Sand Flies Research Articles

Stability and suitability of housekeeping genes in phlebotomine sand flies

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.

Fabrication and characterization of low-cost ceramic membranes from coal fly ash and natural sand

Coal fly ash, an industrial solid waste product, and natural sand, a commonly available and inexpensive natural material, were used to fabricate ceramic membranes that are both affordable and sustainable. These ceramic membranes were fabricated by the uniaxial compaction technique using a manual hydraulic pressing machine. The effects of various fabrication parameters such as the sintering temperature and the amount of natural sand utilized, on the properties of the resulting ceramic membranes were extensively evaluated. X-ray fluorescence (XRF) analysis of the starting materials confirmed the presence of SiO2 and Al2O3, two key inorganic materials required for the fabrication of ceramic membranes. Heavy metals present in the raw coal fly ash were leached out prior to the fabrication process. The coal fly ash and natural sand were mixed in different proportions and the fabricated ceramic membranes were characterized using Fourier transform infrared spectroscopy (FTIR), Scanning electron microscope (SEM), X-ray diffraction (XRD), Energy-dispersive X-ray spectroscopy (EDS), and Thermogravimetric analysis (TGA). The properties of the optimized membranes were further studied to ascertain their mechanical strength, chemical stability, porosity, water absorption, water permeability, and shrinkage behaviour. The membrane fabricated with 20 wt% sand content and at 1000 °C sintering temperature had pore size of 0.64 µm and 34.7 % porosity, exhibited good mechanical strength (7.71 MPa), and exceptional chemical stability. The membrane also showed a remarkable water permeability of 32.23 L/m2/h. This study showed that natural sand and coal fly ash can be efficiently employed to develop a multifunctional filtration membrane with adjustable properties that can be utilized in water purification.

Co-Infection of Cytomegalovirus and Leishmania without Splenomegaly Resulting in Immunosuppression in an Hiv-Negative Patient.

Leishmaniasis is caused by the parasite Leishmania donovani and transmitted by the bite of the sand fly vector Phlebotomus. This disease is endemic in the Bihar region of India. There are three common forms of the disease - cutaneous, mucosal and visceral leishmaniasis. The most common presentation of this disease is prolonged unexplained fever with hepatosplenomegaly. We report an unusual case of pyrexia of unknown origin (PUO) in a patient who was extensively worked up for PUO. She was found to have low CD4 counts even though serial samples were negative for HIV. Workup for PUO revealed a positive result for cytomegalovirus (CMV) IgM and polymerase chain reaction (PCR), fundoscopy suggestive of CMV retinitis and bone marrow biopsy suggestive of visceral leishmaniasis. Interestingly, there was no evidence of hepatosplenomegaly. She was diagnosed as a case of CMV infection and visceral leishmaniasis resulting in immunosuppression and was managed with parenteral ganciclovir followed by oral valganciclovir and amphotericin respectively. She had a dramatic response to the treatment and was discharged after two months of in hospital management. Co-infection of CMV and Leishmania in an immunocompromised patient with HIV-negative status without hepatosplenomegaly posed a diagnostic dilemma and is a rare presentation. This report shows the importance of diagnosis of this co-infectious state, which upon management with ganciclovir and amphotericin lead to resolution of symptoms and pancytopenia. Clinicians should be aware of the unusual presentation to avoid missing the diagnosis of this potentially life-threatening treatable condition. Visceral leishmaniasis without splenomegaly is a rare presentation.Other aetiologies for low CD4 counts in an HIV-negative patient such as cytomegalovirus (CMV) should be considered.Co-infection of CMV and Leishmania resulting in immunosuppression in an HIV-negative patient is rare and poses a diagnostic dilemma.

Population structure analysis of Phlebotomus papatasi populations using transcriptome microsatellites: possible implications for leishmaniasis control and vaccine development

BackgroundPhlebotomus 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.MethodsIn 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.ResultsThe 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.ConclusionsElucidating 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.Graphical

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.

