Agradecemos a Molly firme para la asistencia formulación de dilución y pesticidas. Agradecemos también a Troy Anderson por análisis probit orientación y Zachary Adelman para obtener ayuda con el diseño experimental. Esta investigación fue apoyada en parte por un premio de la Fundación Entomológica y por fondos de la beca Virginia manejo de plagas.

NOTA: Este protocolo incluye dos ensayos de insecticidas para tratar por separado los huevos de chinches y los primeros estadíos. Ambos protocolos se realizaron utilizando los mismos insecticidas, sin embargo, los protocolos tuvieron que ser adaptado para asegurar una exposición insecticida y manipular fácilmente los especímenes. 1. Ensayo de huevo inmersión Insecticida <ol><li> Tire montadas, bien alimentados adultos chinches hembras de una colonia y colocar las hembras (5-10) en una placa de Petri con papel de filtro. Tener al menos diez repeticiones para asegurar que habrá suficientes huevos para el ensayo de insecticida. Compruebe si las hembras han puesto huevos todos los días. <ol><li> Una vez que ponen huevos, tome las hembras de la placa de Petri (6 cm x 5 cm) y ponerlas en nuevas placas de Petri con un nuevo filtro de papel diario y registrar la fecha en las viejas y nuevas placas de Petri. </li><li> Permitir que los huevos a la edad de 4 a 5 días antes de comenzar el ensayo. La susceptibilidad a los insecticidas cambios durante el desarrollo embrionario, so es importante para el control de la edad del huevo. Mantenga todas las etapas de la vida a las 12:12 L: D fotoperíodo, 60% de humedad relativa y 27 ° C. </li></ol></li><li> Una vez que los huevos han envejecido apropiadamente, eliminar los huevos de chinches suavemente de papeles de filtro con unas pinzas de punta blanda. raspar suavemente la parte inferior del huevo unida al papel de filtro con la pinza y luego agarrar suavemente sin aplicar mucha presión sobre el huevo para limitar lanzan cama huevos de insectos. </li><li> Coloque los huevos de chinches retirados en sus respectivos grupos en el interior de placas Petri de plástico. Limitar la electricidad estática de arrojar huevos de chinches por el roce de la parte inferior de cada placa de Petri en el exterior con una hoja de suavizante de telas. Tratar a los grupos de huevos de chinches (5-10 huevos por grupo) a la vez. Considere un grupo un duplicado y el uso de al menos cinco (o más) se replica para este ensayo. </li><li> Preparar diluciones seriadas de insecticidas para obtener una gama de 5 concentraciones diferentes (0,21 a 21 l / ml de imidacloprid (0,1%) / β-ciflutrina (0,5%) y un controlsolución con (agua del grifo) disolvente solamente. Hacer 20 ml de cada concentración para tener suficiente volumen para el ensayo de inmersión. </li><li> tubos cortados de centrífuga (50 ml) a la línea 35 ml medial usando una herramienta rotativa con un accesorio de la rueda de corte y luego se corta un agujero en la tapa del tubo de centrífuga (diámetro = 2,54 cm) usando el mismo herramienta rotativa con un aluminio óxido de molienda accesorio piedra. Deja 5 cm en el borde de la tapa durante el corte, por lo que la tapa será firmemente el tornillo en el tubo (Figura 1). </li><li> Cortar en cuadrados de malla fina lo suficientemente grandes como para caber en la tapa (2 ~ 5 cm). Tornillo de la malla firmemente, eliminando las irregularidades, en los tubos de centrifugación utilizando el tapón del tubo de centrífuga. </li><li> Colocar un grupo de huevos de la cama de errores (un replicado) utilizando un pincel sobre la malla en el interior del tubo de centrífuga. Sumergir el grupo de insectos de la cama en el interior del tubo de centrífuga de corte en una concentración de insecticida durante 5 segundos. </li><li> Retire el tubo de centrífuga de THsolución de insecticida e. Secar la malla