Pacific Hagfish Research Articles

Automated species classification and counting by deep-sea mobile crawler platforms using YOLO

Edge computing on mobile marine platform is paramount for automated ecological monitoring. The goal of demonstrating the computational feasibility of an Artificial Intelligence (AI)-powered camera for fully automated real-time species-classification on deep-sea crawler platforms was searched by running You-Only-Look-Once (YOLO) model on an edge computing device (NVIDIA Jetson Nano), to evaluate the achievable animal detection performances, execution time and power consumption, using all the available cores. We processed a total of 337 rotating video scans (∼180°), taken during approximately 4 months in 2022 at the methane hydrates site of Barkley Canyon (Vancouver Island; BC; Canada), focusing on three abundant species (i.e., Sablefish Anoplopoma fimbria, Hagfish Eptatretus stoutii, and Rockfish Sebastes spp.). The model was trained on 1926 manually annotated video frames and showed high detection test performances in terms of accuracy (0.98), precision (0.98), and recall (0.99). The trained model was then applied on 337 videos.In 288 videos we detected a total of 133 Sablefish, 31 Hagfish, and 321 Rockfish nearly in real-time (about 0.31 s/image) with very low power consumption (0.34 J/image). Our results have broad implications on intelligent ecological monitoring. Indeed, YOLO model can meet operational-autonomy criteria for fast image processing with limited computational and energy loads.

Mitochondria from the systemic heart of Pacific hagfish (Eptatretus stoutii) are insensitive to one hour of anoxia followed by reoxygenation

Pacific hagfish (Eptatretus stoutii) are an ancient agnathan vertebrate known to be anoxia tolerant. To study their metabolic organization and the role of the mitochondria in anoxia tolerance we developed a novel protocol to measure mitochondrial function in permeabilized cardiomyocytes and how this is affected by one hour of anoxia followed by reoxygenation. When measured at 10 °C the mitochondria had a respiration rate of 2.1 ± 0.1pmol/s/mg WW during OXPHOS with saturating concentrations of glutamate, malate, and succinate. This is comparatively low compared to other ectothermic species. The functional characteristics of the mitochondria were quantified with mitochondrial control ratios. These demonstrated that proton leak contributed to just under 50% of the oxygen flux, with the remainder going towards ATP phosphorylation. Finally, when the preparations were exposed to an anoxia-reoxygenation protocol there was no difference in respiration compared to that of a heart sample from the same animal maintained under normoxia for the same time. When Complex I alone or Complex I and II were stimulated following one hour of anoxia there was no decline in oxygen flux observed. However, if Complex II was activated alone there was a significant decline in respiration. This decrease was however also observed in the mitochondria maintained in normoxia for one hour. In conclusion, Pacific hagfish cardiac mitochondria demonstrated a low rate of oxygen consumption, a loosely coupled electron transfer system, and a resistance to one hour of anoxia.

Odorant transport in a hagfish

Odorant transport is of fundamental and applied importance. Using computational simulations, we studied odorant transport in an anatomically accurate model of the nasal passage of a hagfish (probably Eptatretus stoutii). We found that ambient water is sampled widely, with a significant ventral element. Additionally, there is a bilateral element to olfactory flow, which enters the single nostril in two narrow, laminar streams that are then split prior to the nasal chamber by the anterior edge of the central olfactory lamella. An appendage on this lamella directs a small portion (10–14%) of the overall nasal flow to the olfactory sensory channels. Much of the remaining flow is diverted away from the sensory channels by two peripheral channels. The anterior edge of the central olfactory lamella, together with a jet-impingement mechanism, disperses flow over the olfactory surfaces. Diffusion of odorant from bulk water to the olfactory surfaces is facilitated by the large surface area:volume ratio of the sensory channels, and by a resistance-based hydrodynamic mechanism that leads to long residence times (up to 4.5 s) in the sensory channels. With increasing volumetric flow rate, the rate of odorant transfer to the olfactory surfaces increases, but the efficiency of odorant uptake decreases, falling in the range 2–6%. Odorant flux decreases caudally across the olfactory surfaces, suggesting in vivo a preponderance of olfactory sensory neurons on the anterior part of each olfactory surface. We conclude that the hagfish has a subtle anatomy for locating and capturing odorant molecules.

Gross anatomy of the Pacific hagfish, Eptatretus burgeri, with special reference to the coelomic viscera.

Hagfish (Myxinoidea) are a deep-sea taxon of cyclostomes, the extant jawless vertebrates. Many researchers have examined the anatomy and embryology of hagfish to shed light on the early evolution of vertebrates; however, the diversity within hagfish is often overlooked. Hagfish have three lineages, Myxininae, Eptatretinae, and Rubicundinae. Usually, textbook illustrations of hagfish anatomy reflect the morphology of the Myxininae lineage, especially Myxine glutinosa, with its single pair of external branchial pores. Here, we instead report the gross anatomy of an Eptatretinae, Eptatretus burgeri, which has six pairs of branchial pores, especially focusing on the coelomic organs. Dissections were performed on fixed and unfixed specimens to provide a guide for those doing organ- or tissue-specific molecular experiments. Our dissections revealed that the ventral aorta is Y-branched in E. burgeri, which differs from the unbranched morphology of Myxine. Otherwise, there were no differences in the morphology of the lingual apparatus or heart in the pharyngeal domain. The thyroid follicles were scattered around the ventral aorta, as has been reported for adult lampreys. The hepatobiliary system more closely resembled those of jawed vertebrates than those of adult lampreys, with the liver having two lobes and a bile duct connecting the gallbladder to each lobe. Overall, the visceral morphology of E. burgeri does not differ significantly from that of the known Myxine at the level of gross anatomy, although the branchial morphology is phylogenetically ancestral compared to Myxine.

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 (48h) and recovery from hypercapnia conditions (6h), 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.

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. 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. 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. 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.

Molecular identification and postprandial regulation of glucose carrier proteins in the hindgut of Pacific hagfish, Eptatretus stoutii.

Hagfish are an excellent model species in which to draw inferences on the evolution of transport systems in early vertebrates owing to their basal position in vertebrate phylogeny. Glucose is a ubiquitous cellular energy source that is transported into cells via two classes of carrier proteins: sodium-glucose-linked transporters (Sglt; Slc5a) and glucose transporters (Glut; Slc2a). Although previous pharmacological evidence has suggested the presence of both sodium-dependent and -independent transport mechanisms in the hagfish, the molecular identities were heretofore unconfirmed. We have identified and phylogenetically characterized both a Slc5a1-like and Slc2a-like gene in the Pacific hagfish (Eptatretus stoutii), the latter sharing common ancestry with other glucose-transporting isoforms of the Slc2a family. To assess the potential postprandial regulation of these glucose transporters, we examined the abundance and localization of these transporters with qPCR and immunohistochemistry alongside functional studies using radiolabeled d-[14C]glucose. The effects of glucose or insulin injection on glucose transport rate and transporter expression were also examined to determine their potential role(s) in the regulation of intestinal glucose carrier proteins. Feeding prompted an increase in glucose uptake across the hindgut at both 0.5 mM (∼84%) and 1 mM (∼183%) concentrations. Concomitant increases were observed in hindgut Slc5a1 protein expression. These effects were not observed following either of glucose or insulin injection, indicating these postprandial factors are not the driving force for transporter regulation over this timeframe. We conclude that Pacific hagfish utilize evolutionarily conserved mechanisms of glucose uptake and so represent a useful model to understand early-vertebrate evolution of glucose uptake and regulation.

Reproductive biology and ecology of Pacific hagfish (Eptatretus stoutii) and black hagfish (Eptatretus deani).

The reproductive biology of Pacific hagfish Eptatretus stoutii (Lockington, 1878) and black hagfish Eptatretus deani (Evermann & Goldsborough, 1907) was assessed using current and historical data. Our results found that the reproductive characteristics of both hagfish species reflect those of K-selected species, which tend to live long and exhibit slow growth rates, low fecundity (approximately 20 eggs per female) and late maturity. Additionally, females of both species commence maturation prior to males. This study provides a population profile for both species of hagfish, but further assessments are needed to effectively manage a sustainable hagfish fishery.

Understanding ventilation and oxygen uptake of Pacific hagfish (Eptatretus stoutii), with particular emphasis on responses to ammonia and interactions with other respiratory gases.

