Philosophiae Doctor - PhD / Tsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication.

The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of G-protein-coupled olfactory receptors (ORs) and olfactory carriers. Two such carrier gene families are found in insects: the odorant binding proteins (OBPs) and the chemosensory proteins (CSPs). In olfactory sensilla, OBPs and CSPs are believed to deliver hydrophobic air-borne molecules to ORs, but their expression in non-olfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes. ¶ Bioinformatics and experimental approaches were used to characterise the OBP and CSP gene families in a highly social insect, the western honey bee (Apis mellifera). Comparison with other insects reveals that the honey bee has the smallest set of these genes, consisting of only 21 OBPs and 6 CSPs. These numbers stand in stark contrast to the 66 OBPs and 7 CSPs in the mosquito Anopheles gambiae and the 46 OBPs and 20 CSPs in the beetle Tribolium castaneum. The genes belonging to both families are often organised in clusters, and evolve by lineage specic expansions. Positive selection has been found to play a role in generating a greater sequence diversication in the OBP family in contrast to the CSP gene family that is more conserved, especially in the binding pocket. Expression proling under a wide range of conditions shows that, in the honey, bee only a minority of these genes are antenna-specic. The remaining genes are expressed either ubiquitously, or are tightly regulated in specialized tissues or during development. These findings support the view that OBPs and CSPs are not restricted to olfaction, and are likely to be involved in broader physiological functions. ¶ Finally, the detailed expression study and the functional characterization of a member of the CSP family, uth (unable-to-hatch), is reported. This gene is expressed in a maternal-zygotic fashion, and is restricted to the egg and embryo. Blocking the zygotic expression of uth with double-stranded RNA causes abnormalities in all body parts where this gene is highly expressed. The treated embryos are `unable-to-hatch' and cannot progress to the larval stages. Our ndings reveal a novel, essential role for this gene family and suggest that uth is an ectodermal gene involved in embryonic cuticle formation.

Philosophiae Doctor - PhD / Tsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication. In the sensilla shaft the dendrite of OSNs are housed, which are protected by called the sensillum lymph produced by support cells and contains a variety of olfactory proteins, including the odorant binding protein (OBP) and chemosensory proteins (CSP). While on the dendrite of OSNs are expressed olfactory receptors. In my PhD, studies I tried to decipher the sense of smell in tsetse fly. In the second chapter, I demonstrated that G. f. fuscipes is equipped with diverse olfactory sensilla, that various from basiconic, trichoid and coeloconic. I also demonstrated, there is shape, length, number difference between sensilla types and sexual dimorphism. There is a major difference between male and female, while male has the unique basiconic sensilla, club shaped found in the pits, which is absent from female pits. In my third chapter, I investigated the odorant receptors which are expressed on the dendrite of the olfactory sensory neurons (OSNs). G. f. fuscipes has 42 ORs, which were not functionally characterised. I used behaviourally well studied odorants, tsetse repellents, composed of four components blend. I demonstrated that tsetse repellent is also a strong antifeedant for both G. pallidipes and G. f. fuscipes using feeding bioassays as compared to the attractant odour, adding the value of tsetse repellent. However, the attractant odour enhanced the feeding index. Using DREAM (deorphanization of receptors based on expression alterations of mRNA levels). I found that in G. f. fuscipes, following a short in vivo exposure to the individual tsetse repellent component as well as an attractant volatile chemical, OSNs that respond to these compounds altered their mRNA expression in two opposite direction, significant downregulation and upregulation in their number of transcripts corresponding to the OR that they expressed and interacted with odorant. Also, I found that the odorants with opposite valence already segregate distinctly at the cellular and molecular target at the periphery, which is the reception of odorants by OSNs, which is the basis of sophisticated olfactory behaviour. Deorphanization of ORs in none model insect is a challenge, here by combining DREAM with molecular dynamics, as docking score, physiology and homology modelling with Drosophila a well-studied model insects, I was able to predict putative receptors of the tsetse repellent components and an attractant odour. However, many ORs were neutral, showing they were not activated by the odorants, demonstrating the selectivity of the technique as well as the receptors. In my fourth chapter, I investigated the OBPs structures and their interaction with odorants molecules. I demonstrated that OBPs are expressed both in the antenna, as well as in other tissues, such as legs. I also demonstrated that there are variations in the expression of OBPs between tissues as well as sexes. I also demonstrated that odorants induced a fast alteration in OBP mRNA expression, some odorants induced a decrease in the transcription of genes corresponding to the activated OBP and others increased the expression by many fold in OBPs in live insect, others were neutral after 5 hours of exposure. Moreover, with subsequent behavioural data showed that the behavioural response of G. f. fuscipes toward 1-octen-3-ol decreased significantly when 1-octen-3-ol putative OBPs were silenced with feeding of double-stranded RNA (dsRNA). In summary, our finding whereby odorant exposure affects the OBPs mRNA, their physiochemical properties and the silencing of these OBPs affected the behavioural response demonstrate that the OBPs are involved in odour detection that affect the percept of the given odorant. The expression of OBPs in olfactory tissues, antenna and their interaction with odorant and their effect on behavioural response when silenced shows their direct involvement in odour detection and reception. Furthermore, their expression in other tissues such as legs indicates they might also have role in other physiological functions, such as taste.