con los huevos en el interior, colocando el tubo de centrífuga directamente sobre un tejido de laboratorio. </li><li> Retire el grupo de huevos fuera del tubo de centrífuga utilizando un pequeño, fino pincel de cerdas y colocarlos en una placa de Petri limpia con un papel de filtro limpio. </li><li> Registro de mortalidad de los huevos (falta de eclosionar) durante 14 días para asegurar que todos los huevos han tenido tiempo suficiente para salir del cascarón. </li><li> Correcta para la mortalidad en el tratamiento de control utilizando la fórmula de Abbott (mortalidad prueba% - Control% de mortalidad / 100 - mortalidad del control x 100). </li><li> Inserte mortalidad registrada con las concentraciones correspondientes en el software de análisis 15 para calcular valores de LC50. </li></ol> 2. Ensayo de primer instar Insecticida <ol><li> Separar un grupo de insectos de la cama bien alimentados (5 machos y 5 hembras adultas adultos), preferentemente alimentado 1 día antes, y colocarlos en una placa de Petri (6 cm x 5 cm) con papel de filtro prevista mating y la puesta de huevos. Permitir que los chinches de cama adulto para aparearse y poner huevos durante aproximadamente 7-9 días. Para la alimentación, utilice un sistema de alimentación artificial que hace circular el agua caliente en tubos de vidrio que rodean la sangre 16. Para este estudio, el uso de sangre de conejo defibronated. </li><li> Retire los adultos a partir de los platos de Petri (6 cm x 5 cm) y permita que los huevos eclosionan en la primera estadios en las placas de Petri. Se tarda aproximadamente 7-9 días para los huevos de chinches que eclosionan a temperatura ambiente. </li><li> Recoge los primeros estadios sin alimentar de la misma edad utilizando un pincel. La edad puede ser determinada mediante la eliminación de los primeros estadios diaria y registro de la día que eclosionaron. Colocarlos en platos nuevos, limpios de Petri hasta el ensayo insecticida. ninfas de la primera fase larval no son muy resistentes desecante, por lo que el ensayo de insecticidas debe ser completado tan pronto como sea posible. </li><li> Para el ensayo, preparar diluciones en serie de imidacloprid / β-ciflutrina para obtener una gama de 5 concentraciones diferentes y unos Solu de controlción con único disolvente. Los productos formulados etiquetados para el control de chinches de cama en los Estados Unidos fueron utilizados para estos estudios, por lo tanto, el disolvente era agua (según la etiqueta). </li><li> Pipeta alícuotas de 150 l de cada concentración de insecticida y el agua para el grupo de control sobre papeles de filtro individuales (# 1; 4,2 cm de diámetro). Distribuir el insecticida en toda el papel de filtro usando la pipeta mediante la aplicación de las gotitas de alrededor de todo el borde y el centro del papel de filtro. </li><li> Coloque los papeles de filtro tratados de forma individual en la parte superior de los paneles de aglomerado individuales (7 cm 2). </li><li> grupos de liberación de primer estadio (5 a 10 por grupo) en los papeles de filtro individuales mientras aún esté húmedo con insecticida y cubrir el primer estado con un fondo invertido de una placa de Petri. </li><li> Coloque un peso en la parte superior de la placa de Petri para evitar que los primeros chinches instar se escape de la superficie tratada. </li><li> registrar datos de mortalidad de los primeros chinches estadio después de 24 h de exposición a todosconcentraciones de insecticidas. </li><li> Correcta para la mortalidad en el tratamiento de control utilizando la fórmula de Abbott (mortalidad prueba% - Control% de mortalidad / 100 - mortalidad del control x 100). </li><li> Insertar la mortalidad y las concentraciones de grabado en el software de análisis 15 para calcular valores de LC50. </li></ol>

