Amélie Vantaux

Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes

Mosquitoes of the Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O’nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity.

Contribution to Malaria Transmission of Symptomatic and Asymptomatic Parasite Carriers in Cambodia

BackgroundnEliminating falciparum malaria in Cambodia is a top priority, requiring the implementation of novel tools and strategies to interrupt its transmission. To date, few data are available regarding the contributions to malaria transmission of symptomatic and asymptomatic carriers.nnnMethodsnDirect-membrane and skin feeding assays (DMFAs, SFAs) were performed, using Anopheles minimus and Anopheles dirus, to determine infectivity of symptomatic falciparum-infected patients and malaria asymptomatic carriers; a subset of the latter were followed up for 2 months to assess their transmission potential.nnnResultsnBy microscopy and real-time polymerase chain reaction, Plasmodium falciparum gametocyte prevalence rates were, respectively, 19.3% (n = 21/109) and 44% (n = 47/109) on day (D) 0 and 17.9% (n = 5/28) and 89.3% (n = 25/28) in recrudescent patients (Drec) (RT-PCR Drec vs D0 P = .002). Falciparum malaria patient infectivity was low on D0 (6.2%; n = 3/48) and in Drec (8.3%; n = 1/12). Direct-membrane feeding assays and SFAs gave similar results. None of the falciparum (n = 0/19) and 3 of 28 Plasmodium vivax asymptomatic carriers were infectious to mosquitoes, including those that were followed up for 2 months. Overall, P. falciparum gametocytemias were low except in a few symptomatic carriers.nnnConclusionsnOnly symptomatic falciparum malaria patients were infectious to mosquito vectors at baseline and recrudescence, highlighting the need to detect promptly and treat effectively P. falciparum patients.

A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.Currently available platforms to study liver stage of Plasmodium species have limitations. Here, the authors show that primary human hepatocyte cultures in 384-well format support hypnozoite and other liver stage development and are suitable for drug and antibody screens.

Characterization of P. vivax blood stage transcriptomes from field isolates reveals similarities among infections and complex gene isoforms

Our understanding of the structure and regulation of Plasmodium vivax genes is limited by our inability to grow the parasites in long-term in vitro cultures. Most P. vivax studies must therefore rely on patient samples, which typically display a low proportion of parasites and asynchronous parasites. Here, we present stranded RNA-seq data generated directly from a small volume of blood from three Cambodian vivax malaria patients collected before treatment. Our analyses show surprising similarities of the parasite gene expression patterns across infections, despite extensive variations in parasite stage proportion. These similarities contrast with the unique gene expression patterns observed in sporozoites isolated from salivary glands of infected Colombian mosquitoes. Our analyses also indicate that more than 10% of P. vivax genes encode multiple, often undescribed, protein-coding sequences, potentially increasing the diversity of proteins synthesized by blood stage parasites. These data also greatly improve the annotations of P. vivax gene untranslated regions, providing an important resource for future studies of specific genes.

No evidence for manipulation of Anopheles gambiae, An. coluzzii and An. arabiensis host preference by Plasmodium falciparum

Whether malaria parasites can manipulate mosquito host choice in ways that enhance parasite transmission toward suitable hosts and/or reduce mosquito attraction to unsuitable hosts (i.e. specific manipulation) is unknown. To address this question, we experimentally infected three species of mosquito vectors with wild isolates of the human malaria parasite Plasmodium falciparum, and examined the effects of immature and mature infections on mosquito behavioural responses to combinations of calf odour, human odour and outdoor air using a dual-port olfactometer. Regardless of parasite developmental stage and mosquito species, P. falciparum infection did not alter mosquito activation rate or their choice for human odours. The overall expression pattern of host choice of all three mosquito species was consistent with a high degree of anthropophily, with infected and uninfected individuals showing higher attraction toward human odour over calf odour, human odour over outdoor air, and outdoor air over calf odour. Our results suggest that, in this system, the parasite may not be able to manipulate the early long-range behavioural steps involved in the mosquito host-feeding process. Future studies are required to test whether malaria parasites can modify their mosquito host choice at a shorter range to enhance transmission.

The influence of facultative endosymbionts on honeydew carbohydrate and amino acid composition of the black bean aphid Aphis fabae: Influence on honeydew composition

The facultative endosymbionts Hamiltonella defensa and Regiella insecticola are commonly found in aphids. They are linked with various ecological benefits but generally occur at low prevalence, which indicates a possible harbouring cost. Little is known about how the presence of facultative endosymbionts is reflected in honeydew composition. Honeydew is the key mediator of the mutualism between aphids and their tending ants. The present study examines whether endosymbionts have an influence on aphid honeydew quality by comparing the amino acid and carbohydrate concentrations between infected and uninfected aphids. To this end, two genetic lines of the aphid Aphis fabae Scopoli are experimentally infected with different strains of Hamiltonella and Regiella. Infected aphids are shown to have reduced concentrations of amino acids in the honeydew compared with uninfected aphids. However, the presence of endosymbionts has no effect on the absolute amount of carbohydrates produced. Nevertheless, interclonal variation in honeydew composition between aphid genotypes is observed for both carbohydrate and amino acid production. These results imply that the nutritional value of honeydew depends on aphid genotype, as well as on the presence of secondary bacterial endosymbionts, which suggests that there is a physiological cost of harbouring endosymbionts and which could also impact aphid attractiveness to tending ants.

