Biomarkers of the age of mosquitoes are required to determine the risk of transmission of various pathogens as each pathogen undergoes a period of extrinsic incubation in the mosquito host. Using the 2-D Difference Gel Electrophoresis (2-D DIGE) procedure, we investigated the abundance of up to 898
proteins from the
Yellow Fever and dengue virus vector, Aedes aegypti, during ageing. By applying a mixed-effects model of
protein expression, we identified five common patterns of abundance change during ageing and demonstrated an age-related decrease in variance for four of these. This supported a search for specific
proteins with abundance changes that remain tightly associated with ageing for use as ageing
biomarkers. Using MALDI-TOF/TOF mass spectrometry we identified ten candidate
proteins that satisfied strict
biomarker discovery criteria (identified in two out of three multivariate analysis procedures and in two cohorts of mosquitoes). We validated the abundances of the four most suitable candidates (Actin depolymerising factor; ADF, Eukaryotic
initiation factor 5A; eIF5A, insect cuticle
protein Q17LN8, and Anterior fat body
protein; AFP) using semi-quantitative Western analysis of individual mosquitoes of six ages. The redox-response
protein Manganese superoxide dismutase (SOD2) and electron shuttling
protein Electron transfer
oxidoreductase (ETO) were subject to post-translational modifications affecting their charge states with potential effects on function. For the four candidates we show remarkably consistent decreases in abundance during ageing, validating initial selections. In particular, the abundance of AFP is an ideal
biomarker candidate for whether a female mosquito has lived long enough to be capable of dengue virus transmission. We have demonstrated
proteins to be a suitable class of ageing
biomarkers in mosquitoes and have identified candidates for epidemiological studies of
dengue and the evaluation of new disease reduction projects targeting mosquito longevity.