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.