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.

Molecular monitoring of insecticide resistance in major disease vectors in Armenia.

BACKGROUND: Armenia is considered particularly vulnerable to life-threatening vector-borne diseases (VBDs) including malaria, West Nile virus disease and leishmaniasis. However, information relevant for the control of the vectors of these diseases, such as their insecticide resistance profile, is scarce. The present study was conducted to provide the first evidence on insecticide resistance mechanisms circulating in major mosquito and sand fly populations in Armenia. METHODS: Sampling sites were targeted based mainly on previous historical records of VBD occurrences in humans and vertebrate hosts. Initially, molecular species identification on the collected vector samples was performed. Subsequently, molecular diagnostic assays [polymerase chain reaction (PCR), Sanger sequencing, PCR-restriction fragment length polymorphism (RFLP), quantitative PCR (qPCR)] were performed to profile for major insecticide resistance mechanisms, i.e. target site insensitivity in voltage-gated sodium channel (vgsc) associated with pyrethroid resistance, acetylcholinesterase (ace-1) target site mutations linked to organophosphate (OP) and carbamate (CRB) resistance, chitin synthase (chs-1) target site mutations associated with diflubenzuron (DFB) resistance and gene amplification of carboxylesterases (CCEs) associated with resistance to the OP temephos. RESULTS: Anopheles mosquitoes were principally represented by Anopheles sacharovi, a well-known malaria vector in Armenia, which showed no signs of resistance mechanisms. Contrarily, the knockdown resistance (kdr) mutations V1016G and L1014F/C in the vgsc gene were detected in the arboviral mosquito vectors Aedes albopictus and Culex pipiens, respectively. The kdr mutation L1014S was also detected in the sand fly, vectors of leishmaniasis, Phlebotomus papatasi and P. tobbi, whereas no mutations were found in the remaining collected sand fly species, P. sergenti, P. perfiliewi and P. caucasicus. CONCLUSIONS: This is the first study to report on molecular mechanisms of insecticide resistance circulating in major mosquito and sand fly disease vectors in Armenia and highlights the need for the establishment of systematic resistance monitoring practices for the implementation of evidence-based control applications.

Comparative genomics of Leishmania donovani progeny from genetic crosses in two sand fly species and impact on the diversity of diagnostic and vaccine candidates.

Sand fly transmitted Leishmania species are responsible for severe, wide ranging, visceral and cutaneous leishmaniases. Genetic exchange can occur among natural Leishmania populations and hybrids can now be produced experimentally, with limitations. Feeding Phlebotomus orientalis or Phlebotomus argentipes on two strains of Leishmania donovani yielded hybrid progeny, selected using double drug resistance and fluorescence markers. Fluorescence activated cell sorting of cultured clones derived from these hybrids indicated diploid progeny. Multilocus sequence typing of the clones showed hybridisation and nuclear heterozygosity, although with inheritance of single haplotypes in a kinetoplastid target. Comparative genomics showed diversity of clonal progeny between single chromosomes, and extraordinary heterozygosity across all 36 chromosomes. Diversity between progeny was seen for the HASPB antigen, which has been noted previously as having implications for design of a therapeutic vaccine. Genomic diversity seen among Leishmania strains and hybrid progeny is of great importance in understanding the epidemiology and control of leishmaniasis. As an outcome of this study we strongly recommend that wider biological archives of different Leishmania species from endemic regions should be established and made available for comparative genomics. However, in parallel, performance of genetic crosses and genomic comparisons should give fundamental insight into the specificity, diversity and limitations of candidate diagnostics, vaccines and drugs, for targeted control of leishmaniasis.

Reconstructing the post-glacial spread of the sand fly Phlebotomus mascittii Grassi, 1908 (Diptera: Psychodidae) in Europe.

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

Genetic diversity and phylogeography of Phlebotomus argentipes (Diptera: Psychodidae, Phlebotominae), using COI and ND4 mitochondrial gene sequences.

