A diterpene synthase from the sandfly Lutzomyia longipalpis produces the pheromone sobralene.

The phlebotomine sandfly, Lutzomyia longipalpis, a major vector of the Leishmania parasite, uses terpene pheromones to attract conspecifics for mating. Examination of the L. longipalpis genome revealed a putative terpene synthase (TPS), which-upon heterologous expression in, and purification from, Escherichia coli-yielded a functional enzyme. The TPS, termed LlTPS, converted geranyl diphosphate (GPP) into a mixture of monoterpenes with low efficiency, of which ß-ocimene was the major product. (E,E)-farnesyl diphosphate (FPP) principally produced small amounts of (E)-ß-farnesene, while (Z,E)- and (Z,Z)-FPP yielded a mixture of bisabolene isomers. None of these mono- and sesquiterpenes are known volatiles of L. longipalpis. Notably, however, when provided with (E,E,E)-geranylgeranyl diphosphate (GGPP), LlTPS gave sobralene as its major product. This diterpene pheromone is released by certain chemotypes of L. longipalpis, in particular those found in the Ceará state of Brazil. Minor diterpene components were also seen as products of the enzyme that matched those seen in a sandfly pheromone extract.

Plant-feeding phlebotomine sand flies, vectors of leishmaniasis, prefer Cannabis sativa.

Blood-sucking phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as well as arboviral diseases and bartonellosis. Sand fly females become infected with Leishmania parasites and transmit them while imbibing vertebrates' blood, required as a source of protein for maturation of eggs. In addition, both females and males consume plant-derived sugar meals as a source of energy. Plant meals may comprise sugary solutions such as nectar or honeydew (secreted by plant-sucking homopteran insects), as well as phloem sap that sand flies obtain by piercing leaves and stems with their needle-like mouthparts. Hence, the structure of plant communities can influence the distribution and epidemiology of leishmaniasis. We designed a next-generation sequencing (NGS)-based assay for determining the source of sand fly plant meals, based upon the chloroplast DNA gene ribulose bisphosphate carboxylase large chain (rbcL). Here, we report on the predilection of several sand fly species, vectors of leishmaniasis in different parts of the world, for feeding on Cannabis sativa We infer this preference based on the substantial percentage of sand flies that had fed on C. sativa plants despite the apparent "absence" of these plants from most of the field sites. We discuss the conceivable implications of the affinity of sand flies for C. sativa on their vectorial capacity for Leishmania and the putative exploitation of their attraction to C. sativa for the control of sand fly-borne diseases.

Leishmania proteophosphoglycans regurgitated from infected sand flies accelerate dermal wound repair and exacerbate leishmaniasis via insulin-like growth factor 1-dependent signalling.

Leishmania parasites are transmitted to vertebrate hosts by female phlebotomine sand flies as they bloodfeed by lacerating the upper capillaries of the dermis with their barbed mouthparts. In the sand fly midgut secreted proteophosphoglycans from Leishmania form a biological plug known as the promastigote secretory gel (PSG), which blocks the gut and facilitates the regurgitation of infective parasites. The interaction between the wound created by the sand fly bite and PSG is not known. Here we nanoinjected a sand fly egested dose of PSG into BALB/c mouse skin that lead to the differential expression of 7,907 transcripts. These transcripts were transiently up-regulated during the first 6 hours post-wound and enriched for pathways involved in inflammation, cell proliferation, fibrosis, epithelial cell differentiation and wound remodelling. We found that PSG significantly accelerated wound healing in vitro and in mice; which was associated with an early up-regulation of transcripts involved in inflammation (IL-1ß, IL-6, IL-10, TNFα) and inflammatory cell recruitment (CCL2, CCL3, CCL4, CXCL2), followed 6 days later by enhanced expression of transcripts associated with epithelial cell proliferation, fibroplasia and fibrosis (FGFR2, EGF, EGFR, IGF1). Dermal expression of IGF1 was enhanced following an infected sand fly bite and was acutely responsive to the deposition of PSG but not the inoculation of parasites or sand fly saliva. Antibody blockade of IGF1 ablated the gel's ability to promote wound closure in mouse ears and significantly reduced the virulence of Leishmania mexicana infection delivered by an individual sand fly bite. Dermal macrophages recruited to air-pouches on the backs of mice revealed that IGF1 was pivotal to the PSG's ability to promote macrophage alternative activation and Leishmania infection. Our data demonstrate that through the regurgitation of PSG Leishmania exploit the wound healing response of the host to the vector bite by promoting the action of IGF1 to drive the alternative activation of macrophages.