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</ol>

Los autores no tienen nada que revelar.

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 removal did not cause egg mortality. Thus, any mortality recorded for the insecticide assay should be due to the insecticide treatment. The same preliminary experiment can be conducted using first instars to ensure that methods for collecting do not kill the experimental insects. All experiments conducted later should include control replications that do not receive insecticide but are treated with the solvent (for these experiments, tap water was the solvent). 
Preliminary experiments should be conducted with the recommended label rate for formulated products and then serially diluted to determine the correct concentrations to produce a concentration response curve of mortality ranging from 20-80%. Previous insecticide studies on the same insects or compounds may also be insightful when trying to determine the appropriate concentration. Concentrations will need to be modified dependent upon the insect, life stage, and compound used. New solutions should be made each time an experiment is conducted. 
The exposure of first instars to wet residues was necessary to get high mortality. Although this life stage is considered more susceptible to insecticides than older nymphs and adults, high mortality was still difficult to obtain with formulated products. Submerging the first instars into insecticides similar to the eggs was not plausible because of the resultant high mortality, thus this was a limitation to the study. The alternative first instar bioassay was manipulated to maintain first instars on surfaces to force constant contact with the treatment.
Dry residue assays and bottle assays commonly used for insecticide efficacy and resistance studies do not provide methodology for testing against multiple insect life stages17. These assays are often used to determine resistance in insects, and not to test insecticide efficacy. There are limited studies on egg dipping toxicological studies and none that have reported a standard methodology previously that can be repeated for multiple insect species.
This study provides a protocol for testing insecticide efficacy against bed bug eggs and first instars. Efficacy should be evaluated in different life stages, if not all, for a more holistic view of the efficacy of a treatment program. This consideration is important because an insect's susceptibility to insecticides changes with age and development 18. While age is an important factor in insecticide susceptibility, a factor in egg insecticide susceptibility is the eggshell. The eggshell provides a barrier to insecticides and makes insect eggs particularly difficult to control19. 
These toxicological assays provide researchers a way to evaluate insecticide toxicity against various life stages of insects. Insecticidal solutions are prepared to evaluate mortality and to determine LC50 concentrations. While this protocol was used for formulated products, the steps can be manipulated for technical grade compounds and for a variety of insects and life stages to evaluate toxicity.

Las chinches son una plaga urbana significativo que puede causar la pérdida de sangre, irritaciones de la piel, el insomnio, la depresión y la ansiedad en sus huéspedes humanos 1, 2, 3, 4, 5, 6. Los gastos de eliminación y el control de insectos de la cama son altos y muchas veces requieren varias visitas a un hogar por una empresa de control de plagas 7. Múltiples visitas suelen ser necesarios debido al comportamiento críptico de las chinches y las dificultades asociadas con matarlos. En particular, los huevos de chinches son difíciles de controlar debido a su pequeño tamaño y la protección del embrión de la cáscara del huevo. En la actualidad, una práctica común para muchas empresas de control de plagas en los Estados Unidos es tratar a un hogar para las chinches utilizando un insecticida químico. Es bién sabidoque los huevos son difíciles de controlar, por lo que muchas empresas han implementado un plazo de dos semanas para re-tratamientos 8. Esto permite que los huevos de chinches tiempo suficiente para salir del cascarón, de modo que el primer instar emergerá y supuestamente más fácil de matar con insecticidas. Sin embargo, hay una escasez de estudios que evalúan la eficacia de insecticidas líquidos contra la cama huevos de errores y primeros estadios. huevos de insectos se han documentado que la etapa de la vida más difícil de controlar en las plagas de insectos urbanas, aparte de insectos de la cama, además de muchas plagas agrícolas. La mayor parte de estas dificultades se han atribuido a la cáscara del huevo, mientras que algunos estudios han reportado resistencia a los insecticidas. Resistencia en estado de huevo se ha documentado en Triatoma infestans 9, Pediculus humanus capitis 10, Plutella xylostella 11, 12 RHYZOPERTHA dominica, <em&gt; Cimex lectularius 13, y varios escarabajos de productos almacenados (es decir, Oryzaephilus surinamensis, Tribolium castaneum, Cryptolestes ferrugineus y dominica RHYZOPERTHA). 14 El desarrollo de resistencia en la fase de huevo y etapas inmaduras, así como las complicaciones asociadas con la penetración de insecticidas a través de la cáscara de huevo, requiere la necesidad de bioensayos de eficacia insecticida contra estas etapas de la vida. Este protocolo proporciona un procedimiento paso a paso para determinar la eficacia de los insecticidas en la etapa de huevo y el primer estadio de las chinches comunes, Cimex lectularius. Ambos de estos protocolos son ensayos de concentración-respuesta para permitir la cuantificación de los valores de LC50. Los ensayos de concentración-respuesta se utilizan comúnmente para estudios toxicológicos, sin embargo, este protocolo ha sido adaptado para tratar fácilmente los grupos de cama huevos de errores y primeros estadios. Estos ensayos pueden adaptarsepara los huevos de varias especies de insectos &#39;y etapas de la vida inmaduros.