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

Molecular surveillance of insecticide resistance in Phlebotomus argentipes targeted by indoor residual spraying for visceral leishmaniasis elimination in India.

Molecular surveillance of resistance is an increasingly important part of vector borne disease control programmes that utilise insecticides. The visceral leishmaniasis (VL) elimination programme in India uses indoor residual spraying (IRS) with the pyrethroid, alpha-cypermethrin to control Phlebotomus argentipes the vector of Leishmania donovani, the causative agent of VL. Prior long-term use of DDT may have selected for knockdown resistance (kdr) mutants (1014F and S) at the shared DDT and pyrethroid target site, which are common in India and can also cause pyrethroid cross-resistance. We monitored the frequency of these marker mutations over five years from 2017-2021 in sentinel sites in eight districts of north-eastern India covered by IRS. Frequencies varied markedly among the districts, though finer scale variation, among villages within districts, was limited. A pronounced and highly significant increase in resistance-associated genotypes occurred between 2017 and 2018, but with relative stability thereafter, and some reversion toward more susceptible genotypes in 2021. Analyses linked IRS with mutant frequencies suggesting an advantage to more resistant genotypes, especially when pyrethroid was under-sprayed in IRS. However, this advantage did not translate into sustained allele frequency changes over the study period, potentially because of a relatively greater net advantage under field conditions for a wild-type/mutant genotype than projected from laboratory studies and/or high costs of the most resistant genotype. Further work is required to improve calibration of each 1014 genotype with resistance, preferably using operationally relevant measures. The lack of change in resistance mechanism over the span of the study period, coupled with available bioassay data suggesting susceptibility, suggests that resistance has yet to emerge despite intensive IRS. Nevertheless, the advantage of resistance-associated genotypes with IRS and under spraying, suggest that measures to continue monitoring and improvement of spray quality are vital, and consideration of future alternatives to pyrethroids for IRS would be advisable.

Morphological and molecular evidence of sandfly (Diptera: Psychodidae: Phlebotomine) and its relevance to recent cases of leishmaniasis from Jammu region of North India.

Present study was conducted to carry out morphological and molecular confirmation of sandflies collected at the Department of Parasitology, Faculty of Veterinary and Animal Sciences, R.S. Pura, Jammu, India. Larva was maggot like with large head, thorax and abdomen with typical black head, 12 abdominal segments and last abdominal segment carried two pairs of caudal bristles with matchstick hairs on each segment. The adult fly possessed head, abdomen and thorax. Head consisted of pair of long, hairy and beaded antenna, proboscis and one pair of prominent black eyes. Thorax possessed a pair of wings and three pairs of legs, wings were hairy and pointed with 2nd longitudinal vein branched twice. The abdominal segments were covered with small hairs and last abdominal segment was having a pair of anal recti. These identification characteristics confirmed the fly under study as Phlebotomus argentipes that confirms its occurance in this region. Molecular characterization of identified flies was carried out on positive morophological flies. Confirmation of Phlebotomus species was ascertained by amplifying the 18S ribosomal RNA gene sequence using PCR. Clear amplification was observed for Phlebotomus argentipes (538 bp). After sequencing/genotyping, Phlebotomus argentipes (OP646634) isolate of present study was clustering in same clade with Phlebotomus argentipes sequences obtained from GeneBank from other locations across globe, irrespective of their geographical location, thus providing the molecular evidence of this species present in Jammu region of North India.

Detection of Leishmania DNA in Phlebotomine Sand Flies in Tsatee, a Community in the Volta Region, Ghana.

