Effect of ionic strength and seawater cations on hagfish slime formation

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The effect of ionic strength on hagfish slime was evaluated by mixing freshly harvested hagfish exudate into Milli-Q water and seawater, representing the complete absence of ions and the natural environment for hagfish slime formation, respectively. The resulting fiber network was investigated under the microscope and the water retention of the slime was measured. Milli-Q water resulted in the formation of a confined and compact slime mass, showing a close arrangement and narrow spacings between the slime threads instead of a widespread and expanded network as observed in seawater (Fig. 2a). The slime formed in Milli-Q did not span the entire available water volume of 20 ml upon deployment in the glass flask, which resulted in a condensed ‘blob’. Furthermore, almost all skeins unraveled in Milli-Q, which stands in contrast to observations in seawater where skeins have been shown to remain coiled27. Although more skeins unraveled in Milli-Q, the slime showed a substantially reduced ability to entrap water (Fig. 2b). Slime in Milli-Q initially retained 7.5 g of water, which is roughly 50% less compared to the 14.7 g retained in seawater (Fig. 2c). We suggest that this effect is caused by altered network formation dynamics. In the following sections, we will discuss the effects of ionic strength and seawater cations on skein unraveling, vesicle decondensation, and mucin viscosity and will suggest their implications on whole slime formation and functionality.

Figure 2
Figure 2

Effect of seawater and Milli-Q on hagfish slime thread network and water retention properties. (a) Hagfish slime formed with seawater (left) and Milli-Q (right). In seawater the expanded thread network spans the entire volume whereas in Milli-Q a confined ‘blob’-like network is formed. (b) Images of slime draining during water retention measurements. (the image series in the top line in (b) was previously published25). (c) Water retention measurements of slime formed in seawater and Milli-Q.

Unravelling dynamics of thread skeins in Milli-Q and seawater

Skein unraveling dynamics in ion-free Milli-Q are less controlled and faster than in seawater. Figure 3a shows an image sequence of a skein unraveling in Milli-Q (Supplementary Information Movie S2). The skein swells radially and completes unravelling in about two seconds, even in the absence of substantial external flows. Similar to seawater, the unraveling started at the apical tip of the skein21,25, but then the skein continued to uncoil from both sides in an uncontrolled manner. As the uncoiling is localized to the position of the skein, a confined and narrow thread mesh remains on the uncoiling spot. In contrast, unraveling in seawater can take several minutes when observed under the microscope21,25 (Fig. 3b) and the uncoiling threads are able to span a larger area, especially in the presence of flows. A direct comparison of the skein unraveling characteristics in seawater and in Milli-Q is shown in Fig. 3c. The slower unraveling of skeins of the Atlantic hagfish (M. glutinosa) compared to skeins of the Pacific hagfish (E. stoutii) in seawater was similarly observed by Bernards et al.21, who suggested that a slime that deploys too fast could have drawbacks for burrowing animals such as the Atlantic hagfish.

Figure 3
Figure 3

Characteristic unraveling times of single M. glutinosa skeins in ion-free Milli-Q water and in seawater observed under a light microscope. (a) Image series of a skein unraveling in Milli-Q captured with a high speed camera. (b) Skein unraveling in seawater. (c) Comparison of skeins unraveling with seawater (left) and Milli-Q (right), showing the radial swelling in Milli-Q and the controlled unraveling from the apical tip in seawater. Scale bars = 100 μm.

In Milli-Q the combination of radial swelling and uncontrolled unraveling from two sides probably causes the uncoiling skeins to tangle. Tangled threads are limited in their ability to spread out after they have unraveled, which in turn impairs the formation of a widespread and expanded network, resulting in a lower water retention.

