Biology subjects

Puchot, N.

Publications and source records attributed to Puchot, N..

7 recordsLinked to original sources

bioRxiv2026

High-quality proteins and RNAs extracted from exact same samples for proteomics and RNA-Seq analyses

Back to the 1990 the single step method developed by Chomczynski and Sacchi for RNA isolation was extended for sequential isolation of RNA, DNA and proteins from a same sample. Although the quality of the extracted RNA turned compatible with RNA-Seq analyses, the extraction of the protein fraction from the same sample was time-consuming and resulting in low yield and quality of proteins not compatible with LC-MS proteomic analyses. Here we report a novel procedure by isolating in parallel the protein fraction and the RNA fraction from the same exact minute mosquito samples. We provide evidence that each cognate fractions are compatible with LC-MS proteomic analysis on the one hand and RNA-Seq analysis on the other hand. This protocol is simple, time efficient and adequate for studies involving limited sample size and could be applied easily to a broad range of animal and human samples.

molecular biology↗

bioRxiv2024

Colonization of Anopheles coustani, a neglected malaria vector in Madagascar

Anopheles coustani has long be recognized as a secondary malaria vector in Africa. It has recently been involved in the transmission of both Plasmodium falciparum and Plasmodium vivax in Madagascar. As most secondary malaria vector, An. coustani is mainly biting outdoor, which renders the control of this mosquito species difficult by the classical malaria control measures as the use of bed nets or indoor residual spraying of insecticides. The absence of a colony hinders a better understanding of its biology and vector competence towards the development of adapted mosquito control strategies. Here, we report the first successful establishment of an An. coustani colony from mosquito collected in Madagascar. We used a forced copulation procedure as this mosquito species will not mate in cages. We describe our mosquito colonization procedure with detailed biological features as larval to adult development and survival, recorded over the first six critical generations. The procedure should be easily applicable to An. coustani from different African countries, facilitating local investigation on An. coustani vector competence and insecticide resistance using the colony as a reference.

ecology↗

bioRxiv2022

Establishment of a colony of Anopheles darlingi from French Guiana for vector competence studies on malaria transmission

Anopheles darlingi is a major vector of both Plasmodium falciparum and Plasmodium vivax in South and Central America. However, vector competence and physiology of this mosquito species have been scarcely studied due to difficulties in rearing it in the laboratory. Here, we report the successful establishment of a robust colony, from mosquito collection in French Guiana. We describe our mosquito colonization procedure with relevant information on environmental conditions, mating ability, larval development and survival, recorded over the first six critical generations. Experimental infection showed that our An. darlingi colony has a moderate permissiveness to in vitro produced gametocytes of the P. falciparum NF54 strain originating from Africa. This colony, that has reached its 20th generation, will allow further characterization of An. darlingi life-history traits and of Plasmodium-mosquito interactions with South American malaria parasites.

microbiology↗

bioRxiv2021

Differential transcriptomic response of Anopheles arabiensis to Plasmodium vivax and Plasmodium falciparum infection

Plasmodium vivax malaria is now recognized as the second most dangerous parasitic threat to human health with the regular decrease of Plasmodium falciparum worldwide over recent decades. A very limited numbers of studies address the interaction of P. vivax with its Anopheles mosquito vectors. Those studies were conducted in P. vivax endemic countries with P.vivax local major vectors for which limited genomic and genetic tools are available. Despite the presence of P. vivax in several African countries and increasing reports on its occurrence in many others, there is virtually no data on the molecular responses of Anopheles arabiensis, a major African mosquito vector, to P. vivax, which limits the development of further "mosquito-targeted" interventions aimed at reducing P. vivax transmission. Taking advantage of the situation of Madagascar where P. falciparum, P. vivax and An. arabiensis are present, we explore the molecular responses of An. arabiensis towards these two human malaria parasites. RNA sequencing on RNAs isolated from mosquito midguts dissected at the early stage of infection (24 hours) was performed using mosquitoes fed on the blood of P. vivax and P. falciparum gametocyte carriers in a field station. From a de novo assembly of An. arabiensis midgut total RNA transcriptome, the comparative analysis revealed that a greater number of genes were differentially expressed in the mosquito midgut in response to P. vivax (209) than to P. falciparum (81). Among these, 15 common genes were identified to be significantly expressed in mosquito midgut 24 hours after ingesting P. vivax and P. falciparum gametocytes, including immune responsive genes and genes involved in amino-acid detoxification pathways. Importantly, working with both wild mosquitoes and field circulating parasites, our analysis revealed a strong mosquito genotype by parasite genotype interaction. Our study also identified 51 putative long non-coding RNAs differentially expressed in An. arabiensis mosquito infected midgut. Among these, several mapped to the published An. arabiensis genome at genes coding immune responsive genes such as gambicin 1, leucine-rich repeat containing genes, either on sense or antisense strands. This study constitutes the first comparison of An. arabiensis molecular interaction with P. vivax and P. falciparum, investigating both coding and long non-coding RNAs for the identification of potential transcripts, that could lead to the development of novel approaches to simultaneously block the transmission of vivax and falciparum malaria.

microbiology↗

bioRxiv2019

Hemocyte-targeted gene expression in the female malaria mosquito using the hemolectin promoter from Drosophila