Heme synthesis through the life cycle of the heme auxotrophic parasite Leishmania major.

Heme is an essential molecule synthetized through a broadly conserved 8-step route that has been lost in trypanosomatid parasites. Interestingly, Leishmania reacquired by horizontal gene transfer from γ-proteobacteria the genes coding for the last 3 enzymes of the pathway. Here we show that intracellular amastigotes of Leishmania major can scavenge heme precursors from the host cell to fulfill their heme requirements, demonstrating the functionality of this partial pathway. To dissect its role throughout the L. major life cycle, the significance of L. major ferrochelatase (LmFeCH), the terminal enzyme of the route, was evaluated. LmFeCH expression in a heterologous system demonstrated its activity. Knockout promastigotes lacking lmfech were not able to use the ferrochelatase substrate protoporphyrin IX as a source of heme. In vivo infection of Phlebotomus perniciosus with knockout promastigotes shows that LmFeCH is not required for their development in the sandfly. In contrast, the replication of intracellular amastigotes was hampered in vitro by the deletion of lmfech. However, LmFeCH-/- parasites produced disease in a cutaneous leishmaniasis murine model in a similar way as control parasites. Therefore, although L. major can synthesize de novo heme from macrophage precursors, this activity is dispensable being an unsuited target for leishmaniasis treatment.-Orrego, L. M., Cabello-Donayre, M., Vargas, P., Martínez-García, M., Sánchez, C., Pineda-Molina, E., Jiménez, M., Molina, R., Pérez-Victoria, J. M. Heme synthesis through the life cycle of the heme auxotrophic parasite Leishmania major.

NSs Protein of Sandfly Fever Sicilian Phlebovirus Counteracts Interferon (IFN) Induction by Masking the DNA-Binding Domain of IFN Regulatory Factor 3.

Sandfly fever Sicilian virus (SFSV) is one of the most widespread and frequently identified members of the genus Phlebovirus (order Bunyavirales, family Phenuiviridae) infecting humans. Being transmitted by Phlebotomus sandflies, SFSV causes a self-limiting, acute, often incapacitating febrile disease ("sandfly fever," "Pappataci fever," or "dog disease") that has been known since at least the beginning of the 20th century. We show that, similarly to other pathogenic phleboviruses, SFSV suppresses the induction of the antiviral type I interferon (IFN) system in an NSs-dependent manner. SFSV NSs interfered with the TBK1-interferon regulatory factor 3 (IRF3) branch of the RIG-I signaling pathway but not with NF-κB activation. Consistently, we identified IRF3 as a host interactor of SFSV NSs. In contrast to IRF3, neither the IFN master regulator IRF7 nor any of the related transcription factors IRF2, IRF5, and IRF9 were bound by SFSV NSs. In spite of this specificity for IRF3, NSs did not inhibit its phosphorylation, dimerization, or nuclear accumulation, and the interaction was independent of the IRF3 activation or multimerization state. In further studies, we identified the DNA-binding domain of IRF3 (amino acids 1 to 113) as sufficient for NSs binding and found that SFSV NSs prevented the association of activated IRF3 with the IFN-ß promoter. Thus, unlike highly virulent phleboviruses, which either destroy antiviral host factors or sequester whole signaling chains into inactive aggregates, SFSV modulates type I IFN induction by directly masking the DNA-binding domain of IRF3.IMPORTANCE Phleboviruses are receiving increased attention due to the constant discovery of new species and the ongoing spread of long-known members of the genus. Outbreaks of sandfly fever were reported in the 19th century, during World War I, and during World War II. Currently, SFSV is recognized as one of the most widespread phleboviruses, exhibiting high seroprevalence rates in humans and domestic animals and causing a self-limiting but incapacitating disease predominantly in immunologically naive troops and travelers. We show how the nonstructural NSs protein of SFSV counteracts the upregulation of the antiviral interferon (IFN) system. SFSV NSs specifically inhibits promoter binding by IFN transcription factor 3 (IRF3), a molecular strategy which is unique among phleboviruses and, to our knowledge, among human pathogenic RNA viruses in general. This IRF3-specific and stoichiometric mechanism, greatly distinct from the ones exhibited by the highly virulent phleboviruses, correlates with the intermediate level of pathogenicity of SFSV.

