Genes Involved in Virulence of the Entomopathogenic Fungus Beauveria Bassiana Chapter 3
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A multidisciplinary approach to study virulence of the entomopathogenic fungus Beauveria bassiana towards malaria mosquitoes Claudio A. Valero Jiménez Thesis committee Promotors Prof. Dr B.J. Zwaan Professor of Genetics Wageningen University Prof. Dr W. Takken Personal chair at the Laboratory of Entomology Wageningen University Co-promotors Dr. C.J.M. Koenraadt Assistant professor, Laboratory of Entomology Wageningen University Dr. J.A.L. van Kan Assistant professor, Laboratory of Phytopathology Wageningen University Other members Prof. Dr M.E. Schranz, Wageningen University Dr V.I.D. Ros, Wageningen University Dr M. Rep, University of Amsterdam Prof. Dr J. Eilenberg, University of Copenhagen, Denmark This research was conducted under the auspices of the C.T. de Wit Graduate School for Production Ecology and Resource Conservation A multidisciplinary approach to study virulence of the entomopathogenic fungus Beauveria bassiana towards malaria mosquitoes Claudio A. Valero Jiménez Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 21 September 2016 at 4 p.m. in the Aula. Claudio A. Valero Jiménez A multidisciplinary approach to study virulence of the entomopathogenic fungus Beauveria bassiana towards malaria mosquitoes. 131 pages. PhD thesis, Wageningen University, Wageningen, NL (2016) With references, with summary in English ISBN 978-94-6257-854-8 DOI 10.18174/385465 Table of Contents Chapter 1................................................................................................................................7 Introduction Chapter 2..............................................................................................................................17 Genes involved in virulence of the entomopathogenic fungus Beauveria bassiana Chapter 3..............................................................................................................................39 Natural variation in virulence of the entomopathogenic fungus Beauveria bassiana against malaria mosquitoes Chapter 4..............................................................................................................................55 Comparative genomics of Beauveria bassiana: uncovering signatures of virulence against insects Chapter 5..............................................................................................................................85 Experimental evolution to increase the efficacy of the entomopathogenic fungusBeauveria bassiana against malaria mosquitoes: effects on mycelial growth and virulence Chapter 6............................................................................................................................105 General discussion Summary.............................................................................................................................121 Acknowledgements.............................................................................................................125 Curriculum Vitae................................................................................................................127 Educational Statement.......................................................................................................129 Chapter 1 Introduction Introduction Chapter 1 Malaria disease Malaria is a vector-borne disease that causes a great threat to human health. The disease is present throughout the tropics, especially in sub-Saharan Africa. Around 214 million cases were reported in 2015, with an estimated mortality of 438 thousand people in 2014 (World Health Organization, 2015). Of these, around 70% where children under five years of age. Malaria is caused by parasites of the Plasmodium genus, most cases being caused by P. falciparum or P. vivax, and to a lesser extent by P. ovale, P. malariae, and P. knowlesi (White et al., 2014). Malaria parasites are transmitted by female mosquitoes of the genus Anopheles, but only 70 to 80 of more than 480 Anopheles species are able to do so (Manguin et al., 2008). Mosquitoes of the An. gambiae complex are considered the main vectors of human malaria in Africa and the complex is composed of eight species, namely: An. gambiae s.s., An. arabiensis, An. quadriannulatus, An. melas, An. merus, An. bwambae, An. coluzzii and An. amharicus (Coetzee, 2004; Coetzee et al., 2013). Malaria transmission is initiated when an infected female Anopheles mosquito inoculates sporozoites into the subcutaneous tissue of a human host while blood-feeding. From there, sporozoites travel to the liver, where they undergo several transformations and eventually develop