Pathogenicity of microsclerotia from Metarhizium robertsii against Aedes aegypti larvae and antimicrobial peptides expression by mosquitoes during fungal-host interaction.

Aedes aegypti is a vector of various disease-causing arboviruses. Chemical insecticide-based methods for mosquito control have increased resistance in different parts of the world. Thus, alternative control agents such as the entomopathogenic fungi are excellent candidates to control mosquitoes as part of an ecofriendly strategy. There is evidence of the potential of entomopathogenic fungal conidia and blastospores for biological control of eggs, larval and adult stages, as well as the pathogenicity of fungal microsclerotia against adults and eggs. However, there are no studies on the pathogenicity of microsclerotia against either aquatic insects or insects that develop part of their life cycle in the water, such as the A. aegypti larvae. In this study, we assayed the production of microsclerotia and their pathogenicity against A. aegypti larvae of two isolates of Metarhizium robertsii, i.e., CEP 423 isolated in La Plata, Argentina, and the model ARSEF 2575. Both isolates significantly reduced the survival of A. aegypti exposed to their microsclerotia. The fungus-larva interaction resulted in a delayed response in the host. This was evidenced by the expression of some humoral immune system genes such as defensins and cecropin on the 9th day post-infection, when the fungal infection was consolidated as a successful process that culminates in larvae mortality. In conclusion, M. robertsii microsclerotia are promising propagules to be applied as biological control agents against mosquitoes since they produce pathogenic conidia against A. aegypti larvae.

Compatibility between Leptolegnia chapmanii and diflubenzuron and neem oil for the control of Aedes aegypti / Compatibilidad de Leptolegnia chapmanii con diflubenzurón y aceite de neem parael control de Aedes aegypti

Abstract Leptolegnia chapmanii is pathogenic to mosquito larvae. The compatibility betweenL. chapmanii and two insect growth regulators (IGR), diflubenzuron and neem oil, was evaluated. L. chapmanii was grown on culture media containing different concentrations of eachIGR. The mycelial growth was significantly reduced with the highest concentrations of IGR(F6,98= 268, p < 0.05). However, the production of zoospores (F6,56= 0.93, p > 0.05) and the lar-val mortality of Aedes aegypti (F6,56= 0.95, p > 0.05) were not significantly different amongtreatments. Furthermore, the percentage of adult emergence in the presence of different con-centrations of diflubenzuron or a neem formulation was determined, and the pathogenic activityof zoospores was evaluated at the concentrations that inhibit the emergence at 30, 50 and 90%.The pathogenicity of zoospores was not significantly different among treatments (F6,14= 0.54,p > 0.05), and the larval mortalities were above 90% in all cases.

Resumen Leptolegnia chapmanii es un patógeno de larvas de mosquitos. Se evaluó la com-patibilidad entre L. chapmanii y dos reguladores del crecimiento de insectos (IGR, por sus siglasen inglés), diflubenzurón y aceite de neem. L. chapmanii creció en medios de cultivo con diferentes concentraciones de estos IGR. El crecimiento micelial fue menor con las concentraciones más altas (F6,98= 268; p < 0,05). No hubo diferencias significativas en la producción de zoosporas (F6,56= 0,93; p > 0,05) ni en la mortalidad de las larvas de Aedes aegypti (F6,56= 0,95; p > 0,05).Se determinó el porcentaje de emergencia de adultos en presencia de diferentes concentra-ciones de diflubenzurón o de un formulado a base del aceite de neem. También se evaluó lapatogenicidad de las zoosporas a las concentraciones de los IGR que inhibieron la emergenciadel 30, 50 y 90% de adultos. No hubo diferencias significativas en la actividad de las zoosporasal comparar los tratamientos (F6,14= 054; p > 0,05). La mortalidad de las larvas fue superior al90%.

Compatibility between Leptolegnia chapmanii and diflubenzuron and neem oil for the control of Aedes aegypti.

