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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. [ABSTRACT FROM AUTHOR]

Background: Sand flies, belonging to the Psychodidae family, represent small, hairy insects that serve as significant vectors in various important medical and veterinary diseases. Despite being recognized by the World Health Organization as an endemic area for leishmaniasis, Southeast Asia lacks comprehensive information on the species composition and biology of sand flies. To address this, the current study aimed to survey sand fly biodiversity. Methods: Sand flies from six provinces in Southern Vietnam were collected using CDC light traps. Sand flies were subsequently identified morphologically and confirmed molecularly using mitochondrial cytochrome oxidase c subunit I (COI) and cytochrome b (cytb) sequences. BLASTN searches were conducted, and the species identity of sand flies was further confirmed through a Barcode of Life Database (BOLD) search utilizing COI sequences. Subsequently, nucleotide sequences were subjected to a panel of analyses including intraspecific variation, phylogenetic relationships and haplotype network. The average densities of collected sand flies (sand flies/trap/night) and species richness were also recorded. Results: A total of 753 sand flies were collected. After excluding damaged specimens, six sand fly species, namely Phlebotomus stantoni, Sergentomyia khawi, Se. silvatica, Se. barraudi, Se. bailyi and Grassomyia indica, were identified. All conspecific sand fly sequences, including Ph. stantoni, Se. barraudi, Gr. indica, Se. bailyi, Se. khawi and Se. silvatica, clustered with their reference sequences, corroborating the results of morphology-based identification, BLASTN analysis and BOLD search. For intraspecific variation of sand flies obtained from the current study, COI diversity indices were consistently higher than those of cytb. Conclusions: This study provides the first updates on morphological and molecular characterization of sand flies in Southern Vietnam. This acquired knowledge on sand fly species composition is essential for controlling sand fly-borne diseases in this potentially endemic region. [ABSTRACT FROM AUTHOR]

Phlebotomus argentipes is the established vector of leishmaniasis in the Indian sub-continent. Antibodies to sand fly salivary antigens are biomarkers for vector-host exposure in leishmaniasis-endemic regions. Ph. argentipes transmits Leishmania donovani in Sri Lanka, primarily causing cutaneous leishmaniasis (CL). Our study compared the performance of salivary gland homogenate (SGH) from a lab-reared local strain of Ph. argentipes females to a composite recombinant salivary biomarker (rPagSP02 + rPagSP06) in a CL-endemic population. Sera from 546 healthy individuals, 30 CL patients, and 15 non-endemic individuals were collected. Western blot analysis of Ph. argentipes SGH identified immunogenic bands between 15 kDa and 67 kDa, with bands of predicted molecular weight ∼of 15 kDa (SP02) and ∼28–30 kDa (SP06) as the major antibody targets. Indirect ELISAs using SGH or rPagSP02 + rPagSP06 antigens showed high sensitivity (96.7%) and specificity (100%), detecting comparable seropositivity in endemic populations. rPagSP02 + rPagSP06 exhibited enhanced discriminatory ability, supported by a strong positive correlation (r = 0.869) with SGH. Our findings indicate that the composite rPagSP02 + rPagSP06 salivary biomarker effectively identifies Ph. argentipes exposure in individuals living in Sri Lanka, showing promising potential for use in surveillance. These findings should be further validated to confirm the epidemiological applications in leishmaniasis-endemic regions. [ABSTRACT FROM AUTHOR]

Background: Several new species of Leishmania have recently emerged in Europe, probably as the result of global changes and increased human migration from endemic areas. In this study, we tested whether two sand fly species, the Western Mediterranean Phlebotomus perniciosus and the Eastern Mediterranean P. tobbi, are competent vectors of L. donovani, L. major and L. martiniquensis. Methodology/principal findings: Sand flies were infected through the chick skin membrane using Leishmania species and strains of various geographical origins. Leishmania infections were evaluated by light microscopy and qPCR, and the representation of morphological forms was assessed from Giemsa-stained gut smears. Neither P. perniciosus nor P. tobbi supported the development of L. martiniquensis, but L. major and L. donovani in both species survived defecation of blood meal remnants, colonized the stomodeal valve and produced metacyclic stages. The results with L donovani have shown that infection rates in sand flies can be strain-specific; therefore, to determine vector competence or refractoriness, it is optimal to test at least two strains of Leishmania. Conclusions, significance: Both sand fly species tested are potential vectors of L. donovani and L. major in Mediterranean area. However, further studies will be needed to identify European vectors of L. martiniquensis and to test the ability of other European sand fly species to transmit L. major, L. donovani, L. tropica and L. infantum. Author summary: Leishmaniases are serious human and animal diseases caused by protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae), transmitted by blood-feeding insects–phlebotomine sand flies (Diptera: Psychodidae). They are mainly distributed in tropical and subtropical regions, but recently the ranges of the vectors have spread more into temperate regions. In Europe, species of Leishmania that were previously absent have emerged, mainly due to the mass migration of the refugees from endemic areas. It is therefore important to know whether local sand flies can transmit these "new" Leishmania species. In our study, we tested two widely distributed sand fly species, P. perniciosus and P. tobbi. In both of them, L. major and L. donovani completed their development, thus P. perniciosus and P. tobbi are potential vectors of these parasites. The third Leishmania species tested, L. martiniquensis, which has been repeatedly recorded in central Europe, did not survive in P. perniciosus and P. tobbi; its vectors have yet to be confirmed. [ABSTRACT FROM AUTHOR]

We investigated gene expression patterns in Lutzomyia and Phlebotomus sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed Lutzomyia longipalpis and Phlebotomus papatasi samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and Leishmania infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in L. longipalpis larvae and RiboL32 in adults. In P. papatasi, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for L. longipalpis or P. papatasi were effective in PCR with Lutzomyia migonei, Phlebotomus duboscqi, Phlebotomus perniciosus, and Sergentomyia schwetzi cDNA. Furthermore, L. longipalpis RiboL32 and P. papatasi α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR. [ABSTRACT FROM AUTHOR]

The state of Himachal Pradesh is the most endemic region for cutaneous leishmaniasis transmission in northern India. Phlebotomus adlerius longiductus and Phlebotomus Larrousisus major have a wide distribution in Himachal Pradesh (India) and mostly found in mountainous areas. There is very little data available on the identification and distribution of sand flies in Himachal Pradesh. Therefore, the present study was carried out with the aim of identifying and distinguishing the sand flies species found in endemic focus of CL in Himachal Pradesh (HP), India, using PCR and sequencing methods. Collections of sand flies were made from endemic villages of cutaneous leishmaniasis in Shimla, Kullu and Kinnaur districts of HP during 2017–2019. A total of 291 sand flies were collected and all the collected sandflies were identified morphologically. In addition, genus specific identification of 52 sand flies was carried by Polymerase Chain Reaction (PCR). The PCR amplified products were sequenced for confirmation of sand-fly species. The sequences obtained were submitted to NCBI GenBank under accession no. MT126505 to MT126510 and compared for similarities to the NCBI database. The morphological identification revealed Phlebotomus adlerius longiductus and Phlebotomus Larrousisus major. Of 291 sand flies,>99% were Phlebotomus adlerius longiductus with Phlebotomus Larrousisus major in low numbers. The sequences obtained were compared for similarities to NCBI database and showed 99–100% similarity with Phlebotomus longiductus (Parrot). The phylogenetic tree analysis showed that the isolates were geographically closest to the sand flies from Bhutan and China. The study revealed that the P. longuductus (Parrot) is the predominant species found in the CL endemic area in HP. The study confirms the identity of P. longiductus (Parrot) through molecular tools as well as morphologically. As, P. longiductus (Parrot) has been reported to transmit L.donovani, it is important to distinguish the sand-fly species using molecular tool. [ABSTRACT FROM AUTHOR]

Background: Phlebotomus papatasi is considered the primary vector of Leishmania major parasites that cause zoonotic cutaneous leishmaniasis (ZCL) in the Middle East and North Africa. Phlebotomus papatasi populations have been studied extensively, revealing the existence of different genetic populations and subpopulations over its large distribution range. Genetic diversity and population structure analysis using transcriptome microsatellite markers is important to uncover the vector distribution dynamics, essential for controlling ZCL in endemic areas. Methods: In this study, we investigated the level of genetic variation using expressed sequence tag-derived simple sequence repeats (EST-SSRs) among field and colony P. papatasi samples collected from 25 different locations in 11 countries. A total of 302 P. papatasi sand fly individuals were analyzed, including at least 10 flies from each region. Results: The analysis revealed a high-level population structure expressed by five distinct populations A through E, with moderate genetic differentiation among all populations. These genetic differences in expressed genes may enable P. papatasi to adapt to different environmental conditions along its distribution range and likely affect dispersal. Conclusions: Elucidating the population structuring of P. papatasi is essential to L. major containment efforts in endemic countries. Moreover, the level of genetic variation among these populations may improve our understanding of Leishmania–sand fly interactions and contribute to the efforts of vaccine development based on P. papatasi salivary proteins. [ABSTRACT FROM AUTHOR]