Ionic strength determines skein unraveling

Seawater and Milli-Q resulted in distinct network properties and skein unraveling. We studied the effect of ionic strength on skein unraveling and found that ionic strength slows down unraveling speed and reduces swelling of the skeins. Radial swelling and uncontrolled, fast unraveling of skeins was observed in low-ionic strength sodium chloride solutions (10 mM NaCl, I = 0.01 M) and in diluted seawater (10% seawater, I = 0.06 M), similar to Milli-Q (Supplementary Information Movie S3). The presence of increased ionic strength in the form of sodium chloride (100 mM NaCl, 500 mM NaCl), seawater, or artificial seawater lacking certain cations in contrast resulted in controlled (only from the apical end) and slow unraveling of the skeins (Supplementary Information Movie S4). We have two hypotheses how a high ionic strength could slow down and control skein unraveling. The two hypotheses are not mutually exclusive and both give a reasoning to slower unraveling in a high ionic strength environment.

First, the fast unraveling and radial swelling of the skeins in ion-free water could be caused by thread swelling given the large osmotic gradient. Salt dependence in thread skein uncoiling between distilled water and seawater was already observed and a dependence of uncoiling on seawater-induced swelling of thread skeins was suggested4,28. Milli-Q could cause an excessive swelling of the keratin-like29,30,31 hagfish slime threads. Unlike in hard α-keratins, where intermediate filaments (IFs) are embedded in a isotropic, high-sulfur matrix32, hagfish slime threads constitute matrix-free IFs and are therefore highly sensitive to hydration33,34. Fudge and Gosline34 showed that hagfish threads increased 45% in diameter compared to the dry state when hydrated with deionized water. As spontaneous unraveling of the skeins is considered to be propelled by a stored strain energy in the coiled thread, it seems probable that an excessive swelling of the thread adds to this stored entropy-elasticity. The osmolarity of seawater – as well as the high osmolarity of the residual fluid (888 mOsm) of the slime exudate9 – could reduce the swelling of the threads compared to Milli-Q and thus limit the strain energy, resulting in a slower and controlled unraveling, which can then be accelerated by external mixing flows and attaching mucus strands.

The second hypothesis is that ionic strength reduces the dissolution speed of the seawater-soluble glue, which was found to mediate unraveling in E. stoutii skeins21 and similarly observed on M. glutinosa skeins25. It is possible that the glue dissolves faster in the presence of low ionic strength solutions and that its dissolution speed is reduced at high ionic strength. Deionized water seems to be sufficient to loosen the glue from binding to itself, as already shown in electron microscopy images25. However, a low ionic strength could prevent a further dissolution of the glue from the threads as observed by Bernards et al.21. Both hypotheses could also explain why skeins are stable in high-osmolarity stabilization buffers8,19,20; given a suggested increased insolubility of the glue and/or an osmotically dehydrated slime thread with reduced strain energy.

Regardless if one or both suggested hypotheses are considered the main reason for the observed differences in skein unraveling, the different unraveling patterns governed by ionic strength have implications on whole slime functionality.

Effects of ionic strength on slime network formation

Similar to skein unraveling, the effect of ionic strength on whole slime functionality was studied by mixing hagfish slime exudate with solutions of various sodium chloride (NaCl) concentrations and dilutions of natural seawater. The presence of 10 to 100 mM NaCl resulted in a substantially increased initial load compared to Milli-Q (Fig. 4a) and the fiber network did not show a clump formation after mixing, supporting the beneficial effect of ionic strength. The observed differences between the treatments were too large to be accounted for simply by differences in the density of the different salt solutions. Although a low ionic strength (10 mM NaCl and up to 10% seawater) showed skein swelling under the microscope, the salts had beneficial effects on the water retention. However, a high ionic strength solely based on sodium chloride (I = 0.5 M) – being close to the ionic strength of natural seawater (I = 0.6–0.7 M) – resulted in a collapsed and dysfunctional slime. Although single skeins did not swell and unraveled controlled under the microscope (Supplementary Information Movie S4), no widespread fiber network formed (Supplementary Information Figure S1a) and no water was retained.

