Universitą degli studi di Pavia
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Siciliano research activity
During my PhD I focused on chemical-based communication in insects, specifically on the Medfly (Ceratitis capitata), in order to improve the monitoring of control techniques of this serious pest.
C. capitata is a serious pest of crops in the Mediterranean area and Central America. Its success derives from its multivoltine reproductive behaviour, the ability to infest more than 250 species of vegetables and fruits and the extremely high adaptability to different climates and conditions. To date, the Sterile Insect Technique (SIT) is the only technique that succeeded in the eradication of this species from infested areas. This particular technique is based on the release in the fields of sterilized males able to compete with wild males for females; mating with sterile individuals lead to a considerable decrease of the population size, by not bearing to any progeny production. Being medfly reproductive behaviour highly related to odour and pheromone perception and being the SIT programme monitoring exclusively based on olfactory cues, the study of olfaction mechanisms in this species could furnish very useful information applicable to the development of new strategies for the eradication of this pest from infested areas.
The medfly genome not being available, I analyzed two EST libraries (previously set in my lab) in order to identify genes putatively involved in odour and pheromone perception. Basing on sequence similarity with other insect species, I was able to identify a set of 28 genes coding for proteins belonging to different families (Odorant binding proteins - OBP, Odorant receptors - OR, Chemosensory proteins - CSP) with a demonstrated role in the olfactory mechanisms. Five putative Pheromone Binding Proteins genes (Pbp), an Obp subgroup, were identified and characterized, outlining their structure and their expression profiles in relation to different body compartments and physiological conditions. All these genes display an average level of similarity with other insect putative homologues, they are expressed mainly in sensorial organs (such as antennae, maxillary palps and tarsi) and their expression is sexual maturation-related, at least for four of them. Only one gene was demonstrated to be antenna-specific.
The second step was the heterologous expression and subsequent purification of these proteins in E. coli cells. So far only CcPBP4 (the antenna-specific one) was successfully purified by ion-exchange and gel filtration chromatography from transformed bacterial cells, while CcPBP1, 2, 3 and 5 expression and purification is now in progress.
In order to identify the main actors in the medfly chemical communication, pheromones produced by sexually mature individuals were isolated and characterized. Volatile compounds emitted by males and females during the “calling” period (when males attract females by visual, olfactive and acoustical cues) were isolated and analyzed by GC. 19 compounds were identified from male emissions, while females didn’t seem to emit during this period. Four of these chemicals remain still unknown. The remaining 15 were demonstrated to elicit an intense antennal response on both sexes when tested on GC coupled Elettroantennograms (GC-EAG).
The last step of this PhD project was the analysis of the interactions between the active compounds belonging to the pheromone blend emitted by medfly males and the purified CcPBPs. Binding studies were performed by active displacement of a fluorescent probe (1-NPN) specific for insect OBPs. CcPBP4 displayed a very high binding affinity to one of the most emitted compound of the pheromone mixture. The results achieved so far allowed us to discover specific protein-ligand interactions, giving us the first summary of pheromone perception in the Medfly and furnishing possible useful tools for applications in the field.
C. capitata is a serious pest of crops in the Mediterranean area and Central America. Its success derives from its multivoltine reproductive behaviour, the ability to infest more than 250 species of vegetables and fruits and the extremely high adaptability to different climates and conditions. To date, the Sterile Insect Technique (SIT) is the only technique that succeeded in the eradication of this species from infested areas. This particular technique is based on the release in the fields of sterilized males able to compete with wild males for females; mating with sterile individuals lead to a considerable decrease of the population size, by not bearing to any progeny production. Being medfly reproductive behaviour highly related to odour and pheromone perception and being the SIT programme monitoring exclusively based on olfactory cues, the study of olfaction mechanisms in this species could furnish very useful information applicable to the development of new strategies for the eradication of this pest from infested areas.
The medfly genome not being available, I analyzed two EST libraries (previously set in my lab) in order to identify genes putatively involved in odour and pheromone perception. Basing on sequence similarity with other insect species, I was able to identify a set of 28 genes coding for proteins belonging to different families (Odorant binding proteins - OBP, Odorant receptors - OR, Chemosensory proteins - CSP) with a demonstrated role in the olfactory mechanisms. Five putative Pheromone Binding Proteins genes (Pbp), an Obp subgroup, were identified and characterized, outlining their structure and their expression profiles in relation to different body compartments and physiological conditions. All these genes display an average level of similarity with other insect putative homologues, they are expressed mainly in sensorial organs (such as antennae, maxillary palps and tarsi) and their expression is sexual maturation-related, at least for four of them. Only one gene was demonstrated to be antenna-specific.
The second step was the heterologous expression and subsequent purification of these proteins in E. coli cells. So far only CcPBP4 (the antenna-specific one) was successfully purified by ion-exchange and gel filtration chromatography from transformed bacterial cells, while CcPBP1, 2, 3 and 5 expression and purification is now in progress.
In order to identify the main actors in the medfly chemical communication, pheromones produced by sexually mature individuals were isolated and characterized. Volatile compounds emitted by males and females during the “calling” period (when males attract females by visual, olfactive and acoustical cues) were isolated and analyzed by GC. 19 compounds were identified from male emissions, while females didn’t seem to emit during this period. Four of these chemicals remain still unknown. The remaining 15 were demonstrated to elicit an intense antennal response on both sexes when tested on GC coupled Elettroantennograms (GC-EAG).
The last step of this PhD project was the analysis of the interactions between the active compounds belonging to the pheromone blend emitted by medfly males and the purified CcPBPs. Binding studies were performed by active displacement of a fluorescent probe (1-NPN) specific for insect OBPs. CcPBP4 displayed a very high binding affinity to one of the most emitted compound of the pheromone mixture. The results achieved so far allowed us to discover specific protein-ligand interactions, giving us the first summary of pheromone perception in the Medfly and furnishing possible useful tools for applications in the field.
