Sand Fly Species Research Articles

Entomological aspects of leishmaniasis transmission in the urban area of Teófilo Otoni, a municipality endemic for the disease in the Brazilian state of Minas Gerais.

Brazil is endemic for both visceral (VL) and cutaneous (CL) clinical forms of leishmaniasis, poverty-associated diseases with worldwide distribution. Leishmania parasites are the etiological agents of leishmaniases, which are transmitted to humans through the bites of infected phlebotomine sand flies. From 2018 to 2023, 15 cases of VL and 129 cases of CL were reported in Téofilo Otoni, an important economic center in the Brazilian state of Minas Gerais. Owing to the lack of data on the entomological fauna, the present study aimed to clarify this main aspect of leishmaniasis. From May, 2021 to April, 2023, entomological captures were performed monthly in ten neighborhoods in Teófilo Otoni. The influence of bioclimatic variables on insect populations was evaluated, and natural infection by Leishmania spp. was investigated using molecular methods. A total of 306 specimens of 12 species of phlebotomine sand fly were collected. The majority (91.6%) were proven or putative vectors of leishmaniasis agents. The population of insects tended to increase during the cooler and drier months. Although Leishmania infection was not detected in any of the samples, the presence of vectors provides conditions for the maintenance and expansion of the transmission cycle of leishmaniasis in Teófilo Otoni.

Description of Trichophoromyia macrisae, a new phlebotomine sand fly species (Diptera: Psychodidae) from Manu Biosphere Reserve, Peru

Phlebotomine sand flies are a highly diverse group of insects capable of transmitting Leishmania parasites. Therefore, the identification of vector species is important to understand local leishmaniasis dynamics. Among the Neotropical sand fly species, the genus Trichophoromyia is predominantly found in the Amazon. A new species of this genus, Trichophoromyia macrisae sp. nov., is described. Specimens were collected using Katchy light traps in forested areas of Manu Biological Station, located within the Manu Biosphere Reserve, in southern Peru. Morphological description was based on 10 male specimens. The disposition of setae in the gonocoxite and the shape of the paramere allows Th. macrisae sp. nov. to be distinguished from other species of genus that have aedeagal ducts > 4 times the length of the sperm pump. The description of Th. macrisae sp. nov. brings the number of species of Trichophoromyia in the Americas to 47 and in Peru to 15.

Phlebotomine sand fly survey, blood meal source identification, and description of Sergentomyia imihra n. sp. in the central Sahara of Algeria

BackgroundPhlebotomine 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.MethodsIn 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.ResultsIn 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.ConclusionsIntegrative 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

Survey of sand fly fauna in six provinces of Southern Vietnam with species identification using DNA barcoding

BackgroundSand 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.MethodsSand 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.ResultsA 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.ConclusionsThis 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.Graphical

The development of L. major, L. donovani and L. martiniquensis, Leishmania currently emerging in Europe, in the sand fly species Phlebotomus perniciosus and P. tobbi.

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. 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. 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.

Toscana Virus in Wild-Caught Sand Flies in Portugal, Findings from the National Vector Surveillance Network, 2023.

Phlebotomine sand flies play a crucial role in both human and veterinary medicine, acting as vectors for Leishmania parasites and most known phleboviruses. In Portugal, the REVIVE program, a comprehensive national surveillance network under the Ministry of Health, has included sand fly surveys since 2016. REVIVE aims to identify existing sand fly species in the country, determine which pathogens are circulating among them, and provide actionable insights for prevention and control measures when necessary. In this way, annually, from May to October, health technicians collect sand flies across mainland Portugal with CDC light traps. The collected sand flies are sent to the Centre for Vectors and Infectious Diseases Research for species identification and molecular screening of pathogens. On 21 September 2023, Toscana virus (TOSV), a well-known phlebovirus in the Mediterranean region due to its capacity to cause neurological disease, was detected in a pool of 30 sand flies collected in Algarve, the southernmost region of Portugal. A 668 bp partial sequence of the nucleoprotein gene shows similarity with TOSV strains from Spain. To our knowledge, this is the first detection of TOSV in its vector in this country, having previously only been reported in vertebrate hosts. These findings highlight the important role of ongoing surveillance efforts in monitoring and understanding the dynamics of sand fly-borne diseases in Portugal.

Bartonella spp. in Phlebotominae Sand Flies, Brazil.