Figure 4
Figure 4

Effect of ionic strength on the water retention properties of hagfish slime and on mucin viscosity. (a) Water retention in different concentrations of sodium chloride (NaCl). (b) Water retention of hagfish slime formed in dilutions of seawater. For comparison between NaCl and seawater measurements, the ionic strength I is indicated in brackets. (c) Dynamic viscosity of hagfish mucin in Milli-Q, seawater, and diluted seawater at a concentration of 0.02 mg/ml measured at room temperature. Pure Milli-Q is given as a reference. (d) Mucin viscosity as a function of runs through the capillary of the viscometer, showing the mechanical sensitivity of hagfish mucin towards shear. The inlet shows a schematic drawing of an Ubbelohde capillary viscometer.

This stands in contrast to seawater, where the slime shows a functional network and superior water retention properties despite a high ionic strength (Fig. 4b). Even in the presence of 1% seawater (I < 0.01 M) the initial load was increased to ≈12 g compared to ≈7 g in Milli-Q and ≈10 g in 10 mM NaCl (Fig. 4b). 5% seawater retained the most water initially over 100% seawater. These findings imply an important role of other seawater cations such as Ca2+ and Mg2+ for slime functionality in a high ionic strength environment, which will be discussed in the following section. Furthermore, it suggests that slime network functionality is not determined by skein unraveling and ionic strength alone and that the dynamics of vesicle rupture and mucin viscosity might be similarly crucial.

Mucin viscosity measurements (Fig. 4c) showed that in Milli-Q hagfish mucin had the highest viscosity (2.14 mPas). At increased ionic strength as the case in 5% and 100% seawater the viscosities dropped to 1.86 mPas and 1.77 mPas, respectively. Theses results are in good agreement with the findings of Fudge et al.11 who measured a viscosity of 1.41 mPas in seawater and about 1.54 mPas in Milli-Q at 9 °C. The higher viscosity of mucin in 5% seawater compared to 100% seawater could explain why 5% seawater showed a higher initial load over 100% seawater in water retention measurements (Fig. 4b). A higher viscosity means a higher resistance to flow, suggesting that liquid should be better retained. A higher viscosity combined with the presence of small amounts of salts in 5% seawater and their beneficial effect on skein unraveling seem to lead to a slime with superior water retention properties in comparison to slime formed under natural conditions. However, this does not infer that slime formed in 5% seawater also has superior defense properties. Although Milli-Q showed a higher viscosity than all dilutions of seawater, water retention in Milli-Q was inferior to seawater (Fig. 2c). In this case the negative effects of the proposed tangling of the uncoiling skeins on network formation probably outbalances the slightly positive effect of viscosity on water retention.

The lower mucin viscosity in seawater compared to Milli-Q probably originates in increased electrostatic charge suppression35,36 of the high ionic strength in seawater. Similar effects were shown for porcine gastric mucin (PGM), which does not gel at high ionic strengths (>0.1 M)36 and for human sputum, which shows reduced spinnability, rigidity, and viscoelasticity after treatment with hypertonic saline solutions37,38,39,40. Polyelectrolyte gels such as mucins are shown to stiffen as they swell in low salt solutions because the counterions in the gel network increase the internal pressure41, thus increasing in viscosity. Furthermore, hagfish mucin viscosity showed a sensitivity towards mechanical shear (Fig. 4d), regardless whether in seawater or in Milli-Q. The sensitivity of hagfish slime towards mechanical stress is well known3,42 and was similarly shown for hagfish mucin using a rotational shear rheometer43. These results support previous observations that hagfish mucin seems to aggregate under shear43 and suggest that network cross-links could be disrupted. For the capillary rheometry experiments the mucin solution had to be pulled up through the glass capillary in order to prepare the measurement, meaning the mucin solution inevitably already experienced one shear event prior to the measurement. This infers that the viscosity of natural hagfish mucin immediately after secretion could be substantially higher than reported so far.