<table><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>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

Las evaluaciones de toxicidad estándar de insecticidas contra plagas de insectos se llevan a cabo principalmente en insectos adultos. Las evaluaciones se basan en una relación dosis-respuesta o curva de concentración-respuesta, cuando se incrementa la mortalidad aumenta a medida que la dosis o concentración de un insecticida. Concentración estándar letal (LC50) y la dosis letal (DL50) pruebas que resultan en un 50% la mortalidad de una población de prueba puede ser un reto para la evaluación de la toxicidad de los insecticidas contra los estadios de vida de insectos no adultos, tales como los huevos y el estadio temprano o estadios ninfales . Sin embargo, esta información es esencial para la comprensión de la eficacia insecticida en todas las etapas de la vida de chinches, que afecta a los esfuerzos de control y tratamiento. Este protocolo utiliza un bioensayo de inmersión estándar modificado para los huevos de chinches y un ensayo de insecticida de contacto para el tratamiento de primeros estadios ninfales. Estos ensayos producen una curva de concentración-respuesta para cuantificar más LC 50 valores para las evaluaciones insecticidas.

Los huevos se sumergieron en 5 concentraciones diferentes de imidacloprid / β-ciflutrina (mu l / ml). Los primeros estadios se colocaron sobre las superficies tratadas de cinco concentraciones diferentes de imidacloprid / β-ciflutrina (Figura 2). Se han utilizado tres poblaciones diferentes de C. lectularius: Harlan, Richmond, y Epic Center (Tabla 1). La cepa Harlan es susceptible a los insecticidas piretroides y fue adquirida en 2005 por el Dr. Harold Harlan. Las cepas de Richmond y de Epic Center, poseen una resistencia a deltametrina. La cepa de Richmond se recogió de un hogar de grupo de ancianos en Richmond, VA en 2008. cepa The Epic Center se recogió en 2008 en Cincinnati, OH. Superior LC 50 valores indicaron que se requería una mayor concentración para matar el 50% de lahuevos población de prueba a partir de cepas de Richmond y de Epic Center. Al igual que en los huevos, los primeros estadios de las poblaciones Richmond y Epic Center requieren concentraciones más altas de insecticida para matar el 50% de la población de prueba. Los valores de LC50 seguidos por letras diferentes son significativamente diferentes. <img alt="Figura 1" class="xfigimg" src="/files/ftp_upload/55092/55092fig1.jpg" /> Figura 1. Fotografía del tubo de centrífuga modificada. Un agujero se ha reducido a la tapa, de manera que un trozo cuadrado de malla puede ser atornillado en el casquillo. Las piezas de malla pueden ser desechados y reemplazados cuando se utilizan diferentes insecticidas. Los huevos de chinches de cama se colocan directamente sobre la malla con un cepillo de pintura y luego se sumergieron en las soluciones de insecticidas para el ensayo de inmersión. <a href="http://ecsource.jove.com/files/ftp_upload/55092/55092fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de este figur</a>mi. <img alt="Figura 2" class="xfigimg" src="/files/ftp_upload/55092/55092fig2.jpg" /> Figura 2. Representación de un gráfico de dosis-respuesta. La respuesta ciento (mortalidad) de la cepa Harlan huevos de chinches de imidacloprid / β-ciflutrina se representa en el eje de ordenadas y la concentración de registro que las chinches se expusieron a se representa en el eje x. mortalidad de los huevos aumentó a medida que aumentó la dosis y la mortalidad alcanzó el 100% a la concentración más alta probada. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Etapa de la vida / Strain </td><td colspan="2"> norte </td><td> LC 50 (95% IC) </td><td> Pendiente ± SE </td><td> X 2 (df) </td></tr><tr><td> Huevos </td><td colspan="2"></td><td></td><td></td><td></td></tr><tr><td> Harlan </td><td colspan="2"> 250 </td><td> 0.41a (<html> 0,276 a 0,548) </td><td> 1,86 ± 0,24 </td><td> 33,42 (23) </td></tr><tr><td> Richmond </td><td colspan="2"> 320 </td><td> 1.23b (0,59-2,10) </td><td> 1,13 ± 0,14 </td><td> 82,57 (30) </td></tr><tr><td> Centro de epic </td><td colspan="2"> 400 </td><td> 2.10b (1,049-4,587) </td><td> 0,95 ± 0,10 </td><td> 149,91 (38) </td></tr><tr><td> En primer lugar Instars </td><td colspan="2"></td><td></td><td></td><td></td></tr><tr><td> Harlan </td><td colspan="2"> 150 </td><td> 0.04A (0,030-0,063) </td><td> 2.16 ± 0.34 </td><td> 38,17 (28) </td></tr><tr><td> Richmond </td><td colspan="2"> 195 </td><td> 4.81b (1,94-10,26) </td><td> 0,66 ± 0,12 </td><td> 45,87 (37) </td></tr><tr><td> Centro de epic </td><td colspan="2"> 190 </td><td> 19.72b (8,18 a 184,48) </td><td> 0,75 ± 0,17 </td><td> 45,39 (36) </td></tr></tbody></table>&quot;&gt; Tabla 1. Comparación de los huevos de chinches y el primer estadio LC 50 valores modificados de Campbell y Miller 2015 13.

