Epidermal threads reveal the origin of hagfish slime.

When attacked, hagfishes produce a soft, fibrous defensive slime within a fraction of a second by ejecting mucus and threads into seawater. The rapid setup and remarkable expansion of the slime make it a highly effective and unique form of defense. How this biomaterial evolved is unknown, although circumstantial evidence points to the epidermis as the origin of the thread- and mucus-producing cells in the slime glands. Here, we describe large intracellular threads within a putatively homologous cell type from hagfish epidermis. These epidermal threads averaged ~2 mm in length and ~0.5 µm in diameter. The entire hagfish body is covered by a dense layer of epidermal thread cells, with each square millimeter of skin storing a total of ~96 cm threads. Experimentally induced damage to a hagfish's skin caused the release of threads, which together with mucus, formed an adhesive epidermal slime that is more fibrous and less dilute than the defensive slime. Transcriptome analysis further suggests that epidermal threads are ancestral to the slime threads, with duplication and diversification of thread genes occurring in parallel with the evolution of slime glands. Our results support an epidermal origin of hagfish slime, which may have been driven by selection for stronger and more voluminous slime.

Hagfishes are deep-sea animals, and they represent one of the oldest living relatives of animals with backbones. To defend themselves against predators, they produce a remarkable slime that is reinforced with fibers and can clog a predator's gills, thwarting the attack. The slime deploys in less than half a second, exuding from specialized glands on the hagfish's body and expanding up to 10,000 times its ejected volume. The defensive slime is highly dilute, consisting mostly of sea water, with low concentrations of mucus and strong, silk-like threads that are approximately 20 centimeters long. Where and how hagfish slime evolved remains a mystery. Zeng et al. set out to answer where on the hagfish's body the slime glands originated, and how they may have evolved. First, Zeng et al. examined hagfishes and found that cells in the surface layer of their skin (the epidermis) produce threads roughly two millimeters in length that are released when the hagfish's skin is damaged. These threads mix with the mucus that is produced by ruptured skin cells to form a slime that likely adheres to predators' mouths. This slime could be a precursor of the slime produced by the specialized glands. To test this hypothesis, Zeng et al. analyzed which genes are turned on and off both in the hagfishes' skin and in their slime glands. The patterns they found are consistent with the slime glands originating from the epidermis. Based on these results, Zeng et al. propose that ancient hagfishes first evolved the ability to produce slime with anti-predator effects when their skin was damaged in attacks. Over time, hagfishes that could produce and store more slime and eject it actively into a predator's mouth likely had a better chance of surviving. This advantage may have led to the appearance of increasingly specialized glands that could carry out these functions. The findings of Zeng et al. will be of interest to evolutionary biologists, marine biologists, and those studying the ecology of predator-prey interactions. Because of its unique material properties, hagfish slime is also of interest to biophysicists, bioengineers and those engaged in biomimetic research. The origin of hagfish slime glands is an interesting example of how a new trait evolved, and may provide insight into the evolution of other adaptive traits.

Scalable purification of recombinant structural proteins, hagfish intermediate filament α and É£, from inclusion bodies for fiber formation.

The purpose of this study was to determine a method to purify recombinant hagfish intermediate filament proteins, alpha and gamma, in a scalable manner. The study succeeded by having an increase in protein recovery of up to 35% when comparing centrifuge purification and the developed tangential flow purification. The proteins were approximately the same purity of 70% pure but further purification increased the purity of the proteins by 16%, based on ImageJ analysis. The developed tangential flow filtration purification and final purification methods could be easily scaled up to meet industry scale purification needs. The scaled-up processes described in this study did not interfere with fiber production or formation, indicating the methods can produce usable proteins for material development.

A glimpse into the past: phylogenesis and protein domain analysis of the group XIV of C-type lectins in vertebrates.