Divalent seawater cations (Ca2+ and Mg2+) are crucial for whole slime functionality

The importance of divalent seawater cations (Ca2+ or Mg2+) to efficiently entrap water in a high ionic strength environment was investigated using artificial seawater (ASW) and modifications thereof, lacking specific cations. Water retentions of hagfish slime formed with natural seawater and with ASW did not show substantial differences (Supplementary Information Figure S2a), despite the differences in cationic composition (Supplementary Information Table S3). In contrast, ASW lacking divalent cations Ca2+ and Mg2+ did not form a functional slime network (Fig. 5a, Supplementary Information Figure S1b), i.e. no water was entrapped as similarly observed for the 500 mM NaCl solution (Fig. 4a). Also, when EDTA – being a strong chelator of di- and trivalent cations – was mixed into seawater, the initial entrapped load dropped significantly (Supplementary Information Figure S2b). In contrast, ASW lacking monovalent seawater cations (Na+ and K+) but containing the divalent seawater cations (Ca2+ and Mg2+) resulted in a slime that efficiently entrapped and retained the water, similar to seawater. We found that when one of the two major divalent cations was present at its natural concentration like in seawater (10 mM Ca2+; 50 mM Mg2+ 26), a functional slime network was formed, which entrapped and retained water (Fig. 5b). The beneficial effect of calcium ions was found to allow slime formation beyond ionic strengths occurring in natural seawater. In the presence of 10 mM Ca2+ functional slime networks formed in solutions containing up to 3 M NaCl (Fig. 5c), corresponding to about 4–5 times the ionic strength occurring in natural seawater. However, slime formation eventually failed at 4 M NaCl. Similarly, Bernards et al.21 showed that skein unraveling is inhibited in 4 M NaCl in Pacific hagfish slime. The initial load slightly decreased with increasing NaCl molarity, which could originate in a lower mucin viscosity due to charge screening and/or in the higher density of the higher molarity fluids. These measurements show the extreme resilience and the limits of hagfish slime to high salt conditions and underline the importance of calcium.

Figure 5
Figure 5

Effect of ionic composition of various versions of artificial seawater (ASW) on slime functionality. (a) Water retention in ASW without divalent cations (Ca2+ and Mg2+) and ASW without monovalent cations (Na+ and K+). (b) Water retention in ASW either without Ca2+ or Mg2+, showing that one divalent ion is sufficient for functionality at a high ionic strength. If none are present at a comparable ionic strength, no water is retained (500 mM NaCl). For comparison the ionic strength (I) for every measurement is provided in brackets. (c) Water retention in NaCl solutions containing 10 mM CaCl2. (d) UV-VIS turbidity measurements of hagfish exudate mixed with NaCl solutions in the absence of calcium. The turbidity at NaCl ≥100 mM originates in the presence of condensed vesicles and vanished once 10 mM CaCl2 are added.

The crucial role of the divalent cation Ca2+ for mucin vesicle rupture was in depth investigated by Herr et al.22. The authors showed that Ca2+ is required for the swelling and rupture in approximately 60% of vesicles in seawater strength osmolarity and suggested that Ca2+-activated transporters in the vesicle membrane are responsible for the need of calcium. The remaining 40% ruptured also in the absence of Ca2+. All vesicles ruptured when exposed to distilled water9. Our observations are in line the findings of Herr et al.22 and show that calcium is needed for complete vesicle decondensation already at NaCl concentrations approximately ≥100 mM (Fig. 5d). The turbidity at 75 mM NaCl did not significantly change upon calcium addition, suggesting that most vesicles swelled even in the absence of calcium. At 50 mM the solution was already viscous and many skeins unraveled, making turbidity measurements difficult (not shown). However, the onset of viscosity and unraveled skeins suggest that in these conditions most vesicles swelled and ruptured.

