© 2018. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2018) 221, jeb172254. doi:10.1242/jeb.172254 RESEARCH ARTICLE Emptying and refilling of slime glands in Atlantic (Myxine glutinosa) and Pacific (Eptatretus stoutii) hagfishes Sarah Schorno1, Todd E. Gillis1 and Douglas S. Fudge1,2,* ABSTRACT skein, which is precisely bundled and organized into stacked Hagfishes are known for their unique defensive slime, which they use thread loops within the cell (Winegard et al., 2014; Downing et al., to ward off gill-breathing predators. Although much is known about the 1984; Spitzer et al., 1984). The gland mucous cells are filled with slime cells (gland thread cells and gland mucous cells), little is known hundreds of tiny mucus-containing vesicles (Luchtel et al., 1991; about how long slime gland refilling takes, or how slime composition Salo et al., 1983). When the musculature around the slime glands changes with refilling or repeated stimulation of the same gland. Slime contracts, it initiates a rapid holocrine secretion event; the two gland glands can be individually electrostimulated to release slime, and this components are forced out through the narrow gland pore and, in the technique was used to measure slime gland refilling times for Atlantic process, their plasma membranes are sheared off (Downing et al., and Pacific hagfish. The amount of exudate produced, the 1981a,b). In seawater, the thread and mucus components interact to composition of the exudate and the morphometrics of slime cells yield an ultra-dilute mucous gel (Downing et al., 1981b; Spitzer were analyzed during refilling, and as a function of stimulation number et al., 1987; Fudge et al., 2005; Ewoldt et al., 2011). when full glands were stimulated in rapid succession. Complete The mass of slime exudate from all the glands of a Pacific hagfish – refilling of slime glands for both species took 3–4 weeks, with Pacific (Eptatretus stoutii) has been estimated to be 3 4% of body mass hagfish achieving faster absolute rates of exudate recovery than (Fudge et al., 2005), and therefore represents a significant energetic Atlantic hagfish. We found significant changes in the composition of commitment. In spite of the large amounts of pre-exudate stored in the exudate and in the morphometrics of slime cells from Pacific the slime glands, hagfish appear to be economical with their use of hagfish during refilling. Over successive stimulations of full Pacific the slime in two important ways. First, the slime is remarkably dilute, hagfish glands, multiple boluses of exudate were released, with with mucus concentrations three orders of magnitude lower than exudate composition, but not thread cell morphometrics, changing those of other mucous secretions, such as gastric mucus (Fudge et al., significantly. Finally, histological examination of slime glands 2005). Second, hagfishes conserve slime by only releasing exudate revealed slime cells retained in glands after exhaustion. from glands in the vicinity of an attack, and not as a whole-body Discrepancies in the volume of cells released suggest that response (Lim et al., 2006; Zintzen et al., 2011). The large number of ∼ mechanisms other than contraction of the gland musculature alone slime glands possessed by hagfishes ( 79 pairs in Pacific hagfish ∼ may be involved in exudate ejection. Our results provide a first look at and 97 pairs in Atlantic hagfish) and their ability to release exudate the process and timing of slime gland refilling in hagfishes, and raise locally likely reduce the chance that a hagfish will be left without new questions about how refilling is achieved at the cellular level. slime to defend itself in subsequent attacks (Fernholm, 1998). Vulnerability to predators will also be affected by how many times a KEY WORDS: Mucous cells, Slime, Thread cells, Defense response, single gland can release exudate before it is empty, and how quickly Striated muscle depleted slime glands can recharge, both major foci of this study. Although the time scale for slime gland recovery after a sliming INTRODUCTION event has not been documented in any species of hagfish, Hagfishes (Myxini) are known for their unique defensive slime Lametschwandtner et al. (1986) speculated about the relative (Downing et al., 1981a; Newby, 1946), which is an effective rates of recovery in Pacific hagfish and Atlantic hagfish (Myxine defense against attacks by gill-breathing predators (Lim et al., 2006; glutinosa) based on differences in the vascular anatomy of the Zintzen et al., 2011). The slime is produced by many slime glands slime glands in these two species. In Atlantic hagfish, capillaries that occur in a line down both sides of the hagfish. The glands are form a cage around the slime gland, whereas in