2013 SOUTHEASTERN NATURALIST 12(3):523–533 Evaluating the Efficiency of Flushed Stomach-tube Lavage for Collecting Stomach Contents from Dogfish Sharks Charles W. Bangley1,2,*, Roger A. Rulifson1,2, and Anthony S. Overton1 Abstract - Concern over the use of lethal techniques to collect basic biological data from sharks has necessitated the development of nonlethal methods of data collection. We evaluated the nonlethal method of removing stomach contents using acrylic tubes. Stomach contents of Spiny Dogfish (Squalus acanthias) captured with bottom trawls and longlines were collected using acrylic tubes flushed with seawater. The largest tube used during the trawl survey was 30 mm in diameter, while a larger tube (37 mm in diameter) was used during longline sampling due to catches of larger dogfish. The average efficiency of stomach content removal was 79.5% overall, and improved to 93% with the addition of the larger tube. Selection of a tube with a diameter 10–20 mm less than mouth width can be reasonably expected to recover over 90% of stomach contents. Stomachtube lavage is a useful and efficient method for nonlethal sampling of stomach contents from Spiny Dogfish, and perhaps other small sharks. Introduction Concerns over the conservation status of some shark species have resulted in researchers exploring alternative nonlethal methods of collecting biological data. As public awareness of threatened shark populations increases, societal and political pressures will necessitate the development and refinement of nonlethal sampling methods (Heupel and Simpfendorfer 2010). A variety of nonlethal methods exist for sampling stomach contents of fishes (Kamler and Pope 2001). One such method was developed by White (1930), who collected stomach contents from Salvelinus fontinalis (Mitchill) (Brook Trout) by inserting a glass tube through the mouth into the stomach and exerting pressure on the stomach. It was occasionally necessary to flush the tube with water to collect the entire stomach contents (White 1930). This method was refined by Van Den Avyle and Roussel (1980), who used a set of acrylic tubes of varying diameters and matched the diameter of the tube as closely as possible to the esophageal diameter of the fish. After insertion, it was possible to visually inspect the stomach for the presence of food by shining a light down the tube. If food was detected, the fish was lifted so that the mouth was facing downward, allowing gravity to remove the stomach contents. This method was tested on three species of Centrarchids, and post-lavage dissections showed that only 1 out of 266 fish still contained stomach contents (Van Den Avyle and Roussel 1980). However, tube lavage methods are not without limitations, and effectiveness can vary by species. Van Den Avyle and Roussel (1980) noted that acrylic 1Department of Biology, East Carolina University, East Fifth Street, Greenville, NC 27858. 2Institute for Coastal Science and Policy, East Carolina University, East Fifth Street, Greenville, NC 27858. *Corresponding author - bangleyc09@students.ecu.edu. C.W. Bangley, R.A. Rulifson, and A.S. Overton 2013 Southeastern Naturalist Vol. 12, No. 3 524 tubes may be less effective when used on species with small mouths and large stomachs, and field studies have demonstrated the effects of morphology on lavage efficiency. Using glass tubes, Gilliland et al. (1981) recovered over 90% of stomach contents by weight from percichthyid basses, but only 75% of stomach contents from Pomoxis annularis Rafinesque (White Crappie), most likely due to differences in stomach morphology. Cailteux et al. (1990) found that the efficiency of stomach tubes when sampling Micropterus salmoides (Lacepéde) (Largemouth Bass) was size-dependent, with the method giving the best results for fish 120–590 mm total length. Quist et al. (2002) only recovered slightly above 50% of stomach contents by weight from Sander vitreus (Mitchill) (Walleye), and attributed the poor results to features of the species’ stomach morphology. Waters et al. (2004) compared the use of gastric lavage methods between omnivorous Ictalurus furcatus (Valeciennes) (Blue Catfish) and the more predatory