De Novo Transcriptome Sequencing in Anopheles funestus Using Illumina RNA-Seq Technology

BACKGROUND: Anopheles funestus is one of the primary vectors of human malaria, which causes a million deaths each year in sub-Saharan Africa. Few scientific resources are available to facilitate studies of this mosquito species and relatively little is known about its basic biology and evolution, making development and implementation of novel disease control efforts more difficult. The An. funestus genome has not been sequenced, so in order to facilitate genome-scale experimental biology, we have sequenced the adult female transcriptome of An. funestus from a newly founded colony in Burkina Faso, West Africa, using the Illumina GAIIx next generation sequencing platform. METHODOLOGY/PRINCIPAL FINDINGS: We assembled short Illumina reads de novo using a novel approach involving iterative de novo assemblies and "target-based" contig clustering. We then selected a conservative set of 15,527 contigs through comparisons to four Dipteran transcriptomes as well as multiple functional and conserved protein domain databases. Comparison to the Anopheles gambiae immune system identified 339 contigs as putative immune genes, thus identifying a large portion of the immune system that can form the basis for subsequent studies of this important malaria vector. We identified 5,434 1:1 orthologues between An. funestus and An. gambiae and found that among these 1:1 orthologues, the protein sequence of those with putative immune function were significantly more diverged than the transcriptome as a whole. Short read alignments to the contig set revealed almost 367,000 genetic polymorphisms segregating in the An. funestus colony and demonstrated the utility of the assembled transcriptome for use in RNA-seq based measurements of gene expression. CONCLUSIONS/SIGNIFICANCE: We developed a pipeline that makes de novo transcriptome sequencing possible in virtually any organism at a very reasonable cost ($6,300 in sequencing costs in our case). We anticipate that our approach could be used to develop genomic resources in a diversity of systems for which full genome sequence is currently unavailable. Our An. funestus contig set and analytical results provide a valuable resource for future studies in this non-model, but epidemiologically critical, vector insect

Travel burden and clinical presentation of retinoblastoma: analysis of 1024 patients from 43 African countries and 518 patients from 40 European countries

BACKGROUND: The travel distance from home to a treatment centre, which may impact the stage at diagnosis, has not been investigated for retinoblastoma, the most common childhood eye cancer. We aimed to investigate the travel burden and its impact on clinical presentation in a large sample of patients with retinoblastoma from Africa and Europe. METHODS: A cross-sectional analysis including 518 treatment-naïve patients with retinoblastoma residing in 40 European countries and 1024 treatment-naïve patients with retinoblastoma residing in 43 African countries. RESULTS: Capture rate was 42.2% of expected patients from Africa and 108.8% from Europe. African patients were older (95% CI -12.4 to -5.4, p<0.001), had fewer cases of familial retinoblastoma (95% CI 2.0 to 5.3, p<0.001) and presented with more advanced disease (95% CI 6.0 to 9.8, p<0.001); 43.4% and 15.4% of Africans had extraocular retinoblastoma and distant metastasis at the time of diagnosis, respectively, compared to 2.9% and 1.0% of the Europeans. To reach a retinoblastoma centre, European patients travelled 421.8 km compared to Africans who travelled 185.7 km (p<0.001). On regression analysis, lower-national income level, African residence and older age (p<0.001), but not travel distance (p=0.19), were risk factors for advanced disease. CONCLUSIONS: Fewer than half the expected number of patients with retinoblastoma presented to African referral centres in 2017, suggesting poor awareness or other barriers to access. Despite the relatively shorter distance travelled by African patients, they presented with later-stage disease. Health education about retinoblastoma is needed for carers and health workers in Africa in order to increase capture rate and promote early referral

Genetic and environmental factors associated with laboratory rearing affect survival and assortative mating but not overall mating success in Anopheles gambiae sensu stricto.

Anopheles gambiae sensu stricto, the main vector of malaria in Africa, is characterized by its vast geographical range and complex population structure. Assortative mating amongst the reproductively isolated cryptic forms that co-occur in many areas poses unique challenges for programs aiming to decrease malaria incidence via the release of sterile or genetically-modified mosquitoes. Importantly, whether laboratory-rearing affects the ability of An. gambiae individuals of a given cryptic taxa to successfully mate with individuals of their own form in field conditions is still unknown and yet crucial for mosquito-releases. Here, the independent effects of genetic and environmental factors associated with laboratory rearing on male and female survival, mating success and assortative mating were evaluated in the Mopti form of An. gambiae over 2010 and 2011. In semi-field enclosures experiments and despite strong variation between years, the overall survival and mating success of male and female progeny from a laboratory strain was not found to be significantly lower than those of the progeny of field females from the same population. Adult progeny from field-caught females reared at the larval stage in the laboratory and from laboratory females reared outdoors exhibited a significant decrease in survival but not in mating success. Importantly, laboratory individuals reared as larvae indoors were unable to mate assortatively as adults, whilst field progeny reared either outdoors or in the laboratory, as well as laboratory progeny reared outdoors all mated significantly assortatively. These results highlight the importance of genetic and environment interactions for the development of An. gambiae's full mating behavioral repertoire and the challenges this creates for mosquito rearing and release-based control strategies

Differential effects of inbreeding and selection on male reproductive phenotype associated with the colonization and laboratory maintenance of Anopheles gambiae.

BACKGROUND: Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness. METHODS: Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits. RESULTS: Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions. CONCLUSIONS: These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic 'supermales' could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings