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Chapter IV - Conclusions
The results of the experiments performed during my three years of PhD course and reported in this thesis provide novel information on the olfactory system of insects, with particular reference to the species selected as models, the aphid Acyrthosiphon pisum and the mosquito Anopheles gambiae.
Specific object of my research is the structural and functional study of Odorant-binding proteins (OBPs). The role recently recognised for OBPs as the elements responsible for discriminating olfactory signals in insects allows us to link the molecular properties of these proteins, measured in the lab, to the physiology of olfaction and eventually to the beavioural choices of the insect. This is particularly important for species that pose major problems to the economy and the health of human.
In both species, selected OBPs have been expressed in heterologous systems and utilised in structural studies and ligand-binding experiments.
In the pea aphid A. pisum, a first investigation, performed before the genome of this species was published, led to the identification of 4 OBPs, whose sequences were found to be remarkably similar or even identical in several different species of aphids. This unexpected finding, as generally OBPs are very divergent across species, was related to the anomalous conditions of aphids with respect to pheromones. In fact, whereas in most insect segregation of the species is accomplished by differentiating their pheromones, in aphids the sex pheromones are in most cases mixtures in different proportions, according to the species, of the same four components, nepetalactone and three isomers of nepetalactol. Therefore, being the same molecules to perceive, also the instruments for detecting them (OBPs) would be the same. This is also true to a great extent for the alarm pheromone, (E)-β-farnesene in most cases.
Another unexpected result of this research concerns the way in which (E)-β-farnesene is recognised. Ligand-binding experiments, performed with the three OBPs and a number of chemicals, has shown that this alarm pheromone is far from being the best ligand for any of these proteins. Instead, (E)-β-farnesene and some analogues, that also proved to be repellent for
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the aphids, bind specifically to one of three OBPs. Therefore, the basis for recognition is not related to the strength of binding of particular ligand, but rather to the selectivity of the ligand for a particular protein. This makes the system more complex and therefore more capable of adapting to new situations, even in relatively primitive insects as the aphids.
On this basis, new compounds which are easier to synthesise and more stable than (E)-β-farnesene were designed and synthesised in our lab. They were used in behaviour experiments by the collaborator in the University of Potenza, to verify their repellent activity, The results showed that all the new compounds were efficient aphid repellents, at least as good as (E)-β-farnesene. The best chemical in this respect proved to be 1-phenyl-4,8-dimethyl-1,3,7-nonatriene (PDN), that exhibited a repellent effect even stronger than the natural compound.
In the mosquito An. gambiae, that contains 60 genes encoding OBPs in its genome, a first investigation revealed those most abundantly expressed at the protein level. To get such information we applied (in collaboration with CISM, University of Firenze) a proteomic approach, using both the traditional technique of 2-D gel and MALDI identification of the spots, and the new approach involving direct digestion of the peptide mixture and analysis by mass spectrometry of the single peptides produced. The results indicated that only two OBPs are expressed in male antennae, but a relatively large number were detected in the female’s. On this basis, that to a large extent confirmed a parallel investigation previously performed at the RNA level, we selected few OBPs to be expressed and used in ligand-binding studies. The OBPs investigated differ from each other in their specificities towards odorants, once again supporting the idea that they are involved in the discrimination of chemical messages.
Some interesting and new elements have emerged from this work:
1. One of the best represented OBPs present in female antennae, OBP47, is much longer than classical OBPs and contains 13 cysteines rather than the six conserved in most of these proteins. We have found that this OBP is glycosylated in the mosquito, being the first example of an insect OBP that contains post-translational modifications (besides disulphide bridges). The bacterial expressed protein was crystallised by Prof. Christian Cambillau,
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Marseille, as part of a collaborative project, revealing novel features in its three-dimensional structure.
2. Binding experiments have revealed some interactions taking place between OBP1 and OBP4. At the same time, these two OBPs have been shown by Prof. Jurgen Krieger, University of Hoehenheim, Germany, to be co-expressed in the same sensillum. The co-localization of OBPs had been previously shown in Drosophila and appears to be a general phenomenon, at least in Diptera. The consequence of this fact is that heterodimers could exhibit different behaviour from their monomeric units, thus enlarging the spectrum of molecules that OBPs could recognise.
3. The OBP most represented in the male antennae, OBP9 was also detected in the male sex organs. It is apparently produced by the accessory glands and then transferred to the female during mating. This phenomenon, that had been previously investigated in another mosquito, Aedes aegypti, was further investigated. An orthologous OBP has been cloned from Aedes albopictus and found to be expressed in the male sex organs. Moreover, this protein contains, as an endogenous ligand, nonanal, a compound described as an attractant in another species of mosquito, Culex quinquefasciatus. In Aedes albopictus this compound might function as a male pheromone.
The results obtained with mosquitoes and above summarised represent a contribution toward undestanding the mechanisms of chemical communication, both between individuals of the same species and between mosquitoes and their hosts.
A detailed knowledge at the structural and functional level of OBPs could help designing new volatiles to be used as mosquito repellents or to produce confusion in the insect’s olfactory system. One of the most promising approach to fight malaria and other mosquito-carried diseases goes through the eradication of their vectors. Semiochemicals, that are safe for the environment and for the human health, should certainly represent the best alternative to the current heavy use of insecticides for the control of insect populations in the environment.
