TORC1 is an essential regulator of nutrient-controlled proliferation and differentiation in Leishmania.

Leishmania parasites undergo differentiation between various proliferating and non-dividing forms to adapt to changing host environments. The mechanisms that link environmental cues with the parasite's developmental changes remain elusive. Here, we report that Leishmania TORC1 is a key environmental sensor for parasite proliferation and differentiation in the sand fly-stage promastigotes and for replication of mammalian-stage amastigotes. We show that Leishmania RPTOR1, interacts with TOR1 and LST8, and identify new parasite-specific proteins that interact in this complex. We investigate TORC1 function by conditional deletion of RPTOR1, where under nutrient-rich conditions RPTOR1 depletion results in decreased protein synthesis and growth, G1 cell cycle arrest and premature differentiation from proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. These parasites are unable to respond to nutrients to differentiate into proliferative retroleptomonads, which are required for their blood-meal induced amplification in sand flies and enhanced mammalian infectivity. We additionally show that RPTOR1-/- metacyclic promastigotes develop into amastigotes but do not proliferate in the mammalian host to cause pathology. RPTOR1-dependent TORC1 functionality represents a critical mechanism for driving parasite growth and proliferation.

Characterisation of the antiviral RNA interference response to Toscana virus in sand fly cells.

Toscana virus (TOSV) (Bunyavirales, Phenuiviridae, Phlebovirus, Toscana phlebovirus) and other related human pathogenic arboviruses are transmitted by phlebotomine sand flies. TOSV has been reported in nations bordering the Mediterranean Sea among other regions. Infection can result in febrile illness as well as meningitis and encephalitis. Understanding vector-arbovirus interactions is crucial to improving our knowledge of how arboviruses spread, and in this context, immune responses that control viral replication play a significant role. Extensive research has been conducted on mosquito vector immunity against arboviruses, with RNA interference (RNAi) and specifically the exogenous siRNA (exo-siRNA) pathway playing a critical role. However, the antiviral immunity of phlebotomine sand flies is less well understood. Here we were able to show that the exo-siRNA pathway is active in a Phlebotomus papatasi-derived cell line. Following TOSV infection, distinctive 21 nucleotide virus-derived small interfering RNAs (vsiRNAs) were detected. We also identified the exo-siRNA effector Ago2 in this cell line, and silencing its expression rendered the exo-siRNA pathway largely inactive. Thus, our data show that this pathway is active as an antiviral response against a sand fly transmitted bunyavirus, TOSV.

Visceral Leishmaniasis-Human Immunodeficiency Virus-Coinfected Patients Are Highly Infectious to Sandflies in an Endemic Area in India.

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.

Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

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.

The Phlebotomine sand fly fauna of Switzerland revisited.

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.

Molecular and morphometric study of Brazilian populations of Psychodopygus davisi.

In this study, we analysed the molecular and morphometric differences of several populations of the putative sand fly vector Psychodopygus davisi (Root, 1934) (Diptera, Psychodidae, Phlebotominae) in Brazil. We amplified the 658 base pair fragments of the DNA barcoding region-cytochrome c oxidase subunit 1 (COI) gene-for 57 specimens of P. davisi and three specimens of Psychodopygus claustrei (Abonnenc, Léger & Fauran, 1979). We merged our data with public sequences of the same species available from GenBank. Then, the combined dataset-87 sequences and 20 localities-was analysed using population structure analysis and different species delimitation approaches. Geometric morphometry of wings was performed for 155 specimens of P. davisi populations from the North, Midwest and Southeast Brazilian regions, analysing the differences in centroid sizes and canonical variates. Molecular analysis indicated high intraspecific genetic distance values for P. davisi (maximum p distance = 5.52%). All algorithms identified P. davisi and P. claustrei as distinct molecular taxonomic units, despite the low interspecific distance (p distance to the nearest neighbour = 4.79%). P. davisi sequences were split into four genetic clusters by population structure analysis and at least five genetic lineages using intermediate scenarios of the species delimitation algorithms. The species validation analysis of BPP strongly supported the five-species model in our dataset. We found high genetic diversity in this taxon, which is in agreement with its wide geographic distribution in Brazil. Furthermore, the wing analysis showed that specimens from the Southeast Region of Brazil are different from those in the North and the Midwest. The evolutionary patterns of P. davisi populations in Brazil suggest the presence of candidate species, which need to be validated in future studies using a more comprehensive approach with both genomic data and morphological characters.