Background: Leishmania infantum is endemic in Europe (and elsewhere) while L. donovani s.s., L. tropica and L. major are not but are present in neighboring countries in North Africa, the Middle East, (the Asian part of) Turkey and the Southern Caucasus. Lists of sand fly vector species in the scientific literature vary with the criteria for vector incrimination, and criteria vary because, for some, evidence is difficult to generate. With minimal criteria, about 20 sand fly species are proven or suspected vectors of L. infantum in Europe and neighboring countries, while for L. tropica and L. major, there are seven and four proven or suspected vector species, respectively, in this area. For L. donovani s.s., present in Cyprus, the Middle East and (the Asian part of) Turkey, no local vectors have been incriminated so far. The aim was to assess the degree of spatial agreement between Leishmania spp. and various vectors species and their relative contribution to the explained variation. Methods: We used multivariate regression modeling to analyze the spatial relationship between autochthonous Leishmania spp. and clinical forms in humans and animals and 14 Phlebotomus spp. in Europe and neighboring countries. Results: There was only fair agreement between parasite and vector distributions. The most parsimonious models describing the distribution of Leishmania spp. and clinical forms included three to six sand fly species and explained between 12% (L. infantum) and 37% (L. donovani) of the observed variation. Selected models included confirmed and suspected vector species as well as unexpected species. Conclusions: The relatively low agreement between Leishmania and vector distributions highlights the need to improve leishmaniasis reporting and vector surveillance in areas where no information is available, both for a better understanding of the epidemiology of infection in endemic areas and to monitor possible spread of infection into non-endemic areas. While some of the unexpected sand fly-Leishmania spp. statistical associations might be spurious, for others, the existence of sporadic or recent reports of infections warrants further vector competence studies that consider strain variation. [ABSTRACT FROM AUTHOR]

Toscana virus (TOSV) is a leading cause of summer viral meningitis in Southern Europe (Central Italy, south of France, Spain and Portugal) and can cause severe neurological cases. Within the Mediterranean basin, it is transmitted by hematophagous sand flies belonging to the Phlebotomus genus. Despite the identification of the primary TOSV vectors, the viral developmental cycle in vector species remains largely unknown. Limited research has been conducted on transmission dynamics and the vector competence and vectorial capacity of the principal TOSV vector, Phlebotomus perniciosus. In this context, we investigated the intra-vector TOSV infection dynamics in Ph. perniciosus, as well as its impact on the vector life history traits. Female sand flies were experimentally infected with TOSV through an artificial blood meal. Systemic dissemination of the virus was observed approximately three days post-infection, potentially resulting in a short extrinsic incubation period. Moreover, the study revealed a longer hatching time for eggs laid by infected females. This research brought additional experimental insights regarding the vector competence of Ph. perniciosus but also provided the first insight into TOSV developmental cycle and its impact on the vector. These findings prompt further exploration of TOSV transmission dynamics, raise new hypotheses on the virus transmission and highlight the importance of follow-up studies. Author summary: Toscana virus (TOSV) is a reemerging sandfly-borne virus causing neuroinvasive infections in humans. This virus is endemic in the Mediterranean basin, with a potential risk of introduction in northern Europe and Asia. Despite decades of research, few studies have focused on the development cycle of TOSV in sand flies and the dynamics of transmission. Here, we provide a comprehensive study of the intra-vector dynamics of TOSV infection and its impact on both vector biology and consequently on transmission. Through experimental infections of the major vector Phlebotomus perniciosus, we not only brought additional experimental insights regarding vector competence but also provided the first insight into the TOSV developmental cycle in the vector by estimating the extrinsic incubation period at six days. Our study reveals an impact of TOSV infection on vector egg hatching time that could lead to a delayed emergence of infected sand flies, with a potential impact on transmission. Our findings encourage further exploration of transmission dynamics, raise new hypotheses on alternative transmission pathways, and emphasize the importance of follow-up studies. [ABSTRACT FROM AUTHOR]

Background: Western Ghats is a biodiversity treasure trove with reports of indigenous leishmaniasis cases. Hence, systematic sand fly surveillance was carried out among the tribal population. The present study reports a novel sand fly species, Phlebotomus (Anaphlebotomus) ajithii n. sp. (Diptera: Psychodidae), discovered in the Western Ghats of India. Methods: A comprehensive sand fly survey was conducted across the Kollam, Thrissur, Idukki, Kasaragod and Malappuram districts of Kerala, India. The survey spanned both indoor and outdoor habitats using standard collection methods over a 3-year, 3-month period. DNA barcoding of samples was performed targeting mitochondrial cytochrome c oxidase subunit I (COI) gene, and the sequence generated was subjected to phylogenetic analysis. Results: Phlebotomus (Anaphlebotomus) ajithii, a new sand fly species, is recorded and described in this communication. The morphological relationship of the new species to other members of the subgenus Anaphlebotomus is discussed. Mitochondrial COI barcode followed by phylogenetic analysis confirmed that specimens of Ph. ajithii belong to the same taxonomic group, while a genetic distance of 11.7% from congeners established it as a distinct species. Conclusions: The Western Ghats, known for its rich biodiversity, has lacked systematic entomological surveys focusing on sand flies. This study aims to fill this gap and reports and describes a new species of sand fly. [ABSTRACT FROM AUTHOR]

Background: Southeast Asia is well known as a hotspot of biodiversity. However, very little is known about cave-dwelling hematophagous insects that are medically important. Taxonomic knowledge and ecology of phlebotomine sand flies are very poorly studied in Laos, as well as in other countries in the region. Herein, we report species diversity data and some notes on the ecology of the detected species from these karstic limestone areas of Laos. Methods: Phlebotomine sand flies were collected using Centers for Disease Control and Prevention (CDC) light traps from limestone cave locations in three districts of Vientiane Province, Laos. Both morphological and molecular techniques were used for sand fly identification. Species diversity and abundance were analyzed according to sites, locations, collection seasons, and trapping positions. Results: A total of 6564 sand flies, of which 5038 were females and 1526 were males, were morphologically identified into 20 species belonging to five genera (Chinius, Idiophlebotomus, Phlebotomus, Sergentomyia, and Grassomyia). The most abundant species were Chinius eunicegalatiae, Phlebotomus stantoni, Sergentomyia hivernus, Se. siamensis, and Idiophlebotomus longiforceps. Cytochrome b analysis results supported the morphological identification and revealed that Se. siamensis was separated from other members of the Se. barraudi group. Two new species, Se. dvoraki n. sp. and Se. marolii n. sp., were described. Sand fly density was generally high except in a cave in Vangvieng, with species richness ranging from 14 to 18 across different caves. Outside caves had higher species richness (R = 20) and diversity (H = 2.50) than cave entrances (R = 18, H = 2.41) and interiors (R = 16, H = 2.13). Seasonal variations showed high sand fly density in Feung and Hinheup during both dry and rainy seasons, while Vangvieng had a notable decrease in density during the dry season (D = 6.29). Conclusions: This study revealed that the diversity of phlebotomine sand fly fauna in Laos, particularly in karstic limestone areas, is greater than previously known. However, the taxonomic status of many species in Laos, as well as Southeast Asia, still needs more in-depth study using both morphological characters and molecular methods. Many species could be found from inside, at the entrance, and outside of caves, indicating a wide range of host-seeking behavior or possible natural breeding in the karstic cave areas. [ABSTRACT FROM AUTHOR]

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

Abstract We investigated gene expression patterns in Lutzomyia and Phlebotomus sand fly vectors of leishmaniases. Using quantitative PCR, we assessed the expression stability of potential endogenous control genes commonly used in dipterans. We analyzed Lutzomyia longipalpis and Phlebotomus papatasi samples from L3 and L4 larval stages, adult sand flies of different sexes, diets, dsRNA injection, and Leishmania infection. Six genes were evaluated: actin, α-tubulin, GAPDH, 60 S ribosomal proteins L8 and L32 (RiboL8 and RiboL32), and elongation factor 1-α (EF1-α). EF1-α was among the most stably expressed along with RiboL8 in L. longipalpis larvae and RiboL32 in adults. In P. papatasi, EF1-α and RiboL32 were the top in larvae, while EF1-α and actin were the most stable in adults. RiboL8 and actin were the most stable genes in dissected tissues and infected guts. Additionally, five primer pairs designed for L. longipalpis or P. papatasi were effective in PCR with Lutzomyia migonei, Phlebotomus duboscqi, Phlebotomus perniciosus, and Sergentomyia schwetzi cDNA. Furthermore, L. longipalpis RiboL32 and P. papatasi α-tubulin primers were suitable for qPCR with cDNA from the other four species. Our research provides tools to enhance relative gene expression studies in sand flies, facilitating the selection of endogenous control for qPCR.

