Booklice Liposcelis bostrychophila Naturally Infected by Rickettsia felis Cause Fever and Experimental Pneumonia in Mammals.

BACKGROUND: Rickettsia felis is emergent in tropical areas. Despite its high morbidity, its natural history has not yet been fully determined. We investigated the role of the common household booklouse, Liposcelis bostrychophila, recently found to harbor R. felis. METHODS: Blood samples from 372 febrile patients from Senegalese villages, as well as nasal and skin samples from 264 asymptomatic individuals, were tested for cat flea-associated and booklice-associated strains of R. felis. Dust samples from beds were collected to isolate booklice and R. felis. Mice were infected with aerosol of R. felis strain from naturally infected booklice. RESULTS: Forty febrile patients (11%) were infected by R. felis, including 26 (7%) by the booklice-associated strain. Nine nasal samples (3.4%) and 28 skin samples (10.6%) contained R. felis, including 7 and 24, respectively, with the booklice-associated strain. The presence of live L. bostrychophila was observed in 32 dust samples (16.8%); R. felis was identified in 62 dust samples (32.5%). Several mice samples were positive for R. felis; interstitial lymphohistiocytic infiltrates were identified in lungs. CONCLUSIONS: Liposcelis bostrychophila may be a reservoir of R. felis. The booklice-associated strain is pathogenic in mammals, causing pneumonia. Human infection may be acquired via inhalation of infected booklice particles.

Role of Anopheles Mosquitoes in Cache Valley Virus Lineage Displacement, New York, USA.

Cache Valley virus (CVV) is a mosquitoborne virus that infects livestock and humans. We report results of surveillance for CVV in New York, USA, during 2000-2016; full-genome analysis of selected CVV isolates from sheep, horse, humans, and mosquitoes from New York and Canada; and phenotypic characterization of selected strains. We calculated infection rates by using the maximum-likelihood estimation method by year, region, month, and mosquito species. The highest maximum-likelihood estimations were for Anopheles spp. mosquitoes. Our phylogenetic analysis identified 2 lineages and found evidence of segment reassortment. Furthermore, our data suggest displacement of CVV lineage 1 by lineage 2 in New York and Canada. Finally, we showed increased vector competence of An. quadrimaculatus mosquitoes for lineage 2 strains of CVV compared with lineage 1 strains.

Evaluation of the residual efficacy and physical durability of five long-lasting insecticidal nets (LLINs) in Senegal.

BACKGROUND: The preventive and curative strategies of malaria are based on promoting the use of long-lasting insecticidal nets (LLINs) and treating confirmed cases with artemisinin-based combination therapy. These strategies have led to a sharp decline in the burden of malaria, which remains a significant public health problem in sub-Saharan countries. The objective of this study was to determine and compare the residual efficacy of LLINs recommended by the World Health Organization. METHODS: The study was conducted in six villages in two sites in Senegal located in the Sahelo-Sudanian area of the Thiès region, 70 km from Dakar and in Mbagame, a semi-urban zone in the Senegal River Valley. A census was conducted of all sleeping places in each household to be covered by LLINs. Five brands of LLIN were distributed, and every six months, retention rates, net use, maintenance, physical integrity, insecticide chemical content, and biological efficacy were examined for each type of LLIN. RESULTS: A total of 3012 LLINs were distributed in 1249 households in both sites, with an average coverage rate of 94% (95% CI 92.68-95.3). After 36 months, the average retention rate was 12.5% and this rate was respectively 20.5%, 15.1%, 10%, 7%, and 3% for Olyset Net®, Dawa Plus® 2.0, PermaNet® 2.0, NetProtect® and Life Net®, respectively. The proportion of LLINs with holes and the average number of holes per mosquito net increased significantly during each follow-up, with a large predominance of size 1 (small) holes for all types of LLINs distributed. During the three-year follow-up, bioassay mortality rates of a susceptible strain of insectary reared Anopheles coluzzii decreased in the following net types: in Dawa Plus® 2.0 (100% to 51.7%), PermaNet® 2.0 (96.6% to 83%), and Olyset Net® (96.6% to 33.3%). Mortality rates remained at 100% in Life Net® over the same time period. After 36 months, the average insecticide content per brand of LLIN decreased by 40.9% for Dawa Plus® 2.0, 31% for PermaNet® 2.0, 39.6% for NetProtect® and 51.9% for Olyset Net® and 40.1% for Life Net. CONCLUSIONS: Although some net types retained sufficient insecticidal activity, based on all durability parameters measured, none of the net types survived longer than 2 years.

