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Phlebotomus argentipes sensu lato Annandale & Brunetti, 1908 is the primary vector of Leishmania donovani MON 37, the causative agent of cutaneous leishmaniasis (CL) in Sri Lanka. Effective vector control is essential for managing leishmaniasis. Although numerous taxonomic studies have been conducted on P. argentipes s.l., population genetics remains insufficiently explored. This study investigated the demographic history and population genetic structure of P. argentipes s.l. in Sri Lanka using sequence data obtained from the earlier investigation of two mitochondrial markers, Cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4). For the genetic analysis, 159 individuals from five leishmaniasis endemic sites were examined. In addition to the individual analyses of COI and ND4 genes, a concatenated dataset combining both mitochondrial fragments was constructed to evaluate overall genetic structure and demographic history. The population structure and demographic history of P. argentipes s.l. were assessed using FST estimates, AMOVA, structure analysis, Mantel test, PCoA, Bayesian inference and coalescent analysis. The highest FST value was 0.0271, indicating low genetic differentiation, with over 98% variation occurring within populations. Mantel tests showed weak, non-significant correlations between genetic and geographic distance, indicating no evidence of isolation by distance, suggesting potential gene flow and no distinct clustering within the Sri Lankan P. argentipes s.l. population. Negative and significant neutrality statistics, together with unimodal mismatch distributions, support historical population expansion, further corroborated by Bayesian skyline plots indicating two distinct demographic events, an ancient expansion around 50,000 years ago (COI) and a more recent one approximately 10,000-13,000 years ago (ND4). Additionally, the mismatch distribution analyses revealed a multimodal expansion pattern at the Medirigiriya and Hambantota sites, which are hot spots for leishmaniasis in Sri Lanka. The present study demonstrates a demographic expansion and genetic homogeneity of P. argentipes s.l. populations in Sri Lanka, supporting the species' ability to colonize new areas and possibly enhance leishmaniasis transmission. This connectivity may facilitate the spread of adaptive traits such as insecticide resistance, even in the absence of local selection pressure, posing a potential challenge for future vector control efforts in Sri Lanka.

Sand flies transmit Leishmania infantum, which is responsible for causing leishmaniasis, as well as many phleboviruses, including the human pathogenic Toscana virus. We screened sand flies collected from a single site between 2017 and 2020 for the presence of both phleboviruses and Leishmania. The sand flies were sampled with attractive carbon dioxide traps and CDC light traps between May and October. We collected more than 50,000 sand flies; 2826 were identified at the species level as Phlebotomus perfiliewi (98%) or Phlebotomus perniciosus (2%). A total of 16,789 sand flies were tested in 355 pools, and phleboviruses were found in 61 pools (6 Toscana virus positive pools, 2 Corfou virus positive pools, 42 Fermo virus positive pools, and 7 Ponticelli virus positive pools, and 4 unidentified phlebovirus positive pools). Leishmania was found in 75 pools and both microorganisms were detected in 16 pools. We isolated nine phleboviruses from another 2960 sand flies (five Ponticelli viruses and for Fermo viruses), not tested for Leishmania; the complete genome of a Fermo virus isolate was sequenced. The simultaneous detection in space and time of the Fermo virus and L. infantum is evidence that supports the co-circulation of both microorganisms in the same location and partial overlap of their cycles. A detailed characterization of the epidemiology of these microorganisms will support measures to limit their transmission.

Background Leishmaniasis is a group of neglected vector-borne diseases transmitted by phlebotomine sand flies. Leishmania parasites must overcome various defenses in the sand fly midgut, including the insects’s immune response. Insect immunity is regulated by the ecdysone hormone, which binds to its nuclear receptor (EcR) and activates the transcription of genes involved in insect immunity. However, the role of ecdysone in sand fly immunity has never been studied. Phlebotomus perniciosus is a natural vector of Leishmania infantum ; here, we manipulated its neuroendocrine system using azadirachtin (Aza), a natural compound known to affect ecdysone synthesis. Methods Phlebotomus perniciosus larvae and adult females were fed on food containing either Aza alone or Aza plus ecdysone, and the effects on mortality and ecdysis were evaluated. Genes related to ecdysone signaling and immunity were identified in P. perniciosus , and the expression of antimicrobial peptides (AMPs), EcR , the ecdysone-induced genes Eip74EF and Eip75B , and the transcription factor serpent were analyzed using quantitative polymerase chain reaction (PCR). Results Aza treatment inhibited molting of first-instar (L1) larvae to L2, with only 10% of larvae molting compared to 95% in the control group. Serpent and Eip74EF , attacin, defensin 1, and defensin 2 genes were downregulated by Aza treatment in larvae. Similarly, Aza-treated adult females also presented suppression of ecdysone signaling-related genes and the AMPs attacin and defensin 2. Notably, all gene repression caused by Aza was reversed by adding ecdysone concomitantly with Aza to the larval or female food, indicating that these genes are effective markers for ecdysone repression. Conclusions These results highlight the critical role of ecdysone in regulating the development and immunity of P. perniciosus , which potentially could interfere with Leishmania infection. Graphical Abstract

