Accurate identification of Culicidae at aquatic developmental stages by MALDI-TOF MS profiling

International audienceBACKGROUND: The identification of mosquito vectors is generally based on morphological criteria, but for aquatic stages, morphological characteristics may be missing, leading to incomplete or incorrect identification. The high cost of molecular biology techniques requires the development of an alternative strategy. In the last decade, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has proved to be efficient for arthropod identification at the species level.METHODS:To investigate the usefulness of MALDI-TOF MS for the identification of mosquitoes at aquatic stages, optimizations of sample preparation, diet, body parts and storage conditions were tested. Protein extracts of whole specimens from second larval stage to pupae were selected for the creation of a reference spectra database. The database included a total of 95 laboratory-reared specimens of 6 mosquito species, including Anopheles gambiae (S form), Anopheles coluzzi (M form), Culex pipiens pipiens, Culex pipiens molestus, Aedes aegypti and 2 colonies of Aedes albopictus.RESULTS:The present study revealed that whole specimens at aquatic stages produced reproducible and singular spectra according to the mosquito species. Moreover, MS protein profiles appeared weakly affected by the diet provided. Despite the low diversity of some MS profiles, notably for cryptic species, clustering analyses correctly classified all specimens tested at the species level followed by the clustering of early vs. late aquatic developmental stages. Discriminant mass peaks were recorded for the 6 mosquito species analyzed at larval stage 3 and the pupal stage. Querying against the reference spectra database of 149 new specimens at different aquatic stages from the 6 mosquito species revealed that 147 specimens were correctly identified at the species level and that early and late developmental stages were also distinguished.CONCLUSIONS:The present work highlights that MALDI-TOF MS profiling may be useful for the rapid and reliable identification of mosquito species at aquatic stages. With this proteomic tool, it becomes now conceivable to survey mosquito breeding sites prior to the mosquitoes' emergence and to adapt anti-vectorial measures according to the mosquito fauna detected

Oxidative phosphorylation is required for powering motility and development of the sleeping sickness parasite Trypanosoma brucei in the tsetse fly vector

The single-celled parasite Trypanosoma brucei is transmitted by hematophagous tsetse flies. Life cycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonizes the glucose-poor insect midgut, ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation (OXPHOS). This process involves respiratory chain complexes and F1Fo-ATP synthase and requires protein subunits of these complexes that are encoded in the parasite's mitochondrial DNA (kDNA). Here, we show that progressive loss of kDNA-encoded functions correlates with a decreasing ability to initiate and complete development in the tsetse. First, parasites with a mutated F1Fo-ATP synthase with reduced capacity for OXPHOS can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonize the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonizing or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1Fo-ATP synthase complex that is completely unable to produce ATP by OXPHOS can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, parasites lacking kDNA entirely can initiate differentiation but die soon after. Together, these scenarios suggest that efficient ATP production via OXPHOS is not essential for initial colonization of the tsetse vector but is required to power trypanosome migration within the fly. IMPORTANCE African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands. Crucially, the parasite must switch from a sugar-based diet while in the mammal to a diet based primarily on amino acids when it develops in the insect. Here, we show that efficient energy production by an organelle called the mitochondrion is critical for the trypanosome's ability to swim and to migrate through the tsetse fly. Surprisingly, trypanosomes with impaired mitochondrial energy production are only mildly compromised in their ability to colonize the tsetse fly midgut. Our study adds a new perspective to the emerging view that infection of tsetse flies by trypanosomes is more complex than previously thought

Особенности ценообразования на рынке экскурсионных услуг Крыма

Цель статьи – анализ специфики ценообразования на экскурсионные услуги в Крыму как важного фактора развития этого рынка

Sudy of relationship between arthropods and rickettsia felis

La lutte anti-vectorielle est l’un des volets le plus important de l’entomologie médicale et nécessite une identification précise des vecteurs. Cette dernière décennie, la technique du MALDI-TOF MS a prouvé son potentiel comme outil rapide et efficace pour l'identification des arthropodes hématophages adultes. Dès lorsNous nous sommes intéressés à la mise au point d’un protocole d’identification des stades aquatiques de moustique par MALDI-TOF MS d’une part. D’autre part la détection d’un pathogène dans un arthropode n’implique pas forcement sa capacité à transmettre. L’incrimination d’un arthropode comme vecteur respecte certaines règles allant de la suspicion à la démonstration de sa compétence vectorielle au laboratoire. Afin de mieux comprendre l’épidémiologie de R. felis nous avons d’abord participé à une investigation conduite à l’Ile de la Réunion, en testant des puces, les seuls vecteurs biologiques connus jusqu’à présent. Ensuite Nous avons démontré le rôle potentiel des moustiques en particulier d’Anopheles gambiae à transmettre Rickettsia felis. Enfin nous avons utilisé le MALDI-TOF MS pour la détermination du statut infectieux d’Anopheles gambiae à R. felis. Nous proposons également un cycle probable de transmission de R. felis à l’homme incluant les psoques et les moustiques.Vector control is one of the most important aspects of medical entomology and requires accurate identification of vectors. Within the past decade, the MALDI-TOF MS technique has proven its potential as a fast and effective tool for identification of adult blood-sucking arthropods. From then on we were interested in the development of an identification protocol of aquatic stages of mosquitoes by MALDI-TOF MS. On the other hand, the detection of a pathogen in an arthropod does not necessarily mean its ability to transmit. Incrimination of an arthropod as vector follows certain rules ranging from suspicion to demonstrate its vector competence in the laboratory. To better understand the epidemiology of R. felis we first participated in an investigation conducted in Reunion, testing fleas, the only biological vectors known to date. We demonstrated the potential role of the mosquito particularly Anopheles gambiae, in the transmission of R. felis. Finally, we used the MALDI-TOF MS for the determination of the Anopheles gambiae infection status to R. felis. We also offer a probable transmission cycle of R. felis to man including psocids and mosquitoes