RNA-Seq analysis of blood meal induced gene-expression changes in Aedes aegypti ovaries.

BACKGROUND: Transmission of pathogens by vector mosquitoes is intrinsically linked with mosquito's reproductive strategy because anautogenous mosquitoes require vertebrate blood to develop a batch of eggs. Each cycle of egg maturation is tightly linked with the intake of a fresh blood meal for most species. Mosquitoes that acquire pathogens during the first blood feeding can transmit the pathogens to susceptible hosts during subsequent blood feeding and also vertically to the next generation via infected eggs. Large-scale gene-expression changes occur following each blood meal in various tissues, including ovaries. Here we analyzed mosquito ovary transcriptome following a blood meal at three different time points to investigate blood-meal induced changes in gene expression in mosquito ovaries. RESULTS: We collected ovaries from Aedes aegypti that received a sugar meal or a blood meal on days 3, 10 and 20 post blood meal for transcriptome analysis. Over 4000 genes responded differentially following ingestion of a blood meal on day 3, and 660 and 780 genes on days 10 and 20, respectively. Proteins encoded by differentially expressed genes (DEGs) on day 3 include odorant binding proteins (OBPs), defense-specific proteins, and cytochrome P450 detoxification enzymes. In addition, we identified 580 long non-coding RNAs that are differentially expressed at three time points. Gene ontology analysis indicated that genes involved in peptidase activity, oxidoreductase activity, extracellular space, and hydrolase activity, among others were enriched on day 3. Although most of the DEGs returned to the nonsignificant level compared to the sugar-fed mosquito ovaries following oviposition on days 10 and 20, there remained differences in the gene expression pattern in sugar-fed and blood-fed mosquitoes. CONCLUSIONS: Enrichment of OBPs following blood meal ingestion suggests that these genes may have other functions besides being part of the olfactory system. The enrichment of immune-specific genes and cytochrome P450 genes indicates that ovaries become well prepared to protect their germ line from any pathogens that may accompany the blood meal or from environmental contamination during oviposition, and to deal with the detrimental effects of toxic metabolites.

Transmission potential of Rickettsia felis infection by Anopheles gambiae mosquitoes.

A growing number of recent reports have implicated Rickettsia felis as a human pathogen, paralleling the increasing detection of R. felis in arthropod hosts across the globe, primarily in fleas. Here Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, were fed with either blood meal infected with R. felis or infected cellular media administered in membrane feeding systems. In addition, a group of mosquitoes was fed on R. felis-infected BALB/c mice. The acquisition and persistence of R. felis in mosquitoes was demonstrated by quantitative PCR detection of the bacteria up to day 15 postinfection. R. felis was detected in mosquito feces up to day 14. Furthermore, R. felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries, although no vertical transmission was observed. R. felis was also found in the cotton used for sucrose feeding after the mosquitoes were fed infected blood. Natural bites from R. felis-infected An. gambiae were able to cause transient rickettsemias in mice, indicating that this mosquito species has the potential to be a vector of R. felis infection. This is particularly important given the recent report of high prevalence of R. felis infection in patients with "fever of unknown origin" in malaria-endemic areas.

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

BACKGROUND: 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.

Aedes albopictus saliva contains a richer microbial community than the midgut.