Abstract Background Leishmania infantum is endemic in Europe (and elsewhere) while L. donovani s.s., L. tropica and L. major are not but are present in neighboring countries in North Africa, the Middle East, (the Asian part of) Turkey and the Southern Caucasus. Lists of sand fly vector species in the scientific literature vary with the criteria for vector incrimination, and criteria vary because, for some, evidence is difficult to generate. With minimal criteria, about 20 sand fly species are proven or suspected vectors of L. infantum in Europe and neighboring countries, while for L. tropica and L. major, there are seven and four proven or suspected vector species, respectively, in this area. For L. donovani s.s., present in Cyprus, the Middle East and (the Asian part of) Turkey, no local vectors have been incriminated so far. The aim was to assess the degree of spatial agreement between Leishmania spp. and various vectors species and their relative contribution to the explained variation. Methods We used multivariate regression modeling to analyze the spatial relationship between autochthonous Leishmania spp. and clinical forms in humans and animals and 14 Phlebotomus spp. in Europe and neighboring countries. Results There was only fair agreement between parasite and vector distributions. The most parsimonious models describing the distribution of Leishmania spp. and clinical forms included three to six sand fly species and explained between 12% (L. infantum) and 37% (L. donovani) of the observed variation. Selected models included confirmed and suspected vector species as well as unexpected species. Conclusions The relatively low agreement between Leishmania and vector distributions highlights the need to improve leishmaniasis reporting and vector surveillance in areas where no information is available, both for a better understanding of the epidemiology of infection in endemic areas and to monitor possible spread of infection into non-endemic areas. While some of the unexpected sand fly-Leishmania spp. statistical associations might be spurious, for others, the existence of sporadic or recent reports of infections warrants further vector competence studies that consider strain variation. Graphical Abstract

Abstract Background Southeast Asia is well known as a hotspot of biodiversity. However, very little is known about cave-dwelling hematophagous insects that are medically important. Taxonomic knowledge and ecology of phlebotomine sand flies are very poorly studied in Laos, as well as in other countries in the region. Herein, we report species diversity data and some notes on the ecology of the detected species from these karstic limestone areas of Laos. Methods Phlebotomine sand flies were collected using Centers for Disease Control and Prevention (CDC) light traps from limestone cave locations in three districts of Vientiane Province, Laos. Both morphological and molecular techniques were used for sand fly identification. Species diversity and abundance were analyzed according to sites, locations, collection seasons, and trapping positions. Results A total of 6564 sand flies, of which 5038 were females and 1526 were males, were morphologically identified into 20 species belonging to five genera (Chinius, Idiophlebotomus, Phlebotomus, Sergentomyia, and Grassomyia). The most abundant species were Chinius eunicegalatiae, Phlebotomus stantoni, Sergentomyia hivernus, Se. siamensis, and Idiophlebotomus longiforceps. Cytochrome b analysis results supported the morphological identification and revealed that Se. siamensis was separated from other members of the Se. barraudi group. Two new species, Se. dvoraki n. sp. and Se. marolii n. sp., were described. Sand fly density was generally high except in a cave in Vangvieng, with species richness ranging from 14 to 18 across different caves. Outside caves had higher species richness (R = 20) and diversity (H = 2.50) than cave entrances (R = 18, H = 2.41) and interiors (R = 16, H = 2.13). Seasonal variations showed high sand fly density in Feung and Hinheup during both dry and rainy seasons, while Vangvieng had a notable decrease in density during the dry season (D = 6.29). Conclusions This study revealed that the diversity of phlebotomine sand fly fauna in Laos, particularly in karstic limestone areas, is greater than previously known. However, the taxonomic status of many species in Laos, as well as Southeast Asia, still needs more in-depth study using both morphological characters and molecular methods. Many species could be found from inside, at the entrance, and outside of caves, indicating a wide range of host-seeking behavior or possible natural breeding in the karstic cave areas. Graphical abstract

In this study, we identified and assembled a strain of American nodavirus (ANV) in the Phlebotomus papatasi‐derived PP9ad cell line. This strain most closely resembles Flock House virus and ANV identified in the Drosophila melanogaster S2/S2R cell line. Through small RNA sequencing and analysis, we demonstrate that ANV replication in PP9ad cells is primarily targeted by the exogenous small interfering RNA (exo‐siRNA) pathway, with minimal engagement from the PIWI‐interacting RNA (piRNA) pathway. In mosquitoes such as Aedes and Culex, the PIWI pathway is expanded and specialised, which actively limits virus replication. This is unlike in Drosophila spp., where the piRNA pathway does not restrict viral replication. In Lutzomyia sandflies (family Psychodidae), close relatives of Phlebotomus species and Drosophila, there appears to be an absence of virus‐derived piRNAs. To investigate whether this absence is due to a lack of PIWI pathway proteins, we analysed the piRNA and siRNA diversity and repertoire in PP9ad cells. Previous assemblies of P. papatasi genome (Ppap_1.0) have revealed a patchy repertoire of the siRNA and piRNA pathways. Our analysis of the updated P. papatasi genome (Ppap_2.1) has shown no PIWI protein expansion in sandflies. We found that both siRNA and piRNA pathways are transcriptionally active in PP9ad cells, with genomic mapping of small RNAs generating typical piRNA signatures. Our results suggest that the piRNA pathway may not respond to virus replication in these cells, but an antiviral response is mounted via the exo‐siRNA pathway. [ABSTRACT FROM AUTHOR]

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood‐feeding insects that transmit the protozoan parasites Leishmania spp. and various arboviruses. The Balkan region, including the Republic of Kosovo, harbours a diverse sand fly fauna. Vector species of Leishmania infantum as well as phleboviruses are endemic; however, recent data are scarce. We performed a cross‐sectional study to update the current sand fly distribution in Kosovo and assess biological as well as environmental factors associated with sand fly presence. CDC light trapping was conducted at 46 locations in 2022 and 2023, specifically targeting understudied regions in Kosovo. Individual morphological species identification was supported by molecular barcoding. The occurrence data of sand flies was used to create distribution maps and perform environmental analyses, taking elevation, wind speed and climate‐related factors into account. In addition, PCR‐based blood meal analysis and pathogen screening were conducted. Overall, 303 specimens of six sand fly species were trapped, predominated by Phlebotomus neglectus (97%). Barcodes from eight of nine known endemic sand fly species were obtained. Combining our data with previous surveys, we mapped the currently known sand fly distribution based on more than 4000 specimens at 177 data points, identifying Ph. neglectus and Ph. perfiliewi as the predominant species. Environmental analyses depicted two geographical groups of sand flies in Kosovo, with notable differences between the species. In total, 223 blood meals of five sand fly species were analysed. Of seven identified host species, the predominant blood meal source was observed to be cattle, but the DNA of dogs and humans, among others, was also detected. This study assessed biological as well as ecological factors of sand fly occurrence, which should help better understand and evaluate potential hot spots of disease transmission in Kosovo. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania, affecting millions of people worldwide. The disease is transmitted by the bite of infected female sand flies, which act as vectors and hosts for the parasites. The interaction between Leishmania parasites and sand flies is complex and dynamic, involving various factors that influence parasite development, survival and transmission. This review examines how the immune response of sand flies affects vector competence and transmission of Leishmania parasites, and what the potential strategies are to prevent or reduce infection. The review also summarizes the main findings and conclusions of the existing literature and discusses implications and recommendations for future research and practice. The study reveals that the immune response of sand flies is a key determinant of vector competence and transmission of Leishmania parasites, and that several molecular and cellular mechanisms are involved in the interaction between parasite and vector. The study also suggests that there are potential strategies for controlling leishmaniasis, such as interfering with parasite development, modulating the vector's immune response or reducing the vector population. However, the study also identifies several gaps and limitations in current knowledge and calls for more comprehensive and systematic studies on vector-parasite interaction and its impact on leishmaniasis transmission and control. [ABSTRACT FROM AUTHOR]

Abstract Background Sand fly females require a blood meal to develop eggs. The size of the blood meal is crucial for fecundity and affects the dose of pathogens acquired by females when feeding on infected hosts or during experimental membrane-feeding. Methods Under standard laboratory conditions, we compared blood meal volumes taken by females of ten sand fly species from four genera: Phlebotomus, Lutzomyia, Migonomyia, and Sergentomyia. The amount of ingested blood was determined using a haemoglobin assay. Additionally, we weighed unfed sand flies to calculate the ratio between body weight and blood meal weight. Results The mean blood meal volume ingested by sand fly females ranged from 0.47 to 1.01 µl. Five species, Phlebotomus papatasi, P. duboscqi, Lutzomyia longipalpis, Sergentomyia minuta, and S. schwetzi, consumed about double the blood meal size compared to Migonomyia migonei. The mean body weight of females ranged from 0.183 mg in S. minuta to 0.369 mg in P. duboscqi. In males, the mean body weight ranged from 0.106 mg in M. migonei to 0.242 mg in P. duboscqi. Males were always lighter than females, with the male-to-female weight ratio ranging from 75% (in Phlebotomus argentipes) to 52% (in Phlebotomus tobbi). Conclusions Females of most species took a blood meal 2.25–3.05 times their body weight. Notably, the relatively tiny females of P. argentipes consumed blood meals 3.34 times their body weight. The highest (Mbl/Mf) ratios were found in both Sergentomyia species studied; females of S. minuta and S. schwetzi took blood meals 4.5–5 times their body weight. This parameter is substantially higher than that reported for mosquitoes and biting midges. Graphical Abstract

