Identificazione dei fattori trasferiti dalle ghiandole salivari all'ospite durante la puntura della zanzara tigre Aedes albopictus

Aedes albopictus is a day-biting, nuisance mosquito originated in South-East Asia and that has recently spread all over the world, including Europe and USA. It is a competent vector of several arthropod borne viral diseases (Arboviruses), such as dengue, chikungunya and zika viruses. While both male and female adults rely on sugar feeding for survival, females feed on blood in order to develop eggs. While probing for blood vessels with proboscis under vertebrate (human) skin, females inject saliva and, if infected, they can transmit the viruses. Ae. albopictus saliva is a cocktail of proteins that facilitate blood feeding by inhibiting blood clotting and inducing vasodilation effects. In humans, it can cause severe allergic reactions. Furthermore, Aedes saliva facilitates viral transmission by modulating host immune responses. The full impact of mosquito salivary proteins on the pathogenesis of arboviruses is still not clearly understood. This knowledge gap originated also by the poor biochemical and molecular characterization of the saliva components. In these regards, the research work presented in this thesis aims at identifying the proteins that are in the Ae. albopictus saliva. With the aim to identify the factors necessary for blood feeding, protein extracts from female salivary glands were prepared from 1 day and 6 days old mosquitoes and from 6 days old mosquitoes after having taken a blood meal. The underlying idea was that the expression of a factor important for blood meal should increase from 1 day old mosquitoes (that are young and not ready for egg development) and 6 days old mosquitoes. Furthermore, its protein concentration should decrease after blood meal, because it is transferred to the vertebrate host. Protein extracts were prepared from four biological replicates for each group and using from 60 to 120 pairs of salivary glands. These extracts were studied using 2-D gel electrophoresis and the LC-MS/MS analysis of selected spots. Overall, these experiments showed that the proteome of salivary glands is “simple”, as the number of spots on the gel is limited; however, a marked difference in the proteome of female salivary glands belonging to the three groups is detectable. Particularly, it appeared that the proteome composition does not change much qualitatively, i.e. the protein spots are almost the same in all the gels, but their relative abundance change between groups. The sequencing of four spots allowed the identification of an apyrase, two D7 proteins and two 30 kDa proteins. The identification of all the spots is ongoing and it will facilitate the understanding of the expression dynamics of salivary glands proteins in relation to sexual maturation and the blood feeding. A second part of the work aimed at identifying the proteins that are present in the saliva of Ae. albopictus. The expression of a factor in the salivary gland does not guarantee its presence in the saliva and, because many work rely on salivary gland extracts as a proxy of saliva, it is important to access if and how they are different. The saliva was collected from 1000 mosquitoes preparing five biological replicates (200 mosquitoes each). The proteins were trypsinized in solution and the proteolytic peptides analysed using LC-MS/MS. The analysis allowed us to identify 19 proteins: 2 Salivary Gland Specific (SGS), 1 Apyrase, 2 Serpins, 4 D7 proteins, 2 Antigen-5, 2 belonging to the 34 kDa and 5 to the 30 kDa classes and 1 peptide of 8kDa. Most of these proteins were known to be expressed in the salivary glands, but what is more important is that this represent only a subset of factors expressed in the Ae. albopictus salivary glands, highlighting that extracts from salivary glands are not similar to the saliva. Concluding, this work presents the first study of the temporal dynamics of the expression of salivary glands factors in the tiger mosquito and the first in depth characterization of saliva composition in a mosquito species.