Mitochondrial DNA Intraspecific Variability in Sergentomyia minuta (Diptera: Psychodidae).

Recently, there has been growing interest in analysis of the geographical variation between populations of different Phlebotomus spp. and American sand flies by comparing the sequences of various genes. However, little is known about the genetic structure of the genus Sergentomyia França & Parrot. No study has been carried out on Sergentomyia minuta Rondani. Most authors recognize this as a species with a high degree of morphological polymorphism, and some suspect that there are two subspecies: Se. minuta minuta Rondani in Europe, having about 40 horizontal cibarial teeth (sticks aligned along a straight line in the cibarial cavity), and Se. minuta parroti Adler & Theodor in North Africa, having about 70 cibarial teeth. Here we analyzed phylogeographic patterns using cytochrome b (Cytb) and cytochrome C oxidase I mtDNA for 29 populations from 10 countries: Algeria, Cyprus, France (continental and Corsica), Greece (continental and Crete), Malta, Montenegro, Morocco, Portugal (continental and Atlantic Savage Islands), Spain, and Tunisia. We analyzed intra- and interpopulation patterns of genetic diversity. Our results from Bayesian inference showed a complex genetic structure of Se. minuta with four haplogroups including many different haplotypes. One haplogroup includes all the specimens from North Africa. A second haplogroup includes a few specimens from the south of France, Spain, and one from Portugal. The third includes many specimens from southern France, all the specimens from Corsica, one from Spain, and all specimen from Portugal except one. A fourth branch includes specimens from the Balkans, Malta, Crete, Cyprus, and curiously some from the Atlantic Savage Islands; settlement of the latter population remains unexplained. However, our results suggest that the settlement of the Mediterranean basin could have occurred at the same time for Se. minuta and both Phlebotomus perniciosus Newstead and Phlebotomus ariasi Tonnoir. The spatial distribution of haplotypes was congruent with phylogenetic findings.

Development of a fluorescent enzyme-linked DNA aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid leishmania detection in sandflies.

A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests. We hypothesize that the AUR version of this assay is more consistent, especially at low analyte levels, because the fluorescent product of AUR is liberated into bulk solution and readily detectable while fluorophores attached to the reporter aptamer might occasionally be hidden behind magnetic beads near the detection limit. Conversely, fluorophores could be quenched by nearby beads or other proximal fluorophores on the high end of analyte concentration, if packed into a small area after magnetic collection when an enzyme-linked system is not used. A highly portable and rechargeable battery-operated fluorometer with on board computer and color touchscreen is also described which can be used for rapid (<1 h) and sensitive detection of Leishmania promastigote protein extracts (∼ 100 ng per sample) in buffer or sandfly homogenates for mapping of L. major parasite geographic distributions in wild sandfly populations.

Divergence of Lutzomyia (Psathyromyia) shannoni (Diptera: Psychodidae: Phlebotominae) is indicated by morphometric and molecular analyses when examined between taxa from the southeastern United States and southern Mexico.

The medically important sand fly Lutzomyia shannoni (Dyar 1929) was collected at eight different sites: seven within the southeastern United States and one in the state of Quintana Roo, Mexico. A canonical discriminant analysis was conducted on 40 female L. shannoni specimens from each of the eight collection sites (n = 320) using 49 morphological characters. Four L. shannoni specimens from each of the eight collection sites (n = 32) were sent to the Barcode of Life Data systems where a 654-base pair segment of the cytochrome c oxidase subunit 1 (CO1) genetic marker was sequenced from each sand fly. Phylogeny estimation based on the COI segments, in addition to genetic distance, divergence, and differentiation values were calculated. Results of both the morphometric and molecular analyses indicate that the species has undergone divergence when examined between the taxa of the United States and Quintana Roo, Mexico. Although purely speculative, the arid or semiarid expanse from southern Texas to Mexico City could be an allopatric barrier that has impeded migration and hence gene flow, resulting in different morphology and genetic makeup between the two purported populations. A high degree of intragroup variability was noted in the Quintana Roo sand flies.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva.

