BACKGROUND: Ticks are obligate hematophagous arthropods transmitting a wide range of pathogens to humans and animals. They also harbor a non-pathogenic microbiota, primarily in the ovaries and the midgut. In the previous study on Ixodes ricinus, we used a culture-independent approach and showed a diverse but quantitatively poor midgut bacterial microbiome. Our analysis also revealed the absence of a core microbiome, suggesting an environmental origin of the tick midgut microbiota. METHODS: A bacterial analysis of the midgut of adult females collected by flagging from two localities in the Czech Republic was performed. Using the culture-independent approach, we tested the hypothesis that the midgut microbiome is of the environmental origin. We also cultured indigenous bacteria from the tick midgut and used these to feed ticks artificially in an attempt to manipulate the midgut microbiome. RESULTS: The midgut showed a very low prevalence and abundance of culturable bacteria, with only 37% of ticks positive for bacteria. The culture-independent approach revealed the presence of Borrelia sp., Spiroplasma sp., Rickettsia sp., Midichloria sp. and various mainly environmental Gram-positive bacterial taxa. The comparison of ticks from two regions revealed that the habitat influenced the midgut bacterial diversity. In addition, the midgut of ticks capillary fed with the indigenous Micrococcus luteus (Gram-positive) and Pantoea sp. (Gram-negative) could not be colonized due to rapid and effective clearance of both bacterial taxa. CONCLUSIONS: The midgut microbiome of I. ricinus is diverse but low in abundance, with the exception of tick-borne pathogens and symbionts. The environment impacts the diversity of the tick midgut microbiome. Ingested extracellular environmental bacteria are rapidly eliminated and are not able to colonize the gut. We hypothesize that bacterial elimination triggered in the midgut of unfed adult females is critical to maintain low microbial levels during blood-feeding.

• Klíčová slova
• Capillary feeding, Culturing, High-throughput sequencing, Ixodes ricinus, Microbiome, Microbiome manipulation, Midgut,
• MeSH
• Borrelia * MeSH
• klíště * mikrobiologie   MeSH
• mikrobiota * MeSH
• Rickettsia * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH

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

Antimicrobial peptides (AMPs) are produced to control bacteria, fungi, protozoa, and other infectious agents. Sand fly larvae develop and feed on a microbe-rich substrate, and the hematophagous females are exposed to additional pathogens. We focused on understanding the role of the AMPs attacin (Att), cecropin (Cec), and four defensins (Def1, Def2, Def3, and Def4) in Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. Larvae and adults were collected under different feeding regimens, in addition to females artificially infected by Leishmania infantum. AMPs' gene expression was assessed by qPCR, and gene function of Att and Def2 was investigated by gene silencing. The gene knockdown effect on bacteria and parasite abundance was evaluated by qPCR, and parasite development was verified by light microscopy. We demonstrate that L. longipalpis larvae and adults trigger AMPs expression during feeding, which corresponds to an abundant presence of bacteria. Att and Def2 expression were significantly increased in Leishmania-infected females, while Att suppression favored bacteria growth. In conclusion, L. longipalpis AMPs' expression is tuned in response to bacteria and parasites but does not seem to interfere with the Leishmania cycle.

• Klíčová slova
• Leishmania, Lutzomyia longipalpis, RNAi gene silencing, antimicrobial peptides, innate immunity, microbiota,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Leishmaniasis, caused by parasites of the genus Leishmania, is a disease that affects up to 8 million people worldwide. Parasites are transmitted to human and animal hosts through the bite of an infected sand fly. Novel strategies for disease control require a better understanding of the key step for transmission, namely the establishment of infection inside the fly. METHODS: The aim of this work was to identify sand fly systemic transcriptomic signatures associated with Leishmania infection. We used next generation sequencing to describe the transcriptome of whole Phlebotomus papatasi sand flies when fed with blood alone (control) or with blood containing one of three trypanosomatids: Leishmania major, L. donovani and Herpetomonas muscarum, the latter being a parasite not transmitted to humans. RESULTS: Of the trypanosomatids studied, only L. major was able to successfully establish an infection in the host P. papatasi. However, the transcriptional signatures observed after each parasite-contaminated blood meal were not specific to success or failure of a specific infection and they did not differ from each other. The transcriptional signatures were also indistinguishable after a non-contaminated blood meal. CONCLUSIONS: The results imply that sand flies perceive Leishmania as just one feature of their microbiome landscape and that any strategy to tackle transmission should focus on the response towards the blood meal rather than parasite establishment. Alternatively, Leishmania could suppress host responses. These results will generate new thinking around the concept of stopping transmission by controlling the parasite inside the insect.