Knockdown resistance alleles ( kdr alleles) within the para voltage-gated sodium channel gene ( Vgsc ) are a common mechanism of DDT and pyrethroid resistance in insect vectors. In the primary Asian visceral leishmaniasis vector, Phlebotomus argentipes , two kdr alleles in codon 1014 of the Vgsc are associated with insecticide resistance, potentially presenting challenges to vector control efforts in the Indian subcontinent. Here, we screened Vgsc -1014 alleles and blood meal origin in P. argentipes females collected between September 2013 and August 2015 in Bangladesh (Mymensingh), to understand how Vgsc -1014 alleles could impact feeding patterns. The sand fly collection took place in parallel with the vector control agency’s biannual indoor residual spraying (IRS) programme. In this region, the wild-type leucine ( wt -leucine) was the most common allele (66.7%), followed by the mutant serine (19.4%) and phenylalanine alleles (13.9%). Only 55 sand fly blood meals (13%) came from humans, with most of bovine origin (61%). However, sand flies that had fed on humans showed strongly contrasting Vgsc -1014 genotypic frequencies compared to those feeding on other blood sources. Whilst most (81%) P. argentipes with human blood possessed kdr genotypes with two mutant alleles, most (81%) sand flies feeding on other blood sources possessed genotypes with wt -leucine alleles ( P < 0.001). Significant spatial variation in kdr frequencies was detected, but there was no clear temporal trend nor effect of sampling year on any results, and no significant impact of recent IRS in any analyses. The association between human feeding and kdr alleles in parallel with pyrethroid spraying indicates a new mechanism of how kdr alleles might impact VL control programs.

Background Recent climate and environmental changes have resulted in the geographical expansion of Mediterranean Leishmania infantum vectors towards northern latitudes and higher altitudes in different European countries, including Italy, where new foci of canine leishmaniasis have been observed in the northern part of the country. Northern Italy is also an endemic area for mosquito-borne diseases. During entomological surveillance for West Nile virus, mosquitoes and other hematophagous insects were collected, including Phlebotomine sand flies. In this study, we report the results of Phlebotomine sand fly identification during the entomological surveillance conducted from 2017 to 2019. Methods The northeastern plain of Italy was divided by a grid with a length of 15 km, and a CO 2 -CDC trap was placed in each geographical unit. The traps were placed ~ 15 km apart. For each sampling site, geographical coordinates were recorded. The traps were operated every two weeks, from May to November. Sand flies collected by CO 2 -CDC traps were identified by morphological and molecular analysis. Results From 2017 to 2019, a total of 303 sand flies belonging to the species Phlebotomus perniciosus ( n  = 273), Sergentomyia minuta ( n  = 5), P. mascittii ( n  = 2) and P. perfiliewi ( n  = 2) were collected, along with 21 unidentified specimens. The trend for P. perniciosus collected during the entomological surveillance showed two peaks, one in July and a smaller one in September. Sand flies were collected at different altitudes, from −2 m above sea level (a.s.l.) to 145 m a.s.l. No correlation was observed between altitude and sand fly abundance. Conclusions Four Phlebotomine sand fly species are reported for the first time from the northeastern plain of Italy. Except for S. minuta , the sand fly species are competent vectors of Leishmania parasites and other arboviruses in the Mediterranean Basin. These findings demonstrate the ability of sand flies to colonize new environments previously considered unsuitable for these insects. Even though the density of the Phlebotomine sand fly population in the plain areas is consistently lower than that observed in hilly and low mountainous areas, the presence of these vectors could herald the onset of epidemic outbreaks of leishmaniasis and other arthropod-borne diseases in areas previously considered non-endemic. Graphical Abstract