BACKGROUND: The group XIV of C-type lectin domain-containing proteins (CTLDcps) is one of the seventeen groups of CTLDcps discovered in mammals and composed by four members: CD93, Clec14A, CD248 and Thrombomodulin, which have shown to be important players in cancer and vascular biology. Although these proteins belong to the same family, their phylogenetic relationship has never been dissected. To resolve their evolution and characterize their protein domain composition we investigated CTLDcp genes in gnathostomes and cyclostomes and, by means of phylogenetic approaches as well as synteny analyses, we inferred an evolutionary scheme that attempts to unravel their evolution in modern vertebrates. RESULTS: Here, we evidenced the paralogy of the group XIV of CTLDcps in gnathostomes and discovered that a gene loss of CD248 and Clec14A occurred in different vertebrate groups, with CD248 being lost due to chromosome disruption in birds, while Clec14A loss in monotremes and marsupials did not involve chromosome rearrangements. Moreover, employing genome annotations of different lampreys as well as one hagfish species, we investigated the origin and evolution of modern group XIV of CTLDcps. Furthermore, we carefully retrieved and annotated gnathostome CTLDcp domains, pointed out important differences in domain composition between gnathostome classes, and assessed codon substitution rate of each domain by analyzing nonsynonymous (Ka) over synonymous (Ks) substitutions using one representative species per gnathostome order. CONCLUSIONS: CTLDcps appeared with the advent of early vertebrates after a whole genome duplication followed by a sporadic tandem duplication. These duplication events gave rise to three CTLDcps in the ancestral vertebrate that underwent further duplications caused by the independent polyploidizations that characterized the evolution of cyclostomes and gnathostomes. Importantly, our analyses of CTLDcps in gnathostomes revealed critical inter-class differences in both extracellular and intracellular domains, which might help the interpretation of experimental results and the understanding of differences between animal models.

In vitro characterisation of calcium influx across skin and gut epithelia of the Pacific hagfish, Eptatretus stoutii.

Although hagfishes osmoconform, concentrations of calcium in their extracellular fluids are maintained at levels lower than those of seawater. Ultimately, calcium homeostasis is a product of relative rates of influx and efflux into the animal, but little is known regarding these processes in hagfish. Using in vitro approaches, calcium influx across gut and skin epithelia of the Pacific hagfish, Eptatretus stoutii, was characterised. Analysis of concentration-dependent kinetics showed that accumulation into skin tissue was linear at mucosal calcium concentrations up to 10 mM. However, movement into the serosal compartment was saturable, with a maximal transport rate of 59 nmol cm-2 h-1 and an affinity (substrate concentration to give half maximal transport rate) of 1139 µM. Calcium accumulation into gut tissue and uptake into the gut serosal compartment was also saturable, with maximal transport rates of 5.1 and 2.8 nmol cm-2 h-1, and affinities of 6417 and 3327 µM, respectively. A strong correlation between epidermal and intestinal calcium tissue uptake was noted in assays where tissues were taken from the same individual. Intestinal calcium influx was inhibited by nickel, but not significantly affected by other putative inorganic (lanthanum, zinc) or organic (diltiazem, verapamil) transport blockers. Inhibitors had no effect on skin calcium handling. These data indicate that both gut and skin epithelia mediate calcium influx in hagfishes, albeit via distinct mechanisms and with differences in responsiveness to potential modifying factors.

Acquisition of alanyl-alanine in an Agnathan: Characteristics of dipeptide transport across the hindgut of the Pacific hagfish Eptatretus stoutii.

This study used 3 H-L -alanyl-L -alanine to demonstrate dipeptide uptake using in vitro gut sacs prepared from the hindgut of the Pacific hagfish Eptatretus stoutii. Concentration-dependent kinetic analysis resulted in a sigmoidal distribution with a maximal (± SE) uptake rate (Jmax -like) of 70 ± 3 nmol cm-2 h-1 and an affinity constant (Km -like) of 1072 ± 81 µM. Addition of high alanine concentrations to transport assays did not change dipeptide transport rates, indicating that hydrolysis of the dipeptide in mucosal solutions and subsequent uptake via apical amino acid transporters was not occurring, which was further supported by a Km distinct from that of amino acid transport. Transport occurred independent of mucosal pH, but uptake was reduced by 42% in low mucosal sodium. This may implicate cooperation between peptide transporters and sodium-proton exchangers, previously demonstrated in several mammalian and teleost species. Finally, apical L -alanyl-L -alanine uptake rates (i.e., mucosal disappearance) were significantly increased following a meal, demonstrating regulation of uptake. Overall, this examination of dipeptide acquisition in the earliest extant Agnathan suggests evolutionarily conserved mechanisms of transport between hagfish and later-diverging vertebrates such as teleosts and mammals.

Behavioural responses of the hagfish Eptatretus stoutii to nutrient and noxious stimuli.

The suitability of a traditional testing paradigm (e.g. choice chamber) for assessing chemosensory behaviour in the Pacific hagfish, Eptatretus stoutii, was examined. Actively-swimming hagfish, tested at night, showed no preference for any region of a T-maze in the absence of a stimulus, but in the presence of an olfactory food cue, spent significantly more time in the zone where the cue was placed. Conversely, hagfish avoided spending time in the zone the fish anaesthetic 3-amino benzoic acid ethylester (MS-222) was placed, and demonstrated significantly more reversal responses in which the fish moved its body backwards. These data suggest that hagfish are an amenable model species for laboratory testing of behaviour.