Wolbachia Natural Infection of Mosquitoes in French Guiana: Prevalence, Distribution, and Genotyping

Wolbachia are the most spread bacterial endosymbionts in the world. These bacteria can manipulate host reproduction or block virus transmission in mosquitoes. For this reason, Wolbachia-based strategies for vector control are seriously considered or have already been applied in several countries around the world. In South America, Wolbachia have been studied in human pathogen vectors such as sand flies and mosquitoes. In French Guiana, the diversity and distribution of Wolbachia are not well known in mosquitoes. In this study, we screened for Wolbachia natural infection in mosquitoes in French Guiana by using 16S rRNA, Wolbachia surface protein (WSP), and multi-locus sequence typing (MLST) molecular assays. A total of 29 out of 44 (65.9%) mosquito species were positive for natural Wolbachia infection according to the PCR results, and two Wolbachia strains co-infected three specimens of Mansonia titillans. Then, we analyzed the phylogenetic relationships among the Wolbachia detected. All of the tested specimens of Aedes aegypti, the major dengue vector of French Guiana, were negative. These results regarding Wolbachia strain, distribution, and prevalence in mosquitoes from French Guiana highlight Wolbachia–mosquito associations and pave the way for a future Wolbachia-based strategy for vector control in this Amazonian territory.

Measuring the Concentration of Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-α) in People infected with Cutaneous leishmaniasis in Tikrit

The study conducted in Salahaddeen Governorate aimed to measure the concentrations of cytokines (interleukin-6 and tumor necrosis factor-alpha) in patients infected with Baghdad boil and to compare these concentrations with those of the control group. The endemic disease of leishmaniasis manifests in three types in Iraq; cutaneous, mucocutaneous and visceral. The most common type is cutaneous leishmaniasis (CL) which causes skin sores. CL attributed to L. tropica is disseminated via the bite of an infected female sand fly. The study was conducted at Salahaddeen Teaching Hospital in Salahaddeen Governorate, Iraq, from March 1, 2021, to September 1, 2022. Detailed data were collected about age, gender, residence, location, number and type of lesions (wet or dry). Sixty clinical cases of cutaneous leishmaniasis were included in the study. The cytokine concentration of interleukin-6 (IL-6) exhibited an increase, reaching 94.7a±7.74 picograms/ml in patients infected with multiple leishmaniasis lesions (7-10 lesions). Conversely, the lowest concentration was observed at 56.3b±7.43 picograms/ml in patients with 1-3 lesions. Furthermore, the concentration of interleukin-6 reached its peak in patients with dry lesions (96.0a±10.9) compared to those with wet lesions (63.3b±12.4). An elevation in tumor necrosis factor (TNF-α) levels was observed across different age groups, with patients infected with cutaneous leishmaniasis typically falling within the age range of 11 to 40. Consistently, the highest TNF-α levels were documented annually in patients with cutaneous leishmaniasis, specifically within the age brackets of 11-20, 21-30 and 31-40, reaching 343.0a±28.3 picograms/ml, 316.7abd±41.2 picograms/ml and 295.9b±51.9 picograms/ml respectively, in comparison to the control group. Conversely, the lowest TNF-α level was noted in patients aged 41 (238.0c±42.6 picograms/ml). Moreover, TNF-α concentration was higher in patients with leishmaniasis in rural areas compared to urban areas. In rural regions, the TNF-α concentration was 436.7a±54.4 picograms/ml.

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

Nocturnal activity and forestry-urban dispersal of phlebotomine sand flies (Diptera: Psychodidae) from an ecotourism park in Amazonian Brazil.

Phlebotomine sand flies are insects of notorious importance in public health, mainly due to their involvement in the transmission of Leishmania protozoa. Their flight activity occurs predominantly in the twilight/night period, being stimulated mainly by the need to search for food and reproduction. Despite being naturally wild, some species are able to invade anthropized environments. Present work aimed to assess the nocturnal activity and forestry-urban dispersal of phlebotomine sand flies from an ecotourism park in Belém, Amazonian Brazil. The study area comprised a horizontal transect, extending from a forest park to the neighboring urban environment, in Belém. Sampling was conducted with night-operating light traps. Nocturnal activity was assessed through time-set captures with a collection bottle rotator in the forest environment. Dispersal was assessed through captures carried out along the transect, starting from the forest edge (0 m), extending to the urban environment (50-200 m), phlebotomine sand flies were identified. Abundance, richness, diversity, and sampling sufficiency were estimated. Fourteen species were recorded in the surveyed environments, with Nyssomyia antunesi, Trichophoromyia brachipyga, and Trichophoromyia ubiquitalis being the most abundant. Nocturnal activity behavior of Ny. antunesi occurred between 8 PM and 4 AM while that of Th. brachipyga and Th. ubiquitalis occurred between 2 AM and 6 AM. In the urban environment, the 150 m site presented the highest abundance. Bichromomyia flaviscutellata was sampled in all sites. Gravid females of Pressatia choti and Bi. flaviscutellata were sampled in the urban environment. Putative differential activity between the species herein assessed and their urban dispersal observed are worthy of note, adding data for supporting vector surveillance at a local scale.

