Processing and mounting phlebotomine sand flies: a consensus guideline

This article provides a comprehensive guide for the processing and mounting of phlebotomine sand fly specimens, which is crucial for species identification and pathogen detection and isolation. It discusses a range of techniques suitable for both field and laboratory settings. The guide includes detailed instructions on sand fly collection, handling, covering, and euthanasia (recommending dry freezing or CO2 over chemicals) as well as conservation strategies, such as cold storage and preservation in ethanol. The quality of preparation of certain anatomical structures (genital organs, head and wings) is essential for their proper microscopic observation and is described in this work. The article also presents detailed sample processing, including the clearing process with agents such as potassium hydroxide then Marc-André solution. The mounting process compares different media, emphasizing their optical properties and preservation potential. Hoyer fluid (also known as chloral gum) is recommended for quick observation, particularly for spermathecae, due to its clarity, although it is not suitable for long-term storage. Other media discussed include polyvinyl alcohol, Euparal® (for limited water tolerance), and Canada balsam (a hydrocarbon-soluble medium), with the latter two offering long-term preservation capabilities. Innovative molecular biology approaches such as DNA sequencing and MALDI-ToF, which require particular attention to sample processing, are also addressed. Furthermore, short video clips illustrating various mounting techniques as well as translations in many different languages are provided, allowing the guideline to reach the diverse needs and expectations of the global scientific community.