Contractile function of the excised hagfish heart during anoxia exposure.

Pacific hagfish, Eptatretus stoutii, can recover from 36 h of anoxia and their systemic hearts continue to work throughout the exposure. Recent work demonstrates that glycogen stores are utilized in the E. stoutii heart during anoxia but that these are not sufficient to support the measured rate of ATP production. One metabolic fuel that could supplement glycogen during anoxia is glycerol. This substrate can be derived from lipid stores, stored in the heart, or delivered via the blood. The purpose of this study was to determine the effect of glycerol on the contractile function of the excised E. stoutii heart during anoxia exposure. When excised hearts, perfused with metabolite free saline (mf-saline), were exposed to anoxia for 12 h, there was no difference in heart rate, pressure generation (max-dP), rate of contraction (max-dP/dtsys), or rate of relaxation (max-dP/dtdia) compared to hearts perfused with mf-saline in normoxia. However, hearts perfused with saline containing glycerol (gly-saline) in anoxia had higher max-dP, max-dP/dtsys, and max-dP/dtdia than hearts perfused with mf-saline in anoxia. Tissue levels of glycerol increased when hearts were perfused with gly-saline in normoxia, but not when perfused with gly-saline in anoxia. Anoxia exposure did not affect the activities of triglyceride lipase, glycerol kinase, or glycerol-3-phosphate dehydrogenase. This study suggests that glycerol stimulates cardiac function in the hagfish but that it is not derived from stored lipids. How glycerol may stimulate contraction is not known. This could be as an energy substrate, as an allosteric factor, or a combination of the two.

Immunohistochemical detection of prolactin-releasing peptide2 in the brain of the inshore hagfish Eptatretus burgeri.

Prolactin-releasing peptide2 (PrRP2) belongs to the RFamide peptide group and is a paralog of prolactin-releasing peptide (PrRP). Recent studies demonstrated that PrRP2, but not PrRP, regulates prolactin release in teleosts. The evolutionary origin of PrRP and PrRP2 dates back to at least early vertebrates because homologs of PrRP/PrRP2 were identified in lampreys, one of the earliest branch of vertebrates class Agnatha. However, PrRP/PrRP2 remains to be identified in hagfish, another representative species of class Agnatha. Here, we examined the distribution of PrRP2 in the brain and pituitary of the inshore hagfish Eptatretus burgeri to obtain further understanding of the neuroendocrine system of PrRP2. PrRP2-immunoreactive (ir) cell bodies were detected in the infundibular nucleus of hypothalamus (HYinf). PrRP2-ir fibers were restricted around PrRP2-ir cell bodies and were not detected in the dorsal wall of the neurohypophysis compared to the abundant PrRP2-ir fiber distribution in the brain and innervation to the pituitary in other vertebrates. To examine possible reciprocal connections of PrRP2 and other neuropeptides, we further conducted dual-label immunohistochemistry of PrRP2 and the PQRFamide (PQRFa) peptide or corticotropin-releasing hormone (CRH). Reciprocal connections are suggested between PrRP2 and PQRFa neurons as well as between PrRP2 and CRH neurons. The present study demonstrates, for the first time, that PrRP2 is expressed in the brain of inshore hagfish. The restricted distribution of PrRP2-ir fibers in the HYinf suggests that PrRP2 does not directly regulate the pituitary gland, but regulates the function of the HYinf where PQRFa and CRH are expressed.

Lipid acquisition and tissue storage in hagfish: new insights from an ancient vertebrate.

Hagfishes are ancient vertebrates, which have the ability to tolerate nearly a year of food deprivation with energy during fasting maintained using lipid stores. While lipid transporters are evolutionarily conserved, there are relatively few studies examining mechanisms of lipid acquisition in teleosts and no reports in the primitive and evolutionarily important agnathans. We examined tissue lipid droplet distribution and used gut sac preparations to characterize uptake mechanisms of the monounsaturated fatty acid, oleic acid (OA; 18:1 cis-9), in the Pacific hagfish. OA absorption was carrier-mediated and demonstrated saturable concentration-dependent uptake with an affinity constant of 55 ± 7 µM, and a maximal rate of uptake of 1311 ± 97 pmol cm- 2 h- 1. Additionally, regulation of intestinal transport was demonstrated as feeding significantly increased uptake. To further examine post-prandial effects on fatty acid transport, hagfish were dosed with bovine insulin, which had no effect on OA intestinal acquisition, but did lower plasma glucose. Overall, this is the first evidence for intestinal fatty acid acquisition in an agnathan, which are ideal models for understanding the evolution of nutrient transport processes in vertebrates, and are particularly suited to lipid research owing to their dependence upon this energy source during prolonged periods of fasting.

