Fano José Randrianambinintsoa

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 CO ₂ 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.

Durant de nombreuses décennies, la faune phlébotomienne (Diptera, Psychodidae, Phlebotominae) Malgaches est demeurée très peu explorée. Deux Grassomyia avaient été signalés puis une espèce décrite sous le nom de Sergentomyia berentiensis. A partir des années 2000, la faune de Madagascar a révélé une richesse et une diversité non soupçonnées jusqu'alors avec la description de plusieurs espèces nouvelles et d'un sous-genre nouveau : Vattieromyia. Cette thèse est une contribution à la connaissance des Phlébotomes de Madagascar et des îles voisines des Seychelles et des Comores.Notre approche a été qualitative et non quantitative. Les phlébotomes collectés à Madagascar, aux Comores et aux Seychelles ont été étudiés morphologiquement puis, pour certains d'entre eux, par biologie moléculaire à diverses fins : associations mâles-femelles et systématique évolutive. Dans ce dernier cas, différents marqueurs ribosomiques, mitochondriaux et nucléaires ont été séquencés selon les problématiques.A Madagascar, les Phlebotomus forment un groupe monophylétique. Nous suggérons, sur des arguments morphologiques et moléculaires, de les individualiser dans un sous-genre nouveau étant donnée la mise en évidence de la paraphylie du sous-genre Anaphlebotomus dans lequel ont été classées les espèces malgaches.Nos travaux révèlent que P. fertei possède une aire de distribution qui couvre la presque totalité du pays. Les séquences de cytochrome b individualisent de nombreuses populations selon leurs origines géographiques mais nous n'avons pas pu individualiser ces populations sur le plan morphologique et morphométrique. Les séquences de l'ITS2 n'individualisent pas ces populations et nous critiquons l'utilisation du cytochrome b, et plus largement des marqueurs mitochondriaux, pour la systématique des Phlébotomes.En ce qui concerne les autres espèces de Phlebotomus, elles possèdent toutes une distribution étroite, réduite à leur lieu de capture. Nous avons décrit deux espèces nouvelles durant cette thèse : P. vaomalalae et P. vincenti. Les études moléculaires et morphologiques révèlent l'existence d'au moins trois espèces nouvelles : deux sympatriques à Andranoilovy (dont une espèce commune avec Berenty) et une à Ankililaoka.Enfin, nous proposons le rattachement de P. huberti au genre Sergentomyia. Cette espèce ne possède pas de soies mésanepisternales et le mâle que nous décrivons dans ce travail possède les caractères génitaux des Sergentomyia. De plus, nous décrivons sur une la seule femelle, une espèce nouvelle proche de S. huberti. Une étude moléculaire menée avec d'autres espèces supposées proches (appartenant au sous-genre Sintonius) nous conduit à proposer la création d'un nouveau sous-genre pour classer ces espèces malgaches.Nous analysons la paléobiogéographie des Phlébotomes de Madagascar et envisageons au moins deux épisodes de peuplement : l'un très ancien (environ 120 millions d'années), « africain » datant de la fragmentation du Gondwana et le second, plus récent (65 millions d'années), provenant d'Asie via un pont formé par le plateau des Seychelles.D'un point de vue épidémiologique, la recherche d'ADN leishmanien s'est révélée négative sur tous les phlébotomes testés.Dans l'archipel des Comores, aucun phlébotome n'avait été rapporté. Au cours de trois campagnes de piégeage menées en 2003, 2007 et 2011, nous rapportons la première mention de phlébotomes dans ces îles et décrivons deux taxons nouveaux S. pessoni et S. goodmani comorensis.Aux Seychelles, nous avons identifié S. clydei à Aldabra. Cette population possède des séquences mitochondriales très différentes des nombreuses populations continentales étudiées. L'origine du peuplement de cette île volcanique demeure mystérieuse, sans adéquation avec les données relatives à l'horloge moléculaire du cytochrome b dont nous doutons de la fiabilité.

Background: Phlebotomine sand flies (Diptera: Psychodidae) are tiny, blood-sucking insects that are of significant public and veterinary health importance for their role in the transmission of Leishmania parasites, bacteria, and arboviruses. Although sand flies have been documented in most Southeast Asian countries, there are no published records confirming their presence in Singapore. Here, we present this fauna with descriptions of new species. Methods: Sand fly species identification was confirmed using an integrative taxonomic approach that combines morphological analysis with DNA barcoding of the mitochondrial cytochrome b (cytb) and cytochrome c oxidase subunit I (COI) genes. Results: We identified eight sand fly species, including four newly described species: Phlebotomus seowpohi n. sp., Sergentomyia leechingae n. sp., Sergentomyia gubleri n. sp., and Sergentomyia retrocalcarae n. sp. Phylogenetic analyses suggest that the new Phlebotomus species, belonging to subgenus Euphlebotomus, is closely related to Phlebotomus argentipes, an important vector of Leishmania donovani from the South Asian region. Conclusions: The potential risk of leishmaniasis in Singapore is compounded by the recent detection of antibodies to Leishmania infantum in local free-roaming dogs. Therefore, continuous monitoring is essential to assess and manage the risk of disease agent transmission, support the development of an early warning system, and enable timely and targeted public health interventions. The findings of this study contribute to the global knowledge on sand flies and enhance our understanding of local fauna diversity and distribution. Graphical

