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.

The roles of plasma accessible and cytosolic carbonic anhydrases in bicarbonate (HCO3-) excretion in Pacific hagfish (Eptatretus stoutii).

Pacific hagfish (Eptatretus stoutii) are marine scavengers and feed on decaying animal carrion by burrowing their bodies inside rotten carcasses where they are exposed to several threatening environmental stressors, including hypercapnia (high partial pressures of CO2). Hagfish possess a remarkable capacity to tolerate hypercapnia, and their ability to recover from acid-base disturbances is well known. To deal with the metabolic acidosis resulting from exposure to high CO2, hagfish can mount a rapid elevation of plasma HCO3- concentration (hypercarbia). Once PCO2 is restored, hagfish quickly excrete their HCO3- load, a process that likely involves the enzyme carbonic anhydrase (CA), which catalyzes HCO3- dehydration into CO2 at the hagfish gills. We aimed to characterize the role of branchial CA in CO2/HCO3- clearance from the plasma at the gills of E. stoutii, under control and high PCO2 (hypercapnic) exposure conditions. We assessed the relative contributions of plasma accessible versus intracellular (cytosolic) CA to gill HCO3- excretion by measuring in situ [14C]-HCO3- fluxes. To accomplish this, we employed a novel surgical technique of individual gill pouch arterial perfusion combined with perifusion of the gill afferent to efferent water ducts. [14C]-HCO3- efflux was measured at the gills of fish exposed to control, hypercapnic (48 h) and recovery from hypercapnia conditions (6 h), in the presence of two well-known pharmacological inhibitors of CA, the membrane impermeant C18 (targets membrane bound, plasma accessible CA) and membrane-permeant acetazolamide, which targets all forms of CA, including extracellular and intracellular cytosolic CAs. C18 did not affect HCO3- flux in control fish, whereas acetazolamide resulted in a significant reduction of 72%. In hypercapnic fish, HCO3- fluxes were much higher and perfusion with acetazolamide caused a reduction of HCO3- flux by 38%. The same pattern was observed for fish in recovery, where in all three experimental conditions, there was no significant inhibition of plasma-accessible CA. We also observed no change in CA enzyme activity (measured in vitro) in any of the experimental PCO2 conditions. In summary, our data suggests that there are additional pathways for HCO3- excretion at the gills of hagfish that are independent of plasma-accessible CA.

Dining on the dead in the deep: Active NH4 + excretion via Na+ /H+ (NH4 + ) exchange in the highly ammonia tolerant Pacific hagfish, Eptatretus stoutii.

AIM: Pacific hagfish are exceptionally tolerant to high environmental ammonia (HEA). Here, we elucidated a cellular mechanism that enables hagfish to actively excrete ammonia against steep ammonia gradients expected to be found inside a decomposing whale carcass. METHODS: Hagfish were exposed to varying concentrations of HEA in the presence or absence of environmental Na+ , while plasma ammonia levels were tracked. 14 C-methylammonium was used as a proxy for NH4 + to measure efflux in whole animals and in isolated gill pouches; the latter allowed us to assess the effects of amiloride specifically on Na+ /H+ exchangers (NHEs) in gill cells. Western blotting and immunohistochemistry were utilized to evaluate the abundance and sub-cellular localization of Rhesus glycoprotein (Rh) channels in the response to HEA. RESULTS: Hagfish actively excreted NH4 + against steep inwardly directed ENH4 + (ΔENH4 + ~ 35 mV) and pNH3 (ΔpNH3 ~ 2000 µtorr) gradients. Active NH4 + excretion and plasma ammonia hypo-regulation were contingent on the presence of environmental Na+ , indicating a Na+ /NH4 + exchange mechanism. Active NH4 + excretion across isolated gill pouches was amiloride-sensitive. Exposure to HEA resulted in decreased abundance of Rh channels in the apical membrane of gill ionocytes. CONCLUSIONS: During HEA exposure, hagfish can actively excrete ammonia against a steep concentration gradient using apical NHEs energized by Na+ -K+ -ATPase in gill ionocytes. Additionally, apical Rh channels are removed from the apical membrane, presumably to reduce ammonia loading from the environment. We suggest that this mechanism allows hagfish to maintain tolerable ammonia levels while feeding inside decomposing carrion, allowing them to exploit nutrient-rich food-falls.

Breathing versus feeding in the Pacific hagfish.