Pylodictus olivaris (Rafinesque) (Flathead Catfish) and found that both diet and gut morphology played a role in creating a significant difference in the efficiency of stomach content removal (14.6% for Blue Catfish, 86.9% for Flathead Catfish). Flushing the stomach with water is a common method for dislodging stomach contents. Foster (1977) described a method known as pulsed gastric lavage, in which a pump connected to a tube inserted through the esophagus of the fish pulsed flushes of water and essentially forced the fish to regurgitate its stomach contents. This method removed 100% and 98% of stomach contents from Esox americanus Gmelin (Grass Pickerel) and Largemouth Bass (Foster 1977), respectively, and 96% from catfishes (Waters et al. 2004). Hartleb and Moring (1995) modified this method by building a trough to hold the fish during flushing, which allowed the stomach contents to flow into a mesh screen for collection. This method was used by Hannan (2009) to remove stomach contents from juvenile Squalus acanthias L. (Spiny Dogfish), though the efficiency of stomach content removal was not reported. Barnett et al. (2010) used stomach flushing to remove the stomach contents of Notorynchus cepedianus (Péron) (Broadnose Sevengill Shark) and successfully removed all contents from seven of eight stomachs that were later dissected to verify effectiveness. Nonlethal stomach sampling of sharks is often accomplished by stomach eversion. As described by Cortés and Gruber (1990), stomach eversion involves anesthetizing the shark, grasping the stomach with a pair of forceps, and inverting it out the mouth. Sharks are capable of everting the entire stomach without permanent damage, and may do so in the wild on a regular basis (Brunnschweiler et al. 2005). Bush (2003) found that 25% of juvenile Sphyrna lewini (Griffin and Smith) (Scalloped Hammerhead) dissected after stomach eversion still contained stomach contents, though these were small teleost bones and crustacean shell fragments that comprised less than 0.05% of the shark body weig ht. The most desirable field sampling method is one that is quick, efficient, and requires a minimum of equipment. Though effective, stomach eversion can be time-consuming, and the flushing techniques described by Foster (1977) and 525 C.W. Bangley, R.A. Rulifson, and A.S. Overton 2013 Southeastern Naturalist Vol. 12, No. 3 Hartleb and Moring (1995) require the use of equipment that may be cumbersome in certain field situations. The stomach-tube method requires only the tubes themselves, but can be confounded by stomach morphology (Gilliland et al. 1981, Quist et al. 2002), which poses a particular challenge in sharks. Shark stomachs are divided into two regions: the cardiac stomach, which leads straight from the esophagus, and the pyloric stomach, which curves upwards from the end of the cardiac stomach and leads into the intestine (Gilbert 1973). Because the stomach tube method is easily performed and requires a minimum of equipment, it remains popular as a nonlethal method of collecting stomach contents despite its limitations (Cailteux et al. 1990, Quist et al. 2002). When originally developing the method, White (1930) used flushing with water to dislodge stomach contents, which may be a way of overcoming the confounding influence of stomach morphology. The goals of this study were to determine the efficiency of acrylic tubes flushed with water in collecting stomach contents from live Spiny Dogfish, identify any techniques that may increase efficiency, and assess whether stomach tube lavage may be an effective nonlethal method for collecting diet data from sharks in the field. Field-Site Description We collected Spiny Dogfish and performed lavage procedures aboard vessels in United States waters in the Northwest Atlantic Ocean. The broad area sampled ranged from Massachusetts Bay to North Carolina waters between Cape Hatteras and Cape Lookout. This area is roughly equivalent to the Virginian marine ecoregion, which includes all coastal waters between Cape Cod and Cape Hatteras and is characterized by a broad continental shelf and the influence of large estuarine systems including Delaware Bay, Chesapeake