Review Article

Processing and mounting phlebotomine sand flies: a consensus guideline

Traitement et montage des phlébotomes : un guide consensuel

¹ Faculté de Pharmacie, Université de Reims Champagne Ardenne, UR ESCAPE-USC ANSES PETARD, 51 rue Cognacq-Jay 51096 Reims Cedex, France
² Pôle de Biologie territoriale, Laboratoire de Parasitologie-Mycologie, Centre Hospitalo-Universitaire, 51092 Reims, France
³ Parasitology-Mycology Department, Avicenne Hospital, AP-HP, Bobigny, Sorbonne Paris Nord University, France; Unité des Virus Émergents (UVE: Aix-Marseille Univ, Università di Corsica, IRD 190, Inserm 1207, IRBA), 13005 Marseille, France
⁴ Parasitology Collection of Basic Pathology, Department of Basic Pathology, Federal University of Paraná, Curitiba 19031, Brazil
⁵ Department of Animal Health, University of Murcia, Campus de Espinardo, 30100 Espinardo, Murcia, Spain
⁶ Department of Infectious Diseases, Vector-borne Diseases Unit, Istituto Superiore di Sanità, 00166 Rome, Italy
⁷ Medical and Veterinary Entomology Unit, Institut Pasteur du Cambodge, Phnom Penh 12201, Cambodia
⁸ Ecology & Emergence of Arthropod-borne Pathogens Unit, Department of Global Health, Institut Pasteur, CNRS UMR2000, 75015 Paris, France
⁹ Section Veterinary Services (1417), Laboratory for Animal Health Virology, Aglantzia, Nicosia 2109, Cyprus
¹⁰ Insects Vectors and Parasites Laboratory, Department of Basic Pathology and Postgraduate program in Microbiology, Parasitology and Pathology, Federal University of Paraná, 81530-900 Curitiba, Brazil
¹¹ Department of Biological Sciences, National University of Singapore, 117558, Singapore
¹² Laboratory of the Leishmaniasis Research Project, Mokolo District Hospital, Mokolo, Cameroon; Laboratory of Cellular Immunology and Parasitology, Department of Biological Sciences, Central Washington University, 98926 Ellensburg, WA, USA
¹³ Department of Parasitology, Faculty of Science, Charles University, 12800 Prague, Czechia
¹⁴ VERG Laboratories, Department of Biology, Faculty of Science, Hacettepe University, Beytepe, Ankara 06800, Türkiye
¹⁵ Faculdade de Saúde Pública da Universidade de São Paulo (FSP/USP), Pós-graduação em Saúde Pública, 01246-904 São Paulo, Brazil
¹⁶ Secció de Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona, & Institut de Salut Global de Barcelona (ISGlobal), Centro de Investigación Biomédica en Red, Enfermedades Infecciosas (CIBERINFEC), 08028 Barcelona, Spain
¹⁷ Laboratory of Infectious Diseases and Public Health, School of Medicine, University of Cyprus, Nicosia, Cyprus & Department of Pediatrics, Archbishop Makarios III Hospital, Nicosia 2115, Cyprus
¹⁸ Faculty of Health Sciences, American University of Beirut, 1107 2020 Beirut, Lebanon
¹⁹ Medical Entomology Unit, Infectious Disease Research Centre, Institute for Medical Research (IMR), National Institutes of Health (NIH), Ministry of Health Malaysia, 40170 Shah Alam, Selangor, Malaysia
²⁰ School of Medicine, Addis Ababa University, 28017 – 1000 Addis Ababa, Ethiopia
²¹ Faculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, 6000 Koper, Slovenia
²² University of Lodz, Faculty of Biology and Environmental Protection, Department of Invertebrate Zoology and Hydrobiology, Banacha 12/16, 90-237 Łódź, Poland
²³ Laboratory of Entomology, Ministry of Health, 9134302 Jerusalem, Israel
²⁴ Center for Pathophysiology, Infectiology and Immunology, Institute of Specific Prophylaxis and Tropical Medicine, Medical University Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria
²⁵ Papua New Guinea Institute of Medical Research (PNGIMR) Institute, PO Box 60, Headquarter, Homate Street, 441 Goroka, Eastern Highlands Province, Papua New Guinea
²⁶ Museum of Natural History, University of the Philippines Los Baños, 4031 Laguna, Philippines
²⁷ National Centre of Infectious and Parasitic Diseases, 1504 Sofia, Bulgaria
²⁸ Ege University, Faculty of Medicine, Department of Parasitology, 35040 Bornova/Izmir, Türkiye
²⁹ Retired, Faculté de Pharmacie, Université de Strasbourg, Strasbourg, 67400 Illkirch-Graffenstaden, France
³⁰ Program for the Study and Control of Tropical Diseases (PECET), Faculty of Medicine, University of Antioquia, 050010 Medellin, Colombia
³¹ MIVEGEC, Univ. Montpellier, CNRS, IRD, 34394 Montpellier, France & Medical Entomology Unit, Institut Pasteur de Madagascar, 101 Antananarivo, Madagascar
³² Laboratorio de Entomología Médica, Departamento de Zoología de Invertebrados, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, 66455, NL, México
³³ Tropical and Infectious Disease Centre, BP Koirala Institute of Health Sciences, Dharan 56700, Nepal
³⁴ ICMR-Vector Control Research Centre, Puducherry 605006, India
³⁵ Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
³⁶ Grupo de estudos em Leishmanioses/Coleção de Flebotomíneos (COLFLEB/Fiocruz-MG), Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Minas Gerais, 30190009, Brazil
³⁷ Center of Excellence in Vector Biology and Vector-Borne Disease, Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
³⁸ Department of Arbovirology, Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Str. 74, 20359 Hamburg, Germany
³⁹ Laboratory Vectors & Parasites, Department of Livestock Sciences and Techniques, Sine Saloum University El Hadji Ibrahima Niasse (SSUEIN) Kaffrine Campus, C.P. 24600, Senegal
⁴⁰ Environmental Health Institute, National Environment Agency, Singapore 138667, Singapore & Department of Biological Sciences, National University of Singapore, 117558, Singapore
⁴¹ Institut Pasteur du Laos, Laboratory of Vector-Borne Diseases, Samsenhai Road, Ban Kao-Gnot, Sisattanak District, 3560 Vientiane, Lao PDR
⁴² National Institute of Hygiene and Epidemiology, 1 Yec-Xanh Street, Hai Ba Trung District, 100000 Hanoi, Vietnam
⁴³ Indonesian Research Center for Veterinary Science, Indonesian Agency for Agricultural Research and Development, Ministry of Agriculture Republic Indonesia, Bogor 16114, Indonesia Department of Parasitology, Faculty of Veterinary Medicine, Airlangga University, Surabaya 60115, Indonesia
⁴⁴ Laboratory of Research in Applied Biology, Polytechnic School of Abomey-Calavi, University of Abomey-Calavi, 01 P.O. Box 2009, 00000 Cotonou, Benin
⁴⁵ Instituto de Microbiología, Colegio de Ciencias Biológicas y Ambientales (COCIBA), Universidad San Francisco de Quito (USFQ), 170901 Quito, Ecuador

^* Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.

Received: 1 December 2025
Accepted: 29 January 2026

Abstract

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.

Résumé

Cet article propose un guide complet pour le traitement et le montage des phlébotomes, étapes cruciales pour l’identification des espèces et la détection ou l’isolement des pathogènes. Il aborde diverses techniques adaptées au terrain comme au laboratoire. Le guide comprend des instructions détaillées sur la collecte, la manipulation, la protection et l’euthanasie des phlébotomes (avec recommandation de la congélation à sec ou du CO₂ plutôt que de l’usage de produits chimiques), ainsi que sur les stratégies de conservation telles que la réfrigération et la conservation dans l’éthanol. La préparation des structures anatomiques spécifiques (organes génitaux, tête et ailes) pour l’observation microscopique est privilégiée dans la description des techniques de dissection. Le document présente également le traitement détaillé des échantillons, notamment la clarification à l’aide de solutions telles que la potasse puis le liquide de Marc-André. Le processus de montage compare différents milieux, en soulignant leurs propriétés optiques et leur potentiel de conservation. Le liquide de Hoyer (également appelé gomme au chloral) est recommandé pour l’observation rapide, en particulier pour les spermathèques, en raison de sa limpidité, bien qu’il ne convienne pas à la conservation à long terme. D’autres milieux de culture sont abordés, notamment l’alcool polyvinylique, l’Euparal^® (pour une tolérance limitée à l’eau) et le baume du Canada (un milieu soluble dans les hydrocarbures), ces deux derniers permettant une conservation à long terme. Les approches innovantes de biologie moléculaire, telles que le séquençage de l’ADN et la spectrométrie de masse MALDI-ToF, qui exigent une attention particulière lors du traitement des échantillons, sont également présentées. De plus, de courtes vidéos illustrant diverses techniques de montage et des traductions en de nombreuses langues sont disponibles, permettant ainsi de répondre aux besoins et attentes variés de la communauté scientifique internationale.