Background Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of various pathogens, mainly Leishmania parasites. In the Old World, the most important genus in term of pathogens transmission is the genus Phlebotomus , which includes many proven or suspected vectors of several Leishmania species, while the genus Sergentomyia remains so far unproven as a vector of human pathogens. Algeria is one of the most affected countries by human leishmaniasis. Methods In the present study, an entomological survey was carried out in two provinces, Ghardaïa and Illizi, located in the north and central Sahara, respectively, where cases of human leishmaniasis are recorded. Our goal was to understand the role of the local sand fly species in the transmission of Leishmania parasites and to analyze their blood meal preferences. Collected sand flies were identified by a combination of morphological and molecular approaches that included DNA-barcoding and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling. In addition, female blood meals were analyzed by peptide mass mapping using MALDI-TOF MS. Results In total, 640 sand fly specimens belonging to Phlebotomus and Sergentomyia genera were collected in the two provinces. Sergentomyia antennata and Se. fallax were most abundant species in Ghardaïa, and Ph. papatasi and Ph. alexandri in Illizi. In addition, a new sand fly species was described in Illizi named Sergentomyia ( Sergentomyia ) imihra n. sp. Blood meal analysis of the engorged females revealed various mammalian hosts, especially goats, but also humans for Phlebotomus papatasi and Ph. alexandri , suggesting that these vector species are opportunistic feeders. Conclusions Integrative approach that combined morphological analysis, sequencing of DNA markers, and protein profiling enabled the recognition and description of a new Sergentomyia species, raising the number of the Algerian sand fly fauna to 27 species. Further sand fly surveillance in the central Sahara is recommended to identify the thus-far unknown males of Se. imihra n. sp. Graphical Abstract

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. Graphical Abstract

Background Phlebotomine sand flies (Diptera: Psychodidae) are haematophagous insects that transmit the protozoan parasite Leishmania infantum (Kinetoplastida: Trypanosomatidae), the main causative agent of both zoonotic visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in the Mediterranean basin. Eight species of sand flies have been previously recorded in Romania: Phlebotomus papatasi , Phlebotomus alexandri , Phlebotomus sergenti , Phlebotomus perfiliewi , Phlebotomus neglectus , Phlebotomus longiductus , Phlebotomus balcanicus and Sergentomyia minuta. Three of them ( P. perfiliewi , P. neglectus and P. balcanicus) were incriminated as vectors of L. infantum. Recent reports of autochthonous CanL in Romania require updates on sand fly distribution and diversity in this country. Methods Between 2013–2014 and 2016–2018, CDC light traps and mouth aspirators were used to collect sand flies in 132 locations from Romania, indoors and around various animal species shelters. Species identification of collected specimens was done using morphological keys, genetic tools and MALDI-TOF protein profiling. Results Sand flies were present in seven localities (5.3%): Eibenthal, Baia Nouă, Gura Văii (south-western Romania, Mehedinţi County); Fundătura, Pâhneşti, Epureni (eastern Romania, Vaslui County); and Schitu (southern Romania, Giurgiu County). Of the total number of collected sand flies ( n  = 251), 209 (83.27%) were Phlebotomus neglectus , 39 (15.53%) P. perfiliewi , 1 (0.40%) P. papatasi , 1 (0.40%) P. balcanicus and 1 (0.40%) P. sergenti (sensu lato). Conclusions We confirmed the presence of five sand fly species previously recorded in Romania. However, their updated distribution differs from historical data. The diversity of sand fly species in Romania and their presence in areas with Mediterranean climatic influences constitutes a threat for the reemergence of vector-borne diseases. In the context of CanL and VL reemergence in Romania, but also due to imported cases of the diseases in both humans and dogs, updates on vector distribution are imperative.

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 Abstract

Nepal is committed to eliminating visceral leishmaniasis as a public health problem by 2030. The scattered distribution of VL cases across wide geo-ecological regions, including areas previously considered unsuitable for the survival of vectors and the transmission of the pathogen, poses a major threat to Nepal’s national VL elimination programme. Regular monitoring and accurate identification of sand fly species are essential for implementing tailored vector control interventions. Hence, this study aimed to update the distribution of sand fly species with a focus on Leishmania vectors and evaluate DNA barcoding as a complementary tool for their identification. Sand flies were collected from 43 districts with active VL cases across the country between 2017 and 2022. The mitochondrial COI gene was amplified for DNA barcoding analysis. The primary vector, Phlebotomus argentipes , was present in all except three districts. Potential vectors, Ph. ( Adlerius ) spp. and Ph. major , were found common in high-altitude regions. The species identification success rate of generated COI barcode sequences based on the “Best Close Match” was 97%, indicating high accuracy in delineating sand flies to the species level. The information on the distribution of phlebotomine sand flies and the potential use of DNA barcoding for their identification could be milestones for sand fly research and help to guide the vector control interventions in support of VL elimination in Nepal.