Bay, and the Albemarle/Pamlico Sound estuary of North Carolina (Fautin et al. 2010). Sampling was concentrated in Massachusetts and North Carolina waters, which represent the summer and winter range, respectively, of the Spiny Dogfish population in the Northwest Atlantic (Stehlik 2007). Methods In March 2010, we collected 31 Spiny Dogfish using a bottom trawl aboard the NOAA/NMFS R/V Henry B. Bigelow in Atlantic nearshore and continental shelf waters between Cape May, NJ and Cape Hatteras, NC. We sampled an additional 14 Spiny Dogfish by longline aboard a commercial fishing vessel in Massachusetts waters in May and June 2011. After capture, total length (TL), fork length (FL), and mouth width (MW) of each shark were recorded in millimeters (mm). We measured mouth width horizontally between the hinges of the jaw using calipers. During trawl sampling, it took approximately one hour to conduct all the lavages after sorting and documenting the tow’s catch. This time period was divided by the number of sharks lavaged to estimate handling time per shark (min). C.W. Bangley, R.A. Rulifson, and A.S. Overton 2013 Southeastern Naturalist Vol. 12, No. 3 526 During trawl sampling, we performed gastric lavage using four acrylic tubes of 360 mm in length and 3 mm thick, with beveled edges at one end (Fig. 1). The outer diameter of each tube measured 30, 25, 20, and 18 mm, respectively. Another tube, 37 mm in diameter, was added during longline sampling to more adequately sample larger sharks. All sharks were held ventral side up with a hand over the snout to induce tonic immobility. At this point, the shark’s mouth would usually open readily, but occasionally needed to be pried open using a flat metal ruler as a lever. The tube with the largest outer diameter that would fit through the esophagus was inserted through the mouth and into the stomach. Once the tube felt as though it could not travel any further, it was pulled out enough so that it was not pressed against the posterior end of the cardiac stomach. We flushed the tube with water using a saltwater deck hose available aboard both vessels. The shark was lifted so that the body angle pointed downward, and stomach contents were captured in a mesh sample bag at the distal end of the tube. This procedure was repeated until no stomach contents were observed exiting the tube in three consecutive flushes. In the final flush, the shark was held in a vertical, head-down position as the tube was removed, and the mouth was checked for the presence of additional food items. During trawl sampling, the 30-mm diameter tube was used to sample all sharks >740 mm TL, the 25-mm tube was used for three sharks between 690 and 710 mm TL, and the 20-mm tube was used to lavage a single shark that measured 560 mm TL. The 37-mm tube was used for 12 of the 14 sharks sampled during the longline survey; the two sharks less than 760 mm TL were lavaged using the 30-mm tube. No captured sharks in either sampling trip were small enough to test the 18-mm tube. Each shark was immediately sacrificed and dissected post-lavage to validate the efficiency of the method. We visually inspected sharks for signs of tissue damage resulting from lavage. For each of these sharks, we recorded the weight (g) for the stomach contents removed by the tube. Remaining stomach contents were recovered by dissection, and the weight was recorded. Removed and remaining weights were summed to determine the total weight of stomach contents. The efficiency (%) by weight was estimated as the ratio between the weight of stomach contents recovered using the tube and the total weight of stomach contents. To determine the effect, if any, of prey type on lavage efficiency, we identified the stomach contents and determined the mean lavage efficiency for each identified prey taxa. Sharks with empty stomachs were excluded from analysis. Figure 1. General design for acrylic tubes used to remove stomach contents from Spiny Dogfish in this survey. 