Bartonella spp. are opportunistic, vectorborne bacteria that can cause disease in both animals and humans. We investigated the molecular occurrence of Bartonella spp. in 634 phlebotomine sand fly specimens, belonging to 44 different sand fly species, sampled during 2017-2021 in north and northeastern Brazil. We detected Bartonella sp. DNA in 8.7% (55/634) of the specimens by using a quantitative real-time PCR targeting the 16S-23S internal transcribed spacer intergenic region. Phylogenetic analysis positioned the Lutzomyia longipalpis sand fly-associated Bartonella gltA gene sequence in the same subclade as Bartonella ancashensis sequences and revealed a Bartonella sp. sequence in a Dampfomyia beltrani sand fly from Mexico. We amplified a bat-associated Bartonella nuoG sequence from a specimen of Nyssomyia antunesi sand fly. Our findings document the presence of Bartonella DNA in sand flies from Brazil, suggesting possible involvement of these insects in the epidemiologic cycle of Bartonella species.

The spatial relationship between leishmaniases and sand flies in Europe and neighboring countries

BackgroundLeishmania 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.MethodsWe 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.ResultsThere 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.ConclusionsThe 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

Phlebotomine sand flies (Diptera: Psychodidae) of Bosnia and Herzegovina: distribution, ecology and environmental preferences

Sand flies (Diptera: Phlebotominae) are the principal vectors for the protozoan parasites Leishmania spp. and for phleboviruses. The sand fly fauna on the Balkan Peninsula, including Bosnia and Herzegovina (BIH), is diverse and the circulation of Leishmania infantum as well as phleboviruses has been proven. However, recent data on the sand fly fauna in BIH are scarce. In this study, we surveyed understudied regions in central and northeastern BIH to update the sand fly distribution and gain insights into the ecological and environmental factors shaping their appearance. CDC light trapping was conducted in 2022 and 2023 and a combination of morphological and molecular methods (cytochrome oxidase I barcoding) was performed for species identifications. We mapped the currently known distribution, modelled climatic suitability patterns and performed environmental analyses by applying machine learning methods. In addition, we analyzed blood meals by host gene sequencing and MALDI-TOF peptide mass mapping and screened for Leishmania spp. DNA and Phlebovirus RNA.Altogether, 591 sand flies of four species were trapped, predominantly Phlebotomus neglectus (97 %), but also Ph. balcanicus, Ph. mascittii, and Ph. papatasi. Records of seven sand fly species known to be endemic were plotted onto distribution maps based on 101 datapoints, identifying Ph. neglectus as the overall predominant species. The environmental analyses of sand fly species indicated variation in altitudinal, thermal, and precipitation conditions across the sand fly-positive sites. Phlebotomus simici, Phlebotomus tobbi, and Sergentomyia minuta are typically found exclusively in Mediterranean and subtropical climate zones, whereas other species typically inhabit continental regions. The Inverse Distance Weighted (IDW) interpolation of sand fly species numbers and Shannon entropy values suggested the southeastern coastal region of BIH as a primary focus for sand fly occurrence. This finding was corroborated by modeled average climatic suitability patterns for sand flies, depicting four distinct meso-regions for sand fly occurrence. The results of the ensemble method highlight the importance of annual precipitation to distinguish between positive and negative sand fly trapping sites in BIH. In total, 55 blood meals of two sand fly species, Ph. neglectus and Ph. balcanicus, were analyzed and five host species identified.Our comprehensive assessment of ecological and environmental preferences of sand flies in BIH may support further entomological surveys and help to better understand and evaluate potential hot spots of disease transmission in the country.

Report of a new species of sand fly, Phlebotomus (Anaphlebotomus) ajithii n. sp. (Diptera: Psychodidae), from Western Ghats, India

BackgroundWestern 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.MethodsA 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.ResultsPhlebotomus (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.ConclusionsThe 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.Graphical abstract

A cross-sectional study on phlebotomine sand flies in relation to disease transmission in the Republic of Kosovo.

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.

Abundance and Leishmania infection patterns of the sand fly Psathyromyia cratifer in Southern Mexico.