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

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.

Laboratory evaluation of rubidium as a long-lasting marker for bloodfeeding sand flies (Diptera: Psychodidae).

The objective of this study was to evaluate the use of the trace element rubidium (Rb) as a long-lasting systemic biomarker for bloodfeeding females of the sand fly Phlebotomus papatasi Scopoli. Baits containing Rb chloride were found to be palatable to hamsters in this study. We were able to detect Rb using a portable X-ray fluorescence analyzer in all sand flies that fed on Rb-treated hamsters for at least 14 d postbloodmeal. We also detected Rb in sand flies that took a bloodmeal from hamsters up to 10 d after the hamsters were withdrawn from a Rb-treated diet. Results of this study constitute proof of concept for the incorporation of Rb chloride into rodent baits for marking bloodfeeding sand flies, and suggest that Rb marking could be used as a technique for evaluating rodent-targeted sand fly control methods and in ecological studies on sand flies.

Proteophosophoglycans regurgitated by Leishmania-infected sand flies target the L-arginine metabolism of host macrophages to promote parasite survival.

All natural Leishmania infections start in the skin; however, little is known of the contribution made by the sand fly vector to the earliest events in mammalian infection, especially in inflamed skin that can rapidly kill invading parasites. During transmission sand flies regurgitate a proteophosphoglycan gel synthesized by the parasites inside the fly midgut, termed promastigote secretory gel (PSG). Regurgitated PSG can exacerbate cutaneous leishmaniasis. Here, we show that the amount of Leishmania mexicana PSG regurgitated by Lutzomyia longipalpis sand flies is proportional to the size of its original midgut infection and the number of parasites transmitted. Furthermore, PSG could exacerbate cutaneous L. mexicana infection for a wide range of doses (10-10,000 parasites) and enhance infection by as early as 48 hours in inflamed dermal air pouches. This early exacerbation was attributed to two fundamental properties of PSG: Firstly, PSG powerfully recruited macrophages to the dermal site of infection within 24 hours. Secondly, PSG enhanced alternative activation and arginase activity of host macrophages, thereby increasing L-arginine catabolism and the synthesis of polyamines essential for intracellular parasite growth. The increase in arginase activity promoted the intracellular growth of L. mexicana within classically activated macrophages, and inhibition of macrophage arginase completely ablated the early exacerbatory properties of PSG in vitro and in vivo. Thus, PSG is an essential component of the infectious sand fly bite for the early establishment of Leishmania in skin, which should be considered when designing and screening therapies against leishmaniasis.

Mechanisms of pH control in the midgut of Lutzomyia longipalpis: roles for ingested molecules and hormones.

Control of the midgut pH in Lutzomyia longipalpis enables the insect's digestive system to deal with different types of diet. Phlebotomines must be able to suddenly change from a condition adequate to process a sugar diet to one required to digest blood. Prior to blood ingestion, the pH in the midgut is maintained at ∼6 via an efficient mechanism. In the abdominal midgut, alkalization to a pH of ∼8 occurs as a consequence of the loss of CO(2) from blood (CO(2) volatilization) and by a second mechanism that is not yet characterized. The present study aimed to characterize the primary stimuli, present in the blood, that are responsible for shutting down the mechanism that maintains a pH of 6 and switching on that responsible for alkalization. Our results show that any ingested protein could induce alkalization. Free amino acids, at the concentrations found in blood, were ineffective at inducing alkalization, although higher concentrations of amino acids were able to induce alkalization. Aqueous extracts of midgut tissue containing putative hormones from intestinal endocrine cells slightly alkalized the midgut lumen when applied to dissected intestines, as did hemolymph collected from blood-fed females. Serotonin, a hormone that is possibly released in the hemolymph after hematophagy commences, was ineffective at promoting alkalization. The carbonic anhydrase (CA) enzyme seems to be involved in alkalizing the midgut, as co-ingestion of acetazolamide (a CA inhibitor) with proteins impaired alkalization efficiency. A general model of alkalization control is presented.

Examination of the interior of sand fly (Diptera: Psychodidae) abdomen reveals novel cuticular structures involved in pheromone release: Discovering the manifold.

