Generation of full genome sequence of 3 members of the Lutzomyia longipalpis species complex from embryonic cell cultures, laboratory colonies and field collection: unlocking genomic novelties in search of parasite control strategies of the future.

Globally, vector-borne diseases account for more than 17% of all infectious diseases every year. One such disease, leishmaniasis, is endemic in 98 countries with an estimated 700,000 to 1 million new cases leading to 26-65,000 deaths each year. Leishmaniasis is a group of vector-borne diseases caused by protozoan parasites from the genus Leishmania and is considered to be one of the most important neglected tropical disease. Sand flies (Diptera: Psychodoidae) belonging to the genus Lutzomyia are vectors in the New World. The most important New World vector is Lu. longipalpis which transmits Le. infantum the parasite that causes visceral leishmaniasis. It has a wide but discontinuous geographical distribution from southern Mexico to northern Argentina, where it inhabits diverse ecological environments. It is widely accepted that Lu. longipalpis is a species complex.

 

Globally, vector-borne diseases account for more than 17% of all infectious diseases every year. One such disease, leishmaniasis, is endemic in 98 countries with an estimated 700,000 to 1 million new cases leading to 26-65,000 deaths each year. Leishmaniasis is a group of vector-borne diseases caused by protozoan parasites from the genus Leishmania and is considered to be one of the most important neglected tropical disease. Sand flies (Diptera: Psychodoidae) belonging to the genus Lutzomyia are vectors in the New World. The most important New World vector is Lu. longipalpis which transmits Le. infantum the parasite that causes visceral leishmaniasis. It has a wide but discontinuous geographical distribution from southern Mexico to northern Argentina, where it inhabits diverse ecological environments. It is widely accepted that Lu. longipalpis is a species complex.

However the members of the complex have not been fully defined even though the evident genetic variability has potential implications for their ability to transmit Leishmania parasites. There is a clear and significant difference between distribution of Leishmaniasis cases in the NE compared to other states of Brazil which may be associated with the predominance of one of the members of the species complex.

Genome analysis of Lu. longipalpis has been slow and unreliable resulting until very recently in a partially complete EST library. One reason for this difficulty in establishing a ‘clean’ sample preparation for whole genome sequencing has been the presence of other species genomic material among the sand fly genome, including infecting parasites, blood-feed host and gut microbiome

We have recently established embryonic cell cultures from 3 members of the Lu. longipalpis complex. Two of these are from the Lu. longipalpis pheromone producing type that is predominant in the NE States of Brazil and the other is from a pheromone type that is widespread in the rest of Brazil and many other South and Central American countries.

This project will provide the opportunity to employ metagenomics approaches to sequence the genome of the members of the complex established as cell lines, and compare them with genomes of colony maintained Lu. longipalpis and examples of Lu. longipalpis collected from the representative field locations. In addition, we have Brazilian Leishmania infantum parasites in culture at Lancaster University as well as the representative members of the species complex so we can infect members of the complex with Le. infantum to pinpoint potential difference in the members of the complex that might be associated with their ability to transmit the parasite.

The project aims to involve Brazil-based field work dependent upon COVID-19 restrictions. Basic molecular biology and bioinformatics methods will be employed to identify and barcode sand flies. Additional DNA extractions and sequencing will take place at Sanger and/or Liverpool sequencing centre, producing data that the student will use to assemble genome sequences. A range of bioinformatic techniques will then be applied to the data, assisted by advice and support from Sanger and/or LSTM support teams and supervisors.

This proposed project aims to:

1. Sequence Lu. longipalpis embryonic cell cultures, laboratory colonies and field collected samples from Brazil in locations of differing leishmaniasis distribution,
2. Sequence laboratory colonised Lu. longipalpis infected with Le. Infantum to provide a reference genome for field isolates both infected and uninfected,
3. Assemble draft genome sequences across the range of Lu. longipalpis samples and complex,
4. Undertake comparative genomic analyses led by the data collected to: i) provide basic understanding of self and non-self genomes and environmental iterations, ii) uncover features of previously undescribed genomes within the complex. 

Where does the project lie on the Translational Pathway?

T1 (Basic Research) – T2 (Human/Clinical Research)

Expected Outputs

After the completion of this PhD project (3-year period), this project would;

 

  1. Provide genome sequences of the Lu. longipalpis complex from both lab and field isolates for use by the wider scientific community,
  2. Provide essential  comparative genomic data to:  i) provide basic understanding of self and non-self genomes and environmental iterations of  the Lu. longipalpis complex,  ii) uncover features of previously undescribed genomes within the complex.
  3. Result in multiple publications in leading peer-reviewed, open access journals

 

Training Opportunities

LSTM provides a unique programme of training opportunities, events and seminars (updated each semester) to encourage students to meet other researchers from a wide range of fieldwork, statistical, bioinformatics, and laboratory specialities to grow their network.  LSTM utilises innovative Technology Enhanced Learning initiatives to ensure that students working in the field are able to interact as fully as possible with training sessions.  The full catalogue of MSc modules delivered at LSTM are available for study and students can attend lectures and seminars.

 

The student will receive the following training: 1. Insectary culture methods and animal host blood sampling strategies: sampling and statistical data handling skills and methods designed for disease surveillance, trap sampling methods and animal processing (arthropods and small warm-blooded hosts) 2. Pathogen isolation and processing in a biosecurity level 2 laboratory and 4. Parasite xenodiagnoses from arthropds,  4. Molecular biology methods and preparation of samples for basic and modern sequencing technologies,  5. Bioinformatics skills from basic sequence analyses to computational and statistical handling of output data from modern sequencing technologies.

 

Skills Required

The potential candidate would require a high-quality degree in Life Sciences (Biology, Veterinary, or Medicine), basic knowledge on molecular biology and parasitology, and have good communication skills in English.  Previous experience in computing or bioinformatics is desired but not essential as full training will be provided.  The PhD candidate is expected to be highly motivated.

 

Key Publications associated with this project

Aunin E, Böhme U, Sanderson T, Simons ND, Goldberg TL, Ting N, Chapman CA, Newbold CI, Berriman M, Reid AJ. Genomic and transcriptomic evidence for descent from Plasmodium and loss of blood schizogony in Hepatocystis parasites from naturally infected red colobus monkeys. PLoS Pathog. 2020 Aug 3;16(8):e1008717. doi: 10.1371/journal.ppat.1008717. Epub ahead of print. PMID: 32745123.

Cwiklinski K, Allen K, LaCourse J, Williams DJ, Paterson S, Hodgkinson JE. (2015). Characterisation of a novel panel of polymorphic microsatellite loci for the liver fluke, Fasciola hepatica, using a next generation sequencing approach. Infect Genet Evol. 32:298-304.

Cwiklinski K, Dalton JP, Dufresne PJ, LaCourse J, Williams DJ, Hodgkinson J, Paterson S. (2015). The Fasciola hepatica genome: gene duplication and polymorphism reveals adaptation to the host environment and the capacity for rapid evolution. Genome Biol. 16:71.

 

 

LSTM Themes and Topics – Key Words

Neglected Tropical Diseases (NTD); Vector borne Diseases; Tropical Disease Biology; International Public Health

 

Deadline: Thursday 11th February 2021; 12:00 noon GMT

Further details on the MRC/DTP and CASE programmes and application guidance and process can be found here