527 C.W. Bangley, R.A. Rulifson, and A.S. Overton 2013 Southeastern Naturalist Vol. 12, No. 3 Student’s t-tests were performed to determine if the % efficiency and shark size measurements (total length, mouth width, stomach content weight) were significantly different between the trawl- and longline-sampled sharks. We used linear correlation analysis to determine the effect of different measures of shark size on the efficiency of stomach content removal, and if any of these measurements would be helpful in selecting the appropriate tube size in the field. Correlations between efficiency, total length, mouth width, and total weight of stomach contents were calculated using Spearman product moment. If any parameters showed significant relationships with efficiency, we used two-tailed t-tests to determine whether those measurements differed significantly between sharks lavaged at ≥90% and less than 90% efficiency. Results In total, 31 Spiny Dogfish were sampled from North Carolina waters, and 14 were sampled in Massachusetts, of which four from North Carolina and one from Massachusetts had empty stomachs. All sharks with empty stomachs were excluded from further analysis. We observed no post-lavage tissue damage to the stomach or any other internal organs. Longline-caught sharks had a larger mean total length (841.31 ± 46.1 mm), while differences in mouth width and stomach content weight were not significant (Table 1). Handling time during trawl sampling ranged from 2.5–4 mins per shark. Efficiency was 79.5% overall, with mean efficiencies of 69.7% and 93.0% in trawl- and longline-caught dogfish, respectively (Table 1). Lavages performed on longline-captured sharks were significantly more efficient than those performed during the trawl survey (P = 0.01). Trends in lavage efficiency related to size measurements could be observed graphically. Before the addition of the 37 mm tube, efficiency declined rapidly at size thresholds of approximately 740 mm TL (Fig. 2A) and 50 mm MW (Fig 2B). No threshold was observed for stomach content weight, but generally efficiency was higher after the addition of the 37 mm tube (Fig. 2C). Food items recovered from the sharks comprised 26 prey taxa, including a variety of fishes and invertebrates. Among the species recovered were flatfish of the Paralichthyidae and Cynoglossidae families, Urophycis regia (Walbaum) Table 1. Mean total length, mouth width, stomach content weight, and lavage efficiency (± standard deviation) for trawl- and longline-caught Spiny dDogfish, with t-test results for differences between sampling methods. Mean ± SD Variable Trawl Longline t-test (P) n 29 13 Total length (mm) 782.1 ± 68.9 841.3 ± 46.1 0.007 Mouth width (mm) 49.6 ± 5.8 47.9 ± 5.9 0.401 Stomach content weight (g) 18.8 ± 23.5 21.2 ± 23.2 0.753 Lavage efficiency (%) 69.7 ± 37.0 93.0 ± 21.00 0.010 C.W. Bangley, R.A. Rulifson, and A.S. Overton 2013 Southeastern Naturalist Vol. 12, No. 3 528 Figure 2. Percent efficiency of s t o m a c h content removal as a function of A) total length (mm), B) mouth width (mm), and C) total stomach content weight (g) for Spiny D o g f i s h s a m p l e d with a maximum tube diameter of 30 mm (n = 29) and 37 mm (n = 13). 529 C.W. Bangley, R.A. Rulifson, and A.S. Overton 2013 Southeastern Naturalist Vol. 12, No. 3 (Spotted Hake), Prionotus carolinus (L.) (Northern Searobin), Ctenogobius boleosoma (Jordan and Gilbert) (Darter Goby), ctenophores (Ctenophora), Rossia sp. (bobtail squid), shrimp (Malacostraca), and sea cucumbers (Holothuroidea). Ctenophores and Ammodytes americanus DeKay (Sand Lance) made up the majority of stomach contents observed from the longline samples. Generally, invertebrates were removed at a higher efficiency than fishes, thou gh no definitive connection between fish morphology and lavage efficiency could be determined (Table 2). Field observations during trawl sampling showed that small flatfish would occasionally become trapped between the outer surface of the tube and the lining of the stomach. However, flatfish were also present in stomachs in which 100% of the stomach contents were recovered. The only problematic prey item Table 2. Prey taxa identified in the stomach contents of lavaged Spiny Dogfish, with the number of stomachs in which they occurred, mean lavage efficiency, and standard deviation of lavage efficiency. No. Mean Prey sp. occurrences efficiency SD