Physiology, biomechanics, and biomimetics of hagfish slime.

Hagfishes thwart attacks by fish predators by producing liters of defensive slime. The slime is produced when slime gland exudate is released into the predator's mouth, where it deploys in a fraction of a second and clogs the gills. Slime exudate is composed mainly of secretory products from two cell types, gland mucous cells and gland thread cells, which produce the mucous and fibrous components of the slime, respectively. Here, we review what is known about the composition of the slime, morphology of the slime gland, and physiology of the cells that produce the slime. We also discuss several of the mechanisms involved in the deployment of both mucous and thread cells during the transition from thick glandular exudate to ultradilute material. We review biomechanical aspects of the slime, along with recent efforts to produce biomimetic slime thread analogs, and end with a discussion of how hagfish slime may have evolved.

Biphasic burrowing in Atlantic hagfish (Myxine limosa).

Myxine limosa is a burrowing species of hagfish that occurs in the western North Atlantic in areas with muddy substrate and at depths generally greater than 100 meters. Burrowing of M. limosa has been observed from submersibles, but little is known about the behavior of these animals within the substrate or the biomechanical mechanisms involved. Here, we investigated burrowing in M. limosa by observing individuals as they burrowed through transparent gelatin. A photoelastic setup using crossed polarizers allowed us to visualize stress development in the gelatin as the hagfish moved through it. We found that M. limosa created U-shaped burrows in gelatin using a stereotyped, two-phase burrowing behavior. In the first ('thrash') phase, hagfish drove their head and their anterior body into the substrate using vigorous sinusoidal swimming movements, with their head moving side-to-side. In the second ('wriggle') phase, swimming movements ceased, with propulsion coming exclusively from the anterior, submerged portion of body. The wriggle phase involved side-to-side head movements and movements of the submerged part of the body that resembled the internal concertina strategy used by caecilians and uropeltid snakes. The entire burrowing process took on average 7.6 min to complete and ended with the hagfish's head protruding from the substrate and the rest of its body generally concealed. Understanding the burrowing activities of hagfishes could lead to improved understanding of sediment turnover in marine benthic habitats, new insights into the reproductive behavior of hagfishes, or even inspiration for the design of burrowing robots.

A New Model of Hagfish Slime Mucous Vesicle Stabilization and Deployment.

Hagfishes thwart predators by releasing large volumes of gill-clogging slime, which consists of mucus and silk-like fibers. The mucous fraction originates within gland mucous cells, which release numerous vesicles that swell and rupture when ejected into seawater. Several studies have examined the function of hagfish slime mucous vesicles in vitro, but a comprehensive model of their biophysics is lacking. Here, we tested the hypothesis that vesicles contain polyanionic glycoproteins stabilized by divalent cations and deploy in seawater via exchange of divalent for monovalent cations. We also tested the hypothesis that vesicle swelling and stabilization are governed by "Hofmeister effects". We found no evidence for either hypothesis. Our results show that hagfish mucous granules are only stabilized by multivalent anions, and pH titration experiments underscore these results. Our results lead us to the conclusion that the hagfish slime mucous gel is in fact polycationic in nature.

The best predictions in experimental biology are critical and persuasive.

A powerful way to evaluate scientific explanations (hypotheses) is to test the predictions that they make. In this way, predictions serve as an important bridge between abstract hypotheses and concrete experiments. Experimental biologists, however, generally receive little guidance on how to generate quality predictions. Here, we identify two important components of good predictions - criticality and persuasiveness - which relate to the ability of a prediction (and the experiment it implies) to disprove a hypothesis or to convince a skeptic that the hypothesis has merit. Using a detailed example, we demonstrate how striving for predictions that are both critical and persuasive can speed scientific progress by leading us to more powerful experiments. Finally, we provide a quality control checklist to assist students and researchers as they navigate the hypothetico-deductive method from puzzling observations to experimental tests.

High concentrations of trimethylamines in slime glands inhibit skein unraveling in Pacific hagfish.