Hagfish represent the oldest extant connection to the ancestral vertebrates, but their physiology is not well understood. Using behavioural (video), physiological (respirometry, flow measurements), classical morphological (dissection, silicone injection) and modern imaging approaches (micro-MRI, DICE micro-CT), we examined the interface between feeding and the unique breathing mechanism (nostril opening, high-frequency velum contraction, low-frequency gill pouch contraction and pharyngo-cutaneous duct contraction) in the Pacific hagfish, Eptatretus stoutii. A video tour via micro-MRI is presented through the breathing and feeding passages. We have reconciled an earlier disagreement as to the position of the velum chamber, which powers inhalation through the nostril, placing it downstream of the merging point of the food and water passage, such that the oronasal septum terminates at the anterior end of the velum chamber. When feeding occurs by engulfment of large chunks by the dental plates, food movement through the chamber may transiently interfere with breathing. Swallowing is accelerated by peristaltic body undulation involving the ventral musculature, and is complete within 5 s. After a large meal (anchovy, 20% body mass), hagfish remain motionless, defaecating bones and scales at 1.7 days and an intestinal peritrophic membrane at 5 days. O2 consumption rate approximately doubles within 1 h of feeding, remaining elevated for 12-24 h. This is achieved by combinations of elevated O2 utilization and ventilatory flow, the latter caused by varying increases in velar contraction frequency and stroke volume. Additional imaging casts light on the reasons for the trend for greater O2 utilization by more posterior pouches and the pharyngo-cutaneous duct in fasted hagfish.

Ancient fishes and the functional evolution of the corticosteroid stress response in vertebrates.

The neuroendocrine mechanism underlying stress responses in vertebrates is hypothesized to be highly conserved and evolutionarily ancient. Indeed, elements of this mechanism, from the brain to steroidogenic tissue, are present in all vertebrate groups; yet, evidence of the function and even identity of some elements of the hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis is equivocal among the most basal vertebrates. The purpose of this review is to discuss the functional evolution of the HPA/I axis in vertebrates with a focus on our understanding of this neuroendocrine mechanism in the most ancient vertebrates: the agnathan (i.e., hagfish and lamprey) and chondrichthyan fishes (i.e., sharks, rays, and chimeras). A review of the current literature presents evidence of a conserved HPA/I axis in jawed vertebrates (i.e., gnathostomes); yet, available data in jawless (i.e., agnathan) and chondrichthyan fishes are limited. Neuroendocrine regulation of corticosteroidogenesis in agnathans and chondrichthyans appears to function through similar pathways as in bony fishes and tetrapods; however, key elements have yet to be identified and the involvement of melanotropins and gonadotropin-releasing hormone in the stress axis in these ancient fishes warrants further investigation. Further, the identities of physiological glucocorticoids are uncertain in hagfishes, chondrichthyans, and even coelacanths. Resolving these and other knowledge gaps in the stress response of ancient fishes will be significant for advancing knowledge of the evolutionary origins of the vertebrate stress response.

Hypoxia modifies calcium handling in the Pacific hagfish, Eptatretus stoutii.

Hagfishes may encounter low dissolved oxygen in their natural habitats, a consequence of association with hypoxic sediments and their feeding behaviour. In teleost fish, hypoxia exposure decreases ion uptake, speculated to be a mechanism for energy conservation. Although hagfishes osmoconform, they do regulate extracellular fluid concentrations of divalent cations such as calcium. The current study hypothesised that exposure of hagfish to hypoxia (0.4 kPA, 24 h) would reduce calcium uptake (determined via in vitro isolated skin and gut epithelial transport assays) and calcium accumulation (determined by in vivo whole animal exposures, using radiolabelled calcium (45Ca) to assess newly acquired calcium). A decrease in in vitro epidermal uptake was observed at sub-environmental calcium levels (10 µM), but not at environmental calcium levels (10 mM). No changes in gut calcium uptake were determined. Conversely, hypoxia led to a more rapid in vivo accumulation of calcium in tissues (skin, muscle, liver, heart, plasma, brain), mediated mostly by a significant increase in accumulation at the gill. These differences were only apparent after 1-h of exposure to the radiolabel (i.e., the last hour of the 24-h hypoxia exposure) and were not observed after 3 and 24 h periods of radiolabel exposure. This outcome was the opposite of the hypothesised effect. The reasons for a more rapid accumulation of calcium in hypoxic hagfish are unknown but may relate to roles for calcium in enhancing hypoxia tolerance in hagfishes or could be a consequence of changes in ventilatory frequency.

Comparative biomechanics of hagfish skins: diversity in material, morphology, and movement.