The hagfishes are an ancient and evolutionarily important group, with breathing mechanisms and gills very different from those of other fishes. Hagfish inhale through a single nostril via a velum pump, and exhale through multiple separate gill pouches. We assessed respiratory performance in E. stoutii (31 ppt, 12ºC, 50-120g) by measuring total ventilatory flow ([Formula: see text]) at the nostril, velar (respiratory) frequency (fr), and inspired (PIO2) and expired (PEO2) oxygen tensions at all 12 gill pouch exits plus the pharyngo-cutaneous duct (PCD) on the left side, and calculated ventilatory stroke volume (S[Formula: see text]), % O2 utilization, and oxygen consumption (ṀO2). At rest under normoxia, spontaneous changes in [Formula: see text] ranged from apnea to > 400mlkg-1min-1, due to variations in both fr and S[Formula: see text]; "normal" [Formula: see text] averaged 137mlkg-1min-1, ṀO2 was 718µmolkg-1h-1, so the ventilatory convection requirement for O2 was about 11 L mmol-1. Relative to anterior gill pouches, lower PEO2 values (i.e. higher utilization) occurred in the more posterior pouches and PCD. Overall, O2 utilization was 34% and did not change during hyperventilation but increased to > 90% during hypoventilation. Environmental hypoxia (PIO2 ~ 8% air saturation, 1.67kPa, 13Torr) caused hyperventilation, but neither acute hyperoxia (PIO2 ~ 275% air saturation, 57.6kPa, 430Torr) nor hypercapnia (PICO2 ~ 1% CO2, 1.0kPa, 7.5Torr) significantly altered [Formula: see text]. ṀO2 decreased in hypoxia and increased in hyperoxia but did not change in hypercapnia. Acute exposure to high environmental ammonia (HEA, 10mM NH4HCO3) caused an acute decrease in [Formula: see text], in contrast to the hyperventilation of long-term HEA exposure described in a previous study. The hypoventilatory response to HEA still occurred during hypoxia and hyperoxia, but was blunted during hypercapnia. Under all treatments, ṀO2 increased with increases in [Formula: see text]. Overall, there were lower convection requirements for O2 during hyperoxia, higher requirements during hypoxia and hypercapnia, but unchanged requirements during HEA. We conclude that this "primitive" fish operates a flexible respiratory system with considerable reserve capacity.

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.

Genetic and functional analysis of a Pacific hagfish opioid system.

The actions of endogenous opioids and nociceptin/orphanin FQ are mediated by four homologous G protein-coupled receptors that constitute the opioid receptor family. However, little is known about opioid systems in cyclostomes (living jawless fish) and how opioid systems might have evolved from invertebrates. Here, we leveraged de novo transcriptome and low-coverage whole-genome assembly in the Pacific hagfish (Eptatretus stoutii) to identify and characterize the first full-length coding sequence for a functional opioid receptor in a cyclostome. Additionally, we define two novel endogenous opioid precursors in this species that predict several novel opioid peptides. Bioinformatic analysis shows no closely related opioid receptor genes in invertebrates with regard either to the genomic organization or to conserved opioid receptor-specific sequences that are common in all vertebrates. Furthermore, no proteins analogous to vertebrate opioid precursors could be identified by genomic searches despite previous claims of protein or RNA-derived sequences in several invertebrate species. The presence of an expressed orthologous receptor and opioid precursors in the Pacific hagfish confirms that a functional opioid system was likely present in the common ancestor of all extant vertebrates some 550 million years ago, earlier than all previous authenticated accounts. We discuss the premise that the cyclostome and vertebrate opioid systems evolved from invertebrate systems concerned with antimicrobial defense and speculate that the high concentrations of opioid precursors in tissues such as the testes, gut, and activated immune cells are key remnants of this evolutionary role.

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 10mM. However, movement into the serosal compartment was saturable, with a maximal transport rate of 59nmolcm-2h-1 and an affinity (substrate concentration to give half maximal transport rate) of 1139µM. Calcium accumulation into gut tissue and uptake into the gut serosal compartment was also saturable, with maximal transport rates of 5.1 and 2.8nmolcm-2h-1, and affinities of 6417 and 3327µM, 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.

Characterization of body knotting behavior used for escape in a diversity of hagfishes

AbstractHagfishes possess flexible bodies that allow them to tie and manipulate body knots. These knots are used to remove mucous from the body, escape tight spaces, pull prey from burrows and, since they lack opposable jaws, to create leverage during feeding events. Despite its importance, knotting is poorly understood because this fast and three‐dimensionally complex behavior is difficult to study both in the laboratory and in their natural benthic habitats. To consistently stimulate analyzable knotting behaviors, we developed a novel restraint device to record high‐speed, biplanar video in three species of hagfish from two major taxa (Eptatretinae and Myxininae): Eptatretus stoutii, Eptatretus springeri and Myxine glutinosa. These data were analyzed to characterize knot type and kinematics of knot formation. We found that despite reduced radii of curvature and strain associated with more complex knots, all species of hagfish preferred the simplest overhand knots. While all species were physically capable of tying more complex knots, E. stoutii exhibited a lower ‘behavioral’ stiffness by tying complex knots more frequently and coiling when at rest. The hyper‐flexible, hyper‐redundant hagfish body may require a high level of neural input for control. However, analyses of knotting motions indicate that hagfish seem to employ only four movements (body bends, twists, surface contacts and tail insertions into body loops) but in different combinations depending on the knot being tied. Furthermore, these movements and combinations are conserved across all species tested. We hypothesize that control of knotting in hagfish may employ motor primitives to reduce neural input.

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.

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 36h 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 12h, 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.