama

Image analysis has been increasingly used for the measurement of river flows due to its capabilities to furnish detailed quantitative depictions at a ...

A Novel Method for the Pentosan Analysis Present in Jute Biomass and Its Conversion into Sugar Monomers Using Acidic Ionic Liquid

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, lower vapor pressure, non-flammability, higher heat capacity, and tunable solubility and acidity. Here, we demonstrate a method for the synthesis of C5 sugars (xylose and arabinose) from the pentosan present in jute biomass in a one-pot process by utilizing a catalytic amount of Br&#248;nsted acidic 1-methyl-3-(3-sulfopropyl)-imidazolium hydrogen sulfate IL. The acidic IL is synthesized in the lab and characterized using NMR spectroscopic techniques for understanding its purity. The various properties of BAIL are measured such as acid strength, thermal and hydrothermal stability, which showed that the catalyst is stable at a higher temperature (250 &#176;C) and possesses very high acid strength (Ho 1.57). The acidic IL converts over 90% of pentosan into sugars and furfural. Hence, the presenting method in this study can also be employed for the evaluation of pentosan concentration in other kinds of lignocellulosic biomass.

We present a protocol for the synthesis of C5 sugars (xylose and arabinose) from a renewable non-edible lignocellulosic biomass (i.e., jute) with the presence of Br&#248;nsted acidic ionic liquids (BAILs) as the catalyst in water. The BAILs catalyst exhibited better catalytic performance than conventional mineral acid catalysts (H2SO4 and HCl).

Um novo método para a análise de Pentosan presentes na biomassa de juta e sua conversão em monômeros de açúcar usando ácido líquido iônico

Recently, ionic liquids (ILs) are used for biomass valorization into valuable chemicals because of their remarkable properties such as thermal stability, ...

Visualizing Efficacy of Pesticides Against Disease Vector Mosquitoes in the Field

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform across ecological zones. To best protect public and veterinary health from these insects, the environmental limitations of pesticides need to be investigated to inform effective use of the most appropriate pesticide formulations and techniques. We have developed a research program to evaluate combinations of pesticides, pesticide application equipment, and application techniques in hot-arid desert, hot-humid tropical, warm and cool temperate, and urban locations to derive pesticide use guidelines specific to target insect and environment. To these ends we designed a system of protocols to support efficient, cost-effective, portable, and standardized evaluation of a diverse range of pesticides and equipment across multiple environments. At the core of these protocols is the use of an array of small cages with colony-reared sentinel mosquitoes (adults and immatures) and sand flies (adults), strategically arranged in natural habitats and exposed to pesticide spray. Spatial and temporal patterns of pesticide efficacy are derived from percent mortality in sentinel cages, then mapped and visualized in a geographic information system. Maps of sentinel mortality data may be statistically compared to evaluate relative efficacy of a pesticide across multiple environments, or to study multiple pesticides in a single environment. Protocols may be modified to accommodate a variety of scenarios, including, for example, the vertical orientation of sentinels in canopy habitats or simultaneous testing of ground and aerial application methods.

The efficacy of public health pesticides targeting nuisance and disease-vector insects is not uniform across different ecological zones. Here we present a system of techniques using captive vector insects as sentinels for pesticide efficacy to derive electronic maps supporting the standard evaluation of pesticides across multiple environments.

Visualizando a eficácia de pesticidas contra mosquitos vetores de doenças no campo

Efficacy of public health pesticides targeting nuisance and disease-vector insects such as mosquitoes, sand flies, and filth-breeding flies is not uniform ...