A novel method for standardized application of fungal spore coatings for mosquito exposure bioassays

<p>Abstract</p> <p>Background</p> <p>Interest in the use of fungal entomopathogens against malaria vectors is growing. Fungal spores infect insects via the cuticle and can be applied directly on the insect to evaluate infectivity. For flying insects such as mosquitoes, however, application of fungal suspensions on resting surfaces is more realistic and representative of field settings. For this type of exposure, it is essential to apply specific amounts of fungal spores homogeneously over a surface for testing the effects of fungal dose and exposure time. Contemporary methods such as spraying or brushing spore suspensions onto substrates do not produce the uniformity and consistency that standardized laboratory assays require. Two novel fungus application methods using equipment developed in the paint industry are presented and compared.</p> <p>Methods</p> <p>Wired, stainless steel K-bars were tested and optimized for coating fungal spore suspensions onto paper substrates. Different solvents and substrates were evaluated. Two types of coating techniques were compared, i.e. manual and automated coating. A standardized bioassay set-up was designed for testing coated spores against malaria mosquitoes.</p> <p>Results</p> <p>K-bar coating provided consistent applications of spore layers onto paper substrates. Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity. Evaporative Shellsol T solvent dried quickly and resulted in high spore infectivity to mosquitoes. Smooth proofing papers were the most effective substrate and showed higher infectivity than cardboard substrates. Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival. The standardized mosquito exposure bioassay was effective and consistent in measuring effects of fungal dose and exposure time.</p> <p>Conclusions</p> <p>K-bar coating is a simple and consistent method for applying fungal spore suspensions onto paper substrates and can produce coating layers with accurate effective spore concentrations. The mosquito bioassay was suitable for evaluating fungal infectivity and virulence, allowing optimizations of spore dose and exposure time. Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.</p

External development of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in the subterranean termite Heterotermes tenuis Desenvolvimento dos fungos entomopatogênicos Beauveria bassiana E Metarhizium anisopliae no cupim subterrâneo Heterotermes tenuis

The subterranean termite Heterotermes tenuis is one of the main pests of sugarcane and eucalyptus in Brazil, and the use of entomopathogenic fungi, alone or associated to chemicals, is an efficient and environmentally favorable method for its control. Studies related to the fungal development on these insects are important due to the effect of insect behavior on entomopathogens. The objective of this work was to describe the external development of Beauveria bassiana and Metarhizium anisopliae on H. tenuis using Scanning Electron Microscopy (SEM), determining the duration of the different phases of fungal infection. Two fixation techniques for preparing SEM samples were also evaluated. Worker specimens of H. tenuis were inoculated with a 1 x 10(9) conidia mL-1 suspension of the fungi and maintained at 25±1ºC and 70±10% relative humidity. Insects were collected from 0 to 144 hours after inoculation and prepared on SEM stubs for each of the two fixation techniques. The results obtained with the two techniques were compared and duration of the different phases of the infection process were estimated from SEM observations and compared for three fungal isolates. B. bassiana and M. anisopliae have similar development cycles on the termite, but some important differences exist. The penetration, colonization and conidiogenesis phases are relatively faster for M. anisopliae than for B. bassiana, which results in a faster rate of insect mortality. The fixation technique with OsO4 vapor is suitable for preparation of insects to be used in SEM observation of the developmental stages of entomopathogenic fungi.<br>O cupim subterrâneo Heterotermes tenuis , uma das principais pragas da cana-de-açúcar e eucalipto no Brasil, e o uso de fungos entomopatogênicos, isoladamente ou associados a produtos químicos, é um método eficiente e ambientalmente seguro para seu controle. Estudos relacionados ao desenvolvimento fúngico nestes insetos são importantes devido ao efeito do comportamento dos insetos sobre entomopatógenos. O objetivo deste trabalho foi descrever o desenvolvimento de Beauveria bassiana e Metarhizium anisopliae sobre H. tenuis por meio da Microscopia Eletrônica de Varredura (MEV), determinando a duração das fases de infecção fúngica. Também foram avaliadas duas técnicas de fixação para o preparo de amostras para MEV. Operários de H. tenuis foram inoculados com suspensões fúngicas de 1 x 10(9) conídios mL-1 e mantidos a 25 ± 1ºC e umidade relativa de 70 ± 10%. Foram coletados insetos de 0 a 144 horas após a inoculação e preparados pelas duas técnicas de fixação. Foram comparados os resultados obtidos com as duas técnicas e estimadas e comparadas as durações das fases do processo de infecção para três isolados fúngicos por meio de observações em MEV. B. bassiana e M. anisopliae têm ciclos de desenvolvimento semelhantes sobre H. tenuis, mas algumas diferenças importantes existem. As fases de penetração, colonização e conidiogênese são relativamente mais rápidas para M. anisopliae que para B. bassiana, o que resulta em uma taxa mais rápida de mortalidade do inseto. A técnica de fixação com vapor de OsO4 é satisfatória para preparação de insetos para observação do desenvolvimento de fungos entomopatogênicos em MEV