A user-friendly software to easily count Anopheles egg batches

BACKGROUND: Studies on malaria vector ecology and development/evaluation of vector control strategies often require measures of mosquito life history traits. Assessing the fecundity of malaria vectors can be carried out by counting eggs laid by Anopheles females. However, manually counting the eggs is time consuming, tedious, and error prone. METHODS: In this paper we present a newly developed software for high precision automatic egg counting. The software written in the Java programming language proposes a user-friendly interface and a complete online manual. It allows the inspection of results by the operator and includes proper tools for manual corrections. The user can in fact correct any details on the acquired results by a mouse click. Time saving is significant and errors due to loss of concentration are avoided. RESULTS: The software was tested over 16 randomly chosen images from 2 different experiments. The results show that the proposed automatic method produces results that are close to the ground truth. CONCLUSIONS: The proposed approaches demonstrated a very high level of robustness. The adoption of the proposed software package will save many hours of labor to the bench scientist. The software needs no particular configuration and is freely available for download on: http://w3.ualg.pt/∼hshah/eggcounter/

SNP discovery and molecular evolution in Anopheles gambiae, with special emphasis on innate immune system

<p>Abstract</p> <p>Background</p> <p><it>Anopheles </it>innate immunity affects <it>Plasmodium </it>development and is a potential target of innovative malaria control strategies. The extent and distribution of nucleotide diversity in immunity genes might provide insights into the evolutionary forces that condition pathogen-vector interactions. The discovery of polymorphisms is an essential step towards association studies of susceptibility to infection.</p> <p>Results</p> <p>We sequenced coding fragments of 72 immune related genes in natural populations of <it>Anopheles gambiae </it>and of 37 randomly chosen genes to provide a background measure of genetic diversity across the genome. Mean nucleotide diversity (π) was 0.0092 in the <it>A. gambiae </it>S form, 0.0076 in the M form and 0.0064 in <it>A. arabiensis</it>. Within each species, no statistically significant differences in mean nucleotide diversity were detected between immune related and non immune related genes. Strong purifying selection was detected in genes of both categories, presumably reflecting strong functional constraints.</p> <p>Conclusion</p> <p>Our results suggest similar patterns and rates of molecular evolution in immune and non-immune genes in <it>A. gambiae</it>. The 3,214 Single Nucleotide Polymorphisms (SNPs) that we identified are the first large set of <it>Anopheles </it>SNPs from fresh, field-collected material and are relevant markers for future phenotype-association studies.</p

Different distribution of malaria parasite in left and right extremities of vertebrate hosts translates into differences in parasite transmission

International audienceMalaria, a vector-borne disease caused by Plasmodium spp., remains a major global cause of mortality. Optimization of disease control strategies requires a thorough understanding of the processes underlying parasite transmission. While the number of transmissible stages (gametocytes) of Plasmodium in blood is frequently used as an indicator of host-to-mosquito transmission potential, this relationship is not always clear. Significant effort has been made in developing molecular tools that improve gametocyte density estimation and therefore prediction of mosquito infection rates. However a significant level of uncertainty around estimates remains. The weakness in the relationship between gametocyte burden, measured from a blood sample, and the mosquito infection rate could be explained by a non-homogeneous distribution of gametocytes in the bloodstream. The estimated gametocyte density would then only be a single snapshot that does not reflect the host infectivity. This aspect of Plasmodium infection, however, remains largely neglected. In both humans and birds, we found here that the gametocyte densities differed depending on which side of the body the sample was taken, suggesting that gametocytes are not homogeneously distributed within the vertebrate host. We observed a fluctuating asymmetry, in other words, the extremity of the body with the highest density of parasites is not always the same from one individual to another. An estimation of gametocyte density from only one blood sample, as is commonly measured, could, therefore, over-or underestimated the infectivity of gametocyte carriers. This might have important consequences on the epidemiology of the disease since we show that this variation influences host-to-mosquito transmission. Vectors fed on the least infected body part had a lower parasite burden than those fed on the most infected part. The heterogeneous distribution of gametocytes in bloodstream should be considered to improve diagnosis and test new malaria control strategies

Plasmodium falciparum Produce Lower Infection Intensities in Local versus Foreign Anopheles gambiae Populations

Both Plasmodium falciparum and Anopheles gambiae show great diversity in Africa, in their own genetic makeup and population dynamics. The genetics of the individual mosquito and parasite are known to play a role in determining the outcome of infection in the vector, but whether differences in infection phenotype vary between populations remains to be investigated. Here we established two A. gambiae s.s. M molecular form colonies from Cameroon and Burkina Faso, representing a local and a foreign population for each of the geographical sites. Experimental infections of both colonies were conducted in Cameroon and Burkina Faso using local wild P. falciparum, giving a sympatric and allopatric vector-parasite combination in each site. Infection phenotype was determined in terms of oocyst prevalence and intensity for at least nine infections for each vector-parasite combination. Sympatric infections were found to produce 25% fewer oocysts per midgut than allopatric infections, while prevalence was not affected by local/foreign interactions. The reduction in oocyst numbers in sympatric couples may be the result of evolutionary processes where the mosquito populations have locally adapted to their parasite populations. Future research on vector-parasite interactions must take into account the geographic scale of adaptation revealed here by conducting experiments in natural sympatric populations to give epidemiologically meaningful results