Philosophiae Doctor - PhD / Tsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication. In the sensilla shaft the dendrite of OSNs are housed, which are protected by called the sensillum lymph produced by support cells and contains a variety of olfactory proteins, including the odorant binding protein (OBP) and chemosensory proteins (CSP). While on the dendrite of OSNs are expressed olfactory receptors. In my PhD, studies I tried to decipher the sense of smell in tsetse fly. In the second chapter, I demonstrated that G. f. fuscipes is equipped with diverse olfactory sensilla, that various from basiconic, trichoid and coeloconic. I also demonstrated, there is shape, length, number difference between sensilla types and sexual dimorphism. There is a major difference between male and female, while male has the unique basiconic sensilla, club shaped found in the pits, which is absent from female pits. In my third chapter, I investigated the odorant receptors which are expressed on the dendrite of the olfactory sensory neurons (OSNs). G. f. fuscipes has 42 ORs, which were not functionally characterised. I used behaviourally well studied odorants, tsetse repellents, composed of four components blend. I demonstrated that tsetse repellent is also a strong antifeedant for both G. pallidipes and G. f. fuscipes using feeding bioassays as compared to the attractant odour, adding the value of tsetse repellent. However, the attractant odour enhanced the feeding index. Using DREAM (deorphanization of receptors based on expression alterations of mRNA levels). I found that in G. f. fuscipes, following a short in vivo exposure to the individual tsetse repellent component as well as an attractant volatile chemical, OSNs that respond to these compounds altered their mRNA expression in two opposite direction, significant downregulation and upregulation in their number of transcripts corresponding to the OR that they expressed and interacted with odorant. Also, I found that the odorants with opposite valence already segregate distinctly at the cellular and molecular target at the periphery, which is the reception of odorants by OSNs, which is the basis of sophisticated olfactory behaviour. Deorphanization of ORs in none model insect is a challenge, here by combining DREAM with molecular dynamics, as docking score, physiology and homology modelling with Drosophila a well-studied model insects, I was able to predict putative receptors of the tsetse repellent components and an attractant odour. However, many ORs were neutral, showing they were not activated by the odorants, demonstrating the selectivity of the technique as well as the receptors. In my fourth chapter, I investigated the OBPs structures and their interaction with odorants molecules. I demonstrated that OBPs are expressed both in the antenna, as well as in other tissues, such as legs. I also demonstrated that there are variations in the expression of OBPs between tissues as well as sexes. I also demonstrated that odorants induced a fast alteration in OBP mRNA expression, some odorants induced a decrease in the transcription of genes corresponding to the activated OBP and others increased the expression by many fold in OBPs in live insect, others were neutral after 5 hours of exposure. Moreover, with subsequent behavioural data showed that the behavioural response of G. f. fuscipes toward 1-octen-3-ol decreased significantly when 1-octen-3-ol putative OBPs were silenced with feeding of double-stranded RNA (dsRNA). In summary, our finding whereby odorant exposure affects the OBPs mRNA, their physiochemical properties and the silencing of these OBPs affected the behavioural response demonstrate that the OBPs are involved in odour detection that affect the percept of the given odorant. The expression of OBPs in olfactory tissues, antenna and their interaction with odorant and their effect on behavioural response when silenced shows their direct involvement in odour detection and reception. Furthermore, their expression in other tissues such as legs indicates they might also have role in other physiological functions, such as taste.