Leptolegnia chapmanii is pathogenic to mosquito larvae. The compatibility between L. chapmanii and two insect growth regulators (IGR), diflubenzuron and neem oil, was evaluated. L. chapmanii was grown on culture media containing different concentrations of each IGR. The mycelial growth was significantly reduced with the highest concentrations of IGR (F6,98=268, p<0.05). However, the production of zoospores (F6,56=0.93, p>0.05) and the larval mortality of Aedes aegypti (F6,56=0.95, p>0.05) were not significantly different among treatments. Furthermore, the percentage of adult emergence in the presence of different concentrations of diflubenzuron or a neem formulation was determined, and the pathogenic activity of zoospores was evaluated at the concentrations that inhibit the emergence at 30, 50 and 90%. The pathogenicity of zoospores was not significantly different among treatments (F6,14=0.54, p>0.05), and the larval mortalities were above 90% in all cases.

Adulticidal Activity of Metarhizium anisopliae s.l. (Hypocreales: Clavicipitaceae) Native Strains From Argentina Against Aedes aegypti (Diptera: Culicidae).

The adulticidal activity of six fungal strains of Metarhizium anisopliae (Metschn.) Sorokin s.l. (Hypocreales: Clavicipitaceae) against Aedes aegypti (L.) (Diptera: Culicidae) was assessed. These strains (CEP 085, CEP 087, CEP 120, CEP 350, CEP 404, and CEP 423) were isolated from soil samples or nondipteran hosts collected from areas in Argentina where Ae. aegypti is distributed. Bioassays were performed with four conidial concentrations plus a control of each fungal strain to determine the lethal concentrations (LC50/LC90), the median survival times (ST50), the mean percentage of the surviving individuals, and the mean percentage of mycotized cadavers. The strains were able to infect and kill adult Ae. aegypti. Significant differences were found among the LC50 values, with CEP 423 as the most virulent strain with the lowest LC50 (2.4 × 106 conidia/ml). At 1 × 107 conidia/ml: no significant differences were found in the Kaplan-Meier survival functions among the strains; the ST50 ranged from 5 (CEP 085) to 8 d (CEP 350); and the mean percentage of the surviving adults was between 13.3 (CEP 085, CEP 350 and CEP 423) and 46.7% (CEP 087). Significant differences were also found among strains in the mean percentage of cadavers with fungal sporulation. Strain CEP 423 produced the highest percentage of mycotized adults (70%). The concentration of the inoculum significantly affected the survival of individuals and the percentage of mycotized cadavers within each strain. Metarhizium anisopliae s.l. CEP 423 was selected as the most promising candidate for further research aiming to develop a mycoinsecticide against Ae. aegypti.

Effect of UV-B Irradiation on Water-Suspended Metarhizium anisopliae s.l. (Hypocreales: Clavicipitaceae) Conidia and Their Larvicidal Activity in Aedes aegypti (Diptera: Culicidae).

Ultraviolet (UV) radiation is a key limiting factor for biological pest control with entomopathogenic fungi. While little is known about the impact of UV on Metarhizium anisopliae Metchnikoff (Sorokin) (Hypocreales: Clavicipitaceae) conidia in aquatic mosquito-breeding sites, this study determined the effect of UV-B on the viability and virulence of M. anisopliae sensu lato (s.l.) strain IP 46 in the laboratory against Aedes aegypti (L.) (Diptera: Culicidae) larvae. Conidia were treated in cups under defined water depths (0, 1, 2, and 3 cm) to six different UV-B doses (0, 0.657, 1.971, 3.942, 7.884, 11.826, or 15.768 kJ m-2) at 27 ± 2°C. The ability of treated conidia to germinate up to 24 h postexposure on PDAY + benomyl + chloramphenicol medium at 25 ± 1°C was adversely affected by higher doses of UV-B radiation regardless of the water depth. Germination, however, did not fall below 70% regardless of the test conditions. In fact, conidial virulence against second-instar larvae was not affected by either the water depth (F3,84 = 0.3, P = 0.85) or any tested levels of UV-B radiation (F6,21 ≤ 1.2, P ≥ 0.39) including those distinctly higher than might be expected for tropical sites. These findings strengthen previous observations that IP 46 has significant potential for use against A. aegypti larvae, even when exposed to elevated UV-B irradiance levels in the small breeding sites that are common for this important vector.