Natural plant diet impacts phenotypic expression of pyrethroid resistance in Anopheles mosquitoes

Success in reducing malaria transmission through vector control is threatened by insecticide resistance in mosquitoes. Although the proximal molecular mechanisms and genetic determinants involved are well documented, little is known about the influence of the environment on mosquito resistance to insecticides. The aim of this study was to assess the effect of plant sugar feeding on the response of Anopheles gambiae sensu lato to insecticides. Adults were fed with one of four treatments, namely a 5% glucose control solution, nectariferous flowers of Barleria lupulina, of Cascabela thevetia and a combination of both B. lupulina+C. thevetia. WHO tube tests were performed with 0.05% and 0.5% deltamethrin, and knockdown rate (KD) and the 24 h mosquito mortality were measured. Plant diet significantly influenced mosquito KD rate at both concentrations of deltamethrin. Following exposure to 0.05% deltamethrin, the B. lupulina diet induced a 2.5 fold-increase in mosquito mortality compared to 5% glucose. Species molecular identification confirmed the predominance of An. gambiae (60% of the samples) over An. coluzzii and An. arabiensis in our study area. The kdr mutation L1014F displayed an allelic frequency of 0.75 and was positively associated with increased phenotypic resistance to deltamethrin. Plant diet, particularly B. lupulina, increased the susceptibility of mosquitoes to insecticides. The finding that B. lupulina-fed control individuals (i.e. not exposed to deltamethrin) also displayed increased 24 h mortality suggests that plant-mediated effects may be driven by a direct effect of plant diet on mosquito survival rather than indirect effects through interference with insecticide-resistance mechanisms. Thus, some plant species may weaken mosquitoes, making them less vigorous and more vulnerable to the insecticide. There is a need for further investigation, using a wider range of plant species and insecticides, in combination with other relevant environmental factors, to better understand the expression and evolution of insecticide resistance

Mosquito ageing modulates the development, virulence and transmission potential of pathogens

Host age variation is a striking source of heterogeneity that can shape the evolution and transmission dynamic of pathogens. Compared with vertebrate systems, our understanding of the impact of host age on invertebrate–pathogen interactions remains limited. We examined the influence of mosquito age on key life-history traits driving human malaria transmission. Females of Anopheles coluzzii, a major malaria vector, belonging to three age classes (4-, 8- and 12-day-old), were experimentally infected with Plasmodium falciparum field isolates. Our findings revealed reduced competence in 12-day-old mosquitoes, characterized by lower oocyst/sporozoite rates and intensities compared with younger mosquitoes. Despite shorter median longevities in older age classes, infected 12-day-old mosquitoes exhibited improved survival, suggesting that the infection might act as a fountain of youth for older mosquitoes specifically. The timing of sporozoite appearance in the salivary glands remained consistent across mosquito age classes, with an extrinsic incubation period of approximately 13 days. Integrating these results into an epidemiological model revealed a lower vectorial capacity for older mosquitoes compared with younger ones, albeit still substantial owing to extended longevity in the presence of infection. Considering age heterogeneity provides valuable insights for ecological and epidemiological studies, informing targeted control strategies to mitigate pathogen transmission

Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes

Low linkage disequilibrium in wild Anopheles gambiae s.l. populations

<p>Abstract</p> <p>Background</p> <p>In the malaria vector <it>Anopheles gambiae</it>, understanding diversity in natural populations and genetic components of important phenotypes such as resistance to malaria infection is crucial for developing new malaria transmission blocking strategies. The design and interpretation of many studies here depends critically on Linkage disequilibrium (LD). For example in association studies, LD determines the density of Single Nucleotide Polymorphisms (SNPs) to be genotyped to represent the majority of the genomic information. Here, we aim to determine LD in wild <it>An. gambiae s.l</it>. populations in 4 genes potentially involved in mosquito immune responses against pathogens (<it>Gambicin</it>, <it>NOS</it>, <it>REL2 </it>and <it>FBN9</it>) using previously published and newly generated sequences.</p> <p>Results</p> <p>The level of LD between SNP pairs in cloned sequences of each gene was determined for 7 species (or incipient species) of the <it>An. gambiae </it>complex. In all tested genes and species, LD between SNPs was low: even at short distances (< 200 bp), most SNP pairs gave an r²< 0.3. Mean r²ranged from 0.073 to 0.766. In most genes and species LD decayed very rapidly with increasing inter-marker distance.</p> <p>Conclusions</p> <p>These results are of great interest for the development of large scale polymorphism studies, as LD generally falls below any useful limit. It indicates that very fine scale SNP detection will be required to give an overall view of genome-wide polymorphism. Perhaps a more feasible approach to genome wide association studies is to use targeted approaches using candidate gene selection to detect association to phenotypes of interest.</p