Background: Checklists of zoological groups are useful to document species names in a specific area or even worldwide. They serve for various purposes, including ecological studies, conservation reports, policy and decision-making, and species identification. Phlebotomine sand flies (Diptera, Psychodidae) are the vectors of pathogens such as Leishmania, Bartonella and some arboviruses (Toscana, Naples, Sicily), and checklists for sand flies have primarily been published mainly for a state, department, or country. A checklist for American sand flies was published in 2017, but, until then, no effort has been made to compile a comprehensive list of species worldwide. The present study aims to fill this gap of knowledge. Methods: The present checklist is provided based on a literature overview and biological collections records and includes unpublished data from the authors. The species are presented according to the classification, then alphabetically by Eastern and Western Hemispheres. Distribution by country and type locality of each species are provided. Discussions on the taxonomic status or occurrence of each species are provided when needed. Results: A total of 23 genera in the Western Hemisphere, formerly the New World (Bichromomyia, Brumptomyia, Deanemyia, Dampfomyia, Edentomyia, Evandromyia, Expapillata, Hertigia, Lutzomyia, Martinsmyia, Micropygomyia, Migonemyia, Nyssomyia, Oligodontomyia, Pintomyia, Pressatia, Psathyromyia, Psychodopygus, Sciopemyia, Trichophoromyia, Trichopygomyia, Viannamyia, and Warileya) and 17 genera in the Eastern Hemisphere, formerly the Old World (Australophlebotomus, Chinius, Demeillonius, Grassomyia, Idiophlebotomus, Libanophlebotomus, Libanophlebotomites, Mesophlebotomites, Paleomyia, Parvidens, Phlebotoiella, Phlebotomiella, Phlebotomites, Phlebotomus, Sergentomyia, Spelaeomyia, Spelaeophlebotomus), including the fossil species, are listed herein. The updated list, excluding nomina dubia/species inquirenda includes 1063 sand fly species, with 549 and 514 species for New World and Old World, respectively. Only New Zealand and the Pacific Islands (excluding New Caledonia) do not record the presence of sand flies. The dataset for this study is publicly available in the SiBBr and GBIF. Conclusions: This is the first detailed list of valid names of phlebotomine sand flies worldwide, including records from each country where they have been documented. Graphical

Phlebotomine sand flies (Diptera: Psychodidae) are small, blood-sucking insects that are of significant public and veterinary health importance for their role in the transmission of Leishmania parasites, bacteria and arboviruses. Although sand flies have been documented in most Southeast Asian countries, there are no published records confirming their presence in Singapore. Here, we provide the first documented evidence on the presence of sand flies in Singapore. Using an integrated taxonomic approach that combines morphological analysis with DNA barcoding of the mitochondrial cytochrome b ( cytb ) and cytochrome c oxidase subunit I ( COI ) genes, we identified eight sand fly species, including four newly described species – Phlebotomus seowpohi n. sp., Sergentomyia leechingae n. sp., Sergentomyia gubleri n. sp., and Sergentomyia retrocalcarae n. sp.. Phylogenetic analyses suggest that the new Phlebotomus species, belonging to subgenus Euphlebotomus , is closely related to Phlebotomus argentipes , an important vector of Leishmania donovani from the South Asian region. The discovery of phlebotomine sand flies in Singapore underscores the importance of biosurveillance of biting arthropods. Given Singapore’s status as a major travel hub, there is a potential risk of leishmaniasis being introduced either by residents returning, or visitors arriving from endemic regions. This risk is compounded by the recent detection of local canine leishmaniasis. Thus, continuous monitoring is essential to assess and manage the risk of disease transmission, support the development of an early warning system, and enable timely and targeted public health interventions. The findings from this study contributes to the global knowledge on sand fly and enhance our understanding of local sand fly diversity and distribution. Author Summary: Phlebotomine sand flies are small, blood-sucking insects recognized as significant vectors for various diseases including leishmaniasis. This study provides the first evidence of their presence in Singapore. Using an integrated taxonomic approach combining morphological analysis with DNA barcoding, we identified eight sand fly species that include four new species: Phlebotomus seowpohi n. sp., Sergentomyia leechingae n. sp., Sergentomyia gubleri n. sp., and Sergentomyia retrocalcarae n. sp. The presence of sand flies in Singapore highlights an important biosurveillance need, as the region’s status as a major travel hub increases the risk of leishmaniasis being introduced. Importantly, recent findings of canine leishmaniasis in local dogs amplify the threat of disease transmission. Continuous monitoring and understanding of sand fly diversity and ecology are essential for developing effective public health strategies and early warning systems to manage this risk.

