Phlebotomus papatasi is the vector of Leishmania major, causing cutaneous leishmaniasis in the Old World. We investigated whether P. papatasi immunity genes were expressed toward L. major, commensal gut microbes, or a combination of both. We focused on sand fly transcription factors dorsal and relish and antimicrobial peptides (AMPs) attacin and defensin and assessed their relative gene expression by qPCR. Sand fly larvae were fed food with different bacterial loads. Relish and AMPs gene expressions were higher in L3 and early L4 larval instars, while bacteria 16S rRNA increased in late L4 larval instar, all fed rich-microbe food compared to the control group fed autoclaved food. Sand fly females were treated with an antibiotic cocktail to deplete gut bacteria and were experimentally infected by Leishmania. Compared to non-infected females, dorsal and defensin were upregulated at early and late infection stages, respectively. An earlier increase of defensin was observed in infected females when bacteria recolonized the gut after the removal of antibiotics. Interestingly, this defensin gene expression occurred specifically in midguts but not in other tissues of females and larvae. A gut-specific defensin gene upregulated by L. major infection, in combination with gut-bacteria, is a promising molecular target for parasite control strategies.

• Klíčová slova
• Leishmania, defensin, gut-specific response, insect immunity, sand fly,
• Publikační typ
• časopisecké články MeSH

Background: Leishmaniasis is a globally important yet neglected parasitic disease transmitted by phlebotomine sand flies. With new candidate vaccines in or near the clinic, a controlled human challenge model (CHIM) using natural sand fly challenge would provide a method for early evaluation of prophylactic efficacy. Methods : We evaluated the biting frequency and adverse effects resulting from exposure of human volunteers to bites of either Phlebotomus papatasi or P. duboscqi, two natural vectors of Leishmania major. 12 healthy participants were recruited (mean age 40.2 ± 11.8 years) with no history of significant travel to regions where L. major-transmitting sand flies are prevalent. Participants were assigned to either vector by 1:1 allocation and exposed to five female sand flies for 30 minutes in a custom biting chamber. Bite frequency was recorded to confirm a bloodmeal was taken. Participant responses and safety outcomes were monitored using a visual analogue scale (VAS), clinical examination, and blood biochemistry. Focus groups were subsequently conducted to explore participant acceptability. Results: All participants had at least one successful sand fly bite with none reporting any serious adverse events, with median VAS scores of 0-1/10 out to day 21 post-sand fly bite. Corresponding assessment of sand flies confirmed that for each participant at least 1/5 sand flies had successfully taken a bloodmeal (overall mean 3.67±1.03 bites per participant). There was no significant difference between P. papatasi and P. duboscqi in the number of bites resulting from 5 sand flies applied to human participants (3.3±0.81 vs 3.00±1.27 bites per participant; p=0.56) . In the two focus groups (n=5 per group), themes relating to positive participant-reported experiences of being bitten and the overall study, were identified. Conclusions: These results validate a protocol for achieving successful sand fly bites in humans that is safe, well-tolerated and acceptable for participants. Clinicaltrials.gov registration: NCT03999970 (27/06/2019).