The baggy skins of hagfishes confer whole-body flexibility that enables these animals to tie themselves into knots without injury. The skin's looseness is produced by a subcutaneous blood sinus that decouples the skin and body core and permits the core to contort dramatically without loading the skin in tension or shear. Hagfish skin represents a biological composite material comparable in strength and stiffness to the conventionally taut skins of other fishes. However, our understanding of hagfish skin is restricted to only one of 78 species: The Pacific hagfish Eptatretus stoutii. To determine if other hagfish share similar characteristics with E. stoutii, we measured material properties and compared histological data sets from the skins of four hagfish species: E. springeri, E. stoutii, Myxine glutinosa, and M. hubbsi. We also compared these material properties data with skins from the American eel, Anguilla rostrata. We subjected skin samples from all species to uniaxial tensile tests in order to measure strength, stiffness, extensibility, and toughness of skins stretched along longitudinal and circumferential axes. We also used a series of equibiaxial tensile tests on skin samples from E. stoutii, M. glutinosa, and A. rostrata to measure stiffness of skins simultaneously strained along both axes. Significant results of uniaxial and biaxial tests show that the skins from Eptatretus are anisotropic, being stiffer in the longitudinal axis, and more extensible than the isotropic skins of Myxine. Skins of A. rostrata were stiffer in the circumferential axis and they were stronger, tougher, and stiffer than all hagfish skins examined. The skins of Eptatretus are histologically distinct from Myxine skins and possess arrays of fibers that stain like muscle. These interspecific differences across hagfish skins show a phylogenetic pattern with knotting kinematics and flexibility; both genera belong to distinct but major subfamilies within the Myxinidae, and Eptatretus is known for creating and manipulating a greater diversity of knotting styles than Myxine.

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.

The good, the bad and the slimy: experimental studies of hagfish digestive and nutritional physiology.

The hagfishes provide valuable insight into the physiology of feeding, digestion and nutrient absorption by virtue of unusual and unique features of their biology. For example, members of this group undergo long periods of fasting, and are the only vertebrates known to absorb organic nutrients across their epidermal surface. Such properties engender significant attention from researchers interested in feeding and feeding-related processes; however, the practical realities of employing the hagfish as an experimental organism can be challenging. Many of the key tools of the experimental biologist are compromised by a species that does not readily feed in captivity, is difficult to instrument and which produces copious quantities of slime. This Commentary provides critical insight into the key aspects of hagfish feeding and digestive processes, and highlights the pitfalls of this group as experimental organisms. We also suggest key research gaps that, if filled, will lead to better understanding of hagfishes, and we consider how this group may advance our knowledge of feeding, digestion and nutrient absorption processes.

Ventilatory sensitivity to ammonia in the Pacific hagfish (Eptatretus stoutii), a representative of the oldest extant connection to the ancestral vertebrates.

Ventilatory sensitivity to ammonia occurs in teleosts, elasmobranchs and mammals. Here, we investigated whether the response is also present in hagfish. Ventilatory parameters (nostril flow, pressure amplitude, velar frequency and ventilatory index, the last representing the product of pressure amplitude and frequency), together with blood and water chemistry, were measured in hagfish exposed to either high environmental ammonia (HEA) in the external sea water or internal ammonia loading by intra-vascular injection. HEA exposure (10 mmol l-1 NH4HCO3 or 10 mmol l-1 NH4Cl) caused a persistent hyperventilation by 3 h, but further detailed analysis of the NH4HCO3 response showed that initially (within 5 min) there was a marked decrease in ventilation (80% reduction in ventilatory index and nostril flow), followed by a later 3-fold increase, by which time plasma total ammonia concentration had increased 11-fold. Thus, hyperventilation in HEA appeared to be an indirect response to internal ammonia elevation, rather than a direct response to external ammonia. HEA-mediated increases in oxygen consumption also occurred. Responses to NH4HCO3 were greater than those to NH4Cl, reflecting greater increases over time in water pH and PNH3  in the former. Hagfish also exhibited hyperventilation in response to direct injection of isotonic NH4HCO3 or NH4Cl solutions into the caudal sinus. In all cases where hyperventilation occurred, plasma total ammonia and PNH3  levels increased significantly, while blood acid-base status remained unchanged, indicating specific responses to internal ammonia elevation. The sensitivity of breathing to ammonia arose very early in vertebrate evolution.

Unravelling hagfish slime.