These findings imply that a low ionic strength (approx. ≤100 mM) allows a hypo-osmotic swelling and rupture of most mucin vesicles similar to Milli-Q but results in a controlled skein unraveling as the ionic strength could be sufficient to suppress excessive thread swelling. Combined, this seems to form a somewhat functional fiber network that retains more water than Milli-Q (Fig. 5b). At a high ionic strength (approx. ≥100 mM) vesicle decondensation is limited to about 40% of the vesicles. The reduced amount of ruptured vesicles and mucin strands does not seem to be able to sufficiently drive the unraveling of the skeins. A strongly impaired and collapsed network forms with many skeins remaining coiled, resulting in an almost absent water retention. Therefore, at high ionic strength the presence of Ca2+ seems crucial to rupture all the vesicles within the deployment time frame, allowing to transmit mixing forces to the threads27 and thus form a functional slime network.

The presence of 50 mM Mg2+ resulted in an only slightly inferior water retention to Ca2+ (Fig. 6b). Although it was found that Mg2+ only increased rupture in vesicles at about double the seawater concentration in seawater strength osmolarity22, it seems that for whole slime functionality Mg2+ has a similar effect to Ca2+. However, the origin of this beneficial effect is so far elusive. The similar water retentions between seawater and dilutions of seawater (Fig. 5a) imply that hagfish slime functionality is not limited to a narrow window of ion composition as long as specific divalent ions (Ca2+ and Mg2+) are present at concentrations similar to seawater. It was shown that >3 mM Ca2+ resulted in a significant increase in vesicle rupture22. Although in 1%/5% seawater there is only about 0.1/0.5 mM Ca2+, there might be a beneficiary effect of having additionally 0.5/2.5 mM Mg2+ present. Additionally, the low osmolarities of these dilutions could support a hypo-osmotic vesicle rupture and at the same time reduce thread swelling, allowing for controlled unraveling without tangling.

Figure 6
Figure 6

Dynamic cation concentrations during slime formation. The concentration of seawater cations (Na+, K+, Ca2+, Mg2+) of three seawater fractions – (a) seawater, (b) unbound seawater, and (c) bound and drained mucin-rich seawater – were analysed. The figure on the left represents a schematic drawing of the experiment. Concentrations are in parts per million (ppm).

Dynamic interactions of hagfish slime with seawater cations

Since hagfish slime deploys rapidly, it must distinctly interact with ions in the direct environment. To capture the dynamic processes between hagfish slime and seawater cations, the cation flux was investigated immediately after and five minutes after slime formation. Three fractions of liquids (a) seawater (Norway), the (b) unbound and the (c) bound & retained fraction were analyzed (Fig. 6). Hagfish slime significantly depleted potassium ions (K+) from seawater and released calcium ions (Ca2+). It was found that most K+ was depleted in the unbound fraction (−24 ppm, p < 0.02). Some K+ was again added to fraction (c) after five minutes of draining as fraction (c) showed an only 19 ppm (p < 0.04) lower concentration compared to seawater. Calcium followed an opposite trend as the unbound fraction showed some more calcium (+15 ppm, p < 0.08) whereas the bound and drained fraction showed significantly more calcium compared to seawater (+23 ppm, p < 0.02). Sodium and magnesium levels did not change significantly.