• Klíčová slova
• Controlled human infection models; leishmaniasis, focus groups, sand flies; public engagement, vaccines,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Identification of blood sources of hematophagous arthropods is crucial for understanding the transmission cycles of vector-borne diseases. Many different approaches towards host determination were proposed, including precipitin test, ELISA, DNA- and mass spectrometry-based methods; yet all face certain complications and limitations, mostly related to blood degradation. This study presents a novel method for blood meal identification, peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry. METHODOLOGY/PRINCIPAL FINDINGS: To identify blood meal source, proteins from abdomens of engorged sand fly females were extracted, cleaved by trypsin and peptide fragments of host hemoglobin were sequenced using MALDI-TOF MS. The method provided correct host identification of 100% experimentally fed sand flies until 36h post blood meal (PBM) and for 80% samples even 48h PBM. In females fed on two hosts, both blood meal sources were correctly assigned for 60% of specimens until 36h PBM. In a validation study on field-collected females, the method yielded unambiguous host determination for 96% of specimens. The suitability of PMM-based MALDI-TOF MS was proven experimentally also on lab-reared Culex mosquitoes. CONCLUSIONS/SIGNIFICANCE: PMM-based MALDI-TOF MS analysis targeting host specific hemoglobin peptides represents a sensitive and cost-effective method with a fast and simple preparation protocol. As demonstrated here on phlebotomine sand flies and mosquitoes, it allows reliable and rapid blood source determination even 48h PBM with minimal material input and provides more robust and specific results than other currently used methods. This approach was also successfully tested on field-caught engorged females and proved to be a promising useful tool for large-scale screening of host preferences studies. Unlike other methods including MALDI-TOF protein profiling, it allows correct identification of mixed blood meals as was demonstrated on both experimentally fed and field-collected sand flies.

• MeSH
• biochemická analýza krve metody   MeSH
• druhová specificita MeSH
• hemoglobiny chemie   MeSH
• lidé MeSH
• ověření koncepční studie MeSH
• peptidy chemie   MeSH
• Psychodidae chemie   fyziologie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• stravovací zvyklosti MeSH
• tandemová hmotnostní spektrometrie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemoglobiny MeSH
• peptidy MeSH

Laboratory colonies of phlebotomine sand flies are necessary for experimental study of their biology, behaviour and mutual relations with disease agents and for testing new methods of vector control. They are indispensable in genetic studies and controlled observations on the physiology and behaviour of sand flies, neglected subjects of high priority. Colonies are of particular value for screening insecticides. Colonized sand flies are used as live vector models in a diverse array of research projects, including xenodiagnosis, that are directed toward control of leishmaniasis and other sand fly-associated diseases. Historically, labour-intensive maintenance and low productivity have limited their usefulness for research, especially for species that do not adapt well to laboratory conditions. However, with growing interest in leishmaniasis research, rearing techniques have been developed and refined, and sand fly colonies have become more common, enabling many significant breakthroughs. Today, there are at least 90 colonies representing 21 distinct phlebotomine sand fly species in 35 laboratories in 18 countries worldwide. The materials and methods used by various sand fly workers differ, dictated by the availability of resources, cost or manpower constraints rather than choice. This paper is not intended as a comprehensive review but rather a discussion of methods and techniques most commonly used by researchers to initiate, establish and maintain sand fly colonies, with emphasis on the methods proven to be most effective for the species the authors have colonized. Topics discussed include collecting sand flies for colony stock, colony initiation, maintenance and mass-rearing procedures, and control of sand fly pathogens in colonies.

Les colonies de laboratoire de phlébotomes sont nécessaires pour une étude expérimentale de leur biologie, leur comportement et leurs relations mutuelles avec des agents pathogènes et pour tester de nouvelles méthodes de lutte antivectorielle. Elles sont indispensables dans les études génétiques et les observations contrôlées sur la physiologie et le comportement des phlébotomes, sujets négligés de haute priorité. Les colonies ont une valeur particulière pour le criblage des insecticides. Les phlébotomes en élevage sont utilisés comme modèles de vecteurs vivants dans un éventail varié de projets de recherche, y compris le xénodiagnostic, qui visent le contrôle de la leishmaniose et d'autres maladies associées aux phlébotomes. Historiquement, la maintenance à forte intensité de main-d'œuvre et la faible productivité ont limité leur utilité pour la recherche, en particulier pour les espèces qui ne s'adaptent pas bien aux conditions de laboratoire. Mais, avec un intérêt croissant pour la recherche sur la leishmaniose, les techniques d'élevage ont été développées et affinées, et les colonies de phlébotomes sont devenues plus fréquentes, permettant de nombreuses percées significatives. Aujourd'hui, il y a au moins 90 colonies représentant 21 espèces distinctes de phlébotomes dans 35 laboratoires et 18 pays à travers le monde. Les matériaux et les méthodes utilisés par divers chercheurs sur les phlébotomes diffèrent, dictés par la disponibilité des ressources et les contraintes de coûts ou de main-d'œuvre plutôt que par le choix. Cet article n'est pas destiné à être un examen complet, mais plutôt une discussion sur les méthodes et les techniques les plus utilisées par les chercheurs pour initier, établir et maintenir les colonies de phlébotomes, en mettant l'accent sur les méthodes démontrées les plus efficaces pour les espèces que les auteurs ont établies en colonies. Les sujets abordés comprennent la collecte de phlébotomes pour les stocks de colonies, l'initiation des colonies, les procédures de maintenance et d'élevage et le contrôle des agents pathogènes des phlébotomes dans les colonies.