Leishmania (Leishmania) infantum DNA detection in Nyssomyia neivai in Vale do Ribeira, Paraná, Brazil.

BACKGROUND: The incidence of visceral leishmaniasis (VL) has increased in the Southern region of Brazil in recent years, especially in the State of Paraná. New species have been suggested with potential to act as vector in VL endemic areas. OBJECTIVES: Identify the Leishmania species in sand fly specimens collected from 2016 to 2018 in the municipality of Itaperuçu, Vale do Ribeira, Paraná, Brazil. METHODS: Light traps were used for collections and for the analysis of sand fly were used the multiplex polymerase chain reaction (PCR) methodology and subsequent sequencing. FINDINGS: Among the collected specimens, 88.62% were attributed to the species Nyssomyia neivai, which were grouped into 176 pools. Three positive pools were detected: two with Leishmania (Viannia) braziliensis and one with L. (Leishmania) infantum. The positivity rate for the parasite was 0.25% based on the presence of at least one infected insect in the pool. MAIN CONCLUSIONS: The detection of L. infantum in Ny. neivai draws attention due to its abundance and anthropophily in the State of Paraná. Moreover, this finding is considered as an alert and suggests that the vector competence of Ny. neivai and the criteria for its incrimination should be carried out, given its wide distribution in southern of Brazil.

Identification of Ochrobactrum as a bacteria with transstadial transmission and potential for application in paratransgenic control of leishmaniasis.

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.

Phlebotomine sand fly (Diptera: Phlebotominae) diversity in the foci of cutaneous leishmaniasis in the Surxondaryo Region of Uzbekistan: 50 years on.

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.

Leishmania donovani Transmission Cycle Associated with Human Infection, Phlebotomus alexandri Sand Flies, and Hare Blood Meals, Israel1.

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA-positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression.

Identification of cuticle and midgut fungal microflora of phlebotomine sandflies collected in Tunisia.

Phlebotomine sand flies (Diptera: Psychodidae) are the proven vectors of Leishmaniases which are widespread parasitosis in many tropical and subtropical countries. The development of infective metacyclic Leishmania (Kinetoplastida: Trypanosomatidae) promastigotes stage is restricted to the vector midgut. Recently, several studies have assessed the influence of the sand fly midgut fungal microflora on the development of invective Leishmania stage. The aim of this study was to identify the fungal microflora from the cuticle and midgut of wild caught sandflies. A total of 50 sandflies were caught in two different leishmaniasis foci of center Tunisia and analyzed using an in vitro isolation of fungi followed by a morphological and molecular identification of fungal isolates. The morphological identification of sandflies specimens revealed five Species: Phlebotomus (P.) papatasi (n = 25), P. perniciosus (n = 15) P. riouxi (n = 6), P. longicuspis (n = 3) and P. sergenti (n = 1). Forty positive fungal cultures were isolated from 34 sand flies (19 males and 15 females) distributed as following: P. papatasi (n = 16), P. perniciosus (n = 11), P. riouxi (n = 4), P. longicuspis (n = 2) and P. sergenti (n = 1). Thirty-five cultures were isolated from the cuticles and five from the guts. A total of 15 fungi genera belonging to 8 families were identified with the predominance of Aspergillus genus followed by Penicillium genus. Among the 15 fungi genera, five were common between males and females specimens. Lecytophora canina and Leishmania major co-infection was detected in the gut of a female P. papatasi. Our preliminary findings highlight the high diversity of fungal microflora from the sand flies midguts.

Species diversity and detection of pathogens in phlebotomine sand flies collected from forest management areas of Quintana Roo, Mexico.

Sand flies have expanded their areas of distribution, thereby increasing the risk of pathogen transmission in non-endemic areas. To establish efficient prevention and control strategies for the transmission of vector-borne pathogens, it is important to understand seasonal dynamics of their vectors. In Mexico, there are several areas where the contact between sand flies, hosts and reservoirs favours the transmission of the pathogen. We compared sand fly communities in a forest management area and a conserved area in Noh-Bec, Quintana Roo, Mexico. The analysis included species diversity, activity peaks and molecular detection of pathogens. Sand flies were collected from November to December 2021 and April to May 2022, during 84 night-traps. The conserved area showed higher numbers and greater species heterogeneity of sand flies as compared with the other sites. The ß-diversity analysis revealed that sites disturbed by logging (S1, S2, S3) had greater similarity (90%) in their sand fly species composition than a conserved area (S4) (similarity = 36%). Although none of the specimens were infected with Leishmania, we detected Wolbachia (19.4%) in all four sites, as well as Bartonella (3.25%) only in the disturbed sites. Further studies on the dynamics of sand fly populations and their association with pathogens are necessary.