Abstract Background Abiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the detection of new L. infantum infection cases in Valdeorras (Ourense, Northwestern Spain) over a 20-year period and how different climatic variables and preventive measures may have affected it. Methods Indirect immunofluorescence antibody tests (IFAT) were performed on serum samples collected from dogs attending the ‘Servicios Veterinarios de Sil’ veterinary clinic (Valdeorras, Northwestern Spain) between May 2003 and April 2023 to detect L. infantum exposure. The percentage of new cases of L. infantum infection was calculated from May of one year to April of the following year. Climatic conditions in the region, global sales of ectoparasiticides and the number of vaccines against L. infantum delivered in the veterinary clinic from 2003 to 2022 were recorded. Statistical analyses were conducted to determine the associations between these factors and the percentage of new cases of L. infantum infection. Results A total of 2909 dogs were assessed, and 3785 IFAT tests were performed between May 2003 and April 2023. The mean percentage of new seropositive cases over the 20-year period studied was 21.65 ± 10.8%, with a decline from the beginning to the end of the period studied. The percentage was significantly higher between May 2003 and April 2008 compared with the other periods (May 2008 to April 2013, May 2013 to April 2018 and May 2018 to April 2023). There was a positive correlation between the percentage of new cases of L. infantum infection and the maximum relative humidity in winter. Conversely, there was a negative correlation between the percentage of new cases and sales of ectoparasiticides and vaccination against L. infantum. Conclusions This study is one of the longest evaluations of the evolution of L. infantum infection in a fixed location and its association with external factors including climatic conditions and preventive measures. The results confirm that Valdeorras is a high-risk area for L. infantum infection. The use of ectoparasiticides and vaccines against L. infantum has been shown to play a significant role in preventing L. infantum infection, highlighting the crucial role of veterinarians in the fight against this disease. Graphical Abstract

Background: Sand fly females require a blood meal to develop eggs. The size of the blood meal is crucial for fecundity and affects the dose of pathogens acquired by females when feeding on infected hosts or during experimental membrane-feeding. Methods: Under standard laboratory conditions, we compared blood meal volumes taken by females of ten sand fly species from four genera: Phlebotomus, Lutzomyia, Migonomyia, and Sergentomyia. The amount of ingested blood was determined using a haemoglobin assay. Additionally, we weighed unfed sand flies to calculate the ratio between body weight and blood meal weight. Results: The mean blood meal volume ingested by sand fly females ranged from 0.47 to 1.01 µl. Five species, Phlebotomus papatasi, P. duboscqi, Lutzomyia longipalpis, Sergentomyia minuta, and S. schwetzi, consumed about double the blood meal size compared to Migonomyia migonei. The mean body weight of females ranged from 0.183 mg in S. minuta to 0.369 mg in P. duboscqi. In males, the mean body weight ranged from 0.106 mg in M. migonei to 0.242 mg in P. duboscqi. Males were always lighter than females, with the male-to-female weight ratio ranging from 75% (in Phlebotomus argentipes) to 52% (in Phlebotomus tobbi). Conclusions: Females of most species took a blood meal 2.25–3.05 times their body weight. Notably, the relatively tiny females of P. argentipes consumed blood meals 3.34 times their body weight. The highest (Mbl/Mf) ratios were found in both Sergentomyia species studied; females of S. minuta and S. schwetzi took blood meals 4.5–5 times their body weight. This parameter is substantially higher than that reported for mosquitoes and biting midges. [ABSTRACT FROM AUTHOR]

Phlebotomine sand flies are the vectors of leishmaniasis, a neglected tropical disease. High-quality reference genomes are an important tool for understanding the biology and eco-evolutionary dynamics underpinning disease epidemiology. Previous leishmaniasis vector reference sequences were limited by sequencing technologies available at the time and inadequate for high-resolution genomic inquiry. Here, we present updated reference assemblies of two sand flies, Phlebotomus papatasi and Lutzomyia longipalpis. These chromosome-level assemblies were generated using an ultra-low input library protocol, PacBio HiFi long reads, and Hi-C technology. The new P. papatasi reference has a final assembly span of 351.6 Mb and contig and scaffold N50s of 926 kb and 111.8 Mb, respectively. The new Lu. longipalpis reference has a final assembly span of 147.8 Mb and contig and scaffold N50s of 1.09 Mb and 40.6 Mb, respectively. Benchmarking Universal Single-Copy Orthologue (BUSCO) assessments indicated 94.5% and 95.6% complete single copy insecta orthologs for P. papatasi and Lu. longipalpis. These improved assemblies will serve as an invaluable resource for future genomic work on phlebotomine sandflies. [ABSTRACT FROM AUTHOR]

Background: Sand flies serve as crucial vectors in various medical and veterinary diseases. Sand fly-borne diseases pose a significant public health burden globally, as the causative agents can infect a diverse range of hosts, leading to severe consequences such as leishmaniasis and sand fly fever. Additionally, the widespread use of insecticides for agricultural purposes and mosquito control is not specifically targeted at sand flies, potentially leading to resistance development. We investigated sand fly species, their potential role as vectors of various parasitic agents, and insecticide resistance in the endemic regions of Natawi and Sadao districts in Songkhla, Thailand. Methods: Sand flies were collected using CDC light traps. The collected sand flies were then identified to species level using molecular techniques. Subsequent analyses included the detection of pathogens and the identification of pyrethroid resistance mutations within the voltage-sensitive sodium channel (Vgsc) domain IIS6 gene, followed by sequence analysis. Results: The study identified nine sand fly species belonging to the genera Phlebotomus and Sergentomyia. The DNA of Sergentomyia khawi was the only species found to test positive for one sample of Leishmania orientalis in Sadao district. This finding represents the first detection of L. orientalis in Thailand. Moreover, three samples of Leishmania martiniquensis and four samples of Trypanosoma sp. were found in the Natawi district. No I1011M, L1014F/S, V1016G, or F1020S mutations were detected in Vgsc gene. Conclusions: The results of this study provide valuable information on sand fly species and the continuous circulation of Leishmania spp. and Trypanosoma spp. in Songkhla, southern Thailand. Moreover, the development of geo-spatial information on vectors, parasites, and insecticide resistance in sand flies has the potential to provide well-informed risk assessments and evidence-based guidance for targeted vector control in Thailand. These results can serve as a foundation for integrating the One Health approach, which is crucial for disease control, considering the diverse ecological interactions among human and/or animal reservoir hosts, parasites, and sand fly vectors. [ABSTRACT FROM AUTHOR]

Background: Vector control based on indoor residual spraying (IRS) is one of the main components of the visceral leishmaniasis (VL) elimination programme in India. Dichlorodiphenyltrichloroethane (DDT) was used for IRS until 2015 and was later replaced by the synthetic pyrethroid alpha-cypermethrin. Both classes of insecticides share the same target site, the voltage-gated sodium channel (Vgsc). As high levels of resistance to DDT have been documented in the local sand fly vector, Phlebotomus argentipes, it is possible that mutations in the Vgsc gene could provide resistance to alpha-cypermethrin, affecting current IRS pyrethroid-based vector control. Methods: This study aimed to compare frequencies of knockdown resistance (kdr) mutations in Vgsc between two sprayed and two unsprayed villages in Bihar state, India, which had the highest VL burden of the four endemic states. Across four villages, 350 female P. argentipes were collected as part of a 2019 molecular xenomonitoring study. DNA was extracted and used for sequence analysis of the IIS6 fragment of the Vgsc gene to assess the presence of kdr mutations. Results: Mutations were identified at various positions, most frequently at codon 1014, a common site known to be associated with insecticide resistance in mosquitoes and sand flies. Significant inter-village variation was observed, with sand flies from Dharampur, an unsprayed village, showing a significantly higher proportion of wild-type alleles (55.8%) compared with the three other villages (8.5–14.3%). The allele differences observed across the four villages may result from selection pressure caused by previous exposure to DDT. Conclusions: While DDT resistance has been reported in Bihar, P. argentipes is still susceptible to pyrethroids. However, the presence of kdr mutations in sand flies could present a threat to IRS used for VL control in endemic villages in India. Continuous surveillance of vector bionomics and insecticide resistance, using bioassays and target genotyping, is required to inform India's vector control strategies and to ensure the VL elimination target is reached and sustained. [ABSTRACT FROM AUTHOR]