• MeSH
• hmyz - vektory metabolismus   parazitologie   MeSH
• krev parazitologie   MeSH
• Leishmania infantum MeSH
• Leishmania major MeSH
• leishmanióza parazitologie   přenos   MeSH
• lidé MeSH
• Phlebotomus metabolismus   parazitologie   MeSH
• stanovení celkové genové exprese * MeSH
• stravovací zvyklosti MeSH
• Trypanosomatina * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Background: Massilia virus (MASV) is a phlebovirus isolated from Phlebotomus perniciosus in various regions of southwestern Europe. It is closely related to human pathogens such as Toscana virus and sandfly fever Naples virus. The natural cycle of phleboviruses is poorly understood. Indeed, experimental studies demonstrate that transovarial and sexual transmission are not efficient enough for the maintenance of the virus in nature and to date there is no convincing evidence that a species of vertebrates is the reservoir of the virus. Here, we studied various transmission routes of MASV taking advantage of experimental colonies representing different species of sand flies. Methodology/Principal findings: In P. perniciosus, four sources of infection were compared: (i) Virus-seeded larval food to the first instar larvae (L1), or (ii) to the fourth instar larvae (L4), (iii) virus-seeded blood meal to adult females, and (iv) virus-seeded sugar meal to adults of both sexes. From 875 adults emerged from infected L1 and L4, only three were positive. In females infected by bloodmeal the infection rate was high before defecation, then it decreased drastically; MASV RNA was detected in only 5 out of 27 post-defecation. Surprisingly, the most efficient route of infection was observed after intake of virus-seeded sugar meal: 72% of females (79/110) and 52% of males (51/99) were found to be MASV RNA-positive. In addition, MASV-infected sandflies regurgitated virus particules into the sugar drop and MASV RNA was detectable in this drop for at least 24 h after regurgitation. MASV RNA was detected in about one third of the P. perniciosus exposed to this sugar drop contaminated by regurgitation. Sugar meal infection was also tested with six other species of sand flies. In males, there were no significant differences in infection rates when compared to P. perniciosus. In females, most species tested showed high infection rate at the beginning but then significant gradual decrease in infection rate during the experiment. Conclusions/Significance: We present the first description of arboviral infection of a dipteran vector using sugar meal. In all seven sand fly species tested, MASV was detected for two weeks post-infection. Our results showed that MASV can be transmitted between P. perniciosus either through co-feeding or via an infected sugar source such as plant sap. These newly described routes of horizontal transmission may play an important role in the circulation of phleboviruses in nature.

• Klíčová slova
• Lutzomyia, Phenuiviridae, Phlebotomus, Phlebovirus, Sergentomyia, Toscana virus, sand fly, virus transmission,
• MeSH
• cukry MeSH
• fomity virologie   MeSH
• hmyz - vektory fyziologie   virologie   MeSH
• horečka pappataci přenos   MeSH
• jídla MeSH
• kontaminace potravin * MeSH
• Phlebovirus růst a vývoj   izolace a purifikace   MeSH
• Psychodidae fyziologie   virologie   MeSH
• stravovací zvyklosti * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cukry 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: An important factor influencing the transmission dynamics of vector-borne diseases is the contribution of hosts with different parasitemia (no. of parasites per ml of blood) to the infected vector population. Today, estimation of this contribution is often impractical since it relies exclusively on limited-scale xenodiagnostic or artificial feeding experiments (i.e., measuring the proportion of vectors that become infected after feeding on infected blood/host). METHODOLOGY: We developed a novel mechanistic model that facilitates the quantification of the contribution of hosts with different parasitemias to the infection of the vectors from data on the distribution of these parasitemias within the host population. We applied the model to an ample data set of Leishmania donovani carriers, the causative agent of visceral leishmaniasis in Ethiopia. RESULTS: Calculations facilitated by the model quantified the host parasitemias that are mostly responsible for the infection of vector, the sand fly Phlebotomus orientalis. Our findings indicate that a 3.2% of the most infected people were responsible for the infection of between 53% and 79% (mean - 62%) of the infected sand fly vector population. SIGNIFICANCE: Our modeling framework can easily be extended to facilitate the calculation of the contribution of other host groups (such as different host species, hosts with different ages) to the infected vector population. Identifying the hosts that contribute most towards infection of the vectors is crucial for understanding the transmission dynamics, and planning targeted intervention policy of visceral leishmaniasis as well as other vector borne infectious diseases (e.g., West Nile Fever).

• MeSH
• hmyz - vektory parazitologie   MeSH
• kohortové studie MeSH
• Leishmania donovani izolace a purifikace   MeSH
• leishmanióza viscerální parazitologie   přenos   MeSH
• lidé MeSH
• logistické modely MeSH
• parazitemie přenos   MeSH
• Phlebotomus parazitologie   MeSH
• Psychodidae parazitologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Etiopie epidemiologie   MeSH