Pathogenicity of the Fungus, Aspergillus clavatus, Isolated from the Locust, Oedaleus senegalensis, Against Larvae of the Mosquitoes Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus

The use of insect pathogenic fungi is a promising alternative to chemical control against mosquitoes. Among the Hyphomycetes isolated from insects for mosquito control, the genus Aspergillus remains the least studied. In September 2005, four fungi were isolated from the Senegalese locust, Oedaleus senegalensis Kraus (Orthoptera: Acrididae), collected in Dakar, Senegal. One of these fungi, identified as Aspergillus clavatus, Desmazières (Eurotiales: Trichocomaceae) was highly pathogenic against larvae of the mosquitoes Aedes aegypti L., Anopheles gambiae s.l. Giles and Culex quinquefasciatus Say (Diptera: Culicidae). An application of 1.2 mg/ml dry conidia yielded 100% mortality after 24 hours against both Ae. aegypti and Cx. quinquefasciatus while with An. gambiae it was 95%. With unidentified species in the genus Aspergillus, mortality after 24 h was <5% against all the larval species. Application of A. clavatus produced in a wheat powder medium using doses ranging between 4.3 to 21×107 spores/ml, caused 11 to 68% mortality against Cx. quinquefasciatus at 24h, and 37 to 100% against Ae. aegypti. Microscopic observations showed fungal germination on both Ae. aegypti and Cx. quinquefasciatus larvae. Histological studies revealed that A. clavatus penetrated the cuticle, invaded the gut and disintegrated its cells. Some Cx. quinquefasciatus larvae, treated with A. clavatus reached the pupal stage and produced infected adults. However, the infection was mainly located on the extremity of their abdomen. These results suggest that A. clavatus could be an effective tool to manage mosquito proliferation

Test de pathogénicité du champignon Aspergillus clavatus sur le puceron Acyrthosiphon pisum (Aphididae)

Pea aphid is a pest of many cultivated and wild plants, but also a vector of several viral diseases. To control this pest, the most widely used methods are physical, chemical and more recently an integrated approach that includes biological control. With the use of pathogenic agents against insects, the use of entomopathogenic fungi is one of the most promising. The present study demonstrated the possibility of using an entomopathogenic fungus Aspergillus clavatus against aphids. In laboratory conditions (8/16 photoperiod, average temperature 25°C), the insects were in contact with different concentrations ranging from 10^2 to 10^4 spores/cm2 deposited on filter paper in Petri dishes, or applied directly to young plants with doses ranging from 10^4 to 10^6 spores/ml. In 24 hours, mortality was 0 to 31.5% in Petri dishes. For treatment plants, the cumulative mortality in 5 days was 55 to 79%. Microscopic observations showed that the aphids were infected by contact and fungus has a mycosis effect. From these preliminary results, investigations should be made to study the action of the fungus on the reproduction of aphids. Therefore, A. clavatus could be introduced along with other fungi found in the literature as a biological control agent against aphids

Using micro-injection technique to assess fungal toxicity in mosquito control

Topical application of insecticidal compounds allows directly exposing these substances on insect tissues and measuring their toxicity while ignoring many factors. However, this technique remains difficult to apply on mosquito larvae considering their aquatic lifestyle. Micro-injection could be used for the direct deposition of toxic compounds in the larvae. Capillaries exhibiting an injection tip with an external diameter of 0.5 mm have been designed from silica tubes. For each treatment, a capillary is mounted on a pump connected to a flow rate regulator. Culex quinquefasciatus larvae were injected with 10^7 spores/ml of entomopathogenic fungi (Aspergillus clavatus, Metarhizium anisopliae, Metarhizium sp.). Mortalities were recorded daily during 72h. The distribution of spores stained with methylene blue and injected into the body of larvae was also observed according to the system described. Results showed that spores were distributed over the whole body. The injection of Aspergillus clavatus, Metarhizium anisopliae and Metarhizium sp spores induced corrected mortalities of 62%, 53% and 57% after 72h, and differed statistically from control groups. Finally, post-mortem emergences of filaments from dead larvae were observed in the case of the three fungal strains confirming spore viability. Injection of inactivated spores (or inert bodies of similar size) could help to reject the hypothesis of a response due to the presence of foreign bodies

