Ultra-deep exploration of transcription in Anopheles gambiae

Jaroslaw Krzywinski (PI); jarek@liv.ac.uk
Martin Donnelly
Liverpool School of Tropical Medicine, UK

Executive summary

Catastrophic numbers of mosquito-borne disease cases and deaths remain one
of the unsolved major health problems in developing countries. Malaria, arboviral
diseases and filariasis kill more than one million people and/or debilitate hundreds of
millions every year. Management of these diseases relies heavily on the use of
insecticides. However, the evolution and spread of insecticide resistance can
compromise the effectiveness of control campaigns and disease incidence continues to
be extremely high. Approximately 90% of deaths caused by mosquito-borne disease
are attributable to malaria in sub-Saharan Africa, where children under the age of five
are the primary victims and the mosquito Anopheles gambiae is the major vector. Novel
control strategies are urgently needed to combat mosquito-borne diseases, and to meet
this challenge we must grasp the biological complexities of the vectors ranging from
molecular to population level interactions.

We propose to develop a comprehensive picture of the Anopheles gambiae
transcriptome using high-coverage RNA-seq through two discrete, complementary
study aims.

Aim 1 will be to characterise the transcripts expressed during development and
to utilise this ontogenetic series to drastically improve genome annotation through
examining correlated patterns of gene expression. We propose next-generation
sequencing of sexed whole-insect transcriptomes at 16 time points spanning from 10-
hour old embryos to 20-day old adults. We will identify transcribed features and
comprehensively characterize their expression dynamics throughout development.

Aim 2 is to discover additional genes that are induced following physiological
stress. To document global transcriptional response to a range of physiological
stressors we propose to conduct RNA-seq experiments on females subject to (1) sublethal insecticide exposure and (2) sub-optimal relative humidity (RH), and (3) females
post-blood feeding and during vitellogenesis/oogenesis.

The RNA-seq reads from both sets of experiments will be mapped to the PEST
and the S molecular form genome sequences to provide an insight into dynamic
changes in transcription levels and transcript models will be created to provide a nearly
exhaustive catalogue of transcribed features in each sample. Finally, the RNA-seq data
will be integrated into Vectorbase as new tracks.

Our study will have a profound impact on the vector research community.
Discovery of a complete gene set is a pre-requisite for an understanding of the
molecular basis of the development of An. gambiae and its response to physiological
and environmental stressors. We expect that deep insight into these processes will
allow identification of novel anti-vector targets and will result in effective interventions in
disease transmission. Sexed transcriptomes are expected to shed light on mosquito sex
determination and sexual differentiation processes that may be targeted in genetic
control approaches. A highly improved An. gambiae transcript dataset will also allow
efficient implementation of comparative methods to the annotation of additional
Anopheles genomes that are currently in a sequencing pipeline. Further, it will be highly
informative for improvement of annotations of the already sequenced culicine, sandfly,
tsetse fly and other vectors genomes.