Feeding in Eptatretus cirrhatus: effects on metabolism, gut structure and digestive processes, and the influence of post-prandial dissolved oxygen availability.

Hagfishes are characterised by feeding behaviours that may include long intervals between meals, and a hypoxic feeding environment inside decaying carrion. The effects of feeding on metabolism (oxygen consumption rate), gut mass and morphology (gut somatic index, gut epithelium mucosal thickness), and digestive function (maltase and peptidase activity) were examined in the New Zealand hagfish, Eptatretus cirrhatus. The influence of post-prandial hypoxia on oxygen consumption rate was also investigated to replicate the immersive feeding environment. Fed hagfish displayed a 1.9-fold increase in peak oxygen consumption relative to sham controls. This elevation in post-prandial oxygen consumption continued for 72 h, during which the energy cost of digesting the meal (specific dynamic action; SDA) was 2.1 kJ. Oxygen consumption rate increased when the post-prandial environment was hypoxic, a response suggesting a lack of hypoxia tolerance in this species. Feeding did not alter gut somatic index (percentage of digesta-free gut mass to whole body mass), but there was an increase in the mucosal thickness of the gut epithelium. Maltase activity in the gut was unchanged by feeding, but the activity of gut peptidases was increased significantly, consistent with a protein-based diet. These data indicate that some postprandial responses of New Zealand hagfish are similar in nature to those seen in other animals, but this species does not exhibit the extreme post-prandial physiological and biochemical changes that are observed in other intermittently-feeding vertebrates.

Globular-shaped variable lymphocyte receptors B antibody multimerized by a hydrophobic clustering in hagfish.

In hagfish and lampreys, two representative jawless vertebrates, the humoral immunity is directly mediated by variable lymphocyte receptors B (VLRBs). Both monomeric VLRBs are structurally and functionally similar, but their C-terminal tails differ: lamprey VLRB has a Cys-rich tail that forms disulfide-linked pentamers of dimers, contributing to its multivalency, whereas hagfish VLRB has a superhydrophobic tail of unknown structure. Here, we reveal that VLRBs obtained from hagfish plasma have a globular-shaped multimerized form (approximately 0.6 to 1.7 MDa) that is generated by hydrophobic clustering instead of covalent linkage. Electron microscopy (EM) and single-particle analysis showed that the multimerized VLRBs form globular-shaped clusters with an average diameter of 28.7 ± 2.2 nm. The presence of VLRBs in the complex was confirmed by immune-EM analysis using an anti-VLRB antibody. Furthermore, the hydrophobic hagfish C-terminus (HC) was capable of triggering multimerization and directing the cellular surface localization via a glycophosphatidylinositol linkage. Our results strongly suggest that the hagfish VLRB forms a previously unknown globular-shaped antibody. This novel identification of a structurally unusual VLRB complex may suggest that the adaptive immune system of hagfish differs from that of lamprey.

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.

Functional redundancy of glucose acquisition mechanisms in the hindgut of Pacific hagfish (Eptatretus stoutii).

This study examined the mechanisms of glucose acquisition in the hindgut of Pacific hagfish (Eptatretus stoutii) using in vitro gut sac techniques. The intestine was determined to have the capacity to digest maltose into glucose along the entirety of the tract, including the foregut. Glucose uptake was biphasic and consisted of a high-affinity, low-capacity concentration-dependent component conforming to Michaelis-Menten kinetics (Km 0.37mM, Jmax 8.48nmol/cm2/h) as well as a diffusive component. There was no observed difference in glucose flux rate along the length of the intestine, similar to other nutrients investigated in the hagfish intestine. A reduced sodium (<1mM) environment did not result in a change in glucose uptake rates, likely due to a functional redundancy of glucose transporters. There was no observed effect of phloretin, yet the sodium glucose-linked transporter (SGLT)-specific inhibitor phlorizin significantly reduced glucose uptake at all concentrations tested (0.0001-1mM). Additionally, the glucose transporter (GLUT) inhibitor cytochalasin b significantly reduced glucose transport rates. The effects of these pharmacological inhibition experiments suggest the presence of multiple types of glucose transport proteins. This study clarifies the uptake strategies used by hagfish to acquire glucose at the intestine and provides insight into the evolution of such transport systems in early-diverging vertebrates.