• MeSH
• bydlení zvířat klasifikace   normy   MeSH
• doprava metody   MeSH
• hmyz - vektory klasifikace   růst a vývoj   parazitologie   fyziologie   MeSH
• kladení vajíček MeSH
• králíci MeSH
• křečci praví MeSH
• kur domácí MeSH
• morčata MeSH
• myši MeSH
• Psychodidae klasifikace   růst a vývoj   parazitologie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• křečci praví MeSH
• morčata MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Indie MeSH

BACKGROUND: In mosquitoes, it has previously been shown that rearing conditions of immature stages have an effect on the vector competence of adults. Here, we studied the impact of different larval rearing temperatures (27 °C versus 32 °C) on the sand fly Phlebotomus sergenti Parrot, 1917 and its susceptibility to two parasites: Leishmania tropica Wright, 1903, a dixenous trypanosomatid transmissible from sand flies to humans, and Psychodiella sergenti Lantova, Volf & Votypka, 2010, a monoxenous sand fly gregarine. RESULTS: Increased rearing temperature (32 °C) affected the larval developmental times and size of P. sergenti adults but had no effect on the susceptibility of P. sergenti to L. tropica. No differences were found in Leishmania infection rates or in the intensities of Leishmania infection. Interestingly, increased larval rearing temperature significantly suppressed the development of gregarines. All 117 control sand flies tested were infected with Ps. sergenti, and the mean number of gamonts per individual was 29.5. In contrast, only three of 120 sand flies maintained at 32 °C were infected and the mean number of gamonts per individual was just 0.04. CONCLUSIONS: We demonstrated that the increased rearing temperature of P. sergenti larvae had no impact on the development of L. tropica in adult sand flies but had a profound effect on the gregarine Ps. sergenti. We suggest that increasing the larval rearing temperature by 5 °C is a simple and effective way to clean sand fly colonies infected by gregarines.

• Klíčová slova
• Effect of temperature, Gregarines, Leishmania tropica, Phlebotomus sergenti, Psychodiella sergenti, Vector competence,
• MeSH
• Apicomplexa fyziologie   MeSH
• dezinsekce MeSH
• hmyz - vektory parazitologie   MeSH
• interakce hostitele a parazita MeSH
• larva fyziologie   MeSH
• Leishmania tropica fyziologie   MeSH
• Phlebotomus růst a vývoj   parazitologie   MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. METHODS/PRINCIPAL FINDINGS: A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. CONCLUSIONS: Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

• MeSH
• hmyz - vektory parazitologie   fyziologie   MeSH
• Leishmania infantum fyziologie   MeSH
• leishmanióza epidemiologie   parazitologie   přenos   MeSH
• lidé MeSH
• podnebí MeSH
• populační dynamika MeSH
• Psychodidae parazitologie   fyziologie   MeSH
• roční období MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Středomoří epidemiologie   MeSH