Large scale systemic control short-circuits pathogen transmission by interrupting the sand rat (Psammomys obesus)-to-sand fly (Phlebotomus papatasi) Leishmania major transmission cycle.

Systemic control uses the vertebrate hosts of zoonotic pathogens as "Trojan horses," killing blood-feeding female vectors and short-circuiting host-to-vector pathogen transmission. Previous studies focused only on the effect of systemic control on vector abundance at small spatial scales. None were conducted at a spatial scale relevant for vector control and none on the effect of systemic control on pathogen transmission rates. We tested the application of systemic control, using Fipronil-impregnated rodent baits, in reducing Leishmania major (Kinetoplastida: Trypanosomatidae; Yakimoff & Schokhor, 1914) infection levels within the vector, Phlebotomus papatasi (Diptera: Psychodidae; Scopoli, 1786) population, at the town-scale. We provided Fipronil-impregnated food-baits to all Psammomys obesus (Mammalia:Muridae; Cretzschmar, 1828), the main L. major reservoir, burrows along the southern perimeter of the town of Yeruham, Israel, and compared sand fly abundance and infection levels with a non-treated control area. We found a significant and substantial treatment effect on L. major infection levels in the female sand fly population. Sand fly abundance was not affected. Our results demonstrate, for the first time, the potential of systemic control in reducing pathogen transmission rates at a large, epidemiologically relevant, spatial scale.

Host preference and human blood index of Phlebotomus orientalis, an exophilic sand fly vector of visceral leishmaniasis in eastern Sudan.

Visceral leishmaniasis (VL, kala azar), caused by Leishmania donovani, transmitted by Phlebotomus orientalis, is a serious systemic disease that causes high morbidity and mortality rates in Sudan and other parts of East Africa and the world. Despite progress in understanding the epidemiology of the disease in East Africa, little is known about the host preference of P. orientalis in kala azar endemic villages of Sudan, which have some of the highest VL incidence rates in the world. The present study used host choice experiments and blood-meal identification approaches to determine the host preference of P. orientalis in kala azar endemic villages in Gedarif state, eastern Sudan. In the host choice experiment, tent traps were used to compare the attractiveness of cows, donkeys, sheep and goats for host-seeking P. orientalis. In the blood-meal identification study, blood-fed P. orientalis females, captured inside houses and peri-domestic habitats, were subjected to molecular typing using cytochrome b gene (cyt b) amplification and sequence analysis. Cows and donkeys were the most attractive to blood-seeking P. orientalis, followed by goats. Similarly, the blood-meal analysis of P. orientalis showed that the vector preferentially feeds on cows, followed by donkeys, humans and goats. The human blood index of P. orientalis was 19.4% (42/216), indicating a high zoophilic habit of the vector, both inside and outside the houses. Although the order of host preference varied by location, it was clear that cows are the most preferred host of P. orientalis in the area. Results are discussed in relation to the role of domestic/livestock animals in VL zoopotentiation and zooprophylaxis. Inference is made on the potential impact of insecticide treatment of cows in control of the vector and the transmission of VL in Sudan and other parts of East Africa.

Diversity of sandflies in Vidarbha region of Maharashtra, India, a region endemic to Chandipura virus encephalitis.

Background & objectives: Sandflies are implicated as vectors of Chandipura virus (CHPV) (Vesiculovirus: Rhabdoviridae). The virus is prevalent in central India including Vidarbha region of Maharashtra. CHPV causes encephalitis in children below 15 yr of age with case fatality rates ranging from 56 to 78 per cent. The present study was undertaken to determine the sandfly fauna in the CHPV endemic Vidharba region. Methods: A year round survey of sandflies was conducted at 25 sites in three districts of Vidarbha region. Sandflies were collected from their resting sites using handheld aspirators and identified using taxonomical keys. Results: A total of 6568 sandflies were collected during the study. Approximately 99 per cent of the collection belonged to genus Sergentomyia, which was represented by Ser. babu, Ser. bailyi and Ser. punjabensis. Genus Phlebotomus was represented by Ph. argentipes and Ph. papatasi. Ser. babu was the predominant species (70.7%) collected during the study. Ph. argentipes was detected in four villages with 0.89 per cent, whereas Ph. papatasi was detected in only one village with 0.32 per cent of the total collection. CHPV could not be isolated despite processing all the sandflies for virus isolation in cell culture. Interpretation & conclusions: The present study showed influence of higher temperature and relative humidity on sandfly population dynamics. An important observation during the study was the absence or decline in the population of Ph. papatasi and Ph. argentipes in the study area. Surge in Sergentomyia population and their breeding/resting in close vicinity to humans pose a concern as they are known to harbour CHPV and other viruses of public health importance.