Diversity of Sand Flies and Incidence of Cutaneous Leishmaniasis in Humans and Domestic Animals at Lower Dir, Pakistan

Diversity of Sand Flies and Incidence of Cutaneous Leishmaniasis in Humans and Domestic Animals at Lower Dir, Pakistan

Diversity, vertical stratification and detection of Leishmania DNA in sand flies (Diptera: Psychodidae) collected in the Mapinguari National Park, Brazilian Amazon

This study aimed to assess the diversity patterns of sand fly fauna across different strata and detect Leishmania DNA in these insects in the Mapinguari National Park in Rondônia and Amazonas states, Northern Brazil. Sand flies were collected with “HP” light traps in the canopy (15 m) and at ground level (1 m) on two trails, during August and November 2021 and March and August 2022. Polymerase chain reaction and DNA sequencing were used to identify Leishmania species. A total of 8,040 individuals (2,303♂ – 28.64 %, 5,737♀ – 71.36 %) were collected and 53 species and 13 genera were identified. The most abundant species were Psychodopygus chagasi (21.00 %), Trichophoromyia ubiquitalis (13.61 %) Psychodopygus davisi (11.69 %), Nyssomyia fraihai (10.58 %), Nyssomyia antunesi (6.68 %) and Nyssomyia richardwardi (5.55 %). Species diversity was greater in the canopy (Shannon index H' = 10.8 common species) when compared to ground level (H' = 10.5 common species). We observed a minimum infection rate of 0.45 % (22/4,868 females), in which Leishmania braziliensis DNA was found in Ps. chagasi and Le. lainsoni DNA in Ps. chagasi, Ny. richardwardi, Ps. ayrozai and Th. ubiquitalis. The minimum infection rate of Leishmania in the canopy was 0.47 % (19/4,031) and in the ground was 0.52 % (3/567). In the present study, we observed Le. lainsoni DNA in females of Ny. richardwardi for the first time. The data presented in this study contribute to understanding sand fly diversity and its distribution between the states of Rondônia and Amazonas. They may be useful for implementing targeted control measures to reduce the spread of leishmaniasis and implement entomological surveillance strategies.

Study on Freeze–Thaw Resistance of Cement Concrete with Manufactured Sand Based on BP Neural Network

In this study, experiments were conducted on the freeze–thaw performance of manufactured sand cement concrete with different sand ratios and fly ash contents. The research found that during 200 freeze–thaw cycles, as the fly ash content increased, the concrete exhibited a higher mass loss rate and a decline in the relative dynamic modulus of elasticity. This was due to the lower activity of SiO2 and Al2O3 in the fly ash, which reduced the hydration products. Incorporating an optimal amount of manufactured sand can increase the density of concrete, thereby improving its resistance to freeze–thaw cycles. However, when the content of manufactured sand was high, its large surface area could interfere with the hydration process and reduce strength, thereby diminishing the freeze–thaw resistance of the concrete. Given that studying the freeze–thaw resistance of manufactured sand concrete is time-consuming and influenced by many factors, a prediction model based on a BP (back propagation) neural network was developed to estimate the mass loss rate and the relative dynamic modulus of elasticity following freeze–thaw cycles. After validation, the model was found to be highly reliable and could serve as a foundation for mix design decisions and freeze–thaw performance prediction of manufactured sand cement concrete.