Hagfish defend themselves from fish predators by producing large volumes of gill-clogging slime when they are attacked. The slime consists of seawater and two major components that are ejected from the slime glands: mucus and threads. The threads are produced within specialized cells and packaged into intricately coiled bundles called skeins. Skeins are kept from unraveling via a protein adhesive that dissolves when the skeins are ejected from the slime glands. Previous work revealed that hagfish slime glands have high concentrations of methylamines including trimethylamine N-oxide (TMAO), trimethylglycine (betaine) and dimethylglycine (DMG); however, the function of these compounds in the slime glands is unknown. We hypothesized that methylamines have stabilizing effects on the skeins that prevent premature unraveling in the gland. To test this hypothesis, we quantified the effect of methylamines on skein unraveling in Pacific hagfish and found that TMAO and betaine have inhibitory effects on skein unraveling in vitro Furthermore, we found that TMAO is a more effective inhibitor of unraveling than betaine, but the presence of TMAO synergistically boosts the inhibitory action of betaine. Glycine and DMG were far less effective inhibitors of unraveling at natural concentrations. Our results support the hypothesis that high levels of trimethylamines in the slime glands may act to hold the coiled thread skeins together within gland thread cells, and they may do so by stabilizing adhesive proteins. These results advance our knowledge of skein stabilization and deployment and provide yet another example of trimethylamines functioning to stabilize proteins in a marine organism.

Cellular mechanisms of slime gland refilling in Pacific hagfish (Eptatretus stoutii).

Hagfishes use their defensive slime to ward off gill-breathing predators. Slime gland refilling is a surprisingly slow process, and previous research has shown that the composition of the slime exudate changes significantly during refilling, which likely has consequences for the functionality of the slime. This study set out to expand our understanding of slime gland refilling by examining the cellular processes involved in refilling of the glands, as well as determining where in the gland the main slime cells - the gland thread cells and gland mucous cells - arise. Slime glands were electro-stimulated to exhaust their slime stores, left to refill for set periods of time, and harvested for histological and immunohistochemical examination. Whole slime glands, gland thread cell morphometrics and slime cell proportions were examined over the refilling cycle. Slime glands decreased significantly in size after exhaustion, but steadily increased in size over refilling. Gland thread cells were the limiting factor in slime gland refilling, taking longer to replenish and mature than gland mucous cells. Newly produced gland thread cells underwent most of their growth near the edge of the gland, and larger cells were found farthest from the edge of the gland. Immunohistochemical analysis also revealed proliferating cells only within the epithelial lining of the slime gland, suggesting that new slime cells originate from undifferentiated cells lining the gland. Our results provide an in-depth look at the cellular dynamics of slime gland refilling in Pacific hagfish, and provide a model for how slime glands refill at the cellular level.

Emptying and refilling of slime glands in Atlantic (Myxine glutinosa) and Pacific (Eptatretus stoutii) hagfishes.

Hagfishes are known for their unique defensive slime, which they use to ward off gill-breathing predators. Although much is known about the slime cells (gland thread cells and gland mucous cells), little is known about how long slime gland refilling takes, or how slime composition changes with refilling or repeated stimulation of the same gland. Slime glands can be individually electrostimulated to release slime, and this technique was used to measure slime gland refilling times for Atlantic and Pacific hagfish. The amount of exudate produced, the composition of the exudate and the morphometrics of slime cells were analyzed during refilling, and as a function of stimulation number when full glands were stimulated in rapid succession. Complete refilling of slime glands for both species took 3-4 weeks, with Pacific hagfish achieving faster absolute rates of exudate recovery than Atlantic hagfish. We found significant changes in the composition of the exudate and in the morphometrics of slime cells from Pacific hagfish during refilling. Over successive stimulations of full Pacific hagfish glands, multiple boluses of exudate were released, with exudate composition, but not thread cell morphometrics, changing significantly. Finally, histological examination of slime glands revealed slime cells retained in glands after exhaustion. Discrepancies in the volume of cells released suggest that mechanisms other than contraction of the gland musculature alone may be involved in exudate ejection. Our results provide a first look at the process and timing of slime gland refilling in hagfishes, and raise new questions about how refilling is achieved at the cellular level.

Unraveling inter-species differences in hagfish slime skein deployment.

Hagfishes defend themselves from fish predators by producing defensive slime consisting of mucous and thread components that interact synergistically with seawater to pose a suffocation risk to their attackers. Deployment of the slime occurs in a fraction of a second and involves hydration of mucous vesicles as well as unraveling of the coiled threads to their full length of ∼150 mm. Previous work showed that unraveling of coiled threads (or 'skeins') in Atlantic hagfish requires vigorous mixing with seawater as well as the presence of mucus, whereas skeins from Pacific hagfish tend to unravel spontaneously in seawater. Here, we explored the mechanisms that underlie these different unraveling modes, and focused on the molecules that make up the skein glue, a material that must be disrupted for unraveling to proceed. We found that Atlantic hagfish skeins are also held together with a protein glue, but compared with Pacific hagfish glue, it is less soluble in seawater. Using SDS-PAGE, we identified several soluble proteins and glycoproteins that are liberated from skeins under conditions that drive unraveling in vitro Peptides generated by mass spectrometry of five of these proteins and glycoproteins mapped strongly to 14 sequences assembled from Pacific hagfish slime gland transcriptomes, with all but one of these sequences possessing homologs in the Atlantic hagfish. Two of these sequences encode unusual acidic proteins that we propose are the structural glycoproteins that make up the skein glue. These sequences have no known homologs in other species and are likely to be unique to hagfishes. Although the ecological significance of the two modes of skein unraveling described here are unknown, they may reflect differences in predation pressure, with selection for faster skein unraveling in the Eptatretus lineage leading to the evolution of a glue that is more soluble.