The males of many species of New World Phlebotomines produce volatile terpenoid chemicals, shown in Lutzomyia longipalpis s.l. to be sex/aggregation pheromones. Pheromone is produced by secretory cells which surround a cuticular reservoir which collects the pheromone and passes it through a cuticular duct to the surface of the insect. The pheromone then passes through specialised cuticular structures on the abdominal surface prior to evaporation. The shape and distribution of the specialised structures are highly diverse and differ according to species. In this study we used SEM to examine the interior cuticular pheromone collection and transport structures of 3 members of the Lu. longipalpis s.l. species complex and Migonemyia migonei. We found a new structure which we have called the manifold which appears to be a substantial extension of the interior tergal cuticle connected in-line with the cuticular duct and reservoir. The manifold of the Campo Grande member of the complex is longer and wider than the Jacobina member whereas the manifold of the Sobral member was shorter than both other members of the complex. Overall, the secretory apparatus of the Sobral member was smaller than the other two. The manifold of M. migonei was very different to those found in Lu. longipalpis s.l. and was positioned in a pit-like structure within the tergal cuticle. The secretory reservoir was connected by a short duct to the manifold. Differences in the size and shape of the manifold may be related to the chemical structure of the pheromone and may have taxonomic value. Examination of the interior cuticle by SEM may help to locate the secretory apparatus of vector species where pheromonal activity has been inferred from behavioural studies but the external secretory structures or pheromones have not yet been found.

Afrotropical sand fly-host plant relationships in a leishmaniasis endemic area, Kenya.

The bioecology of phlebotomine sand flies is intimately linked to the utilization of environmental resources including plant feeding. However, plant feeding behavior of sand flies remains largely understudied for Afrotropical species. Here, using a combination of biochemical, molecular, and chemical approaches, we decipher specific plant-feeding associations in field-collected sand flies from a dry ecology endemic for leishmaniasis in Kenya. Cold-anthrone test indicative of recent plant feeding showed that fructose positivity rates were similar in both sand fly sexes and between those sampled indoors and outdoors. Analysis of derived sequences of the ribulose-1,5-bisphosphate carboxylase large subunit gene (rbcL) from fructose-positive specimens implicated mainly Acacia plants in the family Fabaceae (73%) as those readily foraged on by both sexes of Phlebotomus and Sergentomyia. Chemical analysis by high performance liquid chromatography detected fructose as the most common sugar in sand flies and leaves of selected plant species in the Fabaceae family. Analysis of similarities (ANOSIM) of the headspace volatile profiles of selected Fabaceae plants identified benzyl alcohol, (Z)-linalool oxide, (E)-ß-ocimene, p-cymene, p-cresol, and m-cresol, as discriminating compounds between the plant volatiles. These results indicate selective sand fly plant feeding and suggest that the discriminating volatile organic compounds could be exploited in attractive toxic sugar- and odor- bait technologies control strategies.

A sand fly salivary protein acts as a neutrophil chemoattractant.

Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

Insights into the salivary N-glycome of Lutzomyia longipalpis, vector of visceral leishmaniasis.

During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host's immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with Man5GlcNAc2 being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.

Sergentomyia schwetzi: Salivary gland transcriptome, proteome and enzymatic activities in two lineages adapted to different blood sources.

During the blood feeding, sand fly females inject saliva containing immunomodulatory and anti-haemostatic molecules into their vertebrate hosts. The saliva composition is species-specific, likely due to an adaptation to particular haemostatic pathways of their preferred host. Research on sand fly saliva is limited to the representatives of two best-studied genera, Phlebotomus and Lutzomyia. Although the members of the genus Sergentomyia are highly abundant in many areas in the Old World, their role in human disease transmission remains uncertain. Most Sergentomyia spp. preferentially attack various species of reptiles, but feeding on warm-blooded vertebrates, including humans and domestic animals, has been repeatedly described, especially for Sergentomyia schwetzi, of which salivary gland transcriptome and proteome is analyzed in the current study. Illumina RNA sequencing and de novo assembly of the reads and their annotation revealed 17,293 sequences homologous to other arthropods' proteins. In the sialome, all proteins typical for sand fly saliva were identified-antigen 5-related, lufaxin, yellow-related, PpSP15-like, D7-related, ParSP25-like, and silk proteins, as well as less frequent salivary proteins included 71kDa-like, ParSP80-like, SP16-like, and ParSP17-like proteins. Salivary enzymes include apyrase, hyaluronidase, endonuclease, amylase, lipase A2, adenosine deaminase, pyrophosphatase, 5'nucleotidase, and ribonuclease. Proteomics analysis of salivary glands identified 631 proteins, 81 of which are likely secreted into the saliva. We also compared two S. schwetzi lineages derived from the same origin. These lineages were adapted for over 40 generations for blood feeding either on mice (S-M) or geckos (S-G), two vertebrate hosts with different haemostatic mechanisms. Altogether, 20 and 40 annotated salivary transcripts were up-regulated in the S-M and S-G lineage, respectively. Proteomic comparison revealed ten salivary proteins more abundant in the lineage S-M, whereas 66 salivary proteins were enriched in the lineage S-G. No difference between lineages was found for apyrase activity; contrarily the hyaluronidase activity was significantly higher in the lineage feeding on mice.

