Effect of ionic strength and seawater cations on hagfish slime formation.

The defensive slime of hagfish consists of a polyanionic mucin hydrogel that synergistically interacts with a fiber network forming a coherent and elastic hydrogel in high ionic strength seawater. In seawater, the slime deploys in less than a second entrapping large quantities of water by a well-timed thread skein unravelling and mucous gel swelling. This rapid and vast hydrogel formation is intriguing, as high ionic strength conditions generally counteract the swelling speed and ratio of polyelectrolyte hydrogels. In this work we investigate the effect of ionic strength and seawater cations on slime formation dynamics and functionality. In the absence of ionic strength skeins swell radially and unravel uncontrolled, probably causing tangling and creating a confined thread network that entraps limited water. At high ionic strength skeins unravel, but create a collapsed and dense fiber network. High ionic strength conditions therefore seem crucial for controlled skein unraveling, however not sufficient for water retention. Only the presence of naturally occurring Ca2+ or Mg2+-ions allowed for an expanded network and full water retention probably due to Ca2+-mediated vesicle rupture and cross-linking of the mucin. Our study demonstrates that hagfish slime deployment is a well-timed, ionic-strength, and divalent-cation dependent dynamic hydrogel formation process.

Hagfish slime exudate stabilization and its effect on slime formation and functionality.

Hagfish produce vast amounts of slime when under attack. The slime is the most dilute hydrogel known to date, and is a highly interesting material for biomaterial research. It forms from a glandular secrete, called exudate, which deploys upon contact with seawater. To study slime formation ex vivo and to characterize its material properties, stabilization of the sensitive slime exudate is crucial. In this study, we compared the two main stabilization methods, dispersion in high osmolarity citrate/PIPES (CP) buffer and immersion in oil, and tested the influence of time, temperature and pH on the stability of the exudate and functionality of the slime. Using water retention measurements to assess slime functionality, we found that CP buffer and oil preserved the exudate within the first 5 hours without loss of functionality. For longer storage times, slime functionality decreased for both stabilization methods, for which the breakdown mechanisms differed. Stabilization in oil likely favored temperature-sensitive osmotic-driven swelling and rupture of the mucin vesicles, causing the exudate to gel and clump. Extended storage in CP buffer resulted in an inhibited unraveling of skeins. We suggest that a water soluble protein glue, which mediates skein unraveling in functional skeins, denatures and gradually becomes insoluble during storage in CP buffer. The breakdown was accentuated when the pH of the CP buffer was raised from pH 6.7 to pH 8.5, probably caused by increased denaturation of the protein glue or by inferior vesicle stabilization. However, when fresh exudate was mixed into seawater or phosphate buffer at pH 6-9, slime functionality was not affected, showing pH insensitivity of the slime formation around a neutral pH. These insights on hagfish exudate stabilization mechanisms will support hagfish slime research at a fundamental level, and contribute to resolve the complex mechanisms of skein unraveling and slime formation.