Hagfish Houdinis: biomechanics and behavior of squeezing through small openings.

Hagfishes are able to squeeze through small openings to gain entry to crevices, burrows, hagfish traps and carcasses, but little is known about how they do this, or what the limits of this ability are. The purpose of this study was to describe this ability, and to investigate possible mechanisms by which it is accomplished. We investigated the hypothesis that the passive movement of blood within a hagfish's flaccid subcutaneous sinus allows it to squeeze through narrow apertures that it would not be able to if it were turgid. To test this hypothesis, we analyzed videos of Atlantic hagfish (Myxine glutinosa) and Pacific hagfish (Eptatretus stoutii) moving through narrow apertures in the lab. We measured changes in body width as the animals moved through these openings and documented the behaviors associated with this ability. We found that hagfishes are able to pass through narrow slits that are less than one half the width of their bodies. Our results are consistent with the idea that a flaccid subcutaneous sinus allows hagfish to squeeze through narrow apertures by facilitating a rapid redistribution of venous blood. In addition, we describe nine distinct behaviors associated with this ability, including a form of non-undulatory locomotion also seen in snakes and lampreys. Our results illuminate a behavior that may be a critical component of the hagfish niche, as a result of its likely importance in feeding and avoiding predators.

Identification of Wet-Spinning and Post-Spin Stretching Methods Amenable to Recombinant Spider Aciniform Silk.

Spider silks are outstanding biomaterials with mechanical properties that outperform synthetic materials. Of the six fibrillar spider silks, aciniform (or wrapping) silk is the toughest through a unique combination of strength and extensibility. In this study, a wet-spinning method for recombinant Argiope trifasciata aciniform spidroin (AcSp1) is introduced. Recombinant AcSp1 comprising three 200 amino acid repeat units was solubilized in a 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/water mixture, forming a viscous α-helix-enriched spinning dope, and wet-spun into an ethanol/water coagulation bath allowing continuous fiber production. Post-spin stretching of the resulting wet-spun fibers in water significantly improved fiber strength, enriched ß-sheet conformation without complete α-helix depletion, and enhanced birefringence. These methods allow reproducible aciniform silk fiber formation, albeit with lower extensibility than native silk, requiring conditions and methods distinct from those previously reported for other silk proteins. This provides an essential starting point for tailoring wet-spinning of aciniform silk to achieve desired properties.

The effects of actomyosin disruptors on the mechanical integrity of the avian crystalline lens.

PURPOSE: Actin and myosin within the crystalline lens maintain the structural integrity of lens fiber cells and form a hexagonal lattice cradling the posterior surface of the lens. The actomyosin network was pharmacologically disrupted to examine the effects on lenticular biomechanics and optical quality. METHODS: One lens of 7-day-old White Leghorn chickens was treated with 10 µM of a disruptor and the other with 0.01% dimethyl sulfoxide (vehicle). Actin, myosin, and myosin light chain kinase (MLCK) disruptors were used. The stiffness and the optical quality of the control and treated lenses were measured. Western blotting and confocal imaging were used to confirm that treatment led to a disruption of the actomyosin network. The times for the lenses to recover stiffness to match the control values were also measured. RESULTS: Disruptor-treated lenses were significantly less stiff than their controls (p≤0.0274 for all disruptors). The disruptors led to changes in the relative protein amounts as well as the distributions of proteins within the lattice. However, the disruptors did not affect the clarity of the lenses (p≥0.4696 for all disruptors), nor did they affect spherical aberration (p = 0.02245). The effects of all three disruptors were reversible, with lenses recovering from treatment with actin, myosin, and MLCK disruptors after 4 h, 1 h, and 8 min, respectively. CONCLUSIONS: Cytoskeletal protein disruptors led to a decreased stiffness of the lens, and the effects were reversible. Optical quality was mostly unaffected, but the long-term consequences remain unclear. Our results raise the possibility that the mechanical properties of the avian lens may be actively regulated in vivo via adjustments to the actomyosin lattice.