Leishmania parasites, which are spread by infected female sand flies, are the cause of the disease leishmaniasis. Although cutaneous leishmaniasis has been found to occur in the Volta Region, there is limited data on vector species and reservoirs. This study focused on the Tsatee community, in the South Dayi District of the Volta Region, and is aimed at identifying the sand fly fauna and detecting the presence of Leishmania DNA by the use of primers that target the conserved region of Leishmania spp. minicircle DNA of the parasite kinetoplast. The miniature light traps and hand aspirators provided by the Centers for Disease Control and Prevention (CDC) were used to collect outdoor and indoor sand flies for five months in a guinea woodland and semideciduous forest area. From the collections, 4,580 phlebotomine sand flies were obtained and identified, and females were examined for Leishmania DNA using PCR. The male flies were 1,202 (26.24%), non-blood-fed females were 3,321 (72.51%), and 57 (1.25%) were blood-fed females. It was observed that Sergentomyia species constituted 99.91% of the total collected sand flies with S. africana (76.77%) as the predominant species. Phlebotomus rodhaini (0.09%) was the only Phlebotomus species identified from the study area. From 283 non-blood-fed sand fly pools and 57 individual blood-fed species screened, Leishmania DNA was detected in 12 (4.24%) pools and 8 (14.04%) individuals, respectively. It was observed that Leishmania DNA was detected in all the sand fly species identified except S. collarti. This study reports the first detection of Leishmania DNA in P. rodhaini in Ghana, with an infection rate of 33.33% (95% CI, 1.23-88.32). The findings suggest that the role of Phlebotomus in disease transmission in the study area cannot be discounted. Future studies should include continuous surveillance, blood meal preferences, and vector competence of the various infected phlebotomine sand flies to create effective control measures.

Trichophoromyia auraensis: evidence for cryptic species and first record in the state of Maranhão, Brazil.

Trichophoromyia auraensis (Mangabeira, 1942) (Diptera, Psychodidae, Phlebotominae) has a wide geographic distribution in the western region of the Amazon biome, where it is a putative Leishmania vector. Here, we reported for the first time a population of this species in the Brazilian state of Maranhão, in the eastern Amazon, from which we DNA-barcoded and compared with previously processed specimens from Acre State, in the western Amazon. For this, we analyzed the DNA barcoding fragment (658 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear internal transcribed spacer 2 (ITS2) of Trichophoromyia species using phylogenetic gene trees, and species delimitation algorithms. The analyses of COI barcodes showed high values of genetic distance (mean K2P = 5.17) and well-supported clades/MOTUs for the eastern and western populations of T. auraensis, which may indicate a possible complex of cryptic species. The western population of this taxon merged with the close-related sand fly Trichophoromyia velezbernali Posada-López, Galvis and Galati, 2018 from Colombia, which may be associated with the recent speciation history and introgression between these populations. These evidences should be evaluated with a more comprehensive sampling in terms of analyzed populations and molecular markers.

On abnormal Phlebotomus perniciosus (Diptera: Psychodidae: Phlebotominae) from the center of Tunisia.

Phlebotomus perniciosus is the most important vector of Leishmania infantum in the Western part of the Mediterranean basin. Atypical specimens of Ph. perniciosus called (pna) with a parameral sheath simply curved, not bifurcated, have been reported in many locations. In this study, we describe abnormal Ph. perniciosus male specimens. Sand flies were collected in center Tunisia and identified morphologically. Cytochrome b PCR-sequencing was carried out for abnormal Ph. perniciosus male specimens in order to confirm the morphological identification and assess the intraspecific genetic polymorphism. Abnormal Ph. perniciosus specimens were characterized by a multifurcated parameral sheath. A parsimonious haplotype network based on cyt b locus analysis showed that typical and abnormal Ph. perniciosus described in our investigation were grouped together in the same branch. Thus, genetic outcomes confirmed that the new phenotype is only an original morphotype of Ph. perniciosus.

Low genetic heterogeneity of Leishmania major in different geographical regions of Iran.