Pacific hagfish, connected to the skin surface by a short duct, and are surrounded by capillary loops also descend into the gland interior, which may striated muscle and a connective tissue capsule (Downing et al., increase the rate of nutrient delivery to the slime glands and 1981b; Blackstad, 1963; Koch et al., 1991). The slime glands decrease the recovery time in this species (Lametschwandtner contain two main secretory cells: gland thread cells and gland et al., 1986). mucous cells. The gland thread cells each contain a single, In this study, we aimed to answer several fundamental questions elaborately coiled protein-rich thread formation, known as a about the refilling of slime glands in hagfishes. Using a population of captive Atlantic and Pacific hagfishes, we measured the time it takes for slime glands to refill after they have been emptied of slime 1Department of Integrative Biology, University of Guelph, Guelph, ON, N1G 2W1, exudate. We also investigated the possibility that a single slime Canada. 2Schmid College of Science and Technology, Chapman University, Orange, CA 92866, USA. gland can eject multiple boluses of exudate and, if so, whether the composition of the exudate changes with successive ejections. In *Author for correspondence ([email protected]) addition, we examined the effects of recovery time after a sliming D.S.F., 0000-0001-9569-1374 event on the composition of exudate in a gland, the relative proportions of threads and mucus in the exudate, as well as the Received 13 October 2017; Accepted 12 February 2018 morphometrics of the thread skeins. Journal of Experimental Biology 1 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb172254. doi:10.1242/jeb.172254 MATERIALS AND METHODS individuals of similar mass from each species (Pacific: 51.54± Experimental animals 2.53 g; Atlantic: 42.14±1.23 g), with ten individuals used for each Pacific hagfish [Eptatretus stoutii (Lockington 1878)] were of the ten time intervals examined. For each recovery time treatment, collected from Bamfield Marine Station in Bamfield, BC, all glands on the left side of each specimen were exhausted Canada, and Atlantic hagfish (Myxine glutinosa Linnaeus 1758) completely (i.e. they no longer produced exudate) at time zero using were collected from Passomoquody Bay, NB, Canada. All hagfishes repeated electrostimulation, while glands on the right side were utilized in this study were adults, but the age and sex of each unstimulated (‘full’) and served as an internal control. Experimental individual was unknown. The two species were housed together in a hagfish were kept separate from others by placing them in a smaller 2000 l environmentally controlled aquatic recirculating system floating container in the tank. Hagfish were isolated for various time filled with chilled artificial seawater (34 ppm, 10°C) at the Hagen intervals (4 days, 8 days, etc.) and removed from the tank at desired Aqualab at the University of Guelph, Guelph, ON, Canada. Hagfish days post-sliming to have their slime exudate collected. Each were isolated in floating bins within the tank for a minimum of individual had one refilling time point (left side of hagfish) and full 1 month (30 days) prior to our experiments, to make sure their slime glands (right side of hagfish) from which slime was collected. After glands were completely full. Hagfish were fed squid to satiety once recording hagfish body mass, we collected slime from each side of per month. All housing and feeding conditions were approved by the animal separately onto pre-weighed paper towels to determine the University of Guelph Animal Care Committee (Animal the mass of slime produced by the refilling side and the full side of Utilization Protocol #2519). the animal. Hagfish anesthetization and slime collection Gland tissue preparation Hagfish were anesthetized by placing them in 3 l artificial seawater Exhausted and unstimulated glands were harvested from five Pacific (Coralife, Energy Savers Unlimited, Inc., Carson, CA, USA) with hagfish for histological analysis. Hagfish were anesthetized then 3 ml of a clove oil (Sigma-Aldrich, Oakville, ON, Canada) euthanized by severing the notochord and dorsal nerve cord using a anesthetic solution (1:9 clove oil to 95% ethanol) and left until large pair of surgical scissors. Hagfish were dissected according to a they ceased to respond to touch. Once fully anesthetized, the hagfish protocol developed by Winegard (2012), which allows the slime were removed from the artificial seawater, rinsed with deionized glands to be separated from the myotomal muscle in which they are water, patted dry using Kim Wipes (Kimberly-Clark Corporation, embedded while still attached to the skin at the gland pore. Irving, TX, USA) and placed on a