Microbial Pre-exposure and Vectorial Competence of Anopheles Mosquitoes

Anopheles female mosquitoes can transmit Plasmodium, the malaria parasite. During their aquatic life, wild Anopheles mosquito larvae are exposed to a huge diversity of microbes present in their breeding sites. Later, adult females often take successive blood meals that might also carry different micro-organisms, including parasites, bacteria, and viruses. Therefore, prior to Plasmodium ingestion, the mosquito biology could be modulated at different life stages by a suite of microbes present in larval breeding sites, as well as in the adult environment. In this article, we highlight several naturally relevant scenarios of Anopheles microbial pre-exposure that we assume might impact mosquito vectorial competence for the malaria parasite: (i) larval microbial exposures; (ii) protist co-infections; (iii) virus co-infections; and (iv) pathogenic bacteria co-infections. In addition, significant behavioral changes in African Anopheles vectors have been associated with increasing insecticide resistance. We discuss how these ethological modifications may also increase the repertoire of microbes to which mosquitoes could be exposed, and that might also influence their vectorial competence. Studying Plasmodium–Anopheles interactions in natural microbial environments would efficiently contribute to refining the transmission risks

Microbial Pre-exposure and Vectorial Competence of Anopheles Mosquitoes

International audienceAnopheles female mosquitoes can transmit Plasmodium, the malaria parasite. During their aquatic life, wild Anopheles mosquito larvae are exposed to a huge diversity of microbes present in their breeding sites. Later, adult females often take successive blood meals that might also carry different micro-organisms, including parasites, bacteria, and viruses. Therefore, prior to Plasmodium ingestion, the mosquito biology could be modulated at different life stages by a suite of microbes present in larval breeding sites, as well as in the adult environment. In this article, we highlight several naturally relevant scenarios of Anopheles microbial pre-exposure that we assume might impact mosquito vectorial competence for the malaria parasite: (i) larval microbial exposures; (ii) protist co-infections; (iii) virus co-infections; and (iv) pathogenic bacteria co-infections. In addition, significant behavioral changes in African Anopheles vectors have been associated with increasing insecticide resistance. We discuss how these ethological modifications may also increase the repertoire of microbes to which mosquitoes could be exposed, and that might also influence their vectorial competence. Studying Plasmodium-Anopheles interactions in natural microbial environments would efficiently contribute to refining the transmission risks

Identification of blood meal sources in the main African malaria mosquito vector by MALDI-TOF MS

International audienceBackground: The identification of blood meal sources in malaria vectors is critical to better understanding host/vector interactions and malaria epidemiology and control. Currently, the identification of mosquito blood meal origins is based on time-consuming and costly techniques such as precipitin tests, ELISA and molecular tools. Although these tools have been validated to identify the blood meal and trophic preferences of female Anopheles mosquitoes, they present several limitations. Recently, matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successfully used as a quick and accurate tool for arthropod identification, including mosquitoes. The aim of the present work was to test whether MALDI-TOF MS could also be applied to identification of blood meal sources from engorged mosquitoes. Methods: Abdomen proteins extracted from Anopheles gambiae (stricto sensu, S molecular form) that were either unengorged or artificially engorged on seven distinct types of vertebrate blood (human, horse, sheep, rabbit, mouse, rat, dog) were submitted for MALDI-TOF MS. Results: The comparison of mass spectrometry (MS) spectra from mosquito abdomens collected 1 h post-feeding, were able to discriminate blood meal origins. Moreover, using Aedes albopictus specimens, abdominal protein MS spectra from engorged mosquitoes were found specific to host blood source and independent of the mosquito species. A sequential analysis revealed stability of mosquito abdominal protein spectra up to 24 h post-feeding. Conclusions: These results indicate that MALDI-TOF MS could determine feeding patterns of freshly engorged mosquitoes up to 24 h post-blood meal. The MALDI-TOF MS technique appears to be an efficient tool for large epidemiological surveillance of vector-borne diseases and outbreak source identification