Therapeutic and Transmission-Blocking Efficacy of Dihydroartemisinin/Piperaquine and Chloroquine against Plasmodium vivax Malaria, Cambodia

We assessed the efficacy of standard 3-day courses of chloroquine and dihydroartemisinin/piperaquine against Plasmodium vivax malaria. Compared with chloroquine, dihydroartemisinin/piperaquine was faster in clearing asexual P. vivax parasites and blocking human-to-mosquito transmission. This drug combination was also more effective in preventing potential recurrences for >2 months.

Author Correction: A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum

The original version of this Article contained an error in the spelling of Richard Thomson-Luque, which was incorrectly given as Richard Thomson Luque. This error has now been corrected in both the PDF and HTML versions of the Article.

Field assessment of potential sugar feeding stations for disseminating bacteria in a paratransgenic approach to control malaria

BackgroundUsing bacteria to express and deliver anti-parasite molecules in mosquitoes is among the list of genetic tools to control malaria. The introduction and spread of transgenic bacteria through wild adult mosquitoes is one of the major challenges of this strategy. In prospect of future field experiments, an open field study with blank (without bacteria) attractive sugar bait (ASB) was performed under the assumption that transgenic bacteria would be spread to all sugar fed mosquitoes.MethodsTwo types of ASB stations were developed, one with clay pots (CP) placed at mosquito resting sites and one with window entry traps (WET) placed inside inhabited houses. The ASB consisted in either glucose, honey or fruit cocktail solutions. In addition, mark-release-recapture (MRR) experiment of mosquitoes after feeding them with glucose was also conducted to check the proportion of the mosquito population that can be reached by the two ASB stations as well as its suitability to complement the ASB stations for disseminating bacteria.ResultsOverall, 88% of the mosquitoes were collected in the WET_ASB. The CP_ASB stations were much less attractive with the highest average of 82u2009±u200911 mosquitoes/day in the CP near the wood piles. The proportions of sugar fed mosquitoes upon ASB were low in both type of ASB stations,u2009~u20092% andu2009~u200914% in WET and CP, respectively. Honey solution was the most attractive solution compared to the glucose and the fruit cocktail solutions. The recapture rate in the MRR experiment was low:u2009~u20094.1% over 7xa0days.ConclusionThe WET_ASB looks promising to disseminate transgenic bacteria to endophilic West Africa Anopheles mosquito. However, this feeding station may not be fully effective and could be combined with the CP_ASB to also target outdoor resting mosquitoes. Overall, efforts are needed to improve the mosquito-feeding rates upon ASB.

Can malaria parasites manipulate the odour-mediated host preference of their mosquito vectors?

Malaria parasites can manipulate mosquito feeding behaviours such as motivation and avidity to feed on vertebrate hosts in ways that increase parasite transmission. However, in natural conditions, not all vertebrate blood-sources are suitable hosts for the parasite. Whether malaria parasites can manipulate mosquito host choice in ways that enhance parasite transmission toward suitable hosts and/or reduce mosquito attraction to unsuitable hosts (i.e. specific manipulation) is unknown. To address this question, we experimentally infected three species of mosquito vectors (Anopheles coluzzii, Anopheles gambiae, and Anopheles arabiensis) with wild isolates of the human malaria parasite Plasmodium falciparum, and examined the effects of immature (oocyst) and mature (sporozoite) infections on mosquito behavioural responses (activation rate and odour choice) to combinations of calf odour, human odour and outdoor air using a dual-port olfactometer. Regardless of parasite developmental stage and mosquito species, P. falciparum infection did not alter mosquito activation rate or their choice for human odours. The overall expression pattern of host choice of all three mosquito species was consistent with a high degree of anthropophily, with both infected and uninfected individuals showing higher attraction toward human odour over calf odour, human odour over outdoor air, and outdoor air over calf odour. Our results suggests that, in this system, the parasite may not be able to manipulate the early long-range behavioural steps involved in the mosquito host-feeding process, including initiation of host-seeking and host orientation. Future studies examining mosquito host-feeding behaviours at a shorter range (i.e. the “at-host” foraging activities) are required to test whether malaria parasites can modify their mosquito host choice to enhance transmission toward suitable hosts and/or reduce biting on unsuitable hosts.