To examine the genetic diversity of Leishmania major, 100 Giemsa-stained positive slides were collected from endemic foci of Iran (Northeast, Central, and Southwest provinces) over two consecutive years during 2019-2021. The Leishmania ITS-rDNA gene was amplified and Leishmania sp. was recognized by PCR-RFLP and sequencing. In addition, 178 registered ITS-rDNA sequences from other geographical regions of Iran were retrieved from GenBank, including different host species (human, sandfly and rodent). A total of 40 new haplotypes were discovered using the ITS-rDNA sequence analysis. IR29 (20.6%) and IR34 (61%) were the two most common haplotypes, represented by a star-like feature in the overall population. Analysis of the molecular variance test revealed low genetic diversity of L. major in human cases (Haplotype diversity; 0.341), rodent (Hd; 0.387) and sandfly (Hd; 0.390) sequences. The lowest genetic diversity of L. major was observed in Southwest/Southeast Iran (Hd: 0.104-0.286). The statistically Fst value indicated that L. major is not genetically differentiated between geographic regions of Iran, except for the Northeast-Southwest (Fst: 0.29055) and Central-Southwest (Fst: 0.30294) population pairs. The current study as the first investigation discloses new perspectives for further evaluation in the identification local transmission paradigms and initiating effective prevention strategies.

Molecular identification of Phlebotomus kandelakii apyrase and assessment of the immunogenicity of its recombinant protein in BALB/c mice.

Sand fly salivary proteins have immunomodulatory and anti-inflammatory features; hence, they are proven to perform important roles in the early establishment of Leishmania parasite in the vertebrate host. Among them, salivary apyrase with anti-hemostatic properties has a crucial role during the blood meal process. In the present study, a Genome-Walking method was used to characterize a full-length nucleotide sequence of Phlebotomus (P.) kandelakii apyrase (Pkapy). Bioinformatics analyses revealed that Pkapy is a ~ 36 kDa stable and hydrophilic protein that belongs to the Cimex family of apyrases. Moreover, recombinant proteins of Pkapy and P. papatasi apyrase (Ppapy) were over-expressed in Escherichia coli BL2 (DE3) and their antigenicity in BALB/c mice was evaluated. Dot-blot and ELISA results indicated that both recombinant apyrases could induce antibodies in BALB/c. Moreover, a partial cross-reactivity between Pkapy and Ppapy was found. In vitro stimulation of splenocytes from immunized mice with the recombinant proteins indicated cross-reactive T cell proliferative responses. Cytokine analysis revealed significant production of IFN-γ (p < 0.001) and IL-10 (p < 0.01) in response to Pkapy. In conclusion, the full-length nucleotide sequence and molecular characteristics of Pkapy were identified for the first time. Immunologic analyses indicated that Pkapy and Ppapy are immunogenic in BALB/c mice and show partial cross-reactive responses. The immunity to Pkapy was found to be a Th1-dominant response that highlights its potential as a component for an anti-Leishmania vaccine.

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.

The Role of the Leishmania infantum Infected Dogs as a Potential Reservoir Host for Toscana Virus in a Zoonotic Visceral Leishmaniasis Focus of Northern Tunisia.

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of zoonotic visceral leishmaniasis (ZVL) located in Northern Tunisia from June to October 2020. At the end of the exposition period, infected and healthy dogs were examined for TOSV and L. infantum infections by xenodiagnosis using a colony of Phlebotomus perniciosus. Pools of freshly engorged P. perniciosus at days 0 and those at days 7 post-feeding were screened for TOSV and L. infantum by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. In the exposure site, P. pernicious is the most abundant sandfly species. The infection rates of sandflies with TOSV and L. infantum were 0.10 and 0.05%, respectively. Leishmania infantum DNA and TOSV RNA were detected in P. perniciosus females fed on dog B and C, respectively. The isolation of TOSV in Vero cells was achieved from two pools containing P. perniciosus fed on dog C. No pathogens were detected in P. perniciosus females fed on dog A and on control dog. We report for the first time the reservoir competence of dog with ZVL in the transmission of TOSV to sandfly vectors in natural settings, in addition to its role as a main reservoir host of L. infantum.

Evaluation of expression variations in virulence-related genes of Leishmania major after several culture passages compared with Phlebotomus papatasi isolated promastigotes.

