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>

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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 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.

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><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

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 susceptível a piretróides e foi adquirida em 2005 pelo Dr. Harold Harlan. As estirpes de Richmond e épico Centro são ambos resistentes a deltametrina. A cepa Richmond foi recolhida a partir de uma casa de grupo de idosos em Richmond, VA em 2008. O épico Centro estirpe foi coletado em 2008, em Cincinnati, OH. LC superior 50 valores indicaram que uma concentração mais elevada foi necessária para matar 50% daovos da população de teste de estirpes Richmond e épico Center. De forma semelhante aos ovos, os primeiros estádios das populações Richmond e épica Centro necessárias concentrações mais elevadas de insecticida para matar 50% da população de teste. Os LC 50 valores seguidos por letras diferentes são significativamente diferentes. <img alt="figura 1" class="xfigimg" src="/files/ftp_upload/55092/55092fig1.jpg" /> Figura 1. fotografia do tubo de centrifugação modificado. Um buraco foi cortada na tampa, de modo que um pedaço quadrado de malha pode ser aparafusado na tampa. As peças em malha pode ser descartado e substituído quando utilizando diferentes insecticidas. Os ovos de percevejos são colocados diretamente sobre a malha usando um pincel e, em seguida, submersa nas soluções inseticidas para o ensaio de imersão. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55092/55092fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figur</a>e. <img alt="Figura 2" class="xfigimg" src="/files/ftp_upload/55092/55092fig2.jpg" /> Figura 2. Representação de um gráfico de dose-resposta. A resposta por cento (mortalidade) da estirpe Harlan ovos de percevejos de imidacloprid / β-ciflutrina é representada no eixo y e a concentração de registo que os percevejos foram expostas a é representado no eixo dos x. a mortalidade de ovos aumentou à medida que a dose aumentou e a mortalidade atingiu 100% à concentração mais alta testada. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Fase de vida / Strain </td><td colspan="2"> n </td><td> LC 50 (95% CI) </td><td> Slope ± SE </td><td> X 2 (df) </td></tr><tr><td> Ovos </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> ,276-,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> épica Centro </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> primeiros estádios </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> épica Centro </td><td colspan="2"> 190 </td><td> 19.72b (8,18-184,48) </td><td> 0,75 ± 0,17 </td><td> 45.39 (36) </td></tr></tbody></table>&quot;&gt; Tabela 1. Comparação de ovo de percevejos e primeira LC instar 50 valores modificados a partir de Campbell e 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

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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Nanyang Technological University

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Environment

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

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and rates of mortality in populations, which has a variety of applications in ecology, including agricultural ecosystems. Horizontal, or cohort-based, life tables provide for the most direct and accurate method of quantifying vital population rates because they follow a group of individuals in a population from birth to death. Here, protocols are presented for conducting and analyzing cohort-based life tables in the field that takes advantage of the sessile nature of the immature life stages of a global insect pest, Bemisia tabaci. Individual insects are located on the underside of cotton leaves and are marked by drawing a small circle around the insect with a non-toxic pen. This insect can then be observed repeatedly over time with the aid of hand lenses to measure development from one stage to the next and to identify stage-specific causes of death associated with natural and introduced mortality forces. Analyses explain how to correctly measure multiple mortality forces that act contemporaneously within each stage and how to use such data to provide meaningful population dynamic metrics. The method does not directly account for adult survival and reproduction, which limits inference to dynamics of immature stages. An example is presented that focused on measuring the impact of bottom-up (plant quality) and top-down (natural enemies) effects on the mortality dynamics of B. tabaci in the cotton system.

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Life tables allow quantification of the sources and rates of mortality in insect populations and contribute to understanding, predicting and manipulating population dynamics in agroecosystems. Methods for conducting and analyzing cohort-based life tables in the field for an insect with sessile immature life stages are presented.

Metodologia para desenvolvimento de tabelas de vida para insetos sésseis no campo utilizando a mosca branca, Bemisia tabaci, em algodão como um modelo de sistema

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

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.

Caracterização funcional de Carboxylesterases em casa resistente a inseticida 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.

Metodologia para testar agentes de controle e inseticidas contra o café 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 ...

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.

Bioensaios de Resistência a Inseticidas Monitoramento

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

Biologia

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.

Usando Individual Sensillum Gravação para detectar respostas Olfativo Neurônio de percevejos para Semioquímicos

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

Neurociência

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 testar a toxicidade de romance químicas inseticidas 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.

Bioensaio de aplicação tópico para quantificar toxicidade de inseticida para mosquitos e moscas de frutas

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

Avaliação dos efeitos fitoquímicos em Helicoverpa armigera por meio de ensaio alimentar

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 em destaque: Desenvolvimento e desafios de ensaios de alimentação para manejo de pragas de insetos 

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.

Desenvolvimento de um Sistema de Ensaio Alimentar para Avaliação do Efeito Inseticida de Fitoquímicos sobre Helicoverpa armigera

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

Um método para avaliar Inseticida Eficácia contra Bed Bug,

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