Candidate biomarkers for mosquito age-grading identified by label-free quantitative analysis of protein expression in Aedes albopictus females

We applied a "shotgun" approach based on nanoliquid chromatography-high resolution mass spectrometry associated to label free quantification (LFQ) to identify proteins varying with age, independently from the physiological state, in Aedes albopictus, a mosquito species which in the last decades invaded temperate regions in North America and Europe, creating concerns for associated high nuisance and risk of arbovirus transmission. The combined "shotgun" and LFQ approach was shown to be highly suitable to simultaneously compare several biological samples, as needed in a study aimed to analyze different age-groups and physiological states of adult mosquito females. The results obtained represent the first wide-scale analysis of protein expression in Ae. albopictus females: >1000 and 665 proteins were identified from few micrograms of crude protein extracts of mosquito heads and thoraxes, respectively. Six of these proteins were shown to significantly vary from 2- to 16-day-old females, independently from their physiological state (i.e. virgin, mated, host-seeking, blood-fed, and gravid). BIOLOGICAL SIGNIFICANCE: Mosquito-borne diseases, such as malaria, dengue and other arboviroses, are a persistent cause of global mortality and morbidity, affecting hundreds of thousands of people. Billions of people living in tropical areas are at risk of being bitten every day by an infective mosquito female and the spread of tropical species such as Aedes albopictus to temperate areas is creating alarm in the northern hemisphere. Mosquito longevity is a critical factor affecting mosquito-borne pathogen transmission cycles and the mosquito capacity to transmit pathogens. However, large scale analyses of the age structure of mosquito field populations is hampered by the lack of optimal age-grading approaches. Our findings open new perspectives for the development of reliable, simple and cheap protein-based assays to age-grade Ae. albopictus females and, most likely, other mosquito species of higher medical relevance, such as the main dengue vector, Aedes aegypti, and the major Afrotropical malaria vectors. These assays would greatly contribute to epidemiological studies aimed at defining the actual vectorial capacity of a given mosquito species. Moreover, they would be very valuable in assessing the effectiveness of mosquito control interventions based on the relative ratio between young and old individuals before and after the intervention.