[Display omitted] • Microbe surface molecules can upregulate a mucin-like gene in Lutzomyia longipalpis. • Expression of the attacin gene is triggered as a generic immune response in sand flies. • Sand fly defensin gene expression is tuned, depending on the ingested microbe molecule. The interaction between pathogens and vectors' physiology can impact parasite transmission. Studying this interaction at the molecular level can help in developing control strategies. We study leishmaniases, diseases caused by Leishmania parasites transmitted by sand fly vectors, posing a significant global public health concern. Lipophosphoglycan (LPG), the major surface glycoconjugate of Leishmania , has been described to have several roles throughout the parasite's life cycle, both in the insect and vertebrate hosts. In addition, the sand fly midgut possesses a rich microbiota expressing lipopolysaccharides (LPS). However, the effect of LPG and LPS on the gene expression of sand fly midgut proteins or immunity effectors has not yet been documented. We experimentally fed Lutzomyia longipalpis and Phlebotomus papatasi sand flies with blood containing purified LPG from Leishmania infantum , Leishmania major , or LPS from Escherichia coli. The effect on the expression of genes encoding gut proteins galectin and mucin, digestive enzymes trypsin and chymotrypsin, and antimicrobial peptides (AMPs) attacin and defensins was assessed by quantitative PCR (qPCR). The gene expression of a mucin-like protein in L. longipalpis was increased by L. infantum LPG and E. coli LPS. The gene expression of a galectin was increased in L. longipalpis by L. major LPG, and in P. papatasi by E. coli LPS. Nevertheless, the gene expression of trypsins and chymotrypsins did not significantly change. On the other hand, both L. infantum and L. major LPG significantly enhanced expression of the AMP attacin in both sand fly species and defensin in L. longipalpis. In addition, E. coli LPS increased the expression of attacin and defensin in L. longipalpis. Our study showed that Leishmania LPG and E. coli LPS differentially modulate the expression of sand fly genes involved in gut maintenance and defence. This suggests that the glycoconjugates from microbiota or Leishmania may increase the vector's immune response and the gene expression of a gut coating protein in a permissive vector. [ABSTRACT FROM AUTHOR]

Background: Abiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the detection of new L. infantum infection cases in Valdeorras (Ourense, Northwestern Spain) over a 20-year period and how different climatic variables and preventive measures may have affected it. Methods: Indirect immunofluorescence antibody tests (IFAT) were performed on serum samples collected from dogs attending the 'Servicios Veterinarios de Sil' veterinary clinic (Valdeorras, Northwestern Spain) between May 2003 and April 2023 to detect L. infantum exposure. The percentage of new cases of L. infantum infection was calculated from May of one year to April of the following year. Climatic conditions in the region, global sales of ectoparasiticides and the number of vaccines against L. infantum delivered in the veterinary clinic from 2003 to 2022 were recorded. Statistical analyses were conducted to determine the associations between these factors and the percentage of new cases of L. infantum infection. Results: A total of 2909 dogs were assessed, and 3785 IFAT tests were performed between May 2003 and April 2023. The mean percentage of new seropositive cases over the 20-year period studied was 21.65 ± 10.8%, with a decline from the beginning to the end of the period studied. The percentage was significantly higher between May 2003 and April 2008 compared with the other periods (May 2008 to April 2013, May 2013 to April 2018 and May 2018 to April 2023). There was a positive correlation between the percentage of new cases of L. infantum infection and the maximum relative humidity in winter. Conversely, there was a negative correlation between the percentage of new cases and sales of ectoparasiticides and vaccination against L. infantum. Conclusions: This study is one of the longest evaluations of the evolution of L. infantum infection in a fixed location and its association with external factors including climatic conditions and preventive measures. The results confirm that Valdeorras is a high-risk area for L. infantum infection. The use of ectoparasiticides and vaccines against L. infantum has been shown to play a significant role in preventing L. infantum infection, highlighting the crucial role of veterinarians in the fight against this disease. [ABSTRACT FROM AUTHOR]

In an area endemic with Indian visceral leishmaniasis (VL), we performed direct xenodiagnosis to evaluate the transmission of Leishmania donovani from patients with VL–human immunodeficiency virus (HIV) coinfection to the vector sandflies, Phlebotomus argentipes. Fourteen patients with confirmed VL-HIV coinfection, with a median parasitemia of 42 205 parasite genome/mL of blood, were exposed to 732 laboratory-reared pathogen-free female P argentipes sandflies on their lower arms and legs. Microscopy revealed that 16.66% (122/732) of blood-fed flies were xenodiagnosis positive. Notably, 93% (13/14) of the VL-HIV group infected the flies, as confirmed by quantitative polymerase chain reaction and/or microscopy, and were 3 times more infectious than those who had VL without HIV. [ABSTRACT FROM AUTHOR]

Sandfly, Phlebotomus papatasi (Diptera: Psychodidae) is the potential vector of leishmania in Egypt. In this study, an attempt was made to investigate the ultrastructure and histopathological alteration in the midgut of Phlebotomus papatasi post-treatment with methoprene (IGR) and B. sphaericus 6184 and 6446 using transmission electron micrographs (TEM). After 48 h, the TEM of the midgut treated with methoprene (IGR) showed sloughing of the periepithelial layer, and overgrowth of the brush border of the midgut wall. After 48 h post-treatment, B. sphaericus 6184 spores were discovered in the lumen and the bacterium began to enter the microvilli. Certain ultrastructural abnormalities were found in the midgut of treated P. papatasi larvae after 48 h of treatment with B. sphaericus 6446, the cytoplasm was distinguished by many vacuoles and a broken brush border. Using the light microscope, IGR-treated third instar larvae showed brush boundary overgrowth and multifocal disintegration of the midgut wall. Focused areas were sloughed and epithelial cells were dissociated from each other in larvae treated with B. sphaericus 6184. The 3rd instar P. papatasi larvae treated with B. sphaericus 6446 showed clear alterations such as total epithelial cell degradation. These results demonstrated that B. sphaericus 6446 showed the best results and great effect on P. papatasi 3rd instar larvae. More research is needed to establish the appropriate control strategies for the many leishmaniasis vectors in their various ecological environments. [ABSTRACT FROM AUTHOR]

The infectious inoculum of a sand fly, apart from its metacyclic promastigotes, is composed of factors derived from both the parasite and the vector. Vector-derived factors, including salivary proteins and the gut microbiota, are essential for the establishment and enhancement of infection. However, the type and the number of bacteria egested during salivation is unclear. In the present study, sand flies of Phlebotomus papatasi were gathered from three locations in hyperendemic focus of zoonotic cutaneous leishmaniasis (ZCL) in Isfahan Province, Iran. By using the forced salivation assay and targeting the 16S rRNA barcode gene, egested bacteria were characterized in 99 (44%) out of 224 sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. Ten top genera of Spiroplasma, Ralstonia, Acinetobacter, Reyranella, Undibacterium, Bryobacter, Corynebacterium, Cutibacterium, Psychrobacter, and Wolbachia constituted >80% of the saliva microbiome. Phylogenetic analysis displayed the presence of only one bacterial species for the Spiroplasma, Ralstonia, Reyranella, Bryobacter and Wolbachia, two distinct species for Cutibacterium, three for Undibacterium and Psychrobacter, 16 for Acinetobacter, and 27 for Corynebacterium, in the saliva. The abundance of microbes in P. papatasi saliva was determined by incorporating the data on the read counts and the copy number of 16S rRNA gene, about 9,000 bacterial cells, per sand fly. Both microbiological and metagenomic data indicate that bacteria are constant companions of Leishmania, from the intestine of the vector to the vertebrate host. This is the first forced salivation experiment in a sand fly, addressing key questions on infectious bite and competent vectors. Author summary: Female sand flies salivate during feeding on vertebrate blood and natural sugars. During salivation, they may release microorganisms associated with the salivary glands and digestive tract, i.e. viruses, bacteria, and other pathogens, into the feeding substrates. However, the type and the number of bacteria egested during salivation is unclear. Here, by sing the forced salivation experiment and targeting the 16S rRNA barcode gene, egested bacteria were characterized in sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. The abundance of microbes in the saliva of each P. papatasi sand fly was determined to be around 9,000 bacterial cells. The lack of a vaccine and the failure of treatment in leishmaniasis provide many motivations to better understand the factors cause the inflammatory response. The findings of this study can improve our insight into measuring the effect of vector-derived bacteria on the improvement or deterioration of leishmaniasis. [ABSTRACT FROM AUTHOR]

Background and Aims: Leishmaniasis is a neglected tropical infection caused by Leishmania parasite that affect human and animal. In Morocco, the cutaneous leishmaniasis has spread substantially to the new areas. The surveillance limited to active foci may underestimate the occurrence of cutaneous leishmaniasis (CL). This study aims to investigate the local transmission of CL in rural districts of Youssoufia province, central Morocco, as a potential focus of CL. Methods: For this purpose, parasitological, molecular and entomological investigations were carried out in this area. Data collection concerns potential vectors and human cases. Thus, 402 patients were examined for suspected leishmaniasis lesions in three localities of the province of Youssoufia. In these same localities, 983 sand flies were collected by CDC light traps and sticky paper during one‐night per month during 6 months. These sand flies were all identified morphologically using the Moroccan identification key. Results: The results showed that among the 25 skin lesions detected in a population of 402 individuals, 18 were confirmed by kDNA nested PCR as CL positive patients, of which only 25% were positive by direct examination. Leishmania tropica and Leishmania major were identified as causative agents of CL in the study area. Direct parasitological examination showed a low sensitivity (27.78%), especially for L. major, although its specificity was evaluated at 100%. Regarding entomological results, both genera of the Moroccan sand fly were collected in the study area: Genus/Phlebotomus (75.28%) and Sergentomyia (24.72%). Phlebotomus (P) papatasi, the proven vector of L. major, was the most abundant species (33.98%), followed by Paralongicollum sergenti (22.58%), the confirmed vector of L. tropica; while Sergentomyia (S) minuta, P. longicuspis, S. fallax and P. kazeruni were collected with, respectively, 17.60%, 16.99%, 7.12% and 1.73%. Conclusion: This study constitutes the first report of CL in the study areas, as well as the coexistence of L. tropica and L. major in these rural localities. Local transmission of CL is highly probable, as indicated by the prevalence of the two proven vectors of L. major and L. tropica. To control the spread of this disease, our results suggest the use of highly sensitive molecular methods to detect CL cases in potential leishmaniasis foci, which will improve surveillance. [ABSTRACT FROM AUTHOR]