The depletion of K+ from seawater suggests that K+ is involved in an ion-exchange process during slime formation rather than for mucus gelation. The elevated Ca2+ levels in the fractions (b) and (c) (Fig. 6) raise the possibility that a K+/Ca2+ exchange process is involved in mucus decondensation during vesicle rupture, suggesting that the calcium is added by the ruptured vesicles. Skeins are unlikely to contribute substantial amounts of intracellular calcium when they unravel because cytoplasmic calcium levels are typically very low, and the skein develops within the cytoplasm of gland thread cells4,5. Apart from the skeins, Ca2+ can only be added by the vesicles as it is almost completely absent in the residual fluid9. A high intragranular calcium ion content of M. glutinosa mucus vesicles was suggested by Herr et al.9. The authors proposed that vesicle swelling is driven by a ‘jack-in-the-box’ mechanism, in which typically Ca2+-ions shield the charges of condensed polyanionic molecules such as mucin inside a vesicle44. This cation is Ca2+ in the case of mice mucin vesicles45 but can also be histamine for heparin or lysozyme for proteoglycans44. Once exposed to seawater, vesicle decondensation is triggered and Ca2+ is replaced by a less effective shielding cation such as Na+ or K+, causing repulsion between the negatively charged mucin polymers and thus fast swelling of the gel46. Our observations of dynamic cation concentrations in slime deployment support the suggestion of Herr et al.9 that hagfish mucin inside the vesicle is kept in a condensed state by Ca2+. Also, it is possible that Ca2+ is exchanged for K+ during vesicle swelling, as similarly reported by Nguyen et al.47 for mucus granules. The small potassium increase in fraction (c) compared to (b) supports the possible role of K+ as a counterion in mucin decondensation. The K+ ions do not seem to be strongly bound by the slime and drain again back into the solution. However, given the fact that sodium is present in seawater at 25× the concentration of potassium and therefore the diffusion of sodium would be faster, it seems unlikely that potassium exchange for calcium would evolve in preference to sodium. Therefore, probably both, potassium and sodium are exchanged for calcium during decondensation but the changes in sodium level could not be measured (see caveat further down) or the sulfonic groups of the mucin have a slight preference for potassium.

Furthermore, the fact that the mucin-rich fraction (c) in Fig. 6 contains higher calcium levels than fraction (b) suggests that Ca2+ binds to hagfish mucin and helps it to gel. The affinity of invertebrate mucus for calcium ions was shown before for mucus of the freshwater snail48. Ca2+-ions knowingly forms reversible cross-links and create salt-bridges between mucin chains, thus forming networks49,50,51. Therefore, the putative gelled mucin network interspaced in the thread network allows to entrap water. Furthermore, competitive binding of divalent cations over monovalent cations to sulfonated polyelectrolytes such as hagfish mucin8 is also well known52,53,54. The calcium probably bound to the mucin in a counterion condensation process55,56,57 and drained from the slime mass together with some of the mucin.

What is the amount of calcium that keeps the mucin condensed in the vesicle? 4 μl of exudate added about 0.3–0.46 mg calcium ions to the seawater. Calculating with an exudate density of about 1 mg/μl10 results in 75–115 μg Ca2+ per mg exudate. Considering that 66% of the exudate are residual fluid and 17% each are mucin vesicles or skeins11 about 0.68 mg mucins were added to the seawater. If all Ca2+ originates from the mucus vesicles 44 –68 wt% of the total mucus dry mass would be calcium. Calcium was shown to reach high intragranular levels between 2.5–3.6 moles calcium/kg dry mass mucus in the giant mucin granules of a slug (Ariolimax columbianus)44, corresponding to about 10–14.4 wt%. Considering that hagfish mucin must swell extremely fast in a defense situation against the high osmotic gradient of seawater, a roughly three to four times higher concentration than reported in slug mucin vesicles does not seem unlikely.

A caveat to the presented data lies in the high levels of sodium and magnesium in seawater, which limited an holistic insight in the dynamic ion flux during slime formation. Both calcium and potassium occur at concentrations of roughly 300–400 ppm whereas magnesium and sodium are present at more than double and ten-fold this concentration, respectively. We worked with concentrations close to the natural concentrations of hagfish exudate (1 mg exudate on 5 ml seawater11). Cation concentrations of 300–400 ppm result in a exudate/cation mass ratios of about 1/1.5–2 per cation as is the case for Ca2+ and K+. In contrast, this ratio is roughly 1/5 for Mg2+ and almost 1/50 for Na+. It is possible that the concentrations of Mg2+ and Na+ varied in the investigated fractions but their variation remained hidden in the small ratio of exudate/ion concentration. Future investigations such as measuring the calcium content only in the mucus and the skein fraction of the exudate or using dilutions of seawater and investigating the vesicles and skeins separately could help to provide a more detailed analysis of intragranular Ca2+ levels and ion flux during slime formation.

