The European honeybee, Apis mellifera, is important economically not just for honey production but also as a pollinator. Bee pollinated plants contribute towards one third of the food eaten worldwide. However, honeybee numbers in some areas are declining. A range of interacting factors are thought to be involved, including pathogens and parasites, loss of forage, pesticide use, bad weather, and limited genetic variability. Pathogens are also known to cause changes in the behaviour of their hosts and these premortality and sublethal effects of disease may well play a role in colony declines and are the focus of this thesis. For individual bees the fungus Metarhizium anisopliae was used as a model pathogen and RT-Q-PCR was used to detect and quantify naturally occurring pathogens. In field colonies the level of infestation of the parasitic mite Varroa destructor was modified as a surrogate for disease load as the amounts of many viruses correlate with mite levels. Survival experiments showed that both disease load and forage availability had an effect on honeybee longevity and feeding the bees pollen increased their survival. Learning experiments showed that both the fungus and some of the bees’ naturally occurring pathogens caused changes in the learning ability of young adult and older forager bees. Young adult bees were better able to learn when infected with the fungus, possibly because it made them more responsive to the sucrose stimulus, whilst older forager bees where less able to learn when infected with the fungus. Harmonic radar was used to show that honeybee flight ability was affected by naturally occurring pathogens, especially deformed wing virus which caused bees to fly shorter distances and for shorter amounts of time than uninfected bees. Observation hives were used to study in-hive behaviour showing that bees with more pathogens were likely to start foraging earlier than healthier bees.

Animal trypanosomiasis is a major hinderance to the growth of livestock farming in sub-Saharan Africa. Chemotherapy using isometamidium, diminazene and ethidium bromide has been the main control method in the absence of a vaccine against this disease. The effectiveness of these few trypanocides is severely threatened by the widespread development of resistance. Therefore, an understanding of the mechanism(s) involved in the development of resistance will assist in the development of screening protocols for easy identification of resistant cases prior to treatment, and also in finding ways to reverse the resistance. We studied the mechanism of resistance to isometamidium in bloodstream forms of Trypanosoma brucei. Resistance to isometamidium in Trypanosoma brucei was found to be composed of a reduced uptake of the drug and the modification of the F1F0 ATPase complex; active drug efflux by ABC transporters was not involved in the resistance mechanism, although efflux of ISM could be observed in both wild-type and resistant lines. Expression of the transporter gene TbAT1, as well as of TbAT-E and TbAT-A, in yeast, each resulted in increased ISM uptake. In addition, the Vmax for the LAPT1 drug transport activity (Low Affinity Pentamidine Transporter) in ISM-resistant trypanosomes (clone ISMR1) was significantly reduced (P<0.05; Student’s t-test) compared to the wild type control. Also, two point mutations, namely G37A and C851A were found in the ATP synthase gamma subunit of the F1F0 ATPase complex of isometamidium-resistant trypanosomes. The resistant clones also lost their mitochondrial DNA and mitochondrial membrane potential and displayed various levels of cross-resistance to ethidium, diminazene, pentamidine and oligomycin. The C851A mutation introduced a stop codon in the open reading frame of the ATP synthase gamma gene. This mutation, when introduced into the wild type Tb427, produced resistance to isometamidium, and cross resistance to diminazene, ethidium, pentamidine and oligomycin. C851A-ATP synthase gamma proves to be a dominant mutation that allows the rapid loss of mitochondrial DNA after just three days exposure of the parasites to 20 nM ISM or ethidium bromide. Finally, following a recent genome-wide loss-of-function RNAi screen that linked TbAQP2 with pentamidine and melarsoprol cross resistance, we were able to demonstrate that TbAQP2 encodes the HAPT1 in T. brucei, thus leaving us with the LAPT1 as the only known T. b. brucei drug transporter of unknown genetic origin. We however identified specific inhibitors for this transporter (LAPT1) that will be of use in its further characterization.

The aim of this study was to investigate the genetic basis of variation in gametocyte sex ratio in the human malaria parasite, Plasmodium falciparum. The gametocyte sex ratio was measured in progeny clones from the 3D7 x HB3 experimental genetic cross and found to be remarkably stable across replicates of different parasite clones. Significant differences in the sex ratio were observed between the parents of the cross. Progeny clones fell into two classes of sex ratio, one similar to that seen in parent 3D7 and the other like parent HB3, suggesting a single gene of major effect controlling sex ratio. Using a genetic map of the progeny and parental clones, QTL analysis revealed two highly significant loci, the first on chromosome 10 (LOD score = 8.8), and the second on chromosome 14 (LOD = 4.0), linked to gametocyte sex ratio. The locus on chromosome 10, spanning approximately 35kb, contained ten genes. This locus, named PfROS1 (Plasmodium falciparum Ratio of Sex 1), explained 95% of the variation in sex ratio. The second locus on chromosome 14, PfROS2 (Plasmodium falciparum Ratio of Sex 2), explained a small proportion of gametocyte sex ratio variation when combined with PfROS1, the two loci explained 99% of the variation in gametocyte sex ratio observed. As PfROS1 explains such a high percentage of the variation observed in the gametocyte sex ratio it represents a single controlling locus to define the sex ratio of gametocytes produced. This is the first report of a genomic locus influencing gametocyte sex ratio in any Plasmodium species. In addition, changes in the sex ratio of clones 3D7 and HB3, over the course of 16 days of gametocyte culture were investigated. The number of gametocytes, and especially male gametocytes, was observed to fall markedly in the last few days of culture, when the majority of gametocytes were stage V (mature). Fluctuations in temperature during the culture process were found to influence sex ratio, suggesting the loss of males was due to exflagellation of mature gametocytes. Parasite clone and day of culture were also significant explanatory variables in influencing sex ratio.