into thousands of merozoites. Once merozoites are released in the blood stream, they infect red blood cells and reproduce asexually. A small amount of asexual parasites develops into gametocytes that may be ingested by an Anopheles mosquito during a blood meal (Miller et al., 2002). Gametocytes reproduce sexually inside the mosquito, developing into a motile ookinete that migrates through the midgut wall, and becomes an oocyst on the mosquito’s midgut. The oocyst grows and bursts, releasing sporozoites, which move to the salivary glands of the mosquito. When a mosquito takes a new blood meal, it will infect a new host, thereby repeating the infection cycle (White et al., 2014). Malaria control Presently, the main interventions to control and eliminate malaria are vector control (stop mosquitoes of infecting malaria), chemoprevention (suppression and prevention of infections) and case management (diagnosis and treatment of infections)(World Health Organization, 2015). Currently, the most effective way of eradicating and preventing malaria is achieved by vector control using insecticides (Raghavendra et al., 2011). This is accomplished through the use of Insecticide-Treated bed Nets (ITNs) and/or through Indoor Residual Spraying of insecticides (IRS). ITNs provide a physical and chemical barrier against mosquitoes and can reduce the incidence of malaria transmission, especially since most malaria mosquitoes have a nocturnal biting behaviour. ITNs are coated with pyrethroids that have a very low toxicity to 8 Introduction mammals, a rapid knock-down effect, and long residual activity (Raghavendra et al., 2011). A review on interventions using ITNs has shown that they can avert the number of malaria cases by 62%-78% (Bhatt et al., 2015). IRS is a method of spraying the inside of houses with residual insecticides to kill and repel malaria mosquitoes. Normally, the insecticides are sprayed on the inside walls of houses, and mosquitoes are repelled or killed when in contact with the insecticides, thus preventing the transmission of the disease (Pluess et al., 2010). IRS has also been shown to be very effective in reducing malaria transmission to similar levels as ITNs (Mabaso et al., 2004; Pluess et al., 2010). Insecticide resistance The progress achieved in malaria reduction using ITNs and IRS as vector control tools is challenged by the development of resistance to insecticides by malaria mosquitoes. For instance, pyrethroid resistance is of high concern since it is the only insecticide class allowed for use in ITNs and one of the four classes used in IRS (Hemingway et al., 2004; Ranson et al., 2011; Takken and Knols, 2009). Recent reports indicate that insecticide resistance is widespread across the globe with 60 countries declaring resistance to at least one class of insecticide, and 49 countries reporting resistance for two or more types of insecticide classes (Ranson and Lissenden, 2016; World Health Organization, 2015). Furthermore, long-term studies have shown that insecticides have an effect on the behaviour of mosquitoes by reducing the rate of mosquito entry into houses that result in a shift to outdoor biting and in time of biting (Mathenge et al., 2001; Mbogo et al., 1996; Reddy et al., 2011; Takken, 2002). Therefore, insecticide resistance can possibly impair the important advances accomplished through chemical malaria vector control, and consequently constrain further success. Entomopathogenic fungi: a tool for biological control of mosquitoes Due to the negative implications of insecticide resistance on the success of malaria control, the development of alternative tools for malaria vector control is urgently needed. There are several malaria vector control tools in development that can supplement current control methods (McGraw and O’Neill, 2013; Takken and Knols, 2009). Such tools include the modification of the environment by reducing the breeding habitats of mosquitoes or house improvement to reduce biting incidence (Imbahale et al., 2012; Kirby et al., 2008). Other vector control tools are based on genetic modification of mosquitoes, for example, the release of sterile male mosquitoes to reduce the number of offspring (Lofgren et al., 1974). Another approach is the usage of chemical compounds to attract and repel mosquitoes in a push-pull system which, when coupled with removal trapping, can reduce house entry of mosquitoes (Menger et al., 9 Chapter 1 2014; Okumu et al., 2010). Finally, an additional control tool is biological control that makes use of natural enemies of insects. For instance, predatory fish as well as bacteria such asBacillus thuringiensis var. israelensis can be used to control larval populations (Fillinger and Lindsay, 2006; Kusumawathie