The main goal of the current study was to investigate the molecular systematics and host–vector interactions of sandfly species (Phlebotomus spp.) in Iran. Sandflies were collected using CDC light traps, sticky traps, and BG-Sentinel traps at seven trapping sites in Iran during 2019 and 2020. The source of blood meals was identified using primer pairs specific to either the 16S rRNA or the mitochondrial cytochrome b (cytb) genes. A total of 736 blood-fed female sandflies were collected. Six species of the genus Phlebotomus (Ph.) and four species of the genus Sergentomyia (Se.) were identified using molecular identification techniques. The most abundant sandfly species were Ph. papatasi (38.45 %) and Se. sintoni (36.28 %). Blood meal analysis identified 19 different host species, specifically, 15 mammalian and four bird species. The most common blood meal sources were Rattus norvegicus , followed by Homo sapiens , Bos primigenius f. taurus , Ovis orientalis f. aries , Meriones persicus , Gallus gallus f. domestica , Rhombomys opimus , and Canis lupus; other host species had incidences of less than 5 %. We observed that Ph. major complex and Se. sintoni had mostly mammalophilic blood-feeding behavior, while Ph. papatasi and Ph. sergenti had mostly anthropophilic and ornithophilic blood-feeding preferences, respectively. The current results confirmed cytb is a suitable gene for regular species level identification, and that phylogenetic analyses of cytb allowed for clear demarcation of sandfly species. [ABSTRACT FROM AUTHOR]

In Nepal, visceral leishmaniasis (VL) has been targeted for elimination as a public health problem by 2026. Recently, increasing numbers of VL cases have been reported from districts of doubtful endemicity including hills and mountains, threatening the ongoing VL elimination program in Nepal. We conducted a multi-disciplinary, descriptive cross-sectional survey to assess the local transmission of Leishmania donovani in seven such districts situated at altitudes of up to 1,764 meters in western Nepal from March to December 2019. House-to-house surveys were performed for socio-demographic data and data on past and current VL cases. Venous blood was collected from all consenting individuals aged ≥2 years and tested with the rK39 RDT. Blood samples were also tested with direct agglutination test, and a titer of ≥1:1600 was taken as a marker of infection. A Leishmania donovani species-specific PCR (SSU-rDNA) was performed for parasite species confirmation. We also captured sand flies using CDC light traps and mouth aspirators. The house-to-house surveys documented 28 past and six new VL cases of which 82% (28/34) were without travel exposure. Overall, 4.1% (54/1320) of healthy participants tested positive for L. donovani on at least one serological or molecular test. Among asymptomatic individuals, 17% (9/54) were household contacts of past VL cases, compared to 0.5% (6/1266) among non-infected individuals. Phlebotomus argentipes, the vector of L. donovani, was found in all districts except in Bajura. L. donovani was confirmed in two asymptomatic individuals and one pool of sand flies of Phlebotomus (Adlerius) sp. We found epidemiological and entomological evidence for local transmission of L. donovani in areas previously considered as non-endemic for VL. The national VL elimination program should revise the endemicity status of these districts and extend surveillance and control activities to curb further transmission of the disease. [ABSTRACT FROM AUTHOR]

Introduction: Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. Methods: To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. Results: A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Discussion: Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. Material & methods: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. Results: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. Conclusion: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups. [ABSTRACT FROM AUTHOR]

Sand flies (Diptera: Psychodidae, Phlebotominae; Newstead, 1911) are widespread in Europe, being particularly common in the Mediterranean region but rare north of the Alps. Thus, Switzerland is an opportune place to investigate the sand fly fauna on both sides of the Alpine crest, in southern sub‐Mediterranean climate and northern oceanic temperate climate. We reinvestigated the Swiss sand fly fauna with the aim to assess changes in composition, altitudinal distribution, abundance and seasonality. Thirty‐eight sites were investigated with light traps and/or interception sticky traps in 4 years. Ninety and 380 specimens were caught by light traps and sticky traps, respectively, at 15 collecting sites. Four species were identified. Phlebotomus mascittii (Grassi, 1908), Phlebotomus perniciosus (Newstead, 1911) and Sergentomyia minuta (Rondani, 1843) were confirmed in Ticino, and P. mascittii for the first time in neighbouring Grisons. Also, Phlebotomus neglectus (Tonnoir, 1921) is for the first time reported, though at a very low density compared to P. perniciosus at the same site. Its presence in Ticino supports the northward spread observed in Italy. Sand flies were detected north of the Alps at one site only, endorsing a historical report. Overall, the low density of P. perniciosus and very low density of P. neglectus suggest that canine leishmaniosis may not be an important disease risk in Switzerland. [ABSTRACT FROM AUTHOR]

Simple Summary: The old-world sand fly, Phlebotomus papatasi (Scopoli 1786), is a major vector of Leishmania major, the predominant pathogen responsible for zoonotic cutaneous leishmaniasis in the Middle East, North Africa, Southern Europe, and Central Asia. DEET and other synthetic insect repellents have been used for personal protection against sand fly bites. However, the frequent use of DEET repellent raised concerns in regards to skin sensitivity, toxicity, and unpleasant odor. There are increasing efforts to evaluate natural products for use in developing more effective organic sand fly repellents. This paper reports the results of a laboratory study on several plant essential oils and saturated fatty acids concerning their repellency against female sand flies. A static air repellency assay was used to measure the responses of sand flies to test materials. The sand fly repellency of each test material was compared with those achieved by commercial repellent DEET and IR35353 at the same test concentration. The study identified two of the tested essential oils as effective spatial repellents at reduced concentrations compared to those of DEET, and two saturated fatty acids were found to produce significant sand fly mortality. The results from this study establish the foundation for developing more effective natural sand fly repellent products. The sand fly, Phlebotomus papatasi (Scopoli, 1786), is a major vector for Leishmania major in the Middle East, which has impacted human health and US military operations in the area, demonstrating the need to develop effective sand fly control and repellent options. Here, we report the results of spatial repellency and avoidance experiments in a static air olfactometer using the female P. papatasi testing essential oils of Lippia graveolens (Mexican oregano), Pimenta dioica (allspice), Amyris balsamifera (amyris), Nepeta cataria (catnip), Mentha piperita (peppermint), and Melaleuca alternifolia (tea tree); the 9–12 carbon saturated fatty acids (nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid); and the synthetic repellents DEET and IR3535. The materials applied at 1% exhibited varying activity levels but were not significantly different in mean repellency and avoidance from DEET and IR3535, except in regards to nonanoic acid. Some materials, particularly nonanoic and undecanoic acids, produced sand fly mortality. The observed trends in mean repellency over exposure time included the following: (1) P. dioica oil, M. alternifolia oil, decanoic acid, undecanoic acid, DEET, and IR3535 exhibited increasing mean repellency over time; (2) oils of N. cataria, A. balsamifera, M. piperita, and dodecanoic acid exhibited relatively constant mean repellency over time; and (3) L. graveolens oil and nonanoic acid exhibited a general decrease in mean repellent activity over time. These studies identified the essential oils of N. cataria and A. balsamifera as effective spatial repellents at reduced concentrations compared to those of DEET. Additional research is required to elucidate the modes of action and potential synergism of repellents and essential oil components for enhanced repellency activity. [ABSTRACT FROM AUTHOR]

Climate change alters the global distribution of leishmaniasis vector sandfly species. However, the possible effect of altering environmental factors on sandfly species varies by species and area. The aim of this study was to project the future potential effect of climate change on the Western Asian occurrence of the sandfly vector species Phlebotomus papatasi and Phlebotomus sergenti and the parasites Leishmania major and Leishmania tropica for 2041–2060 and 2081–2100 compared to their modelled climatic suitability patterns in the reference period 1970–2000. The model results suggest that by 2041–2060 and 2081–2100, in the coastal areas of the Levantine countries and the mid-elevation regions of Western Iran and Southeast Turkey, the climatic suitability of all the studied species is predicted to increase. In contrast, the model results suggest the decline of the populations of the studied vectors as well as the disappearance of the parasites in Iraq, North Saudi Arabia, Kuwait, and the inland regions of Western Asian countries. Considering the present-day population density patterns, it can be said that although in large regions of the region, the climatic suitability values are predicted to decrease, the populated Levantine regions seem to be at risk of climate change-facilitated increase in cutaneous leishmaniasis in the second half of the twenty-first century. The model results suggest that climate change will especially increase the climatic suitability of Leishmania tropica in the Levantine region in the late twenty-first century. [ABSTRACT FROM AUTHOR]