Drinking and water permeability in the Pacific hagfish, Eptatretus stoutii.

Hagfish are osmoconformers, maintaining an internal osmolality that matches their seawater habitats. Hagfish would, therefore, appear to have no physiological need to drink, but previous studies are equivocal regarding whether drinking in hagfish occurs. The current study addressed this knowledge gap, by examining drinking and water permeability in the Pacific hagfish, Eptatretus stoutii. One-third of analysed hagfish were shown to accumulate radiolabelled drinking rate markers (tritiated inulin and polyethylene glycol-4000) in their gut tissues; however, this was attributed to the presence of markers in the blood perfusing the digestive tract, following absorption through paracellular pathways at the gill. No accumulation of marker was observed in hagfish subjected to more dilute (75% seawater) or more concentrated (125% seawater) media. Diffusive water efflux, measured by tritiated water washout, was shown to be very high, with 50% of body water exchanged within 14 to 16 min, depending on exposure salinity. In full-strength seawater, the total exchangeable pool of water was 78% of hagfish mass. We conclude that hagfish do not drink, and their high water permeability is likely to result in rapid osmotic equilibration under circumstances where perturbations may occur.

Characteristics of the brown hagfish Paramyxine atami transthyretin: Metal ion-dependent thyroid hormone binding.

Transthyretin (TTR) is a vertebrate-specific protein involved in thyroid hormone distribution in plasma, and its gene is thought to have emerged by gene duplication from the gene for the ancient TTR-related protein, 5-hydroxyisourate hydrolase, at some early stage of chordate evolution. We investigated the molecular and hormone-binding properties of the brown hagfish Paramyxine atami TTR. The amino acid sequence deduced from the cloned hagfish TTR cDNA shared 33-50% identities with those of other vertebrate TTRs but less than 24% identities with those of vertebrate and deuterostome invertebrate 5-hydroxyisourate hydrolases. Hagfish TTR, as well as lamprey and little skate TTRs, had an N-terminal histidine-rich segment, allowing purification by metal-affinity chromatography. The affinity of hagfish TTR for 3,3',5-triiodo-L-thyronine (T3) was 190 times higher than that for L-thyroxine, with a dissociation constant of 1.5-3.9nM at 4°C. The high-affinity binding sites were strongly sensitive to metal ions. Zn2+ and Cu2+ decreased the dissociation constant to one-order of magnitude, whereas a chelator, o-phenanthroline, increased it four times. The number of metal ions (mainly Zn2+ and Cu2+) was approximately 12/TTR (mol/mol). TTR was also a major T3-binding protein in adult hagfish sera and its serum concentration was approximately 8µM. These results suggest that metal ions and the acquisition of N-terminal histidine-rich segment may cooperatively contribute to the evolution toward an ancient TTR with high T3 binding activity from either 5-hydroxyisourate hydrolase after gene duplication.

Molecular cloning and characterization of hagfish estrogen receptors.

One or more distinct forms of the nuclear estrogen receptor (ER) have been isolated from many vertebrates to date. To better understand the molecular evolution of ERs, we cloned and characterized er cDNAs from the inshore hagfish, Eptatretus burgeri, a modern representative of the most primitive vertebrates, the agnathans. Two er cDNAs, er1 and er2, were isolated from the liver of a reproductive female hagfish. A phylogenetic analysis placed hagfish ER1 into a position prior to the divergence of vertebrate ERs. Conversely, hagfish ER2 was placed at the base of the vertebrate ERß clade. The tissue distribution patterns of both ER subtype mRNAs appeared to be different, suggesting that each subtype has different physiological roles associated with estrogen actions. An estrogen responsive-luciferase reporter assay using mammalian HEK293 cells was used to functionally characterize these hagfish ERs. Both ER proteins displayed estrogen-dependent activation of transcription. These results clearly demonstrate that the hagfish has two functional ER subtypes.

Effect of environmental salinity manipulation on uptake rates and distribution patterns of waterborne amino acids in the Pacific hagfish.