Localized cutaneous leishmaniasis (LCL) is a serious public health problem in Southern Mexico. Six species of Phlebotominae (Diptera: Psychodidae) have been found to be infected with Leishmania (Leishmania) mexicana, the causative agent of LCL in the region. However, little is known about the biology and potential participation of Psathyromyia cratifer in the Leishmania transmission cycle in Mexico, and the Americas. The present study provides evidence of temporal infection caused by Leishmania in Psathyromyia cratifer as well as data on its population dynamics in a LCL endemic area during the well-known transmission cycle of Leishmania in Southern Mexico. Individual specimens of Psathyromyia cratifer were collected in four sites over the course of five months (from November 2020 through March 2021) using animal-baited, human-baited, and light traps. The temporal activity pattern (month + hour) of Psathyromyia cratifer was assessed along with its relationship with environmental variables. Moreover, Leishmania DNA and blood meals were analyzed and detected in female sand flies. This evidenced an infection rate ranging from 8% to 83%, and the record of Homo sapiens and Ototylomys phyllotis as blood hosts of this sand fly species. High abundances of these sand flies in human-baited traps were recorded which revealed the marked anthropophilic behavior of Psathyromyia cratifer. As regards the transmission dynamics of the parasite within the region, it was observed that the potential highest epidemiological risk for Leishmania transmission by Psathyromyia cratifer occurred during the months of January and March. This is the first contribution ever made to both the population dynamic and the temporal Leishmania prevalence patterns in Psathyromyia cratifer. The resulting findings suggest that this sand fly specimen is the sixth potential vector of L. (L.) mexicana in Southern Mexico. Nonetheless, various biology, behavior, and ecology strands are yet to be addressed. The latter, to determine the role it plays in the transmission dynamics of the parasite within the region, and other areas of the country.

Comparative genomics uncovers the evolutionary dynamics of detoxification and insecticide target genes across 11 phlebotomine sand flies.

Sand flies infect more than one million people annually with Leishmania parasites and other bacterial and viral pathogens. Progress in understanding sand fly adaptations to xenobiotics has been hampered by the limited availability of genomic resources. To address this gap, we sequenced, assembled and annotated the transcriptomes of 11 phlebotomine sand fly species. Subsequently, we leveraged these genomic resources to generate novel evolutionary insights pertaining to their adaptations to xenobiotics, including those contributing to insecticide resistance. Specifically, we annotated over 2,700 sand fly detoxification genes and conducted large-scale phylogenetic comparisons to uncover the evolutionary dynamics of the five major detoxification gene families: Cytochrome P450s (CYPs), Glutathione-S-Transferases (GSTs), UDP-Glycosyltransferases (UGTs), Carboxyl/Cholinesterases (CCEs) and ATP-Binding Cassette (ABC) transporters. Using this comparative approach we show that sand flies have evolved diverse CYP and GST gene repertoires, with notable lineage-specific expansions in gene groups evolutionarily related to known xenobiotic metabolizers. Furthermore, we show that sand flies have conserved orthologs of a) CYP4G genes involved in cuticular hydrocarbon biosynthesis, b) ABCB genes involved in xenobiotic toxicity and c) two primary insecticide targets, acetylcholinesterase-1 (Ace1) and Voltage Gated Sodium Channel (VGSC). The biological insights and genomic resources produced in this study provide a foundation for generating and testing hypotheses regarding the molecular mechanisms underlying sand fly adaptations to xenobiotics.

Sand fly blood meal volumes and their relation to female body weight under experimental conditions

BackgroundSand 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.MethodsUnder 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.ResultsThe 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).ConclusionsFemales 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

Sergentomyia khawi: a potential vector for Leishmania and Trypanosoma parasites affecting humans and animals and insecticide resistance status in endemic areas of Songkhla, southern Thailand

BackgroundSand 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.MethodsSand 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.ResultsThe 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.ConclusionsThe 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.Graphic

Description of Trichophoromyia jariensis, a new species of phlebotomine sand fly (Diptera: Psychodidae) from the eastern Amazon.

A new sand fly species, Trichophoromyia jariensis n. sp. Cavalcante, Rodrigues, & Galati, from the state of Amapá, Brazil, is described based on both male and female morphology and cytochrome c oxidase subunit I DNA barcodes. The DNA barcoding analysis clearly associated males and females of this new species.