Determination of the feeding behavior of Phlebotomus sergenti using multiplex PCR and tent-baited traps in a new focus of Anthroponotic Cutaneous Leishmaniasis in the southeast of Iran.

OBJECTIVES: This study aimed to determine feeding behaviors of Phlebotomus sergenti Parrot, in a new focus of Anthroponotic Cutaneous Leishmaniasis in Bam County, southeast Iran. METHODS: Two methods were used to determine the feeding behavior of Phlebotomus sergenti. In the first method, blood-fed sand flies were captured using a mouth aspirator in human and animal dwellings and consequently, blood meal identification was made using Multiplex PCR. The results were used for calculating Host Feeding Index (HFI) and Forage Ratio (FR) parameters. In the second method, human (Homo sapiens), goat (Capra aegagrus), cattle (Bos taurus), chicken (Gallus gallus) and dog (Canis lupus) were used as baits in tent-baited traps to determine the feeding behavior of Phlebotomus sergenti. RESULTS: Multiplex PCR analysis revealed that the most frequent blood in the stomack of sand flies' were from chicken, but the calculation of the FR revealed that this species prefers canine and poultary blood as meal. Human and animal tent-baited traps revealed that most Phlebotomus sergenti were attracted to chicken rather than the other hosts. CONCLUSIONS: Sand flies are attracted to animals for various reasons such as eating blood, mating on their bodies and laying eggs on their feces. Molecular methods are effective and accurate methods to determine the type of host that sandfly fed on, but they do not show host preferences. The results of the molecular analysis, along with the calculation of HFI and FR, can determine the preferred host of sand flies. The current study revealed that dogs, the secondary reservoir of ACL in Iran, is the first preferred host of Phlebotomus sergenti.

Color preference of Sergentomyia minuta (Diptera: Phlebotominae) determined using Flebocollect Do It Yourself light traps based on LED technology.

Whether phlebotomine sand flies show a preference for different light colors remains controversial. As light-capture methods are widely used to study sand flies, knowing the visual stimuli they respond to could help the design of novel control tools to prevent their attraction to hosts. We have detected a significant preference of male Sergentomyia minuta for green and red light sources. Accordingly, male S. minuta were 2.16 and 2.01 times more likely to be lured by Flebocollect model traps with green and red diode-lights, respectively, than the commercial CDC traps. Flebocollect traps are homemade light traps developed through citizen science. Dipterans are widely considered unable to distinguish the color red so this finding was unexpected. To our knowledge, this is the first description of a color preference in a species of the genus Sergentomyia. Our research also confirms the great potential of Flebocollect light traps for use in medical entomology studies.

Field evaluation of a new suction light trap for the capture of phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae), vectors of leishmaniasis.

Phlebotomine sand flies are crepuscular and nocturnal small dipteran insects in the family Psychodidae. Several disease agents, including Leishmania parasites, are transmitted to humans and other vertebrate hosts by the bite of an infected female sand fly. As part of leishmaniasis surveillance programs, light traps have been routinely used in sand fly collections. In this context, new trapping devices are always being required to improve vector monitoring. Here, the efficiency of a new suction light trap, named Silva suction trap or SS trap, was field evaluated in collecting sand flies. Two SS traps, one with green (520 nm, 15,000 mcd) and the other with white (wide spectrum, 18,000 mcd) LEDs, and one CDC-type trap were deployed in a rural forested environment. A total of 4686 phlebotomine sand flies were captured. The most frequent species were females of the Ps. Chagasi series (77.8%) followed by males of Ps. wellcomei (11.6%), Nyssomyia whitmani (3.3%), and Bichromomyia flaviscutellata (2.4%). The CDC-type light trap collected 101.9 ± 20.89 sand flies and 14 species, followed by the white-baited SS trap (87.78 ± 16.36, 14), and the green-baited SS trap (70.61 ± 14.75, 15), but there were no statistically significant differences among traps. A discussion on the considerable advantages of the use of SS traps over CDC traps is included. In this study, the Silva suction trap proved to be efficient and can be an alternative to CDC traps for monitoring adult phlebotomine sand fly populations.