Organización y conservación de la colección de hongos patógenos y simbiontes de insectos y otros artrópodos del CEPAVE (CONICET-UNLP), La Plata, Argentina / Organization and preservation of the collection of pathogenic and fungal symbionts of insects and other arthropods from CEPAVE (CONICET-UNLP), La Plata, Argentina

La colección de hongos patógenos y simbiontes de insectos y otros artrópodos del Centro de Estudios Parasitológicos y de Vectores es única por sus características específicas en la Argentina y por preservar hongos en cultivo, in vitro e in vivo. Esta colección está abierta para actividades de investigación, docencia, servicios y asesoramiento público y privado. Un total de 421 aislamientos pertenecientes a 23 géneros (Ascomycota: 16, Entomophthoromycotina: 4 Glomeromycota: 2 y Oomycota: 1) son preservados en la colección mediante diversos métodos: criopreservación en freezer a −20 °C y −70 °C, agua destilada estéril, papel, arena y liofilización. Las cepas que integran la colección fueron aisladas de insectos y de otros artrópodos; y a partir de suelo (usando insectos trampa o aislamiento directo en medios selectivos). La identificación se realizó mediante estudios de caracterización morfológicos y, en algunos casos, por taxonomía molecular. Esta colección se utiliza como centro de referencia para identificación de especies, certificaciones, estudios de investigación y docencia, transferencia tecnológica, servicios a terceros, depósito de cepas y preservación de la diversidad y conservación del germoplasma. Las cepas preservadas son, en su mayor parte, nativas de distintas regiones de la Argentina. La colección fue originada en el año 1988 y está registrada en la base de la Federación Latinoamericana de Colecciones de Cultivos y en la World Federation of Culture Collections.

The collection of fungal pathogens and symbionts of insects and other arthropods of the Centro de Estudios Parasitológicos y de Vectores, La Plata, Argentina, is unique because it preserves in vivo and in vitro cultures of fungal pathogens. This culture collection is open for research, teaching, consulting services, and strain deposit. It contains 421 strains belonging to 23 genera (16 Ascomycota, 4 Entomophthoromycotina, 2 Glomeromycota and 1 Oomycota), and the cultures are preserved by different methods such as cryopreservation in freezer at −20 °C and −70 °C, paper, distilled water and lyophilization. Fungi were isolated from insects, other arthropods, and soil (by using insect baits and selective media). Species were identified by morphological features and in a few strains by molecular taxonomy (PCR of rDNA). This collection is a reference center for species identification/certifications, research and teaching purposes, strain deposit, transference and consultancy services, and its overall goal is to preserve the fungal germplasm and ex situ diversity. Most of the strains are native of Argentina. The collection was originated in 1988 and is registered in the Latin American Federation for Culture Collections and in the World Federation of Culture Collections.

[Organization and preservation of the collection of pathogenic and fungal symbionts of insects and other arthropods from CEPAVE (CONICET-UNLP), La Plata, Argentina]. / Organización y conservación de la colección de hongos patógenos y simbiontes de insectos y otros artrópodos del CEPAVE (CONICET-UNLP), La Plata, Argentina.

The collection of fungal pathogens and symbionts of insects and other arthropods of the Centro de Estudios Parasitológicos y de Vectores, La Plata, Argentina, is unique because it preserves in vivo and in vitro cultures of fungal pathogens. This culture collection is open for research, teaching, consulting services, and strain deposit. It contains 421 strains belonging to 23 genera (16 Ascomycota, 4 Entomophthoromycotina, 2 Glomeromycota and 1 Oomycota), and the cultures are preserved by different methods such as cryopreservation in freezer at -20°C and -70°C, paper, distilled water and lyophilization. Fungi were isolated from insects, other arthropods, and soil (by using insect baits and selective media). Species were identified by morphological features and in a few strains by molecular taxonomy (PCR of rDNA). This collection is a reference center for species identification/certifications, research and teaching purposes, strain deposit, transference and consultancy services, and its overall goal is to preserve the fungal germplasm and ex situ diversity. Most of the strains are native of Argentina. The collection was originated in 1988 and is registered in the Latin American Federation for Culture Collections and in the World Federation of Culture Collections.