Cutaneous leishmaniasis (CL) is a prevalent infectious disease with considerable morbidity annually. Here, we aimed to investigate the likely variations in gene expression of glycoprotein63 (gp63), heat shock protein 70 (HSP70), histone, arginase, cysteine protease B (CPB), Leishmania homologue of receptors for activated C kinase (LACK), small hydrophilic endoplasmic reticulum-associated protein (SHERP) in metacyclic promastigotes of L. major isolated from Phlebotomus papatasi sand flies and promastigotes excessively cultured in culture medium. The parasites were collected from suspected CL cases in Pasteur Institute of Iran, cultured and inoculated into the female BALB/c mice (2×106 promastigotes). Sand flies were trapped in Qom province, fed with the blood of euthanized infected mice and subsequently dissected in order to isolate the midgut including stomodeal valve. The metacyclic promastigotes were isolated from Ph. papatasi (Pro-Ppap) using peanut agglutinin test (PNA), then continuously cultured in RPMI-1640 medium enriched with fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) to reach stationary phase (Pro-Stat). The gene expression was evaluated in both parasitic stages (Pro-Ppap and Pro-Stat) using qRT-PCR. Out results showed a significant increased gene expression at Pro-Ppap stage for gp63 (P = 0.002), SHERP (P = 0.001) and histone (P = 0.026) genes, in comparison with Pro-Stat stage. Noticeably, significant changes were, also, demonstrated in 10th to 15th passages [gp63 (P = 0.041), arginase (P = 0.016), LACK (P = 0.025)] and in 5th to 20th passage (SHERP) (P = 0.029). In conclusion, the findings of the present study seem to be essential in designing Leishmania studies, in particular regarding host-parasite interaction, immunization and infectivity studies.

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.

DNA barcoding of sand flies (Diptera, Psychodidae, Phlebotominae) from the western Brazilian Amazon.

The subfamily Phlebotominae comprises important insects for public health. The use of complementary tools such as molecular taxonomy is necessary for interspecific delimitation and/or discovery of cryptic species. Here, we evaluated the DNA barcoding tool to identify different species in the southwestern Brazilian Amazon. For this, we collected sand flies in forest fragments along the highway BR-317, in the municipality of Brasiléia, state of Acre, Brazil. The specimens were DNA-barcoded using a fragment of the cytochrome c oxidase subunit I (COI) gene. The sequences were analyzed to generate K2P pairwise genetic distances and a Neighbour-joining tree. The sand fly barcodes were also clustered into Molecular Operation Taxonomic Units (MOTU) using Automatic Barcode Gap Discovery (ABGD) approach. A total of 59 COI sequences comprising 22 nominal species and ten genera were generated. Of these, 11 species had not been sequenced before, thus being new COI sequences to science. Intraspecific genetic distances ranged between 0 and 4.9%, with Pintomyia serrana presenting the highest values of genetic distance, in addition to having been partitioned into three MOTUs. Regarding the distances to the nearest neighbour, all species present higher values in relation to the maximum intraspecific distance, in addition to forming well supported clusters in the neighbour-joining analysis. The DNA barcoding approach is useful for the molecular identification of sand flies from Brasiléia, state of Acre, and was efficient in detecting cryptic diversity of five species which can be confirmed in future studies using an integrative approach. We also generated new COI barcodes for Trichophoromyia auraensis, Nyssomyia shawi, and Psychodopygus paraensis, which may play a role in the transmission of Leishmania spp. in the Brazilian Amazon.

Monitoring of Insecticide Resistance Mutations and Pathogen Circulation in Sand Flies from Emilia-Romagna, a Leishmaniasis Endemic Region of Northern Italy.