Biofilms from entomopathogenic fungi in mosquito control

Mosquitoes (Diptera: Culicidae) are zoonotic vectors of medical and veterinary importance. As part of an integrated vector control, metabolites secreted by entomopathogenic fungi could be developed as biopesticides. In this context, filamentous microorganisms growing on a support as biofilm in a liquid medium would offer several advantages in bioreactor regarding performances and metabolites recovery. The production of toxic metabolites by an entomopathogenic fungus Aspergillus flavus in such conditions was assessed. Three initial inoculum levels, i.e. 10^1, 10^3 and 10^6 spores/ml of PYG medium, have been tested in shake flask with or without support. Toxicity tests were performed on Culex quinquefasciatus larvae using dilutions of 1, 2, 4, 6, 8 and 10% of liquid cultures. The results indicated that A. flavus tends to form pellets in submerged culture; the size and the amount of pellets was affected by the initial inoculum level of spores. Under similar conditions, the filaments fixed on a support and didn’t appear in free form in the liquid. Toxicity tests revealed differences between both free and fixed forms. All combined conditions, LC50s ranging up to dilutions of 2.2 and 4.8% were observed within 48 hours. Secretomes could be compared between these culture conditions by proteomic and metabolomic approaches

Laboratory and semi-field environment tests for the control efficacy of Metarhizium anisopliae formulated with neem oil (suneem) against Anopheles gambiae s.l. adult emergence

peer reviewedMetarhizium anisopliae was evaluated previously in Suneem formulation against malaria vector adults. However, their ability to control aquatic stages is not yet evaluated. In laboratory conditions: the lethal dose (LD90) of the formulation was determined on Anopheles gambiae larvae collected from breeding sites and evaluated into artificial vats at dry and rainy seasons. In laboratory conditions, the LD90 was 5.3 × 106 spores/ml in 48 h. In semi-field environment, the formulation had a great emergence inhibition of mosquito adult (P < 0.0001). The emergences rate at day 8 were 2.25 ± 0.03, 28.00 ± 1.07 and 97.25 ± 1.56 % in dry season for the oil formulation (OF), Suneem (S), and water control respectively. In rainy season, the emergences were 1.25 ± 0.15, 30.25 ± 1.23 and 98 ± 0.76 % respectively. No significant difference was observed between dry and rainy seasons (P=0.3). Therefore, M. anisopliae formulated with Suneem may provide a more sustainable management strategy for malaria vectors control at larval stages

The fight against mosquitoes (Diptera: Culicidae): diversity of approaches and application of biological control

Plusieurs espèces de moustiques (Diptera: Culicidae) sont des vecteurs de zoonoses d’incidence médicale et vétérinaire considérables. Une modification de la distribution géographique de ces vecteurs majoritairement engendrée par des facteurs anthropiques est actuellement accompagnée de (ré-)émergences de maladies infectieuses en Europe et en Amérique du Nord. Depuis l’avènement des insecticides de synthèse lors de la seconde guerre mondiale, les moustiques font l’objet de recherches de plus en plus étendues et approfondies. Dans une vision de lutte intégrée, les moyens de lutte anti-vectorielle se répartissent aujourd’hui selon quatre axes principaux: (1) la gestion environnementale et le contrôle physique, (2) le contrôle chimique, (3) le contrôle génétique, et (4) le contrôle biologique par le biais d’entomophages et de micro-organismes entomopathogènes. Dans ce contexte, ces derniers ont un potentiel intéressant car ils possèdent la capacité d’infecter et de tuer l’hôte avec une sélectivité plus ou moins prononcée. Cet article se propose de resituer le contrôle biologique parmi les autres techniques dans la lutte anti-vectorielle contre les moustiques, et de faire état des potentialités et des perspectives offertes par les bactéries, virus et champignons entomopathogènes. Leur utilisation sous forme de biopesticides est enfin discutée.Many mosquito (Diptera: Culicidae) species are zoonotic vectors responsible for numerous infectious diseases of medical and veterinary importance. Currently, changes in the vectors’ geographical distribution induced chiefly by anthropogenic factors are accompanied by emerging and reemerging infectious diseases in Europe and North America. Since the advent of synthetic insecticides during the Second World War, mosquitoes are the object of considerably expanded and deepened research. In an integrated pest management context, means of control are now mainly classified as: (1) environmental management and physical control, (2) chemical control, (3) genetic control, and (4) biological control by means of entomophagous predators and entomopathogenic microorganisms. In this context, these last have significant potential because of their ability to infect and kill their host with more or less targeted selectivity. This article proposes to emphasize biological control among other techniques in mosquito control, and to assess the potential and the opportunities offered by entomopathogenic bacteria, viruses and fungi. Finally, their use as biopesticides is discussed

