Wamiti L.G. Supervisors  Odiwuor S.O., Mount Kenya University (MKU)  Fathiya K.H. and  Maniania N.K., International Centre of Insect Physiology and Ecology (ICIPE) Interactions Between Entomopathogenic Fungus Metarhizium anisopliae, Parasite Trypanosoma Congolense and Vector Glossina fuscipes

Tsetse flies (Diptera: Glossinidae) are the sole vectors of several species of pathogenic trypanosomes (Genus: Trypanosoma) causing sleeping sickness in humans (HAT) and “nagana” in animals (AAT); (Babokhov, P et al., 2013) Affects 300,000 humans and 3 million cattle deaths annually in 36 African countries.(Catley, A. & Leyland, T, 2011) Total annual agricultural loss is approximated at US$ 4.7 billion annually (Babokhov, P et al., 2013) Main factors responsible for tsetse distribution include land cover, local climate and availability of suitable host (Cecchi G. et al., 2008) Introduction Tsetse fly distribution in African continent

Current control strategies  Aerial and ground spraying  Traps and Targets  Pour –on  Sterile Insect Technique

Entomopathogenic fungus – minimal risks to non-target organisms No environment pollution Easily mass produced Horizontal transmission Alternative control strategies?

Tsetse/day/trap following application of Metarhizium anisopliae in contamination device, mass trapping and control treatments strategies Control strategies Maniania et al. BCST (2006)

Reductions in malaria transmission in Metarhizium-treated mosquitoes Days after feeding on infected blood 1617 ControlWild type Ma Trans- genic Ma ControlWild type Trans- genic Ma % Prevalence ± ± 4 % Feeding86 ± 664 ± 762 ± 582 ± 942 ± 441 ± 3 % Mosquitoes able to transmit malaria 83 ± 238 ± 119 ± 178 ± 117 ± 2 7 ± 1 % Reduction in malaria transmission 054 ± 177 ± 2081 ± 391 ± 1 Ma = Metarhizium anisopliae Fang et al., Science 2011

Horizontal transmission of conidia from fungus-infected to uninfected fly during mating Mating Fungus-contaminated flies Decrease in fecundity Fungal conidia Fly mortality due to fungus Reduced blood meal Horizontal transmission of fungal conidia and effect of infection on feeding and reproduction potential Maniania and Ekesi, J. Invertebrate Pathology (2013)

Gaps  Drugs available are toxic, expensive and resistance is rampant  No conventional vaccine  Chemical pesticides drawbacks- vector resistance, environmental pollution  Mass trapping and SIT require colossal investments  Tsetse vector biological control may be the only viable option, at this time  Effects of entomopathogenic fungus on development of trypanosomes in tsetse and vector competence have not been determined before

objectives  To determine the effect of infection of tsetse fly by Metarhizium anisopliae on the development of T. congolense in G. fuscipes.  To determine the effect of fungal infection by M. anisopliae on G. fuscipes ability to acquire T. congolense.  To elucidate the effects of fungal infection by M. anisopliae on G. fuscipes ability to transmit T. congolense.  To determine the effects of fungal infection by M. anisopliae on G. fuscipes total hemocyte count

materials  Metarhizium anisopliae ICIPE 30 wild type (WT)  Metarhizium anisopliae ICIPE 30 GFP (GZP) Trypanosoma congolense

Sub-culturing of fungi (GFP transformed and Wild Type) Infecting flies with EPF Culturing of trypanosomes in Swiss mice Infecting sterile bovine blood with trypanosomes G. fuscipes fed on blood Harvesting trypanosomes DNA extraction from mid-gut and proboscis and hemocyte count Light and Fluorescent microscopy Mortality counts Methodology

 5ml L-glutathione  0.92ml infected blood  44.08ml bovine blood % infection rates

Treatment Day after treatment 2357 Control8.7 x x x x 10 4 M. anisopliae 30 (WT) 8.7 x x M. anisopliae 30 (GZP) 8.7 x x Effect of infection by M. anisopliae on T. congolense load in G. fuscipes / ml

Treatment % T. congolense-infected flies Day after exposure to parasite-infected blood 1234 Control M. anisopliae 30 (WT) M. anisopliae 30 (GZP) Effect of infection by M. anisopliae on acquisition of T. congolense by G. fuscipes

Presence/Absence of T. congolense in mice Treatment Days after exposure to fungal infection of 11- old parasite-infected fly 3579 Control++++ M. anisopliae 30 (WT)---- M. anisopliae 30 (GZP)---- Effect of infection by M. anisopliae on transmission of T. congolense by G. fuscipes

Effect of infection by M. anisopliae on hemocyte count of T. congolense hemolymph

The T. congolense parasite load per G. fuscipes varied among the flies 4 days after exposing T. congolense parasite-infected flies to M. anisopliae infection. Although the parasite load decreased in all the treatments over time, fungal infection had a significant effect on the prevalence and parasite load in the flies. The vector competence of G. fuscipes flies to acquire and transmit Trypanosoma parasite was significantly affected by fungal infection. when fungus-inoculated flies were offered trypanosome-infected blood, 90% of flies acquired the parasite in the control at day 1 and continued to acquire parasite up to day 4 when the experiment was terminated. However, fewer flies acquired parasite in fungus treatments from day 1 to day 3 and no parasite acquisition was observed on day 4. Discussion

G. fuscipes flies that were previously infected with Trypanosoma, regularly maintained for 11 days (presumably the necessary time for the parasite to migrate to proboscis and becomes infective) and exposed to infection by WT and GZP strains on the 12 th day, were unable to transmit parasite to mice on day 1, 3, 5 and 7 post fungal exposure while flies in the control continued to successfully transmit the parasite during the whole experimental period. Blood feeding dropped from 65% to 35% by day 3 similar to studies on tsetse by Maniania and Ekesi (2013), Maniania et al., (2013) and on mosquitoes, Anopheles by Ondhiaka et al.,(2008) and Culex by Howard et al.,(2010) Four types of hemocytes prohemocytes, plasmatocytes, granulocytes and spindle cells were identified similar to Kaaya and Otieno (1981) Total hemocyte count dropped in fungus and trypanosome infected flies in day 3 and 4 Discussion

 This is the first report demonstrating that development of parasite Trypanosoma congolense in Glossina fuscipes and vector competence to acquire and transmit the parasite was significantly affected by infection of flies with entomopatogenic fungus M. anisopliae.  There was reduction in total hemocyte count in fungus and trypanosome-infected flies as compared to the control and elevated levels of some individual hemocyte with decline in others. Conclusion

 Carry assays using other Glossina species.  Incorporate M. anisopliae in other tsetse management strategies and conduct field trials  Investigate in vitro interactions between M. anisopliae and trypanosomes Future prospects

Acknowledgements  Prof. R. St Leger, University of Maryland, USA  International Atomic Energy Agency (IAEA)  My supervisors (MKU and ICIPE)  Levi Odhiambo for molecular analysis  Jeramiah Ojude and the late Jacob Opere for tsetse rearing and small animals.  Above all ALMIGHTY GOD Thank You