Date Published: April 5, 2019
Publisher: Public Library of Science
Author(s): Alyssa M. Weinrauch, Christina M. Schaefer, Greg G. Goss, José L Soengas.
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.
Digestion is essential for the catabolism and hydrolysis of ingested macronutrients into smaller molecules suitable for transport. It is carried out using mechanical, chemical and enzymatic methods with digestive enzymes released from multiple locations along the alimentary canal. There are a multitude of digestive enzymes for each type of macronutrient, with specifications for substrate and optimal reaction conditions (e.g. acidic vs. alkaline), which correspond to their location in the digestive tract and can be derived from the stomach, exocrine pancreas, or the intestinal mucosa itself (reviewed in ). The capacity for an organism to digest certain foods predominantly depends upon the presence of appropriate enzymes . The complement of digestive enzymes found in bony fishes are consistent with what is found in other vertebrates , however few reports exist focusing on hagfish.
Twenty-four Pacific hagfish (Eptatretus stoutii; 65.3 ± 3.5 g; mean ± standard error of the mean (s.e.m)) were collected using traps baited with hake (Merluccius merluccius) in Trevor Channel, Bamfield, B.C., Canada (N48°50.883-W125°08.380) under a license approved by the Department of Fisheries and Oceans Canada (permit No. XR-136-2017). The animals were immediately transferred to ~5000 L holding tanks with continuously flowing seawater at Bamfield Marine Sciences Station, prior to shipping to the University of Alberta where they were housed in a recirculating artificial salt water system (Instant Ocean SeaSalt; Spectrum Brands, Blacksburg, VA, USA). This 2400 L system is constantly circulated through 6 tanks in a flow-through manner and is maintained at 12 ± 2 °C and 24 ± 2 ppt salinity. Owing to their light sensitivity, hagfish were housed in blackened containers at all times with PVC piping used as habitat enrichment as previously described . Much like some reptiles, hagfish are intermittent feeders, known to regress intestinal function and cellular morphology between feeding periods . It is not uncommon for hagfish to ignore food between feedings for multiple weeks at a time (personal observation), so we opted for a one-month fasting period to mirror natural fasting periods of this animal. Fed animals were given squid, permitted to feed until satiated and to digest for a 2 h period. Previous experiments have demonstrated that heightened physiological perturbations, such as metabolic oxygen consumption, occur 8 h after a feed . We opted to use a 2 h post-fed time frame to examine the tissue enzyme activity near the onset of digestion, rather than at the peak point of many physiological processes to increase the likelihood that tissue enzyme activity would persist post-feeding. All sampling procedures and experimental manipulations were conducted with the approval of the University of Alberta Animal Care Committee (No. AUP0001126 (2017)).
For each of α-amylase, lipase, trypsin and alkaline phosphatase, the anterior and posterior sections differed substantially (see S1 Table; H1 = 49.4, P <0.001; H1 = 9.36, P < 0.002; H1 = 58.4, P < 0.001; H1 = 68.4, P < 0.001). Therefore, the effect of feeding and location was examined independently in each section for these enzymes via a 2-way ANOVA or Mann-Whitney Rank Sum Test (see methods). Notably, the anterior region (B and PCD) was not analyzed for α-amylase, trypsin or alkaline phosphatase owing to the activities being below detectable limits. All other enzymes were analyzed using a 2-way ANOVA taking all sections into consideration. Overall, E. stoutii have digestive enzymes that catabolize each of the major macronutrient classes (carbohydrates, fats, proteins). Much like stomachless teleosts, the lack of a stomach does not appear to impact digestive flexibility or capacity in hagfish [26,27]. Table 1 summarizes the statistical relationships of tissue digestive enzyme activity between anterior and posterior segments of the hagfish alimentary canal. In the event of a significant difference between regions, a secondary effect within an applicable segment is presented. Each enzyme has a unique distribution along the alimentary canal (Table 1) and, for the most part, it seems that the majority of digestive activity takes place within the hindgut as previously suggested ; however, some digestive activity (maltase, lipase and aminopeptidase) was noted in the anterior regions (B and PCD). The typical vertebrate observation of decreasing enzyme activity in the posterior-most segments [28,29] was not evident along the hindgut of hagfish for any studied enzyme. Table 2 highlights the statistical outcome of feeding on digestive enzyme tissue activity in the Pacific hagfish, where differential effects of feeding were observed for certain enzymes. Source: http://doi.org/10.1371/journal.pone.0215027