Host infectiousness to insect vectors is a crucial parameter for understanding the transmission dynamics of insect-borne infectious diseases such as leishmaniases. Despite their importance, critical factors influencing the outwards transmission of Leishmania major, including parasite distribution within the host body and the minimum number of skin amastigotes required for vector infection, remain poorly characterized. To address these gaps, we studied these parameters in the natural North African reservoir host Meriones shawi and in BALB/c mice infected with a low parasite dose. Using qPCR, we quantified Leishmania loads in different zones (regions) of infected ear pinnae, whereas microscale infectiousness was evaluated via microbiopsies and fluorescence microscopy. The amastigote distribution within infected ears was heterogeneous, with pronounced differences between the lesion center, lesion margin, and visually unaffected surrounding skin. Phlebotomus papatasi females that fed in areas where no amastigotes were detected via microscopy did not become infected. In M. shawi, lesion margins have emerged as the most effective source of infection. The number of amastigotes at bite sites where sand fly females became infected ranged from 4--500, with as few as 2--10 amastigotes sufficient to initiate vector infection. This low infection threshold was confirmed by experiments in which P. papatasi was fed through a chick-skin membrane. In contrast, the BALB/c mouse model showed only minor differences in infectiousness between lesion centers and margins. The minimum infectious dose in BALB/c mice was approximately 100 times greater than that in M. shawi, with successful infections occurring at sites containing 1,500-10,000 amastigotes. These findings advance our understanding of Leishmania transmission by addressing critical knowledge gaps and enabling more accurate modelling of cutaneous leishmaniasis epidemiology. Moreover, this study highlights the importance of incorporating natural host models in research, as the dynamics of disease progression and transmission parameters can differ significantly between natural hosts and standard laboratory models.

• MeSH
• Gerbillinae * parasitology   MeSH
• Insect Vectors * parasitology   MeSH
• Skin parasitology   MeSH
• Leishmania major * physiology   pathogenicity   MeSH
• Leishmaniasis, Cutaneous * transmission   parasitology   MeSH
• Disease Models, Animal MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Parasite Load MeSH
• Phlebotomus * parasitology   MeSH
• Disease Reservoirs * parasitology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Toscana virus (TOSV) is an emerging but neglected human pathogen currently circulating around the Mediterranean basin including North Africa. Human illness ranges from asymptomatic or mild flu-like syndromes to severe neurological diseases such as meningitis or meningoencephalitis. Despite its significant impact, understanding of TOSV transmission and epidemiology remains limited. Sand flies (Diptera: Phlebotominae), specifically Phlebotomus perniciosus and Phlebotomus perfiliewi, are believed to be the primary vectors of TOSV. However, the spread of TOSV to new geographical areas and its detection in other sand fly species suggest that additional species play a role in the circulation and transmission of this virus. This study investigated the vector competence of four sand fly species - P. tobbi, P. sergenti, P. papatasi, and Sergentomyia schwetzi - for two TOSV strains: 1500590 (TOSV A lineage) and MRS20104319501 (TOSV B lineage). Sand flies were orally challenged with TOSV via bloodmeals. None of the tested species showed susceptibility to the TOSV A strain. However, for TOSV B strain, P. tobbi demonstrated a high potential as a new vector, exhibiting high infection and dissemination rates. P. sergenti also showed some susceptibility to TOSV B, with the virus dissemination observed in all infected females. These finding suggests that P. tobbi and P. sergenti are new potential vectors for TOSV B. Given that P. tobbi and P. sergenti are the primary vectors of human leishmaniases in the Balkans, Turkey and Middle East, their susceptibility to TOSV could have significant epidemiological consequences. On the other hand, P. papatasi and S. schwetzi appeared refractory to TOSV B infection. Refractoriness of P. papatasi, a highly anthropophilic species distributed from the Mediterranean to the Middle East and India, suggests that this species does not contribute to TOSV circulation.

• MeSH
• Insect Vectors * virology   MeSH
• Humans MeSH
• Phlebotomus * virology   MeSH
• Psychodidae * virology   classification   MeSH
• Sandfly fever Naples virus * physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: We evaluated various membranes for blood-feeding in nine sand fly species from different genera and subgenera. Most of these species are vectors of human-pathogenic Leishmania, whereas Sergentomyia minuta is a herpetophilic sand fly species and a proven vector of Leishmania (Sauroleishmania) tarentolae. METHODS: Female sand flies were offered blood through a range of membranes (chicken, reptilian, and frog skin; synthetic collagen; pig intestine; and duck foot webbing). Two feeding systems (glass feeder, Hemotek) and different blood sources (human, ovine, avian, and reptilian) were used. Feeding trials were conducted under varying thermal and light conditions to determine the optimal parameters. RESULTS: Among the 4950 female S. minuta tested, only a negligible fraction took a blood meal: 2% of the females fed on avian blood, and 0.2% of the females fed on human blood. In eight other species, the chicken membrane was generally more effective than synthetic membranes or pig intestines. For example, Phlebotomus duboscqi refused synthetic membranes, while Lutzomyia longipalpis and P. perniciosus avoided both synthetic membranes and pig intestines. The most effective membrane was duck foot webbing, with four species feeding more readily through it than through the chicken membrane. Additionally, applying coagulated blood plasma to the outer surface of chicken or synthetic membranes significantly increased feeding rates. CONCLUSIONS: Female S. minuta did not reliably feed on blood through the tested membranes, preventing laboratory infection experiments from confirming their vector competence for human-pathogenic Leishmania. However, for future experimental infections of other sand fly species, duck foot webbing has emerged as an effective membrane, and the application of blood plasma to the exterior of membranes may increase the feeding rates.