The early stage of Leishmania development in sand flies is closely connected with bloodmeal digestion. Here we compared various parameters of bloodmeal digestion in sand flies that are either susceptible (Phlebotomus argentipes and P. orientalis) or refractory (P. papatasi and Sergentomyia schwetzi) to Leishmania donovani, to study the effects on vector competence. The volume of the bloodmeal ingested, time of defecation of bloodmeal remnants, timing of formation and degradation of the peritrophic matrix (PM) and dynamics of proteolytic activities were compared in four sand fly species. Both proven vectors of L. donovani showed lower trypsin activity and slower PM formation than refractory species. Interestingly, the two natural L. donovani vectors strikingly differed from each other in secretion of the PM and midgut proteases, with P. argentipes possessing fast bloodmeal digestion with a very high peak of chymotrypsin activity and rapid degradation of the PM. Experimental infections of P. argentipes did not reveal any differences in vector competence in comparison with previously studied P. orientalis; even the very low initial dose (2×103 promastigotes/ml) led to fully developed late-stage infections with colonization of the stomodeal valve in about 40% of females. We hypothesise that the period between the breakdown of the PM and defecation of the bloodmeal remnants, i.e. the time frame when Leishmania attach to the midgut in order to prevent defecation, could be one of crucial parameters responsible for the establishment of Leishmania in the sand fly midgut. In both natural L. donovani vectors this period was significantly longer than in S. schwetzi. Both vectors are equally susceptible to L. donovani; as average bloodmeal volumes taken by females of P. argentipes and P. orientalis were 0.63 μl and 0.59 μl, respectively, an infective dose corresponding to 1-2 parasites was enough to initiate mature infections.

• MeSH
• druhová specificita MeSH
• feces parazitologie   MeSH
• hmyz - vektory imunologie   metabolismus   parazitologie   fyziologie   MeSH
• krev metabolismus   MeSH
• Leishmania donovani růst a vývoj   fyziologie   MeSH
• leishmanióza viscerální imunologie   MeSH
• membrány parazitologie   MeSH
• náchylnost k nemoci MeSH
• přijímání potravy * MeSH
• proteolýza MeSH
• Psychodidae imunologie   metabolismus   parazitologie   fyziologie   MeSH
• trávení * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

BACKGROUND: Sand fly species of the genus Sergentomyia are proven vectors of reptilian Leishmania that are non-pathogenic to humans. However, a consideration of the role of Sergentomyia spp. in the circulation of mammalian leishmaniasis appears repeatedly in the literature and the possibility of Leishmania transmission to humans remains unclear. Here we studied the susceptibility of colonized Sergentomyia schwetzi to Leishmania donovani and two other Leishmania species pathogenic to humans: L. infantum and L. major. METHODS: Females of laboratory-reared S. schwetzi were infected by cultured Leishmania spp. by feeding through a chicken membrane, dissected at different time intervals post bloodmeal and examined by light microscopy for the abundance and location of infections. RESULTS: All three Leishmania species produced heavy late stage infections in Lutzomyia longipalpis or Phlebotomus duboscqi sand flies used as positive controls. In contrast, none of them completed their developmental cycle in Sergentomyia females; Leishmania promastigotes developed within the bloodmeal enclosed by the peritrophic matrix (PM) but were defecated together with the blood remnants, failing to establish a midgut infection. In S. schwetzi, the PM persisted significantly longer than in L. longipalpis and it was degraded almost simultaneously with defecation. Therefore, Leishmania transformation from procyclic to long nectomonad forms was delayed and parasites did not attach to the midgut epithelium. CONCLUSIONS: Sergentomyia schwetzi is refractory to human Leishmania species and the data indicate that the crucial aspect of the refractoriness is the relative timing of defecation versus PM degradation.