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

evaluaciones insecticidas a menudo se dirigen a los insectos adultos, en lugar de estadios inmaduros. A continuación, se presenta un protocolo para la evaluación de insecticidas contra los huevos de insectos de la cama con una comparación a la primera etapa de ninfa chinches. Estos protocolos podrían ajustarse para otros insectos para evaluar la eficacia insecticida en etapas de la vida no adultos.

Un método para evaluar la eficacia insecticida contra las chinches,<em&gt; Cimex lectularius</em&gt;, Los huevos y la primera estadios

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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Nanyang Technological University

Research

JoVE Journal

Environment

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

11.3K vistas.  University of Florida.  evaluaciones insecticidas a menudo se dirigen a los insectos adultos, en lugar de estadios inmaduros. A continuación, se presenta un protocolo para la evaluación de insecticidas contra los huevos de insectos de la cama con una comparación a la primera etapa de ninfa chinches. Estos protocolos podrían ajustarse para otros insectos para evaluar la eficacia insecticida en etapas de la vida no adultos. 

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

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The disease is initiated by feeding activities of the invasive beech scale insect, Cryptococcus fagisuga, which creates entry points for infection by one of the Neonectria species of fungus.&#160;Without scale infestation, there is little opportunity for fungal infection. Using scale eggs to artificially infest healthy trees in heavily BBD impacted stands demonstrated that these trees were resistant to the scale insect portion of the disease complex1.&#160;Here we present a protocol that we have developed, based on the artificial infestation technique by Houston2, which can be used to screen for scale-resistant trees in the field and in smaller potted seedlings and grafts.&#160;The identification of scale-resistant trees is an important component of management of BBD through tree improvement programs and silvicultural manipulation.

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark.&#160;Trees that are resistant to the scale insect are also disease resistant.&#160;Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

Una técnica a la pantalla haya americana para la resistencia al insecto Haya Scale (<em&gt; Fagisuga Cryptococcus</em&gt; Lind.)

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

Investigación

Medio ambiente

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene Md&#945;E7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (Md&#945;E7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of Md&#945;E7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of Md&#945;E7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica

Here, we present a protocol to produce house fly carboxylesterase proteins in vitro with a baculovirus mediated insect cell expression system and later functionally characterize their roles in metabolizing permethrin, thereby, conferring pyrethroid resistance by conducting cell-based MTT assay and in vitro metabolic studies.