In Uzbekistan, the number of reported leishmaniasis cases is rising at the alarming rate. In this work, we studied the phlebotomine sand fly (Diptera: Phlebotominae) diversity in the foci of cutaneous leishmaniasis in the Surxondaryo Region of Uzbekistan and compared it with the data obtained for the same area 50 years ago, when infection prevalence was reportedly low. We found that the implicated vector for zoonotic leishmaniasis, P. papatasi, remained eudominant; the proportion of implicated anthroponotic leishmaniasis vector, P. sergenti, rose significantly from averaged 5.4 to 41.4%; Phlebotomus alexandri, a suspected visceral leishmaniasis vector, was eudominant at two sites, and a second suspected vector for this disease, P. longiductus, was newly recorded in the region. We conclude that the increase in the documented cases of cutaneous leishmaniasis in the Surxondaryo Region of Uzbekistan may be connected to the changes in fauna of sand flies vectoring Leishmania spp. [ABSTRACT FROM AUTHOR]

Background: This study's major aim is to investigate the situation of visceral leishmaniasis vectors, with a focus on their distribution and relationships to the disease in Iran and some other old-world nations. Methods: The terms Iran and the Old World, along with the keywords sand flies, vectors, visceral leishmaniasis, distribution, and Phlebotomus, were searched in electronic databases from 1930 to 2018, including Pub-Med, Web of Science, Google Scholar, and MEDLINE. Results: According to the findings, Phlebotomus tobbi was a mountain species, but it was also found in the plains, rodent nests, and rock crevices. This species was considered to be one of the vectors of visceral leishmaniasis in Iran. Phlebotomus kandelakii has been caught in Afghanistan, Iran, Lebanon, Turkey and Georgia. In Iran, the first infection of Phlebotomus kandelakii and Ph. perfiliewi transcaucasicus with Leishmania infantum were reported in northwest of Iran. Phlebotomus major was one of the complex sand flies, and its members include Ph. major (India, Nepal, and Pakistan), Ph. neglectus (Southern Europe, Crimea and Iran), Ph. syriacus (Southwest Asia, Caucasus), Ph. notus (Afghanistan and Iran), Ph. wenyoni (Iran and Iraq), and Ph. wui (China). The first natural infection of Ph. alexandri with Leishmania infantum was reported in 2006 in endemic foci of visceral leishmaniasis in Fars Province, southern Iran. Conclusion: The findings of this project suggest that sand fly species of the subgenus Larrousisus play a key role in the transmission of all types of visceral leishmaniasis in Iran. [ABSTRACT FROM AUTHOR]

Background: Zoonotic cutaneous leishmaniasis (ZCL) is widely distributed in Iran and around the world. Also, Khuzestan Province is an endemic focus of ZCL. This study aims to investigate the natural infection of sand flies with the Leishmania parasite in Karun County. Methods: Sand flies were collected from Jangiyeh, Qaleh Chanan, Kut-e-Navaser, and Ghazavieh in the spring and summer in the year of 2019, by installing 60 sticky paper traps each time (30 traps outdoors and 30 traps indoors). Two hundred female sand flies with different abdominal conditions (empty, blood-fed, semi-gravid, and gravid) were examined for infection rate using the Nested-PCR method. Results: In this study, seven species of sand flies including Phlebotomus papatasi, Ph. alexandri, Ph. sergenti, Ph. caucasicus, Sergentomyia tiberiadis, Se. sintoni, and Se. antennata were reported from Karun County, with a frequency of 79.64%, 16.96%, 1.07%, 0.18%, 0.36%, 1.61%, and 0.18%, respectively. Only eleven specimens of Ph. papatasi were found to be positive for Leishmania major, with an overall infection rate of 7.8%. The infection of Ph. papatasi was specifically reported in blood-fed, gravid, and semi-gravid specimens, with infection rates of 17.02%, 4.35%, and 14.29%, respectively. Conclusion: In this study, the infection of L. major from Ph. papatasi was reported. The results can be used in planning the control of ZCL in the study area. [ABSTRACT FROM AUTHOR]

Background:Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis. Wolbachia is a symbiotic alphaproteobacteria of arthropods that can be involved in susceptibility or resistance. This study aimed to investigate the relationship between Wolbachia and Deltamethrin susceptibility/resistance in Ph. papatasi. Deltamethrin filter papers (0.00002%) were used to test sand fly field collected from southern Iran. After the test, PCR amplification of the Wolbachia surface protein gene (wsp) was used to measure Wolbachia infection rate in the killed, surviving, and control groups. Result: The rates of infection by Wolbachia strain (wPap, super group A) differed between killed (susceptible) and surviving (resistant) Ph. papatasi specimens. The rate of Wolbachia infection in susceptible individuals was more than twice (2.3) (39% vs. 17%) in resistant individuals with the same genetic background. This difference was highly significant (p < 0.001), indicating a positive association between Wolbachia infection and susceptibility to Deltamethrin. In addition, the results showed that Deltamethrin can act as a PCR inhibitor during detection of Wolbachia in Ph. papatasi. Conclusion: Results of this study show that Wolbachia is associated with Deltamethrin susceptibility level in Ph. papatasi. Also, as Deltamethrin has been identified as a PCR inhibitor, great care must be taken in interpreting Wolbachia infection status in infected populations. The results of this study may provide information for a better understanding of the host-symbiont relationship, as well as application of host symbiosis in pest management. [ABSTRACT FROM AUTHOR]

Purpose: Leishmania major is main causative agent and Phlebotomus papatasi is only proven vector of Zoonotic Cutaneous Leishmaniasis (ZCL) in Iran. Human leishmaniasis is mostly susceptible to climatic conditions and molecular variations of Leishmania parasites within sandflies. Methods: L. major was analyzed based on geographical, environmental, climatic changes and haplotype variations within P. papatasi. Molecular tools and different geographical aspects were employed using Arc-GIS software for mapping the geographic distribution of samples and other statistics tests. Fragments of ITS-rDNA, k-DNA, and microsatellite genes of Leishmania were used for PCR, RFLP, sequencing, and phylogenetic analyses. Results: Totally 81 out of 1083 female P. papatasi were detected with Leishmania parasites: 70 and five were L. major and L. turanica, respectively. Golestan and Fars provinces had the highest (13.64%) and lowest (4.55%) infection rates, respectively. The infection rate among female P. papatasi collected from gerbil burrows was significantly higher (15.15%) than animal shelters, yards, and inside houses (4.48%) (P < 0.0%). Microsatellite was more sensitive (22.72%) than k-DNA (18.8%) and ITS-rDNA (7.48%). More molecular variations of L. major were found in Isfahan province. Conclusions: Arc-GIS software and other statistics tests were employed to find Leishmania positive and haplotype variations among sand flies. Geographical situations, altitude, climate, precipitation, humidity, temperature, urbanization, migrations, regional divergences, deforestation, global warming, genome instability, ecology, and biology of the sand flies intrinsically, and the reservoir hosts and neighboring infected locations could be reasons for increasing or decreasing the rate of Leishmania infection and haplotype variations. [ABSTRACT FROM AUTHOR]

Global changes, such as climate change, represent a worldwide risk for the management of leishmaniosis. The transmission profile can be modified by several mechanisms, mainly through the distribution and ecology of the vectors, causing direct effects on their survival, generation time, reproduction rate and population size. Sergentomyia minuta is an endemic species of the Old World, considered the most abundant in some areas of Spain. Even though S. minuta has traditionally been seen as herpetophilic, several recent researches suggest this species plays a potential role in Leishmania infantum transmission. Furthermore, the Toscana virus has previously been detected in S. minuta and the potential role of this species in the epidemiology of this emerging arbovirus is still uncertain and needs further research. At the end of April 2023, a total of 10 individuals of S. minuta were collected in Torrelodones (Madrid, Central Spain), this being the first collection of sand flies of any species during April in this region. While it is known that climate change can have a significant impact on sand fly distribution and behavior, little is known about the microscale effects of this variation on vector biology and ecology. The early detection of S. minuta in an endemic area of leishmaniosis such as the Madrid autonomous community may act as an early warning of the risk of temporal dynamic changes in Phlebotomus species in response to global change. [ABSTRACT FROM AUTHOR]