Impact of Indoor Residual Spray (IRS) and Insecticide Treated Nets (ITNs) for sand flies (Diptera: Psychodidae) elimination in some remote communities

Impact of Indoor Residual Spray (IRS) and Insecticide Treated Nets (ITNs) for sand flies (Diptera: Psychodidae) elimination in some remote communities

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

Interaction and effects of temperature preference under a controlled environment on the diversity and abundance of the microbiome in Lutzomyia longipalpis (Diptera: Psychodidae)

Characterization of the temperature effects on the abundance and richness of the microbiota of Lutzomyia longipalpis, insect vector of Leishmania infantum in America, is an aspect of pivotal importance to understand the interactions between temperature, bacteria, and Leishmania infection. We developed and used a customized device with a temperature gradient (21–34 °C) to assess the temperature preferences of wild females of Lu. longipalpis collected in a rural area (Ricaurte, Cundinamarca, Colombia). Each replicate consisted of 50 females exposed to the gradient for an hour. At the end of the exposure time, insects were collected and separated by the temperature ranges selected varying from 21 °C to 34 °C. They were organized in 17 pools from which total DNA extracts were obtained, and samples were subjected to 16S rRNA amplicon sequencing analyzes. The most abundant phyla across the different temperature ranges were Proteobacteria (17.22–90.73 %), Firmicutes (5.99–77.21 %) and Actinobacteria (1.56–59.85 %). Results also showed an abundance (30 % to 57.36 %) of Pseudomonas (mainly at temperatures of 21–29 °C and 34 °C) that decreased to 6.55 %-13.20 % at temperatures of 31–33 °C, while Bacillus increase its abundance to 67.24 % at 29–33 °C. Serratia also had a greater representation (49.79 %), specifically in sand flies recovered at 25–27 °C. No significant differences were found at α-diversity level when comparing richness using the Shannon-Wiener, Simpson, and Chao1 indices, while β-diversity differences were found using the Bray-Curtis index (F-value of 3.5073, p-value < 0.013, R-squared of 0,4889), especially in the groups of Lu. longipalpis associated at higher temperatures (29–33 °C). It was also possible to detect the presence of endosymbionts such as Spiroplasma and Arsenophonus in the range of 29–33 °C. Rickettsia was only detected in Lu. longipalpis sand flies recovered between 25–27 °C. It was possible to characterize Lu. longipalpis microbiota in response to intraspecific temperature preferences and observe changes in bacterial communities and endosymbionts at different ranges of said environmental variable, which may be important in its vector competence and environmental plasticity to adapt to new climate change scenarios.

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.

A study on the diversity of phlebotomine sand flies (Diptera, Psychodidae) in karstic limestone areas in Vientiane Province, Laos, with a description of two new species of Sergentomyia França and & Parrot

BackgroundSoutheast 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.MethodsPhlebotomine 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.ResultsA 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).ConclusionsThis 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

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.

New status of Bichromomyia subspecies (Diptera: Psychodidae: Phlebotominae) based on molecular taxonomy.

The sand fly of the genus Bichromomyia (Galati, 1995) includes 3 subspecies considered vectors of Leishmania, which share high morphological similarity. Through information from the Cytochrome Oxidase Subunit I (COI) gene, we provide complementary evidence to support that Bichromomyia olmeca olmeca, and Bichromomyia olmeca bicolor, should be raised to nominal species status. We recovered specimens of Bi. o. olmeca from Quintana Roo, Tabasco, and Oaxaca, Mexico, supply 17 new COI sequences, and also incorporate GenBank sequences for other Bichromomyia species. After a Maximum Likelihood (ML) analysis, all Bichromomyia species clustered with a bootstrap of 100%, although sequences of Bichromomyia flaviscutellata were divided into 2 clusters with an interspecific range distance of 11.16% between them, which confirm cryptic species in Brazil. The genetic distance of Bi. o. olmeca compared to related subspecies ranged between 12.59% and 14.64%. A total of 29 haplotypes (Hd = 0.987; π = 0.08783; S = 136) were recovered from the Bichromomyia sequences. Results of the TC network were consistent with the ML analysis, supporting that subspecies of Bichromomyia are genetically distinct and deserve being raised to valid species category: Bichromomyia olmeca (Vargas & Díaz-Nájera) and Bichromomyia bicolor (Fairchild & Theodor).