BACKGROUND: Past findings demonstrate that arthropods can egest midgut microbiota into the host skin leading to dual colonization of the vertebrate host with pathogens and saliva microbiome. A knowledge gap exists on how the saliva microbiome interacts with the pathogen in the saliva. To fill this gap, we need to first define the microbial composition of mosquito saliva. METHODS: The current study aimed at analyzing and comparing the microbial profile of Aedes albopictus saliva and midgut as well as assessing the impact of Zika virus (ZIKV) infection on the midgut and saliva microbial composition. Colony-reared Ae. albopictus strains were either exposed to ZIKV infectious or noninfectious bloodmeal. At 14 ays postinfection, the 16S V3-V4 hypervariable rRNA region was amplified from midgut and saliva samples and sequenced on an Illumina MiSeq platform. The relative abundance and diversity of midgut and saliva microbial taxa were assessed. RESULTS: We observed a richer microbial community in the saliva compared with the midgut, yet some of the microbial taxa were common in the midgut and saliva. ZIKV infection did not impact the microbial diversity of midgut or saliva. Further, we identified Elizabethkingia spp. in the Ae. albopictus saliva. CONCLUSIONS: This study provides insights into the microbial community of the Ae. albopictus saliva as well as the influence of ZIKV infection on the microbial composition of its midgut and saliva. The identification of Elizabethkingia spp., an emerging pathogen of global health significance, in Ae. albopictus saliva is of medical importance. Future studies to assess the interactions between Ae. albopictus saliva microbiome and ZIKV could lead to novel strategies for developing transmission barrier tools.

Vector competence of Anopheles quadrimaculatus and Aedes albopictus for genetically distinct Jamestown Canyon virus strains circulating in the Northeast United States.

BACKGROUND: Jamestown Canyon virus (JCV; Peribunyaviridae, Orthobunyavirus) is a mosquito-borne pathogen belonging to the California serogroup. The virus is endemic in North America and increasingly recognized as a public health concern. In this study, we determined the vector competence of Anopheles (An.) quadrimaculatus and Aedes (Ae.) albopictus for five JCV strains belonging to the two lineages circulating in the Northeast. METHODS: An. quadrimaculatus and Ae. albopictus were fed blood meals containing two lineage A strains and three lineage B strains. Vector competence of both mosquito species was evaluated at 7- and 14-days post-feeding (dpf) by testing for virus presence in bodies, legs, and saliva. RESULTS: Our results demonstrated that Ae. albopictus mosquitoes are a competent vector for both lineages, with similar transmission levels for all strains tested. Variable levels of infection (46-83%) and dissemination (17-38%) were measured in An. quadrimaculatus, yet no transmission was detected for the five JCV strains evaluated. CONCLUSIONS: Our results demonstrate that establishment of Ae. albopictus in the Northeast could increase the risk of JCV but suggest An. quadrimaculatus are not a competent vector for JCV.

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.

Aedes Albopictus and Cache Valley virus: a new threat for virus transmission in New York State.

We report surveillance results of Cache Valley virus (CVV; Peribunyaviridae, Orthobunyavirus) from 2017 to 2020 in New York State (NYS). Infection rates were calculated using the maximum likelihood estimation (MLE) method by year, region, and mosquito species. The highest infection rates were identified among Anopheles spp. mosquitoes and we detected the virus in Aedes albopictus for the first time in NYS. Based on our previous Anopheles quadrimaculatus vector competence results for nine CVV strains, we selected among them three stains for further characterization. These include two CVV reassortants (PA and 15041084) and one CVV lineage 2 strain (Hu-2011). We analyzed full genomes, compared in vitro growth kinetics and assessed vector competence of Aedes albopictus. Sequence analysis of the two reassortant strains (PA and 15041084) revealed 0.3%, 0.4%, and 0.3% divergence; and 1, 10, and 6 amino acid differences for the S, M, and L segments, respectively. We additionally found that the PA strain was attenuated in vertebrate (Vero) and mosquito (C6/36) cell culture. Furthemore, Ae. albopictus mosquitoes are competent vectors for CVV Hu-2011 (16.7-62.1% transmission rates) and CVV 15041084 (27.3-48.0% transmission rates), but not for the human reassortant (PA) isolate, which did not disseminate from the mosquito midgut. Together, our results demonstrate significant phenotypic variability among strains and highlight the capacity for Ae. albopictus to act as a vector of CVV.