Genetic polymorphisms of common sandflies in selected areas of Henan Province based on DNA barcoding

To characterize the species of common sandflies in Henan Province using DNA barcoding with cytochrome c oxidase subunit I (COI) gene as the molecular marker, and to analyze the genetic polymorphisms of sandflies, so as to provide insights into visceral leishmaniasis prevention and control in Henan Province. Sandfly specimens were sampled from 13 sandflies surveillance sites from 2021 to 2023 in Anyang City, Zhengzhou, Luoyang and Xuchang cities (Zhengzhou-Luoyang-Xuchang areas) where visceral leishmaniasis cases were reported and in Jiaozuo and Xinxiang cities (Jiaozuo-Xinxiang areas) without visceral leishmaniasis cases reported. Genomic DNA was extracted from a single sandfly, and COI gene was amplified. The amplification product was subjected to bidirectional sequencing. Following sequence assembly, the species of sandflies was characterized through sequence alignment using the BLAST tool. The intra-specific and inter-specific genetic distances of sandflies were estimated among different areas using the software Mega 11, and phylogenetic trees were created. The polymorphisms of nucleotide sequences in the sandflies COI gene were estimated using the software DnaSP. The fixation index (FST) of different geographical isolates of sandflies was calculated using the Arlequin software, and the gene flow value (Nm) was used to measure the gene flow in the sandflies populations. In addition, the population genetic structure of different geographical populations of Phlebotomus chinensis was analyzed using the STRUCTURE software. A total of 978 sandflies were collected from 13 sandflies surveillance sites in Zhengzhou-Luoyang-Xuchang areas, Jiaozuo-Xinxiang areas and Anyang City of Henan Province from 2021 to 2023, and 475 sandflies were randomly sampled for subsequent detections. A total of 304 Ph. chinensis, 162 Se. squamirostris and 9 Se. bailyi were identified based on molecular biological detection of the COI gene, and Se. bailyi was reported for the first time in Henan Province. The intraspecific genetic distances of sandflies were 0.000 to 0.040, and the inter-specific genetic distances ranged from 0.133 to 0.161. Phylogenetic analysis revealed that each of the three sandfly species was clustered into a clade. The genetic polymorphisms of Ph. chinensis populations varied among different areas, with the highest haplotype diversity (0.966 ± 0.007) and the greatest nucleotide diversity (0.011) in Zhengzhou-Luoyang-Xuchang areas, and the lowest haplotype diversity (0.720 ± 0.091) and nucleotide diversity (0.004) in Anyang City. The dominant haplotype of Ph. chinensis populations was Pch_Hap_2 in Anyang City and Jiaozuo-Xinxiang areas, with moderate genetic differentiation (0.05 < FST < 0.15) and frequent gene exchange (Nm value > 1) between Ph. chinensis populations sampled from Anyang City, and Jiaozuo-Xinxiang areas. Population genetic structure analysis showed that the dominant component of Ph. chinensis populations was K5 in Anyang City and Jiaozuo-Xinxiang areas. No obvious dominant haplotype was observed in Ph. chinensis populations sampled from Zhengzhou-Luoyang-Xuchang areas, which had very high genetic differentiation (FST > 0.25) and little gene exchange (Nm value < 1) with Ph. chinensis populations from Anyang City, and Jiaozuo-Xinxiang areas, with K3 as the dominant component. In addition, there was no significant difference in the genetic polymorphism level among Se. squamirostris populations from the three areas. There are Ph. chinensis, Se. squamirostris and Se. bailyi in Henan Province, and S. bailyi is recorded for the first time in Henan Province by molecular biological assays. There are different levels of genetic differentiation and gene exchange among P. chinensis populations in different areas of Henan Province.

Trypanosoma infection and bloodmeal analysis in post-feeding sand flies across Thailand

Phlebotomine sand flies are recognized as a primary vector of Leishmania and are also suspected vectors of Trypanosoma. The transmission cycle of these parasites relies on the distribution of sand fly vectors, parasites, and reservoir animals. This study aimed to detect Leishmania and Trypanosoma DNA and identify the sources of bloodmeals in post-feeding sand flies captured across Thailand. A total of 42,911 field female sand flies were collected from 11 provinces across Thailand using CDC light traps. Among these, 253 post-feeding sand flies were selected for analysis. The predominant species in this study was Sergentomyia khawi (33.60 %). The DNA was extracted from individual female sand flies. Polymerase chain reaction (PCR), specific to the internal transcribed spacer 1 (ITS1) and the small subunit ribosomal RNA (SSU rRNA) gene regions were used to detect the presence of Leishmania and Trypanosoma DNA, respectively. Additionally, cytochrome c oxidase subunit I (COI) gene region was utilized to identify the sources of host bloodmeals. Leishmania DNA was not detected in any specimens. The analysis of SSU rRNA sequences revealed the presence of Trypanosoma DNA (11.46 %, 29/253) in sand fly samples. Among these samples, T. noyesi (1.58 %, 4/253) was identified in Idiophlebotomus longiforceps and Phlebotomus asperulus, Trypanosoma Anura01+02/Frog2 (1.18 %, 3/253) in Se. khawi, and Trypanosoma Anura04/Frog1 (8.70 %, 22/253) in Se. khawi, Se. hivernus and Grossomyia indica. Bloodmeal analysis utilizing the COI gene revealed a diverse range of vertebrate hosts’ blood, including bird, bat, frog and sun skink. Our findings confirm the presence of Trypanosoma DNA and identify the sources of bloodmeals from vertebrate hosts in various sand fly species, suggesting their potential as possible vectors for Trypanosoma in Thailand. Furthermore, our study is the first to provide molecular evidence using the COI gene to identify frogs as a host blood source for sand flies in Thailand. Further studies focusing on the isolation of live parasites in sand flies to confirm vector potential and examining the role of animal reservoirs will enhance our understanding of the host-parasite relationship and enable more efficient control for disease transmission.