• Keywords
• Leishmania, Lutzomyia, Phlebotomus, Sergentomyia minuta, Artificial feeding, Vector competence,
• MeSH
• Blood * MeSH
• Humans MeSH
• Membranes MeSH
• Swine MeSH
• Psychodidae * physiology   MeSH
• Feeding Behavior * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Leishmaniasis is a group of neglected vector-borne diseases transmitted by phlebotomine sand flies. Leishmania parasites must overcome various defenses in the sand fly midgut, including the insects's immune response. Insect immunity is regulated by the ecdysone hormone, which binds to its nuclear receptor (EcR) and activates the transcription of genes involved in insect immunity. However, the role of ecdysone in sand fly immunity has never been studied. Phlebotomus perniciosus is a natural vector of Leishmania infantum; here, we manipulated its neuroendocrine system using azadirachtin (Aza), a natural compound known to affect ecdysone synthesis. METHODS: Phlebotomus perniciosus larvae and adult females were fed on food containing either Aza alone or Aza plus ecdysone, and the effects on mortality and ecdysis were evaluated. Genes related to ecdysone signaling and immunity were identified in P. perniciosus, and the expression of antimicrobial peptides (AMPs), EcR, the ecdysone-induced genes Eip74EF and Eip75B, and the transcription factor serpent were analyzed using quantitative polymerase chain reaction (PCR). RESULTS: Aza treatment inhibited molting of first-instar (L1) larvae to L2, with only 10% of larvae molting compared to 95% in the control group. Serpent and Eip74EF, attacin, defensin 1, and defensin 2 genes were downregulated by Aza treatment in larvae. Similarly, Aza-treated adult females also presented suppression of ecdysone signaling-related genes and the AMPs attacin and defensin 2. Notably, all gene repression caused by Aza was reversed by adding ecdysone concomitantly with Aza to the larval or female food, indicating that these genes are effective markers for ecdysone repression. CONCLUSIONS: These results highlight the critical role of ecdysone in regulating the development and immunity of P. perniciosus, which potentially could interfere with Leishmania infection.

• Keywords
• Phlebotomus perniciosus, Antimicrobial peptides, Azadirachtin, Ecdysone,
• MeSH
• Antimicrobial Peptides genetics   pharmacology   MeSH
• Ecdysone * MeSH
• Insect Vectors drug effects   genetics   parasitology   immunology   MeSH
• Insect Proteins genetics   metabolism   MeSH
• Larva * drug effects   immunology   genetics   MeSH
• Limonins * pharmacology   MeSH
• Phlebotomus * drug effects   genetics   parasitology   immunology   MeSH
• Molting drug effects   MeSH
• Signal Transduction * drug effects   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antimicrobial Peptides MeSH
• azadirachtin MeSH Browser
• Ecdysone * MeSH
• Insect Proteins MeSH
• Limonins * MeSH

Leishmania is a genus of the family Trypanosomatidae that unites obligatory parasitic flagellates causing a variety of vector-borne diseases collectively called leishmaniasis. The symptoms range from relatively innocuous skin lesions to complete failures of visceral organs. The disease is exacerbated if a parasite harbors Leishmania RNA viruses (LRVs) of the family Pseudototiviridae. Screening a novel isolate of L. braziliensis, we revealed that it possesses not a toti-, but a bunyavirus of the family Leishbuviridae. To the best of our knowledge, this is a very first discovery of a bunyavirus infecting a representative of the Leishmania subgenus Viannia. We suggest that these viruses may serve as potential factors of virulence in American leishmaniasis and encourage researchers to test leishmanial strains for the presence of not only LRVs, but also other RNA viruses.

• MeSH
• Bunyaviridae classification   genetics   isolation & purification   MeSH
• Phylogeny MeSH
• Leishmania braziliensis * genetics   isolation & purification   MeSH
• Humans MeSH
• Orthobunyavirus genetics   classification   isolation & purification   physiology   MeSH
• RNA Viruses genetics   classification   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

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.