Phlebotomus perniciosus is the most important vector of Leishmania infantum in the Western part of the Mediterranean basin. Atypical specimens of Ph. perniciosus called (pna) with a parameral sheath simply curved, not bifurcated, have been reported in many locations. In this study, we describe abnormal Ph. perniciosus male specimens. Sand flies were collected in center Tunisia and identified morphologically. Cytochrome b PCR-sequencing was carried out for abnormal Ph. perniciosus male specimens in order to confirm the morphological identification and assess the intraspecific genetic polymorphism. Abnormal Ph. perniciosus specimens were characterized by a multifurcated parameral sheath. A parsimonious haplotype network based on cyt b locus analysis showed that typical and abnormal Ph. perniciosus described in our investigation were grouped together in the same branch. Thus, genetic outcomes confirmed that the new phenotype is only an original morphotype of Ph. perniciosus.

Leishmania infantum is the agent of visceral leishmaniasis in the Mediterranean basin. It is transmitted by sandflies of the subgenus Larroussius. Although Phlebotomus perniciosus is the most important vector in this area, an atypical Ph. perniciosus easily confused with Ph. longicuspis has been observed in North Africa. MALDI-TOF MS, an important tool for vector identification, has recently been applied for the identification of sandflies. Spectral databases presented in the literature, however, include only a limited number of Larroussius species. Our objective was to create an in-house database to identify Mediterranean sandflies and to evaluate the ability of MALDI-TOF MS to discriminate close species or atypical forms within the Larroussius subgenus. Field-caught specimens (n = 94) were identified morphologically as typical Ph. perniciosus (PN; n = 55), atypical Ph. perniciosus (PNA; n = 9), Ph. longicuspis (n = 9), Ph. ariasi (n = 9), Ph. mascittii (n = 3), Ph. neglectus (n = 5), Ph. perfiliewi (n = 1), Ph. similis (n = 9) and Ph. papatasi (n = 2). Identifications were confirmed by sequencing of the mtDNA CytB region and sixteen specimens were included in the in-house database. Blind assessment on 73 specimens (representing 1073 good quality spectra) showed a good agreement (98.5%) between MALDI-TOF MS and molecular identification. Discrepancies concerned confusions between Ph. perfiliewi and Ph. perniciosus. Hierarchical clustering did not allow classification of PN and PNA. The use of machine learning, however, allowed discernment between PN and PNA and between the lcus and lcx haplotypes of Ph. longicuspis (accuracy: 0.8938 with partial-least-square regression and random forest models). MALDI-TOF MS is a promising tool for the rapid and accurate identification of field-caught sandflies. The use of machine learning could allow to discriminate similar species.

The genus Uranotaenia (Diptera: Culicidae) has been well documented in Madagascar where it includes 73 species, 89.4% being endemic. However, one problem is that most species are morphologically similar in the adult stage. Here, 713 Uranotaenia specimens collected in the tropical forests of Anorana and Maromizaha between 2008 and 2014 were examined. Using the dichotomous keys for the Uranotaenia fauna of Madagascar published in 2004, three species were identified: Uranotaenia neireti (220), Ur. alboabdominalis (110) and Ur. mayottensis (28). The other specimens (355) were not identifiable and were classified as Uranotaenia sp1. Using wing morphometry, the four taxa were classified into four morphogroups. Within the Uranotaenia sp1 group, specimens from the Anorana forest and those from the Maromizaha forest overlapped. This result suggests that wing morphometric traits could be a good marker to distinguish Uranotaenia species in Madagascar.

A few data are related to the anthropophily of Malagasy Phlebotomine sandflies. Prior studies focussed mainly to inventories and description of new species. Our goal was to emphasize the anthropophily of Malagasy Phlebotomine sandflies. We worked in the Makira region, using two simultaneous methods: human landing catches (HLC) and CDC light traps. We collected sandflies in three rural communities adjacent to the Makira Natural Park. In each community, three different biotopes were sampled: within community settlements; at the edge of forest, typically in agricultural land; and within the forest. We collected 61 sandflies belonging to two new species presently described: Phlebotomus artemievi sp. nov. and Sergentomyia maroantsetraensis sp. nov. These sandflies were caught exclusively in the forest edge biotope. None were captured within communities or within forests. HLC provided 97% of the collected sandflies, corresponding to a human-biting rate of 15 females per human per night. CDC provided only two females. Ph. artemievi sp. nov. was predominantly captured by HLC and appears to be highly anthropophilic. Here, we update the behavioural ecology of sandflies and describe two new species. Further research is required to understand their vector competence and their ability to transmit arboviruses and other pathogens such as Leishmania.