The continuously expanding distribution of sand flies, proven vectors of Leishmania and of several phleboviruses, is a growing public health issue in Europe. Especially in Italy, visceral leishmaniasis (VL) is occurring with increasing incidence northward, in previously non-endemic provinces. Around the globe, disease elimination efforts largely focus on sand fly vector insecticidal control, often leading to the development of resistance. In Emilia-Romagna (ER), northern Italy, insecticides are heavily applied for agricultural and mosquito control, but not specifically against sand flies. Here, we investigated the sand fly species composition in certain environmental settings in ER provinces and monitored the presence of pyrethroid resistance mutations and pathogen circulation. Phlebotomus perfiliewi, a dominant vector of Leishmania infantum, was detected almost exclusively in the region. No mutations in the voltage-gated sodium channel gene, e.g., knock-down resistance mutations I1011M, L1014F/S, V1016G, or F1020S, were recorded. Pathogen monitoring revealed that almost 40% of the tested sand fly pools were positive for Leishmania, while the presence of Toscana and Fermo phleboviruses was also observed in much lower frequencies (≤3% positive pools). Regular epidemiological and entomological monitoring, alongside resistance surveillance, is highly recommended to ensure the sustainability and efficiency of vector control interventions.

Population Genetic Structure of Phlebotomus sergenti (Diptera: Psychodidae) Collected in Four Regions of Morocco Based on the Analysis of Cyt b and EF-1α Genes.

Phlebotomus (Ph.) sergenti is the main vector of Leishmania (L.) tropica (Trypanosomatida: Trypanosomatidae), the causative agent of anthroponotic cutaneous leishmaniasis in Morocco. This species has an extended geographical distribution, wider than that of the parasite. The main objective of our study was to analyze the genetic diversity of Ph. sergenti collected in four foci in Morocco: Taza, Foum Jemâa, El Hanchane, and Ouarzazate. We studied a set of diversity and population structure indices by sequencing two markers; nuclear EF-1α and mitochondrial Cyt b from 175 individual sand flies. Our results showed a considerable degree of intraspecific polymorphism with a high number of haplotypes identified in both genes. Many polymorphic sites detected in the Cyt b sequences (SCyt b = 45) indicate that it is the most polymorphic marker showing a distinct distribution of haplotypes according to their geographical origin, whereas the EF-1α marker showed no geographical isolation. Analysis by Tajima's D and Fu's Fs tests revealed a possible recent expansion of the populations, especially with the EF-1α marker, showing significant values in Taza and Ouarzazate sequences. The present study revealed significant genetic diversity within Ph. sergenti populations in Morocco. The results warrant further research using a combination of more than two markers including mitochondrial and non-mitochondrial markers, which may provide more information to clarify the genetic status of Ph. sergenti.

Differential expression of Phlebotomus tobbi Adler, Theodor & Lourie, 1930 (Diptera: Psychodidae) genes under different environmental conditions.

Phlebotomus tobbi is a widely distributed sand fly species in Turkey and is the proven vector of Leishmania infantum and several Phleboviruses. Information regarding the genetic basis of phenotypic plasticity is crucial for managing vector-borne diseases, as the changing environmental conditions have consequences for the survival of arthropods and the disease agents they transmit. However, limited data is available on the impacts of environmental conditions on the traits associated with sand fly survival, reproduction, and vectorial competence. The present study aimed to reveal the changes in the expression levels of three selected P. tobbi genes using laboratory-reared and wild-caught populations. A nervous system protein, Cacophony (PtCac), related to the life history traits of sand flies, and two sand fly salivary protein genes, PtSP32 and PtSP38, influence the infection of the vertebrate hosts, were assessed. Sand flies were maintained at 23 °C and 27 °C in the laboratory to evaluate the relationship between temperature and the expressed phenotypes. Field collections were carried out in three climatically distinct regions of Turkey to establish the regional differences in the gene expression levels of natural P. tobbi populations. In the laboratory, PtCac expression increased with the temperature. However, PtCac expression was negatively correlated with local temperature and humidity conditions. No differences were detected in the PtSP32 gene expression levels of both laboratory-reared and wild-caught females, but a negative correlation was observed with relative humidity in natural populations. Although the expression levels of PtSP38 did not differ among the females collected from distinct regions, a positive correlation was detected in the laboratory-reared colony. The findings indicated that changes in environmental conditions could drive the expression levels of P. tobbi genes, which influence population dynamics and the transmission risk of the disease.