• Keywords
• Lutzomyia, Phlebotomus, Endogenous control gene, Gene expression, Gene stability, Reference gene,
• MeSH
• Genes, Essential * MeSH
• Insect Vectors genetics   MeSH
• Genes, Insect MeSH
• Larva genetics   MeSH
• Leishmania genetics   MeSH
• Phlebotomus * genetics   MeSH
• Psychodidae genetics   MeSH
• Gene Expression Profiling methods   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Several new species of Leishmania have recently emerged in Europe, probably as the result of global changes and increased human migration from endemic areas. In this study, we tested whether two sand fly species, the Western Mediterranean Phlebotomus perniciosus and the Eastern Mediterranean P. tobbi, are competent vectors of L. donovani, L. major and L. martiniquensis. METHODOLOGY/PRINCIPAL FINDINGS: Sand flies were infected through the chick skin membrane using Leishmania species and strains of various geographical origins. Leishmania infections were evaluated by light microscopy and qPCR, and the representation of morphological forms was assessed from Giemsa-stained gut smears. Neither P. perniciosus nor P. tobbi supported the development of L. martiniquensis, but L. major and L. donovani in both species survived defecation of blood meal remnants, colonized the stomodeal valve and produced metacyclic stages. The results with L donovani have shown that infection rates in sand flies can be strain-specific; therefore, to determine vector competence or refractoriness, it is optimal to test at least two strains of Leishmania. CONCLUSIONS, SIGNIFICANCE: Both sand fly species tested are potential vectors of L. donovani and L. major in Mediterranean area. However, further studies will be needed to identify European vectors of L. martiniquensis and to test the ability of other European sand fly species to transmit L. major, L. donovani, L. tropica and L. infantum.

• MeSH
• Insect Vectors * parasitology   physiology   MeSH
• Chickens parasitology   MeSH
• Leishmania * physiology   classification   genetics   MeSH
• Leishmaniasis transmission   parasitology   MeSH
• Humans MeSH
• Phlebotomus * parasitology   physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Europe MeSH

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

• Keywords
• comparative genomics, cytochrome P450s, gene family evolution, phlebotomine sand flies, xenobiotic adaptation,
• MeSH
• Phylogeny * MeSH
• Genomics MeSH
• Glutathione Transferase genetics   metabolism   MeSH
• Insecticides * pharmacology   MeSH
• Inactivation, Metabolic genetics   MeSH
• Evolution, Molecular * MeSH
• Psychodidae * genetics   MeSH
• Insecticide Resistance * genetics   MeSH
• Cytochrome P-450 Enzyme System genetics   metabolism   MeSH
• Xenobiotics metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Glutathione Transferase MeSH
• Insecticides * MeSH
• Cytochrome P-450 Enzyme System MeSH
• Xenobiotics MeSH

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

• Keywords
• Lutzomyia, Phlebotomus, Sergentomyia, Blood meal, Haemoglobin, Prediuresis,
• MeSH
• Blood MeSH
• Phlebotomus physiology   MeSH
• Psychodidae * physiology   MeSH
• Feeding Behavior * MeSH
• Body Weight * MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Leishmania major is responsible for zoonotic cutaneous leishmaniasis. Therapy is mainly based on the use of antimony-based drugs; however, treatment failures and illness relapses were reported. Although studies were developed to understand mechanisms of drug resistance, the interactions of resistant parasites with their reservoir hosts and vectors remain poorly understood. Here we compared the development of two L. major MON-25 trivalent antimony-resistant lines, selected by a stepwise in vitro Sb(III)-drug pressure, to their wild-type parent line in the natural vector Phlebotomus papatasi. The intensity of infection, parasite location and morphological forms were compared by microscopy. Parasite growth curves and IC50 values have been determined before and after the passage in Ph. papatasi. qPCR was used to assess the amplification rates of some antimony-resistance gene markers. In the digestive tract of sand flies, Sb(III)-resistant lines developed similar infection rates as the wild-type lines during the early-stage infections, but significant differences were observed during the late-stage of the infections. Thus, on day 7 p. i., resistant lines showed lower representation of heavy infections with colonization of the stomodeal valve and lower percentage of metacyclic promastigote forms in comparison to wild-type strains. Observed differences between both resistant lines suggest that the level of Sb(III)-resistance negatively correlates with the quality of the development in the vector. Nevertheless, both resistant lines developed mature infections with the presence of infective metacyclic forms in almost half of infected sandflies. The passage of parasites through the sand fly guts does not significantly influence their capacity to multiply in vitro. The IC50 values and molecular analysis of antimony-resistance genes showed that the resistant phenotype of Sb(III)-resistant parasites is maintained after passage through the sand fly. Sb(III)-resistant lines of L. major MON-25 were able to produce mature infections in Ph. papatasi suggesting a possible circulation in the field using this vector.

• Keywords
• Antimony resistance, Fitness, Leishmania major, Phlebotomus papatasi, experimental infection, Virulence,
• MeSH
• Gene Amplification MeSH
• Antimony * pharmacology   MeSH
• Genetic Fitness MeSH
• Leishmania major * drug effects   genetics   pathogenicity   MeSH
• Drug Resistance * genetics   MeSH
• Phlebotomus * parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antimony * MeSH