Pathogenicity of Aspergillus clavatus produced in a fungal biofilm bioreactor toward Culex quinquefasciatus (Diptera: Culicidae)

peer reviewedMany entomopathogenic fungi have been demonstrated to be potential agents for efficiently controlling mosquito populations. In the present study, we investigated a bioreactor system to produce metabolites and conidia by combining technological advantages of submerged and solid-state fermentations. The efficiency of fungal products was tested toward mosquitoes. Aspergillus clavatus (Eurotiales: Trichocomaceae) was grown by semi-solid-state fermentation in a bioreactor for up to 7 days. Depending on conidial doses (2.5×10^7, 5×10^7, 7.5×10^7, 10×10^7 and 12.5×10^7 conidia/mL), mortality ranged from 37.2±15.0 to 86.3±5.0% toward larvae and from 35.8±2.0 to 85.2±1.5% toward adults. The metabolites (10, 20, 40, 60, 80 and 100% v/v) yielded mortality from 23.7±15.0 to 100.0±0.1% toward larvae, and two sprayed volumes (5 and 10 mL) reached 45.5±1.4 and 75.6±2.6% mortality, respectively, toward adults

Application de trois champignons entomopathogènes dans la lutte contre les pucerons

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Chemical pesticide application is the most commonly used method for aphid control. Due to rapid developments of resistance to pesticides and environmental pollution, integrated pest management, including biological control was promoted. In this context, entomopathogenic fungi are valuable tools for potential aphid control since various fungal strains are already commercially available. The present study aims to evaluate and compare the insecticidal activity of two Metarhizium and one Aspergillus strains against Acyrthosiphon pisum aphid. Fungi were cultivated on wheat bran media in flasks up to sporulation. The solid media were washed with aqueous solutions containing 0.05% Tween 80 before filtration. The content of conidia was determined by haemocytometer. Doses ranging from 10^3 to 10^7 conidia/ml were then applied on young plants with 20 adult parthenogenetic aphids. Batches were incubated at a 16L/8D photoperiod, 25±2°C and 75-80% relative humidity. Adult mortality was assessed in order to determine LD50, LD90 and LT50 values, and the number of nymphs produced was daily recorded. Five days after treatment, mortality rates ranged from 30 to 91% depending on the fungal strain and tested dose. Corresponding LD50 and LD90 values were 1.23 x 10^3 and 1.34 x 10^7, 3.67 x 10^3 and 9.71 x 10^7, 4.95 x 10^2 and 5.65 x 10^7 conidia/ml for Metarhizium sp., Metarhizium anisopliae and Aspergillus clavatus respectively. At the higher dose, the LT50 were reached within 2, 4 and 3 days respectively, whereas the LT50 were never reached in the controls. By contrast, the intrinsic growth rates were significantly different from uninfected aphids only in the case of A. clavatus with 10^6 and 10^7 conidia/ml doses five days after exposure. In conclusion, these fungal isolates induced A. pisum mortality with a similar impact and A. clavatus infection appeared to alter the adults’ fitness. This suggests that these fungi may be candidates for aphid control. Further investigations should be made in order to assess their host range specificity. Toxic metabolites leading to death have to be identified and their safety towards non-target organisms confirmed. Finally, their persistence in the environment as well as the compatibility with over means of aphid control must be verified in a view of a broad integrated pest management

Effect of entomopathogenic Aspergillus strains against the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae)

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Entomopathogenic fungi are valuable tools for potential aphid control. As part of a selection process, laboratory bioassays were carried with five different concentrations of Aspergillus clavatus (Desmazières), Aspergillus flavus (Link) and Metarhizium anisopliae ((Metschnikoff) Sorokin) spores against the pea aphid, Acyrthosiphon pisum (Harris). Aspergillus isolates induced higher mortalities than M. anisopliae which is a well-known entomopathogen in the literature. Lethal concentrations (LC50 and LC90) were 1.23 x 10^3 and 1.34 x 10^7 spores/ml for A. flavus, 4.95 x 10^2 and 5.65 x 10^7 spores/ml for A. clavatus, and 3.67 x 10^3 and 9.71 x 10^7 spores/ml for M. anisopliae five days after treatment. Mycelia development and sporulation on adult cadavers was observed 48 hours after incubation. The intrinsic growth rate of A. pisum decreased with increased spore concentration for all fungal strains suggesting an increase in pathogen fitness related to a consumption of host resources. In conclusion, Aspergillus species could be useful in aphid control as pest control agents despite their saprophytic lifestyle. This is also in our knowledge the first report of A. clavatus and A. flavus strains pathogenic to aphids