Hemocytes, the immune cells in mosquitoes, participate in immune defenses against pathogens including malaria parasites. Mosquito hemocytes can also be infected by arthropod-borne viruses but the pro- or anti-viral nature of this interaction is unknown. Although there has been progress on hemocyte characterization during pathogen infections in mosquitoes, the specific contribution of hemocytes to immune responses and the hemocyte-specific functions of immune genes and pathways remain unresolved due to the lack of genetic tools to manipulate gene expression in these cells specifically. Here, we used the Gal4-UAS system to characterize the activity of the Drosophila hemocyte-specific hemolectin promoter in the adults of Anopheles gambiae, the malaria mosquito. We established an hml-Gal4 driver line that we further crossed to a fluorescent UAS responder line, and examined the expression pattern in the adult progeny driven by the hml promoter. We show that the hml regulatory region drives hemocyte-specific transgene expression in a subset of hemocytes, and that transgene expression is triggered after a blood meal. The hml promoter drives transgene expression in differentiating prohemocytes as well as in differentiated granulocytes. Analysis of different immune markers in hemocytes in which the hml promoter drives transgene expression revealed that this regulatory region could be used to study phagocytosis as well as melanization. Finally, the hml promoter drives transgene expression in hemocytes in which onyongnyong virus replicates. Altogether, the hml promoter constitutes a good tool to drive transgene expression in hemocyte only and to analyze the function of these cells and the genes they express during pathogen infection in Anopheles gambiae.

genetics↗

bioRxiv2019

Differential contribution of Anopheles coustani and Anopheles arabiensis to the transmission of Plasmodium falciparum and Plasmodium vivax in two neighboring villages of Madagascar

BackgroundMalaria is still a heavy public health concern in Madagascar. Few studies combining parasitology and entomology have been recently conducted despite the need for such information to design proper vector control measures. In a region of moderate to intense transmission of both Plasmodium falciparum and Plasmodium vivax, we conducted a combined parasitology and entomology survey in two nearby villages, across a malaria transmission season from December 2016 to April 2017.\n\nMethodology/Principal findingsCommunity-based surveys were conducted in the two close by villages at three time points during a single malaria transmission season. Plasmodium carriage in the human populations was determined by Rapid Diagnostic Tests (RDTs), microscopy and real-time PCR. Anthropophilic mosquitoes were captured by human landing captures and presence of Plasmodium sporozoites was assessed by robust Real Time PCR. Overall human malaria prevalence was 8.0% by RDT, 4.8% by microscopy and 11.9% by PCR, mainly due to P. falciparum detected in 92.2% of the PCR positive samples and Plasmodium vivax (5.7%). No significant differences in Plasmodium human carriage was observed between the 2 villages at any time point. Of the 1553 anopheline mosquitoes tested, 13 were found carrying Plasmodium sporozoites, the majority of them being captured outdoor. The mosquito sporozoite indices were not significantly different between the two villages. However, our entomological analysis revealed that Anopheles coustani was the main vector in one village, being responsible of 25.5 infective bites during the whole survey, whereas it was Anopheles arabiensis in the other village with 15 infective bites. In addition, we found a significant higher number of endophagic An. coustani and An. arabiensis in one village compared to the other.\n\nConclusions/SignificanceDespite similar human malaria prevalence in two close by villages, the entomological survey demonstrated the contribution of two different mosquito species in each village, and importantly the role of a suspected secondary malaria vector, An. coustani, as the main vector in one village. This, along with its higher endophagic rate in that village, highlights the importance of combining parasitology and entomology surveys for better targeting the actual local malaria vector. Such study should contribute to the malaria pre-elimination goal established under the 2018-2022 National Malaria Strategic Plan.\n\nAuthor SummaryMalaria is still a major health concern in many countries in sub-Saharan Africa such as Madagascar. In this study, we determined the contribution of malaria vectors in the transmission of Plasmodium parasites in two nearby villages in an area of moderate to high malaria transmission in Madagascar. We collected, during a single malaria transmission season, parasitological data in the human population and entomological data in the mosquito population, in order to evaluate Plasmodium carriage in these two populations. The results showed that despite similarity in human malaria prevalence and in vector species diversity in each village, the contribution of vectors was different between the two villages. An. arabiensis was the major vector in Ambohitromby while it was An. coustani that played this role in Miarinarivo. Importantly, this study is the first that clearly demonstrates that An. coustani could act as a major local vector in Madagascar. Such study should help deploying adapted malaria vector control and contributing to the malaria pre-elimination goal established under the 2018-2022 National Malaria Strategic Plan.

microbiology↗

bioRxiv2018

Evolution of sexually-transferred steroids in Anopheles mosquitoes

Human malaria, which remains a major public health problem, is transmitted by a subset of Anopheles mosquitoes belonging to only three out of eight subgenera: Anopheles, Cellia and Nyssorhynchus. Unlike almost every other insect species, it was shown that males of some Anopheles species produce and transfer steroid hormones to females during copulation and that this transfer mediates reproductive changes. Steroids are consequently seen as a potential target for malaria vector control. Here, we analysed the evolution of sexually-transferred steroids and their effects on female reproductive traits across Anopheles by using a set of 16 mosquito species (5 Anopheles, 8 Cellia, and 3 Nyssorhynchus), including malaria vector and non-vector species. We show that male steroid production and transfer are specific to the Cellia subgenus and that there is no correlation between mating-induced effects in females and sexually-transferred steroids. In the light of our results, male steroid production, transfer and post-mating effects in females do not correlate with their ability to transmit human malaria, which overturns the suggestion from previous studies and suggests that manipulation of steroid-response pathways in the field should be considered with caution in order to benefit malaria vector control strategies.

evolutionary biology↗