Zika virus infects Aedes aegypti ovaries.

Pathogens are transmitted from one host to another either by vertical transmission (VT) or horizontal transmission (HT). Mosquito-borne arboviruses (arthropod-borne viruses), including several clinically important viruses such as dengue, Zika, West Nile and chikungunya viruses persist in nature by both VT and HT. VT may also serve as an essential link in the transmission cycle during adverse environmental conditions. VT rates (VTRs) vary between virus families and even among viruses within the same genus. The mechanism behind these differences in VTRs among viruses is poorly understood. For efficient VT to occur, viruses must infect the mosquito germline. Here, we show that Zika virus infects mosquito ovaries and is transmitted vertically at a low rate. The infected progeny derive from mosquitoes with infected ovaries. The prevalence of ovary infection increases after a second non-infectious blood meal following an infectious blood meal.

Transmission potential of Mayaro virus by Aedes albopictus, and Anopheles quadrimaculatus from the USA.

BACKGROUND: Mayaro virus (MAYV; Alphavirus, Togaviridae) is an emerging pathogen endemic in South American countries. The increase in intercontinental travel and tourism-based forest excursions has resulted in an increase in MAYV spread, with imported cases observed in Europe and North America. Intriguingly, no local transmission of MAYV has been reported outside South America, despite the presence of potential vectors. METHODS: We assessed the vector competence of Aedes albopictus from New York and Anopheles quadrimaculatus for MAYV. RESULTS: The results show that Ae. albopictus from New York and An. quadrimaculatus are competent vectors for MAYV. However, Ae. albopictus was more susceptible to infection. Transmission rates increased with time for both species, with rates of 37.16 and 64.44% for Ae. albopictus, and of 25.15 and 48.44% for An. quadrimaculatus, respectively, at 7 and 14 days post-infection. CONCLUSIONS: Our results suggest there is a risk of further MAYV spread throughout the Americas and autochthonous transmission in the USA. Preventive measures, such as mosquito surveillance of MAYV, will be essential for early detection.

Possible influence of Plasmodium/Trypanosoma co-infections on the vectorial capacity of Anopheles mosquitoes.

OBJECTIVE: In tropical Africa, trypanosomiasis is present in endemic areas with many other diseases including malaria. Because malaria vectors become more anthropo-zoophilic under the current insecticide pressure, they may be exposed to trypanosome parasites. By collecting mosquitoes in six study sites with distinct malaria infection prevalence and blood sample from cattle, we tried to assess the influence of malaria-trypanosomiasis co-endemicity on the vectorial capacity of Anopheles. RESULTS: Overall, all animal infections were due to Trypanosoma vivax (infection rates from 2.6 to 10.5%) in villages where the lowest Plasmodium prevalence were observed at the beginning of the study. An. gambiae s.l. displayed trophic preferences for human-animal hosts. Over 84 mosquitoes, only one was infected by Plasmodium falciparum (infection rate: 4.5%) in a site that displayed the highest prevalence at the beginning of the study. Thus, Anopheles could be exposed to Trypanosoma when they feed on infected animals. No Plasmodium infection was observed in the Trypanosoma-infected animals sites. This can be due to an interaction between both parasites as observed in mice and highlights the need of further studies considering Trypanosoma/Plasmodium mixed infections to better characterize the role of these infections in the dynamic of malaria transmission and the mechanisms involved.

Leucine-Rich Immune Factor APL1 Is Associated With Specific Modulation of Enteric Microbiome Taxa in the Asian Malaria Mosquito Anopheles stephensi.