Eco-mechanics of lamellar autotomy in larval damselflies.

In larval damselflies, the self-amputation (autotomy) of the caudal lamellae permits escape from predatory larval dragonflies. Lamellar joint size declines among populations with increasing risk of dragonfly predation, but the breaking force required for autotomy and the biomechanical factors that influence breaking force are unknown. If autotomy enhances survival in larval damselflies, then predation by larval dragonflies should select for joints that require less force to break. We test this adaptive hypothesis by evaluating whether breaking force is negatively related to local predation risk from larval dragonflies. We also test a cuticle structure hypothesis, which predicts that breaking force is positively related to joint size and to joint cuticle thickness because of a structural support relationship between joint and lamella. The peak force necessary for lamellar autotomy was assessed on individual larval Enallagma damselflies collected from populations that varied in risk of predation. Easier lamellar autotomy occurred in larvae from sites with higher predation risk because damselflies from fishless ponds (where predatory larval dragonflies are likely more abundant) had lower breaking forces than those from ponds with fish (where larval dragonfly predation is likely reduced). Furthermore, breaking force was a positive function of joint size and also of total cuticle cross-sectional area after controlling for joint size. This suggests that autotomy may evolve in larval damselflies under selection from small grasping predators such as larval dragonflies by favouring smaller joint size or reduced cuticle area of lamellar joints.

Spontaneous unraveling of hagfish slime thread skeins is mediated by a seawater-soluble protein adhesive.

Hagfishes are known for their ability to rapidly produce vast quantities of slime when provoked. The slime is formed via the interaction between seawater and two components released by the slime glands: mucin vesicles from gland mucous cells, which swell and rupture in seawater to form a network of mucus strands, and intermediate filament-rich threads, which are produced within gland thread cells as tightly coiled bundles called skeins. A previous study showed that the unraveling of skeins from Atlantic hagfish (Myxine glutinosa) requires both the presence of mucins and hydrodynamic mixing. In contrast, skeins from Pacific hagfish (Eptatretus stoutii) unravel in the absence of both mucins and mixing. We tested the hypothesis that spontaneous unraveling of E. stoutii skeins is triggered by the dissolution of a seawater-soluble protein adhesive and the release of stored strain energy within the coiled thread. Here we show that, as predicted by this hypothesis, unraveling can be initiated by a protease under conditions in which unraveling does not normally occur. We also demonstrate, using high resolution scanning electron microscopy, that the treatment of skeins with solutions that cause unraveling also leads to the disappearance of surface and inter-thread features that remain when skeins are washed with stabilizing solutions. Our study provides a mechanism for the deployment of thread skeins in Pacific hagfish slime, and raises the possibility of producing novel biomimetic protein adhesives that are salt, temperature and kosmotrope sensitive.

Defensive slime formation in Pacific hagfish requires Ca2+- and aquaporin-mediated swelling of released mucin vesicles.

Hagfishes defend themselves from fish predators via the rapid deployment of a fibrous slime that adheres to and clogs gills. The slime transforms from a thick glandular exudate to a fully hydrated product in a fraction of a second through a process that involves the swelling and rupture of numerous mucin vesicles. Here we demonstrate that the vesicle membrane plays an important role in regulating the swelling of mucin granules, and provide evidence that the membrane contains proteins that facilitate the movement of ions and water molecules. By exposing isolated mucin vesicles to varying combinations of inorganic ions, organic compounds and membrane channel inhibitors, we found that the majority of hagfish mucin vesicles require Ca(2+) to rupture. We also show that Ca(2+)-dependent rupture can be pharmacologically inhibited, which suggests a role for Ca(2+)-activated membrane transporters. We demonstrate that the aquaporin inhibitor mercuric chloride reduces the rate of vesicle swelling by an order of magnitude, which suggests that aquaporins facilitate the influx of water during vesicle deployment. Molecular evidence of two aquaporin homologues expressed in the slime glands further supports this idea. We propose a model of hagfish slime mucin vesicle rupture that involves Ca(2+)-activated transporters and aquaporins, and suggest that the presence of these proteins is an adaptation for increasing the speed of vesicle rupture and, consequently, the speed of the sliming response of hagfishes.

Self-assembly enhances the strength of fibers made from vimentin intermediate filament proteins.