• MeSH
• hmyz - vektory parazitologie   MeSH
• Leishmania donovani růst a vývoj   patogenita   fyziologie   MeSH
• leishmanióza viscerální parazitologie   přenos   MeSH
• lidé MeSH
• Psychodidae parazitologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The Leishmania protozoan parasites cause devastating human diseases. Leishmania have been considered to replicate clonally, without genetic exchange. However, an accumulation of evidence indicates that there are inter-specific and intra-specific hybrids among natural populations. The first and so far only experimental proof of genetic exchange was obtained in 2009 when double drug resistant Leishmania major hybrids were produced by co-infecting sand flies with two strains carrying different drug resistance markers. However, the location and timing of hybridisation events in sand flies has not been described. METHODOLOGY/PRINCIPAL FINDINGS: Here we have co-infected Phlebotomus perniciosus and Lutzomyia longipalpis with transgenic promastigotes of Leishmania donovani strains carrying hygromycin or neomycin resistance genes and red or green fluorescent markers. Fed females were dissected at different times post bloodmeal (PBM) and examined by fluorescent microscopy or fluorescent activated cell sorting (FACS) followed by confocal microscopy. In mixed infections strains LEM3804 and Gebre-1 reached the cardia and stomodeal valves more rapidly than strains LEM4265 and LV9. Hybrids unequivocally expressing both red and green fluorescence were seen in single flies of both vectors tested, co-infected with LEM4265 and Gebre-1. The hybrids were present as short (procyclic) promastigotes 2 days PBM in the semi-digested blood in the endoperitrophic space. Recovery of a clearly co-expressing hybrid was also achieved by FACS. However, hybrids could not sustain growth in vitro. CONCLUSIONS/SIGNIFICANCE: For the first time, we observed L. donovani hybrids in the sand fly vector, 2 days PBM and described the morphological stages involved. Fluorescence microscopy in combination with FACS allows visualisation and recovery of the progeny of experimental crosses but on this occasion the hybrids were not viable in vitro. Nevertheless, genetic exchange in L. donovani has profound epidemiological significance, because it facilitates the emergence and spread of new phenotypic traits.

• MeSH
• červený fluorescenční protein MeSH
• fluorescence MeSH
• fluorescenční mikroskopie MeSH
• hmyz - vektory parazitologie   MeSH
• hybridizace genetická * MeSH
• křížení genetické MeSH
• Leishmania donovani cytologie   genetika   růst a vývoj   patogenita   MeSH
• leishmanióza viscerální parazitologie   MeSH
• lidé MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• průtoková cytometrie MeSH
• Psychodidae parazitologie   MeSH
• stadia vývoje fyziologie   MeSH
• transfekce MeSH
• trávicí systém cytologie   parazitologie   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• luminescentní proteiny MeSH
• zelené fluorescenční proteiny MeSH

The stage-regulated HASPB and SHERP proteins of Leishmania major are predominantly expressed in cultured metacyclic parasites that are competent for macrophage uptake and survival. The role of these proteins in parasite development in the sand fly vector has not been explored, however. Here, we confirm that expression of HASPB is detected only in vector metacyclic stages, correlating with the expression of metacyclic-specific lipophosphoglycan and providing the first definitive protein marker for this infective sand fly stage. Similarly, SHERP is expressed in vector metacyclics but is also detected at low levels in the preceding short promastigote stage. Using genetically modified parasites lacking or complemented for the LmcDNA16 locus on chromosome 23 that contains the HASP and SHERP genes, we further show that the presence of this locus is essential for parasite differentiation to the metacyclic stage in Phlebotomus papatasi. While wild-type and complemented parasites transform normally in late-stage infections, generating metacyclic promastigotes and colonizing the sand fly stomodeal valve, null parasites accumulate at the earlier elongated nectomonad stage of development within the abdominal and thoracic midgut of the sand fly. Complementation with HASPB or SHERP alone suggests that HASPB is the dominant effector molecule in this process.

• MeSH
• antigeny protozoální biosyntéza   MeSH
• esenciální geny MeSH
• geneticky modifikované organismy MeSH
• genový knockout MeSH
• Leishmania major růst a vývoj   MeSH
• Phlebotomus parazitologie   MeSH
• protozoální proteiny biosyntéza   MeSH
• stanovení celkové genové exprese MeSH
• testy genetické komplementace MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny protozoální MeSH
• HASPB protein, Leishmania MeSH Prohlížeč
• protozoální proteiny MeSH