In the last few years, the Atomic Force Microscopy (AFM) has been used to study biological samples under physiological conditions through force measurements and image acquisition. The first measurements of elastic properties of biological material with the Atomic Force Microscope (AFM) date from 1992. The most common elastic property studied in biology is the Young’s modulus, which ranges from 1 up to 10 kPa for living cells, from 0.2 up to 1 GPa to amyloids, and more than 100 GPa to steel. The AFM is a powerful tool to elucidate mechanical properties of biological materials, and it has never been used to investigate Aedes aegypti eggs stiffening during its embryogenesis. Those mosquitoes are vectors of dengue and yellow fever and the results could help both basic and applied research.
Mosquito’s eggshells are white, permeable and soft at oviposition, and they turn black, impermeable and stiff with time. It has recently been shown that the change in permeability occur many hours after darkening. It is known that stiffening happens when eggs turn black, but until now no report had shown precisely when and how much egg darkens and elasticity changes.
In this work we have used the AFM tip as a micro-indenter to measure the elasticity changes of A. aegypti eggs after its oviposition and during its initial embryogenesis. Mosquito’s embryos are covered by a shell composed by a homogeneous endochorion and a heterogeneous exochorion, which is much softer than the endochorion and must be removed for appropriate measurements. Darkening of A. aegypti eggs was observed on a stereomicroscope, digital images were generated and the densitometry of eggs was analyzed.
We have developed a software to analyze the approximation force curves. We analyze the first 50 nm of cantilever’s deflection (indentation) to fit the Hertz model and the deflection between 100 and 150 nm to fit the elastic constant. A. aegypti eggs begin to darken 30 minutes after oviposition and 1 hour later their darkening is completed. From 1 to 3 hours after oviposition A. aegypti eggs stiffens progressively. After 3 hours they are as stiff as eggs at the end of embryogenesis. In summary, we verified that eggshell darkening takes 1.5 hours while stiffening is completed after 3 hours, probably as a consequence of sclerotization. The values founded to Young’s modulus are about 1 GPa, and are consistent with the literature predictions for protein aggregates.