Caracterización funcional de carboxilesterasas en casa resistente al insecticida moscas, Musca Domestica

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were ...

Bioquímica

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these insecticides against adult insects or their impact on reproductive output. However, currently available methods assess adult mortality only, limiting the selection of novel insecticides with a different mode of action. In this work, different experimental methods were examined to identify the diverse effects on the CBB under laboratory conditions. For this, green coffee fruits (GFs) were collected and disinfected by immersion in sodium hypochlorite solution followed by UV light irradiation. In parallel, CBB adults from a colony were disinfected by immersion in sodium hypochlorite solution. To assess fruit protection (preinfestation), the fruits were placed in plastic boxes, and the insecticides were applied. Then, the CBB adults were released at a rate of two CBBs per GF. The GFs were left under controlled conditions to evaluate CBB infestation and survival after 1, 7, 15, and 21 days. To evaluate insecticide efficacy after CBB infestation (postinfestation), CBB adults were released to the GFs in a 2:1 ratio for 3 h at 21 &#176;C. Infested fruits showing CBB adults with their abdomens partially exposed were selected and placed in 96-well racks, and the CBBs boring into the fruits were treated directly. After 20 days, the fruits were dissected, and the CBB biological stages inside each fruit were recorded. The GFs served as substrates that mimic natural conditions to evaluate toxic, chemical, and biological insecticides against the CBB.

Methodology to Test Control Agents and Insecticides Against the Coffee Berry Borer Hypothenemus hampei

A method using green coffee fruits (GFs) was developed to test the toxicity of insecticides against the coffee berry borer (CBB). Insecticides or toxic substances were applied to disinfected GFs before or after CBB infestation. Insect mortality, repellency, and reproductive capacity, in addition to other parameters, were evaluated.

Metodología para probar agentes de control e insecticidas contra el barrenador de la baya del café Hypothenemus hampei

Prior to recommending insecticides to treat the coffee berry borer (CBB) Hypothenemus hampei, it is valuable to know the mortality and repellency of these ...

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ingestion of provisions contaminated with pesticides may increase the mortality rate of the larvae, decrease the collection rate and the population of adult solitary bees in the next year from a demographic perspective. But there are limited studies on the effects of pesticides on the larvae of solitary bees. Therefore, understanding how pesticides influence the larvae of solitary bees should be considered an integral part of pesticide ecological risk assessment. This study presents a method to expose the larvae of solitary bee, Osmia excavata, to lethal or sublethal doses of pesticide, tracking larval weight gain, developmental duration, eclosion ability, and food consumption efficiency conversion of ingested food. To demonstrate the effectiveness of this method, the larvae of O. excavata were fed with provisions containing acute lethal and sublethal doses of chlorpyrifos. Then, the above indexes of the treated larvae were investigated. This technique helps to predict and mitigate the risk of pesticides to pollinators.

The present protocol explains a method to feed pesticide-contaminated provisions to the larvae of the solitary bees, Osmia excavata. The procedure examines the ecotoxicity of the pesticide to the larvae of the solitary bees.

Evaluación del efecto de los pesticidas en las larvas de las abejas solitarias

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC50) of the target populations and a series of confidence limits (CL's) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC50 can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC50 using the same methodology.

This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.

Bioensayos para vigilar la resistencia insecticida

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Biología

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

Uso Individual sensillum grabación para detectar respuestas olfativo neurona de chinches de cama a Semioquímicos

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

Neurociencias

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

Protocolos para las pruebas de la toxicidad de la novela química insecticida para mosquitos

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

Bioensayo de aplicación tópica para cuantificar la toxicidad de insecticidas para mosquitos y moscas de la fruta

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

Evaluación de los efectos fitoquímicos sobre Helicoverpa armigera mediante ensayos de alimentación

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

Autor destacado: Desarrollo y desafíos de los ensayos de alimentación para el manejo de plagas de insectos 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Desarrollo de un sistema de ensayo de alimentación para evaluar el efecto insecticida de los fitoquímicos en Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...

Un método para evaluar la eficacia insecticida contra las chinches,

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