Among vertebrates, hagfish are the only known iono- and osmoconformers, and the only species thus far documented to absorb amino acids directly across the skin. In the current study, short-term (6h) manipulations of exposure salinities (75-125% seawater) were conducted to determine whether changes in osmotic demands influenced the uptake and tissue distribution of waterborne amino acids (alanine, glycine and phenylalanine), in the Pacific hagfish, Eptatretus stoutii. No changes in erythrocyte or muscle amino acid accumulation rates were noted, but the patterns of plasma amino acid accumulation were suggestive of regulation. Contrary to expectations, glycine transport across the skin in vitro was enhanced in the lowest exposure salinity, but no other salinity-dependent changes were demonstrated. Overall, this study indicates that uptake and distribution of amino acids varies with salinity, but not in a manner that is consistent with a role for the studied amino acids in maintaining osmotic balance in hagfish.

Immunohistochemical detection of corticotropin-releasing hormone (CRH) in the brain and pituitary of the hagfish, Eptatretus burgeri.

The distribution of corticotropin-releasing hormone (CRH) in the brain and pituitary of the hagfish Eptatretus burgeri, representing the earliest branch of vertebrates, was examined by immunohistochemistry to better understand the neuroendocrine system of hagfish. CRH-immunoreactive (ir) cell bodies were detected in the preoptic nucleus, periventricular preoptic nucleus, infundibular nucleus of the hypothalamus, and in the nucleus "A" of Kusunoki et al. (1982) in the medulla oblongata. In the brain, CRH-ir fibers were detected in almost all areas except for the olfactory bulb and telencephalon. Bundles of CRH-ir fibers were detected in the dorsal wall of the neurohypophysis. However, CRH-ir fibers were distant from adrenocorticotropic hormone (ACTH) cells in the adenohypophysis, as studied by dual-label immunohistochemistry. Cortisol and corticosterone were detected in the plasma by a combination of reverse-phase high performance liquid chromatography and a time-resolved fluoroimmunoassay. These results suggest that in the hagfish, CRH, ACTH, and corticosteroids exist and that CRH released in the neurohypophysis likely reaches the adenohypophysis via diffusion.

Hagfish slime and mucin flow properties and their implications for defense.

When hagfish (Myxinidae) are attacked by predators, they form a dilute, elastic, and cohesive defensive slime made of mucins and protein threads. In this study we propose a link between flow behavior and defense mechanism of hagfish slime. Oscillatory rheological measurements reveal that hagfish slime forms viscoelastic networks at low concentrations. Mucins alone did not contribute viscoelasticity, however in shear flow, viscosity was observed. The unidirectional flow, experienced by hagfish slime during suction feeding by predators, was mimicked with extensional rheology. Elongational stresses were found to increase mucin viscosity. The resulting higher resistance to flow could support clogging of the attacker's gills. Shear flow in contrast decreases the slime viscosity by mucin aggregation and leads to a collapse of the slime network. Hagfish may benefit from this collapse when trapped in their own slime and facing suffocation by tying a sliding knot with their body to shear off the slime. This removal could be facilitated by the apparent shear thinning behavior of the slime. Therefore hagfish slime, thickening in elongation and thinning in shear, presents a sophisticated natural high water content gel with flow properties that may be beneficial for both, defense and escape.

Iron transport across the skin and gut epithelia of Pacific hagfish: Kinetic characterisation and effect of hypoxia.

In most animals, the acquisition of the essential trace metal iron (Fe) is achieved by the gut, but in hagfishes, the skin is a nutrient absorbing epithelium, and thus may also play a role in Fe uptake. In the current study, the absorption of Fe, as Fe(II), across the intestinal and cutaneous epithelia of Pacific hagfish (Eptatretus cirrhatus) was investigated. Both epithelia absorbed Fe, with saturation at lower tested concentrations, superseded by a diffusive component at higher Fe exposure concentrations. Affinity constants (Km) of 9.4 and 137µM, and maximal Fe transport rates (Jmax) of 0.81 and 0.57nmolcm(-2)h(-1) were determined for the skin and the gut, respectively. This characterises the skin as a relatively high-affinity Fe transport epithelium. The majority of the absorbed Fe in the skin remained in the tissue, whereas in the gut, most absorbed Fe was found in the serosal fluid, suggesting distinct mechanisms of Fe handling between the two epithelia. To determine if reduced dissolved oxygen altered Fe transport, hagfish were subjected to hypoxia for 24h, before Fe transport was again assessed. Hypoxia had no effect on Fe transport across gut or skin, likely owing to the relative lack of change in haematological variables, and thus an unaltered Fe demand under such conditions. These data are the first to kinetically characterise the absorption of a nutritive trace metal across the epithelia of hagfish and add to the growing understanding of the role of the skin in nutritive transport in this group.