The Phlebotomine sand flies are important vectors for human diseases, transmitting different types of pathogens comprising of viruses, bacteria and protozoa in different parts of the world. The most devastating of the vectored diseases are the leishmaniases, with 350 million people at risk in 88 countries. There are approximately 35 different sand fly species transmitting different species of Leishmania, the causative protozoa for leishmaniasis; a diasese with at least one non-human reservoir host. The reservoir differs with different vector species. The clinical manifestation and severity of leishmaniasis varies with the different parasites from self-healing cutaneous lesions to life-threatening visceral leshmaniasis. Two other, more recent, factors that aggravate the disease are the presence of co-infection with the Human Immunodeficiency Virus (HIV) and emergence of resistance to available drugs. The distribution of leishmaniasis encompasses the world, being present in regions of Mediterranean basin, Middle East, the Indian subcontinent and South America, however the distribution across socio-economic levels is more biased towards the poorer segment of the population.
The high costs of developing new drugs, along with the low level of income in the affected population has deterred pharmaceutical companies from investing as much in treating this disease as others. While there are available drugs for the treatment of the different forms of leishmaniasis, they are expensive and resistance to them is emerging the parasite. At this point, the main control measure of the disease is based on vector and reservoir host control. Due to the high diversity of the reservoir hosts, which include both wild and domestic animals, available to sand flies, disease control through animal control is considered to not be cost effective.
Vector control remains the main method for disease control yet, in spite of their world-wide impact, sand flies remain relatively understudied, with limited laboratory and genomic resources yet available. While many different Leishmania species can be readily cultured and manipulated in the laboratory, sand flies are notoriously difficult to colonize and maintain. With the upcoming genome assemblies for Phlebotomus papatasi and Lutzomyia longipalpis the available genomic resources for these important vectors will be greatly improved and it is necessary to develop resources that would help with the genome annotation. One such resource that has the potential to improve future genome annotation is a transcript library. In Chapter 2 we generate and analyze a normalized EST library, generated from total RNA and representing the different transcripts expressed at any point in the sand fly lifetime. The EST library was generating using all life stages (both adult and immature) as well as both genders (females and males); for females both sugar fed and blood females were used. The sequences generated from sequencing this library were cleaned and assembled with an in house generated pipeline combining several different available tools. Also in Chapter 2, we identify new proteins potentially involved in digestion and immune response and study their expression levels across different life stages and different feeding conditions. These new proteins are important because of their potential role in the vector-parasite interaction. The Leishmania parasite is contained in the sand fly midgut and has to evade both the innate host immune response as well the digestive process. Targeting processes at these levels might decrease the chances for a successful transmission of the parasite to a mammalian host.
Prior to the genome sequencing, a Bacterial Artificial Chromosomes (BACs) library were generated from Ph. papatasi containing large stretches of genomic DNA. Fifteen of these BACs have been randomly selected and sequenced. In Chapter 3 we take a look at 15 fully sequenced Ph. papatasi BACs in attempt to understand more about what to expect from this genome regarding the number and distribution of repeats and genes. In the same chapter (Chapter 3), we make a first attempt to analyze the newly released genome assembly with regards to possible miss-assemblies, inversions and gaps. It is believed that a better understanding of the sand fly genome will help generate possible targets for vector control and in help with better understanding of these organisms in general.
One class of possible targets for insecticides (vector control method), in sand flies as well as other vectors, are G-Protein Couple Receptors (GPCRs). GPCRs are 7-transmembrane protein that connect the extracellular world to the intracellular one, transducing a high number of external stimuli into intracellular responses. In chapter 4 we analyze and validate a set of predicted GPCRs for three different vector species: Aedes aegypti, a mosquito responsible for transmission of dengue virus, Anopheles gambiae, another mosquito, responsible for the spread of Plasmodium falciparum, a malaria parasite present in Africa and Pediculus humanus, a tick. Our method for identifying GPCRs results in novel sequences for all three vector species that can at a later time considered for their potential as insecticide targets. In conclusion, these studies greatly add to our knowledge about Phlebotomine sand flies genome and transcriptome and are hope to help improve the genome annotation now available in GenBank.