Interactions between host biogenic amines and sand fly salivary yellow-related proteins.

BACKGROUND: During blood feeding, sand flies inoculate salivary proteins that interact with the host haemostatic system. The blocking of biogenic amines such as serotonin and histamine helps to limit vasodilatation and clot formation, and thus enables the insect to finish the blood-feeding process. In sand flies, an amine-binding ability is known only for the yellow-related proteins of Phlebotomus and Lutzomyia vectors, but not yet for members of the genus Sergentomyia. METHODS: The ability of Phlebotomus argentipes and Sergentomyia schwetzi recombinant yellow-related salivary proteins to bind histamine and serotonin was measured by microscale thermophoresis. Both sand fly species were also fed through a chicken-skin membrane on blood mixed with histamine or serotonin in order to check the effects of biogenic amines on sand fly fitness. Additionally, fecundity and mortality were compared in two groups of P. argentipes females fed on repeatedly-bitten and naive hamsters, respectively. RESULTS: The P. argentipes recombinant yellow-related protein PagSP04 showed high binding affinity to serotonin and low affinity to histamine. No binding activity was detected for two yellow-related proteins of S. schwetzi. Elevated concentrations of serotonin significantly reduced the amount of eggs laid by P. argentipes when compared to the control. The fecundity of S. schwetzi and the mortality of both sand fly species were not impaired after the experimental membrane feeding. Additionally, there were no differences in oviposition or mortality between P. argentipes females fed on immunized or naive hamsters. CONCLUSIONS: Our results suggest that in natural conditions sand flies are able to cope with biogenic amines or anti-saliva antibodies without any influence on their fitness. The serotonin binding by salivary yellow-related proteins may play an important role in Phlebotomus species feeding on mammalian hosts, but not in S. schwetzi, which is adapted to reptiles.

Salivary gland homogenates of Lutzomyia longipalpis and its vasodilatory peptide maxadilan cause plasma leakage via PAC1 receptor activation.

OBJECTIVES: Experiments were designed to determine if salivary gland homogenates (SGH) of the sand fly Lutzomyia longipalpis, the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage and to what extent these effects could be due to PAC1 receptor stimulation. METHODS: Using FITC-dextran as a plasma marker, intravital microscopy of the hamster cheek pouch (HCP) and a digital camera were used to assess arteriolar diameter and fluorescence of a selected area (5 mm(2)) representative of the HCP microcirculation. RESULTS: Cheek pouches prepared for intravital microscopy and exposed to topical application of SGH, maxadilan or PACAP-38 developed maximal dilation of arterioles in the range of 20-60 mum within 10 min, and this effect lasted for 30-90 min. The increase in fluorescence intensity induced by each of these compounds was due to plasma leakage from postcapillary venules. The mutant peptide of maxadilan (M-65), a PAC1 receptor antagonist, inhibited both dilation and plasma leakage induced by SGH or maxadilan. Plasma leakage induced by SGH was modestly inhibited by the bradykinin B(2) receptor antagonist HOE-140, but not by the antihistamine mepyramine or the nitric oxide synthase inhibitor L-NA. CONCLUSIONS: SGH of L. longipalpis and its vasodilatory peptide maxadilan caused long-lasting arteriolar dilation and plasma leakage in the cheek pouch via PAC1 receptor activation.