Hagfish slime threads were recently established as a promising biomimetic model for efforts to produce ecofriendly alternatives to petroleum polymers. Initial attempts to make fibers from solubilized slime thread proteins fell short of achieving the outstanding mechanics of native slime threads. Here we tested the hypothesis that the high strength and toughness of slime threads arise from the ability of constituent intermediate filaments to undergo a stress-induced α-to-ß transition. To do this, we made fibers from human vimentin proteins that were first allowed to self-assemble into 10 nm intermediate filaments. Fibers made from assembled vimentin hydrogels underwent an α-to-ß transition when strained and exhibited improved mechanical performance. Our data demonstrate that it is possible to make materials from intermediate filament hydrogels and that mimicking the secondary structure of native hagfish slime threads using intermediate filament self-assembly is a promising strategy for improving the mechanical performance of biomimetic protein materials.

Regulation of hard α-keratin mechanics via control of intermediate filament hydration: matrix squeeze revisited.

Mammalian hard α-keratins are fibre-reinforced biomaterials that consist of 10 nm intermediate filaments (IFs) embedded in an elastomeric protein matrix. Recent work suggests that the mechanical properties of IFs are highly sensitive to hydration, whereas hard α-keratins such as wool, hair and nail are relatively hydration insensitive. This raises the question of how mammalian keratins remain stiff in water. The matrix squeeze hypothesis states that the IFs in hard α-keratins are stiffened during an air-drying step during keratinization, and subsequently locked into a dehydrated state via the oxidation and cross-linking of the keratin matrix around them. The result is that even when hard α-keratins are immersed in water, their constituent IFs remain essentially 'dry' and therefore stiff. This hypothesis makes several predictions about the effects of matrix abundance and function on hard α-keratin mechanics and swelling behaviour. Specifically, it predicts that high matrix keratins in water will swell less, and have a higher tensile modulus, a higher yield stress and a lower dry-to-wet modulus ratio. It also predicts that disruption of the keratin matrix in water should lead to additional swelling, and a drop in modulus and yield stress. Our results are consistent with these predictions and suggest that the keratin matrix plays a critical role in governing the mechanical properties of mammalian keratins via control of IF hydration.

Mechanisms of gill-clogging by hagfish slime.

Hagfishes defend themselves from gill-breathing predators by producing large volumes of fibrous slime when attacked. The slime's effectiveness comes from its ability to clog predators' gills, but the mechanisms by which hagfish slime clogs are uncertain, especially given its remarkably dilute concentration of solids. We quantified the clogging performance of hagfish slime over a range of concentrations, measured the contributions of its mucous and thread components, and measured the effect of turbulent mixing on clogging. To assess the porous structure of hagfish slime, we used a custom device to measure its Darcy permeability. We show that hagfish slime clogs at extremely dilute concentrations like those found in native hagfish slime and displays clogging performance that is superior to three thickening agents. We report an extremely low Darcy permeability for hagfish slime, and an effective pore size of 10-300 nm. We also show that the mucous and thread components play distinct yet crucial roles, with mucus being responsible for effective clogging and low permeability and the threads imparting mechanical strength and retaining clogging function over time. Our results provide new insights into the mechanisms by which hagfish slime clogs gills and may inspire the development of ultra-soft materials with novel properties.

The production of fibers and films from solubilized hagfish slime thread proteins.

Hagfish slime threads, which make up the fibrous component of the defensive slime of hagfishes, consist primarily of proteins from the intermediate filament family of proteins and possess impressive mechanical properties that make them attractive biomimetic models. To investigate whether solubilized intermediate filament proteins can be used to make high-performance, environmentally sustainable materials, we cast thin films on the surface of electrolyte buffers using solubilized hagfish slime thread proteins. The films were drawn into fibers, and the tensile properties were measured. Fiber mechanics depended on casting conditions and postspinning processing. Postsecondary drawing resulted in fibers with improved material properties similar to those of regenerated silk fibers. Structural analyses of the fibers revealed increased molecular alignment resulting from the second draw, but no increase in crystallinity. Our findings show promise for intermediate filament proteins as an alternative source for the design and production of high performance protein-based fibers.

Comparative Animal Mucomics: Inspiration for Functional Materials from Ubiquitous and Understudied Biopolymers.

The functions of secreted animal mucuses are remarkably diverse and include lubricants, wet adhesives, protective barriers, and mineralizing agents. Although present in all animals, many open questions related to the hierarchical architectures, material properties, and genetics of mucus remain. Here, we summarize what is known about secreted mucus structure, describe the work of research groups throughout the world who are investigating various animal mucuses, and relate how these studies are revealing new mucus properties and the relationships between mucus hierarchical structure and hydrogel function. Finally, we call for a more systematic approach to studying animal mucuses so that data sets can be compared, omics-style, to address unanswered questions in the emerging field of mucomics. One major result that we anticipate from these efforts is design rules for creating new materials that are inspired by the structures and functions of animal mucuses.