Distribution and seasonal fluctuation of visceral leishmaniasis vectors sandflie in Lüliang City of Shanxi Province in 2023

To investigate the distribution and seasonal fluctuations of visceral leishmaniasis vectors sandflies in Lüliang City, Shanxi Province, so as to provide insights into assessment of the visceral leishmaniasis transmission risk and formulation of visceral leishmaniasis control measures. A total of 12 natural villages were sampled from Shilou County, Lishi District, Lanxian County, Linxian County and Wenshui County in Lüliang City, Shanxi Province from June to September, 2023, and sandflies were captured using light traps from 7 breeding habitats, including farmers' houses, sheep pens, cattle pens, chicken coops, pig pens, mule and horse pens, and loess-cave dwellings. Following morphological identification of the sandfly species, the distribution of sandflies and the seasonal fluctuations of the sandfly density were analyzed. In addition, the Leishmania was detected in sandflies using a real-time fluorescence quantitative PCR assay. A total of 2 831 sandflies were captured with 156 light traps in Lüliang City from June to September, 2023, including 2 638 female sandflies (93.18%) and 193 male sandflies (6.82%), and the average density was 16.91 sandflies/(light-night). The seasonal fluctuations of the sandfly density all appeared a unimodal distribution in all survey sites, and the sandfly density peaked in July and then declined rapidly. Among all types of breeding habitats, the greatest sandfly density was found in sheep pens [39.04 sandflies/(light-night)]. In addition, 4.08% (2/49) of the sandfly samples were tested positive for Leishmania nucleic acid as revealed by the real-time fluorescence quantitative PCR assay. Sandflies were widely distributed in Lüliang City, Shanxi Province in 2023, and the peak of the sandfly density was observed in July, which had a visceral leishmaniasis transmission risk. Intensified surveillance of visceral leishmaniasis and sandfly vectors is required and targeted vector control is recommended.

Mapping the distribution of sandflies and sandfly-associated pathogens in China.

Understanding and mapping the distribution of sandflies and sandfly-associated pathogens (SAPs) is crucial for guiding the surveillance and control effort. However, their distribution and the related risk burden in China remain poorly understood. We mapped the distribution of sandflies and SAPs using literature data from 1940 to 2022. We also mapped the human visceral leishmaniasis (VL) cases using surveillance data from 2014 to 2018. The ecological drivers of 12 main sandfly species and VL were identified by applying machine learning, and their distribution and risk were predicted in three time periods (2021-2040, 2041-2060, and 2061-2080) under three scenarios of climate and socioeconomic changes. In the mainland of China, a total of 47 sandfly species have been reported, with the main 12 species classified into three clusters according to their ecological niches. Additionally, 6 SAPs have been identified, which include two protozoa, two bacteria, and two viruses. The incidence risk of different VL subtypes was closely associated with the distribution risk of specific vectors. The model predictions also revealed a substantial underestimation of the current sandfly distribution and VL risk. The predicted areas affected by the 12 major species of sandflies and the high-risk areas for VL were found to be 37.9-1121.0% and 136.6% larger, respectively, than the observed range in the areas. The future global changes were projected to decrease the risk of mountain-type zoonotic VL (MT-ZVL), but anthroponotic VL (AVL) and desert-type zoonotic VL (DT-ZVL) could remain stable or slightly increase. Current field observations underestimate the spatial distributions of main sandfly species and VL in China. More active surveillance and field investigations are needed where high risks are predicted, especially in areas where the future risk of VL is projected to remain high or increase.