Summary

In this study we demonstrate the crucial role of ionic strength and seawater cations – especially Ca2+ – for the formation dynamics and functionality of hagfish slime. The findings are summarized and schematically depicted in Fig. 7. We suggest that sufficient ionic strength controls the dynamics of skein unraveling and slime network formation.

Figure 7
Figure 7

Schematic representation of the suggested role of ionic strength and divalent seawater cations (Ca2+) for the formation and functionality of hagfish slime. A low ionic strength causes the thread skeins to swell radially and unravel uncontrolled from both sides, causing tangling of the threads. The vesicles rupture due to the large osmotic gradient. Tangling combined with immediate vesicle rupture creates a confined thread network that fails to entrap large amounts of water. At a high ionic strength skein unraveling is controlled but vesicle rupture is impaired. A dense and collapsed network forms, resulting in an almost absent water retention. At a high ionic strength with Ca2+-ions present, skeins unravel controlled, vesicles rupture Ca2+-mediated, and the mucin probably gels. A widespread and expanded slime network forms that entraps large amounts of water as observed in seawater, resulting in a functional defensive hydrogel. Citations in the figure: *9; 22.

A low ionic strength caused a confined and narrow thread network in contrast to the widespread and expanded network formed in seawater. The thread skeins swelled and unraveled uncontrolled from both sides, probably causing tangling of the threads and thus preventing a widespread network. It is possible that the fast unraveling in ion-free water originates in an excessive swelling of the intermediate filament slime thread, which would possess increased stored strain energy. More stored strain energy would lead to a less controlled and faster unraveling. However, as the mucin vesicles ruptured in the hypo-osmotic environment of deionized water, a somewhat functional network that entraps about 50% of water in comparison to seawater can be formed in the absence of ionic strength.

At increased ionic strength (approx. >100 mM) a collapsed network formed that failed to incorporate water although the thread skeins unraveled controlled. We assume that as a consequence of impaired mucin vesicle rupture at high ionic strength – in the absence of calcium ions – an effective skein unraveling is limited as less mucus strands can attach to the threads to transmit mixing forces, leaving many skeins coiled. Only in the presence of divalent seawater cations Ca2+ and Mg2+ a functional slime network is realized at seawater strength osmolarity. Whereas the reasons for the beneficial effect of Mg2+ remain elusive, Ca2+ was shown to be important to mediate a complete and well-timed vesicle rupture, which supports skein unraveling in the high ionic strength environment, creating an expanded network. The presence of calcium allowed the formation of a functional slime network up to 3 M NaCl, corresponding to 4–5 times the ionic strength of seawater. Furthermore, Ca2+ could be necessary for an ionic gelation of hagfish mucin, which is supported by cation concentration measurements. These measurements further suggest that M. glutinosa mucin vesicles release intragranular Ca2+ during the rapid decondensation and swelling of hagfish mucin. Based on the findings in this work we propose that calcium has three distinct roles in hagfish slime: mucin condensation within vesicle, mucin decondensation via Ca2+-activated transporters in the vesicle membrane at high ionic strength22, and mucin gelation in the deployed slime.

Our results show that a functional defensive slime that entraps and retains water can only be formed in the presence of divalent seawater cations Ca2+ or Mg2+ at a high ionic strength. The insights on the interactions of hagfish slime with seawater ions will improve our understanding of the complex cascade of physico-chemical events underlying the formation of hagfish defensive slime and might support the design of bioinspired fibrous polyelectrolyte hydrogels that efficiently and rapidly form in high ionic strength environments.



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