The commensal gut microbiome is contained by the enteric epithelial barrier, but little is known about the degree of specificity of host immune barrier interactions for particular bacterial taxa. Here, we show that depletion of leucine-rich repeat immune factor APL1 in the Asian malaria mosquito Anopheles stephensi is associated with higher midgut abundance of just the family Enterobacteraceae, and not generalized dysbiosis of the microbiome. The effect is explained by the response of a narrow clade containing two main taxa related to Klebsiella and Cedecea. Analysis of field samples indicate that these two taxa are recurrent members of the wild Anopheles microbiome. Triangulation using sequence and functional data incriminated relatives of C. neteri and Cedecea NFIX57 as candidates for the Cedecea component, and K. michiganensis, K. oxytoca, and K.sp. LTGPAF-6F as candidates for the Klebsiella component. APL1 presence is associated with host ability to specifically constrain the abundance of a narrow microbiome clade of the Enterobacteraceae, and the immune factor may promote homeostasis of this clade in the enteric microbiome for host benefit.

Exposure of Anopheles mosquitoes to trypanosomes reduces reproductive fitness and enhances susceptibility to Plasmodium.

During a blood meal, female Anopheles mosquitoes are potentially exposed to diverse microbes in addition to the malaria parasite, Plasmodium. Human and animal African trypanosomiases are frequently co-endemic with malaria in Africa. It is not known whether exposure of Anopheles to trypanosomes influences their fitness or ability to transmit Plasmodium. Using cell and molecular biology approaches, we found that Trypanosoma brucei brucei parasites survive for at least 48h after infectious blood meal in the midgut of the major malaria vector, Anopheles coluzzii before being cleared. This transient survival of trypanosomes in the midgut is correlated with a dysbiosis, an alteration in the abundance of the enteric bacterial flora in Anopheles coluzzii. Using a developmental biology approach, we found that the presence of live trypanosomes in mosquito midguts also reduces their reproductive fitness, as it impairs the viability of laid eggs by affecting their hatching. Furthermore, we found that Anopheles exposure to trypanosomes enhances their vector competence for Plasmodium, as it increases their infection prevalence. A transcriptomic analysis revealed that expression of only two Anopheles immune genes are modulated during trypanosome exposure and that the increased susceptibility to Plasmodium was microbiome-dependent, while the reproductive fitness cost was dependent only on the presence of live trypanosomes but was microbiome independent. Taken together, these results demonstrate multiple effects upon Anopheles vector competence for Plasmodium caused by eukaryotic microbes interacting with the host and its microbiome, which may in turn have implications for malaria control strategies in co-endemic areas.

Gene copy number and function of the APL1 immune factor changed during Anopheles evolution.

BACKGROUND: The recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in An. stephensi have not yet been specifically examined. METHODS: The APL1 locus was manually annotated to confirm the computationally predicted single APL1 gene in An. stephensi. APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in An. stephensi and An. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium and expression analysis. RESULTS: APL1 is present as a single ancestral gene in most Anopheles including An. stephensi but has expanded to three paralogs in an African lineage that includes only the Anopheles gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in An. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted An. stephensi is rescued by antibiotic treatment, suggesting that pathology due to bacteria is the cause of mortality, and indicating that the unique APL1 gene is essential for host survival. Successful Plasmodium development in An. stephensi depends upon APL1 activity for protection from high host mortality due to bacteria. In contrast, silencing of all three APL1 paralogs in An. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single An. stephensi APL1 gene is regulated by both the Imd and Toll immune pathways, while the two signaling pathways regulate different APL1 paralogs in the expanded APL1 locus. CONCLUSIONS: APL1 underwent loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. We infer that activity of the unique APL1 gene promotes longevity in An. stephensi by conferring protection from or tolerance to an effect of bacterial pathology. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the An. gambiae species complex in Africa.

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.

Rickettsia and Bartonella species in fleas from Reunion Island.

Rickettsia felis, Rickettsia typhi, and Bartonella DNA was detected by molecular tools in 12% of Rattus rattus fleas (Xenopsylla species) collected from Reunion Island. One-third of the infested commensal rodents captured during 1 year carried at least one infected flea. As clinical signs of these zoonoses are non-specific, they are often misdiagnosed.

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

BACKGROUND: 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.