Our amino acid composition and WAXS analyses, and subsequent modelling, predicted that, in contrast to extensibility and toughness, MA silk ultimate strength was primarily influenced by variations in crystallinity independent of MaSp1: MaSp2 expression or any subsequent shifts in amino acid composition. Our WAXS analyses withal suggested that variations in amorphous region nanostructural alignment and crystallinity combine to influence silk strength. Glandular pH, salts and shear stresses during spinning influence the formation of the crystalline nanostructures . Hence, we deduced that between treatment variations in glandular pH, salts and shear stress induce the crystalline region proteins to undergo α-helix→β-sheet nanostructural phase transitions within the spinning duct . These transitions result in an increase in crystalline density , which causes the crystalline region nanostructures to realign under strain . Such an increase in crystalline density should be identifiable in WAXS experiments as a reduction in 2θ and FWHM at the (200) or (120) diffraction peaks . Indeed, our analyses herein revealed such a mechanism occurred in protein deprived N. plumipes silks (S1 Table). Nonetheless, the phenomenon was not detected in the silks of any other species, leading us to conclude that the precise physiological mechanisms inducing property variation differed among the five species.