Hagfish slime is a unique predator defence material containing a network of long fibrous threads each ∼10 cm in length. Hagfish release the threads in a condensed coiled state known as skeins (∼100 µm), which must unravel within a fraction of a second to thwart a predator attack. Here we consider the hypothesis that viscous hydrodynamics can be responsible for this rapid unravelling, as opposed to chemical reaction kinetics alone. Our main conclusion is that, under reasonable physiological conditions, unravelling due to viscous drag can occur within a few hundred milliseconds, and is accelerated if the skein is pinned at a surface such as the mouth of a predator. We model a single skein unspooling as the fibre peels away due to viscous drag. We capture essential features by considering simplified cases of physiologically relevant flows and one-dimensional scenarios where the fibre is aligned with streamlines in either uniform or uniaxial extensional flow. The peeling resistance is modelled with a power-law dependence on peeling velocity. A dimensionless ratio of viscous drag to peeling resistance appears in the dynamical equations and determines the unraveling time scale. Our modelling approach is general and can be refined with future experimental measurements of peel strength for skein unravelling. It provides key insights into the unravelling process, offers potential answers to lingering questions about slime formation from threads and mucous vesicles, and will aid the growing interest in engineering similar bioinspired material systems.

Activity and post-prandial regulation of digestive enzyme activity along the Pacific hagfish (Eptatretus stoutii) alimentary canal.

Hagfishes are living representatives of the earliest-diverging vertebrates and are thus useful for the study of early vertebrate physiology. It has been previously postulated that digestive enzymes account for the majority of digestion because hagfish are agastric with notable zymogen granules in specialized cells of the hindgut. While the presence of some digestive enzymes (amylase, lipase and leucinaminopeptidase) have been confirmed with histochemistry, quantification of enzymatic activity is limited. This study sought to biochemically quantify the tissue activity of six digestive enzymes (α-amylase, maltase, lipase, trypsin, aminopeptidase and alkaline phosphatase) along the length of the Pacific hagfish (Eptatretus stoutii) alimentary canal. In addition, the effect of feeding on the rate of enzyme activity was examined. Overall, maltase and trypsin activities were unchanging with respect to location or feeding status, while the activities of α-amylase and alkaline phosphatase decreased substantially following feeding, but were consistent along the length. Lipase and aminopeptidase activities were elevated in the anterior region of the alimentary canal in comparison to the more posterior regions, but were not altered with feeding. This study indicates hagfish have an assortment of digestive enzymes that likely are the result of a varied diet. The differential expression of these enzymes along the tract and in regards to feeding may be indications of early compartmentalization of digestive function.

Powering the hagfish "bite": The functional morphology of the retractor complex of two hagfish feeding apparatuses.

Hagfish use forceful retractions of a dental plate to shear and ingest food. Retractile force is generated by the retractor muscle complex of the posterior hagfish feeding apparatus (HFA). While gross morphological descriptions exist, the organization of muscle and connective tissue fibers that form the soft tissue retractor complex do not. In this study, we used paraffin histology to prepare serial sections of Pacific (Eptatretus stoutii, Lockington, 1879) and Atlantic (Myxine glutinosa, Linnaeus, 1758) hagfishes in order to describe constituent soft tissue anatomy and fiber orientations. We generated 3D reconstructions in which digitized sections were segmented and fitted to volumetric scans of retractor complexes taken prior to microtomy. These models confirmed that the retractor complex is composed of a perpendicularis muscle that fits within the eye of a needle-shaped clavatus muscle, which anteriorly bears the dental plate tendon, and in turn fits within a sleeve-like tubulatus muscle. Analysis of fiber orientations within these muscles resulted in novel functional hypotheses: (a) The tubulatus muscle represents a novel tubular bipennate muscle with a considerable physiological cross-sectional area. Its activation may indirectly create tension in the dental plate tendon: as the tubulatus muscle forcefully extends, it displaces the terminal bulb and the clavatus muscle posteriorly. (b) Within the HFA terminal bulb, the muscle fibers of the clavatus and perpendicularis muscles are mutually perpendicular and may cocontract to form a swelling stopper knot-like muscular complex that resists being pulled through the tubulatus muscle. (c) While overall feeding apparatus muscle morphology is conserved, the physiological cross-sectional area of the tubulatus muscle in E. stoutii, is relatively larger than that of M. glutinosa, suggesting a more forceful retraction. The tubular bipennate construction of the tubulatus may represent a novel soft robotic actuator design.

The effects of salinity and hypoxia exposure on oxygen consumption, ventilation, diffusive water exchange and ionoregulation in the Pacific hagfish (Eptatretus stoutii).