Sand flies (Diptera: Psychodidae) are significant medical vectors of pathogens that cause illness in humans and animals. The most significant of these is the genus Leishmania (Kinetoplastida: Trypanosomatidae). Leishmaniasis is an important vector-borne disease. They are spread throughout the Old and New Worlds by the bites of female phlebotomine sand flies. Despite this importance, certain provinces where autochthonous leishmaniasis has historically existed and is re-emerging are lacking current data regarding their sand fly fauna. To provide a current update on the local sand fly fauna, a comprehensive entomological survey encompassing eight provinces was conducted subsequent to an analysis of past data regarding the species composition and distribution of sand flies in the area. Thus, the purpose of this study was to ascertain the species composition in various locations across eight provinces spread across four regions in Thailand. In order to gather information about the distribution of the phlebotomine sand fly, researchers used modified Centers for Disease Control and Prevention light traps between February 2021 and February 2022. At these 30 trappings in each province, we conducted 240 trapping nights in total. Collected sand flies were morphologically identified. The adult sand flies collected in this study were 8314 and represented 24 species of four genera, including 16 of Sergentomyia, 6 of Phlebotomus, 1 of Idiophlebotomus, and 1 of Chinius. Among these species, Sergentomyia anodontis had the largest population (25.71%), and population peaks were observed in Uthaithani province (51.81%). The collected sand flies' male-to-female ratio was 1.21:1.00 (4,556:3,758). The study also discovered that Phlebotomus argentipes, a vector of Leishmania spp., was captured in Phayao province, and Phlebotomus major major was majorly found in Uthaithani province. The widespread distribution of sand fly populations in this study may indicate the epidemiologic importance of vector ecology in this habitat. Our results confirm the presence of several sand fly species in different provinces of Thailand and provide valuable information about the distribution and behavior of phlebotomine sand flies, which can be used to develop effective control strategies for the prevention of sand fly-borne diseases. In several Thai provinces, the study also tracked the nocturnal movements of both male and female sand flies. The results showed that phlebotomine sand flies are more active at night, peaking just after dusk. [ABSTRACT FROM AUTHOR]

Wuxiang virus (WUXV) is the first sandfly-borne Phlebovirus isolated from Phlebotomus chinensis collected in China and has been established as a consistent viral presence in the local sandfly populations of both Wuxiang County and Yangquan City. However, its distribution in the Shanxi Province remains unclear. In this study, three novel WUXV strains were isolated from sandflies collected from Jiexiu City, Shanxi Province, China, in 2022. Subsequently, whole-genome sequences of these novel strains were generated using next-generation sequencing. The open reading frame (ORF) sequences of the WUXV strains from the three locations were subjected to gene analysis. Phylogenetic analysis revealed that WUXV belongs to two distinct clades with geographical differences. Strains from Wuxiang County and Yangquan City belonged to clade 1, whereas strains from Jiexiu City belonged to clade 2. Reassortment and recombination analyses indicated no gene reassortment or recombination between the two clades. However, four reassortments or recombination events could be detected in clade 1 strains. By aligning the amino acid sequences, eighty-seven mutation sites were identified between the two clades, with seventeen, sixty, nine, and one site(s) in the proteins RdRp, M, NSs, and N, respectively. Additionally, selection pressure analysis identified 17 positively selected sites across the entire genome of WUXV, with two, thirteen, one, and one site(s) in the proteins RdRp, M, NSs, and N, respectively. Notably, sites M-312 and M-340 in the M segment not only represented mutation sites but also showed positive selective pressure effects. These findings highlight the need for continuous nationwide surveillance of WUXV. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a zoonotic vector-borne disease with worldwide distribution. All current approaches in leishmaniasis control or development of vaccines/cures showed only limited success. Recently, paratransgenesis has been marked as a promising strategy for leishmaniasis control. Thus, the investigations of the gut microbial content of sand flies have gained popularity. Gut microbial composition of the laboratory colony of Phlebotomus papatasi was investigated via microbial culturomics approach which refers to the combination of multiple culture conditions and different selective and/or enriched culture mediums, followed by 16S rDNA sequencing. Investigations were conducted on three offspring generations, with six samplings of immature stages (four larval samplings, one pre-pupa, one pupa) and samplings of adults before and after blood feeding. The aim was to determine if microbiome changes during the sand fly development and to identify bacteria with transstadial potential. The presence of 8 bacterial taxa (Bacillus sp., Terribacillus sp., Staphylococcus sp., Alcaligenes sp., Microbacterium sp., Leucobacter sp., Ochrobactrum sp. and Enterobacter sp.), 2 fungi (Fusarium sp. and Acremonium sp.) and 1 yeast (Candida sp.) were recorded. Gram-positive bacteria were more diverse, but gram-negative bacteria were more abundant. All taxa were recorded among immature stage samples, while only one bacterium was detected in adults. Microbial diversity among larval samples was stable, with a steady decrease in pre-pupa and pupa, resulting in the survival of only Ochrobactrum sp. in adults. Abundance of microbes was higher when larvae were actively feeding, with a gradual decrease after larvae stopped feeding and commenced pupation. Ochrobactrum sp. is the bacteria with transstadial potential, worthy of future in-depth analysis for the application in paratransgenic approach for the control of Leishmania sp. [ABSTRACT FROM AUTHOR]

Background: Phlebotomus argentipes complex is the primary vector for cutaneous leishmaniasis, a burgeoning health concern in contemporary Sri Lanka, where effective vector control is important for proper disease management. Understanding the genetic diversity of the P. argentipes population in Sri Lanka is vital before implementing a successful vector control program. Various studies have indicated that genetic divergence, caused by genetic drift or selection, can significantly influence the vector capacity of arthropod species. To devise innovative control strategies for P. argentipes, exploring genetic diversity and phylogeography can offer valuable insights into vector competence, key genetic trait transfer, and impact on disease epidemiology. The primary objective is to analyze the genetic diversity and phylogeography of the P. argentipes complex in Sri Lanka, based on two mitochondrial genomic regions in modern representatives of P. argentipes populations. Methodology: A total of 159 P. argentipes specimens were collected from five endemic areas of cutaneous leishmaniasis and identified morphologically. Two mitochondrial regions (Cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4) were amplified using the total DNA and subsequently sequenced. Partial sequences of those mitochondrial genes were utilized to analyze genetic diversity indices and to explore phylogenetic and phylogeographic relationships. Principal findings: Among five sampling locations, the highest genetic diversity for COI and ND4 was observed in Hambantota (Hd—0.749, π—0.00417) and Medirigiriya (Hd—0.977, π—0.01055), respectively. Phylogeographic analyses conducted using COI sequences and GenBank retrieved sequences demonstrated a significant divergence of P. argentipes haplotypes found in Sri Lanka. Results revealed that they have evolved from the Indian ancestral haplotype due to historical- geographical connections of the Indian subcontinent with Sri Lanka. Conclusions: Utilizing high-mutation-rate mitochondrial genes, such as ND4, can enhance the accuracy of genetic variability analysis in P. argentipes populations in Sri Lanka. The phylogeographical analysis of COI gene markers in this study provides insights into the historical geographical relationship between India and P. argentipes in Sri Lanka. Both COI and ND4 genes exhibited consistent genetic homogeneity in P. argentipes in Sri Lanka, suggesting minimal impact on gene flow. This homogeneity also implies the potential for horizontal gene transfer across populations, facilitating the transmission of genes associated with traits like insecticide resistance. This dynamic undermines disease control efforts reliant on vector control strategies. [ABSTRACT FROM AUTHOR]

Phlebotomine sand flies (Diptera: Phlebotominae) are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). In Central Europe, Phlebotomus mascittii is the predominant species, but largely understudied. To better understand factors driving its current distribution, we infer patterns of genetic diversity by testing for signals of population expansion based on two mitochondrial genes and model current and past climate and habitat suitability for seven post-glacial maximum periods, taking 19 climatic variables into account. Consequently, we elucidate their connections by environmental-geographical network analysis. Most analyzed populations share a main haplotype tracing back to a single glacial maximum refuge area on the Mediterranean coasts of South France, which is supported by network analysis. The rapid range expansion of Ph. mascittii likely started in the early mid-Holocene epoch until today and its spread possibly followed two routes. The first one was through northern France to Germany and then Belgium, and the second across the Ligurian coast through present-day Slovenia to Austria, toward the northern Balkans. Here we present a combined approach to reveal glacial refugia and post-glacial spread of Ph. mascittii and observed discrepancies between the modelled and the current known distribution might reveal yet overlooked populations and potential further spread. An integrated approach using phylogeographic data, climate modelling, and network analysis unlocks insights into the post-glacial expansion of the sand fly Phlebotomus mascittii in Europe and suggests two potential (parallel) dispersal routes. [ABSTRACT FROM AUTHOR]

Sandflies (Diptera: Psychodidae) are medically important insects prevalent in tropical and temperate regions of the world. About 30 species of these flies have been recorded from the Western Ghats region of Kerala. While carrying out epidemiological investigations on Leishmaniasis in this region, we found two species of field-collected sandflies viz., Phlebotomus argentipes and Sergentomyia monticola infected with Tylenchid nematode parasites. The infection rates were 0.89% and 0.9%, respectively. The parasite density of nematode juvenile stages was more than 1000 in all the specimens. Both males and females were found infected. We performed molecular characterization of these nematode parasites using multiple genetic markers, mitochondrial COI, and two different regions of 18S rDNA. The genetic analysis revealed that the nematode belonged to the genus Howardula of family Allantonematidae, one infesting P. argentipes (major vector species of Leishmania) and the other, S. monticola. This is the first report of natural infection with the entomo-parasitic nematode species, belonging to Allantonematidae among these sandfly species from India. Genetically related and unclassified species of nematodes belonging to this family had been reported elsewhere from termite species. As larval stages of sandflies develop in the organic matter of termite mounds, this finding may have significant implications on their bionomics and control. [ABSTRACT FROM AUTHOR]