Blood meal analysis and molecular detection of mammalian Leishmania DNA in wild-caught Sergentomyia spp. from Tunisia and Saudi Arabia.

Phlebotomine sand flies (Diptera: Phlebotominae) belonging to the genus Phlebotomus are vectors of pathogens such as arboviruses, bacteria, and parasites (Leishmania). Species of the genus Sergentomyia (Se.) transmit Sauroleishmania (Reptile Leishmania) and feed on cold-blooded vertebrates; recently, they have been incriminated in mammalian Leishmania transmission. In addition, they have been reported to feed on warm-blooded vertebrates. This study aimed to (i) screen wild-caught Sergentomyia species for the detection of mammalian Leishmania and (ii) identify the blood meal origin of engorged females. The sand flies were collected using centers for disease control and prevention (CDC) traps, mounted and identified morphologically. Only females of the genus Sergentomyia were screened for Leishmania infection using PCR targeting the 18S ribosomal DNA locus. For positive specimens, Leishmania parasites were typed using nested PCR targeting ribosomal internal transcribed spacer 1 followed by digestion with HaeIII. The PCR-RFLP results were confirmed through sequencing. Blood meal identification was performed through PCR amplification of the vertebrate cytochrome b gene using degenerate primers followed by sequencing. In total, 6026 sand fly specimens were collected between 2009 and 2018. Among these, 511 belonged to five species of Sergentomyia genus: Se. minuta (58.51%), Se. fallax (18.01%), Se. clydei (14.68%), Se. dreyfussi (6.26%), and Se. antennata (2.54%). A total of 256 female Sergentomyia sp. specimens were screened for Leishmania infection. Seventeen (17) were positive (6.64%). Two Leishmania species were identified. Leishmania major DNA was detected in five specimens; this included three Se. fallax, one Se. minuta, and one Se. dreyfussi collected from Tunisia. Leishmania infantum/L. donovani complex was detected in four Se. minuta and three Se. dreyfussi specimens collected from Tunisia. In addition, we identified the blood meal origin of five engorged Se. minuta specimens collected from Tunisia. Sequencing results revealed two blood sources: humans (n = 4) and reptiles (n = 1) indicating possible role of Sergentomyia species in the transmission of human Leishmania. In addition, these species could be involved in the life cycle of L. infantum/L. donovani complex and L. major. The results of the blood meal origin showed that Sergentomyia fed on both cold- and warm-blooded vertebrates. These findings enable a better understanding of the behavior of this sand fly genus. Further studies should focus on the role of Sergentomyia in human Leishmania transmission and possible control of this disease.

Genetic diversity and haplotype analysis of Leishmania tropica identified in sand fly vectors of the genera Phlebotomus and Sergentomyia using next-generation sequencing technology.

Next-generation sequencing (NGS) was used to investigate the genetic diversity of Leishmania tropica in the sand fly vector, targeting the internal transcribed spacer 1 (ITS1) of the genus Leishmania. Bioinformatics analyses were conducted using Galaxy, MEGA version X, DnaSP ver. 6.12.03, and PopART 1.7 software for NGS analysis, phylogenetic tree, genetic diversity, and haplotype networking, respectively. A total of 307 engorged sand flies were trapped, with an overall Leishmania infection rate of 9.4 (29/307) and 6.8% by NGS and ITS1-PCR, respectively. Two Leishmania-infected sand fly genera were identified: Phlebotomus (10.2%, 26/254) and Sergentomyia (5.7% (3/53). The phylogenetic tree showed two clusters, cluster I included the four study sequences along with 25 GenBank-retrieved DNA sequences. Cluster II consisted of three sequences from Iran and Pakistan. The genetic diversity analysis for the 29 L. tropica sequences showed high haplotype (gene) diversity index (Hd) (0.62 ± 0.07) but low nucleotide diversity index (π) (0.04 ± 0.01). Tajima's D, a neutrality test, is more negative in cluster I (D = - 2.0) than in total population (D = - 1.83), but both are equally significant (P < 0.001), indicating that observed variation in cluster I and whole population is less frequent than expected. The median-joining haplotype network produced a total of 11 active haplotypes. In conclusion, L. tropica from sand flies in Palestine is monophyletic that assembled in one main phylogroup and one haplotype.