Hagfishes (Class: Myxini) are marine jawless craniate fishes that are widely considered to be osmoconformers whose plasma [Na+], [Cl-] and osmolality closely resemble that of sea water, although they have the ability to regulate plasma [Ca2+] and [Mg2+] below seawater levels. We investigated the responses of Pacific hagfish to changes in respiratory and ionoregulatory demands imposed by a 48-h exposure to altered salinity (25 ppt, 30 ppt (control) and 35 ppt) and by an acute hypoxia exposure (30 Torr; 4 kPa). When hagfish were exposed to 25 ppt, oxygen consumption rate (MO2), ammonia excretion rate (Jamm) and unidirectional diffusive water flux rate (JH2O, measured with 3H2O) were all reduced, pointing to an interaction between ionoregulation and gas exchange. At 35 ppt, JH2O was reduced, though MO2 and Jamm did not change. As salinity increased, so did the difference between plasma and external water [Ca2+] and [Mg2+]. Notably, the same pattern was seen for plasma Cl-, which was kept below seawater [Cl-] at all salinities, while plasma [Na+] was regulated well above seawater [Na+], but plasma osmolality matched seawater values. MO2 was reduced by 49% and JH2O by 36% during hypoxia, despite a small elevation in overall ventilation. Our results depart from the "classical" osmorespiratory compromise but are in accord with responses in other hypoxia-tolerant fish; instead of an exacerbation of gill fluxes when gas transfer is upregulated, the opposite happens.

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.

Acute temperature effects on metabolic rate, ventilation, diffusive water exchange, osmoregulation, and acid-base status in the Pacific hagfish (Eptatretus stoutii).

The Pacific hagfish (Eptatretus stoutii) is a representative of the most basal extant craniates, and is a marine osmoconformer with an extremely low metabolic rate (MO2 = 475 µmol O2/kg/h at 12 °C). We investigated potential physiological trade-offs associated with compensatory changes in gill ventilation and perfusion when 12 °C-acclimated hagfish were acutely exposed to 7 °C or 17 °C, as reflected in diffusive unidirectional water flux ([Formula: see text], measured with tritiated water: 3H2O), net ammonia flux (Jamm), and plasma ion and acid-base status. [Formula: see text] was high (~ 1.4 L/kg/h at 12 °C) in comparison to marine teleosts and elasmobranchs. MO2 increased linearly with temperature (R2 = 0.991), and was more sensitive (Q10 = 3.22) in the 12-7 °C range than either Jamm (1.86) or [Formula: see text] (1.35), but the pattern reversed from 12 to 17 °C (Q10s: MO2 = 2.77, Jamm = 2.88, [Formula: see text] = 4.01). Heart rate, ventilatory index (a proxy for total ventilation), and coughing frequency also increased but with different patterns. At 17 °C, plasma [Ca2+] and [Mg2+] decreased, although osmolality increased, associated with elevations in plasma [Na+] and [Cl-]. Blood pH and PCO2 were unaffected by acute temperature changes while [HCO3-] increased. Hyperoxia (PO2 > 300 Torr) attenuated the increase in [Formula: see text] at 17 °C, did not affect Jamm, and had diverse effects on plasma ion and acid-base status. Our results suggest a clear osmorespiratory compromise occurring for the diffusive water fluxes as a result of acute temperature changes in this osmoconformer.

The ventilation mechanism of the Pacific hagfish Eptatretus stoutii.

We made anatomical and physiological observations of the breathing mechanisms in Pacific hagfish Eptatretus stoutii, with measurements of nostril flow and pressure, mouth and pharyngo-cutaneous duct (PCD) pressure and velum and heart impedance and observations of dye flow patterns. Resting animals frequently exhibit spontaneous apnea. During normal breathing, water flow is continuous at a high rate (~125 ml kg-1 min-1 at 12°C) powered by a two-phase unidirectional pumping system with a fast suction pump (the velum, ~22 min-1 ) for inhalation through the single nostril and a much slower force pump (gill pouches and PCD ~4.4 min-1 ) for exhalation. The mouth joins the pharynx posterior to the velum and plays no role in ventilation at rest or during swimming. Increases in flow up to >400 ml kg-1 min-1 can be achieved by increases in both velum frequency and stroke volume and the ventilatory index (product of frequency x nostril pressure amplitude) provides a useful proxy for ventilatory flow rate. Two types of coughing (flow reversals) are described. During spontaneous swimming, ventilatory pressure and flow pulsatility becomes synchronised with rhythmic body undulations.

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.

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.

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.