Algae/detritus 2 0.58 0.59 Animal remains 5 0.64 0.49 Invertebrates Ctenophora 8 0.98 0.03 Holothuroidea 2 1.00 0.00 Polychaeta 3 0.94 0.10 Decapoda 2 0.73 0.39 Stomatopoda 1 0.16 - Malacostraca 4 0.86 0.27 Euphausiidae 2 1.00 0.00 Portunidae 1 0.50 - Rossia sp. 2 0.32 0.18 Teuthoidea 3 0.99 0.01 Euspira heros (Say) (Northern Moon Snail) 1 1.00 - Bivalva 2 0.83 0.24 Clypeasteroida 1 0.67 - Unclassified invertebrate 1 1.00 - Teleosts Brevoortia tyrannus (Latrobe) (Atlantic Menhaden) (gizzard) 1 0.16 - Ammodytes americanus 4 1.00 0.00 Polymixia lowei Günther (Barbudo) 1 0.33 - Ophidion sp. 1 0.33 - Urophycis regia 2 0.18 0.02 Urophycis sp. 1 0.82 - Myoxocephalus sp. 1 0.22 - Prionotus carolinus 4 0.66 0.30 Prionotus sp. 1 1.00 - Sygnathidae 1 0.45 - Paralicthyidae 1 1.00 - Citharichthys arctifrons Goode (Gulf Stream Flounder) 1 0.67 - Cynoglossidae 2 0.24 0.07 Ctenogobius boleosoma 3 0.56 0.51 Unclassified teleost 11 0.59 0.39 C.W. Bangley, R.A. Rulifson, and A.S. Overton 2013 Southeastern Naturalist Vol. 12, No. 3 530 during longline sampling was the head of a large Myoxocephalus sp. (sculpin), which became lodged sideways in the end of the tube during lavage. Mouth width and total length showed strong, significant relationships in correlation analysis (R = 0.598, P < 0.0001), and mouth width also correlated strongly with MW-Tube (R = 0.758, P < 0.0001). Stomach weight correlated strongly with all other size measurements (Table 3). Of the relationships between size measurements and lavage efficiency, stomach weight (R = -0.617, P < 0.0001) and MW-Tube (R = -0.353, P = 0.032) were significant (α = 0.05). On the basis of linear correlation results and because stomach content weight would be impractical to measure pre-lavage, the difference between mouth width and tube diameter was the only variable chosen to compare between sharks lavaged at ≥90% and <90% efficiency. The mean difference between mouth width and tube diameter was significantly smaller (P = 0.015) in dogfish lavaged at ≥90% efficiency (15.98 ± 6.20 mm) than those lavaged at <90% efficiency (20.96 ± 5.02 mm). Discussion The results show that acrylic tubes can be an effective method for non-lethally extracting stomach contents from sharks, as long as the size of the tube is appropriate for the size of the shark. Lavage efficiency improved significantly with the addition of the larger tube diameter during longline sampling, resulting in the recovery of >90% of stomach contents. The most important predictive variable was the difference between mouth width and tube diameter. A difference within a range of 9.78–22.18 mm was associated with the recovery of 90% of stomach contents or better. Based on these results, selecting a tube diameter no more than 10–20 mm smaller than the shark’s mouth width will likely provide the best lavage efficiency in the field. The overall high lavage efficiency suggests that dogfish gut morphology alone does not play a large role in limiting the efficiency of stomach content removal. Prey morphology also does not appear to be a major confounding Table 3. Pairwise correlations between efficiency, total length (TL), mouth width (MW), stomach weight (SW), and the difference between mouth width and tube diameter (MW-Tube) for all lavaged dogfish. Variable 1 Variable 2 Correlation (R) P TL Efficiency -0.294 0.077 MW Efficiency -0.320 0.053 MW TL 0.598 less than 0.0001 MW-Tube Efficiency -0.353 0.032 MW-Tube TL 0.056 0.744 MW-Tube MW 0.758 less than 0.0001 SW Efficiency -0.617 less than 0.0001 SW TL 0.405 0.013 SW MW 0.383 0.019 SW MW-Tube 0.230 0.170 531 C.W. Bangley, R.A. Rulifson, and A.S. Overton 2013 Southeastern Naturalist Vol. 12, No. 3 factor. Prey groups such as flatfish, which were expected to be difficult to extract because of morphology, were recovered at 100% efficiency. Larger species were usually present in the stomach contents in dismembered pieces small enough to fit through the tube. In fact, the lowest efficiencies were found in more fusiform fishes. As in Cailteux et al. (1990), the size of the fish was the most important factor influencing efficiency. The precipitous decline in efficiency at TL 785 mm and MW 50 mm in the trawl-caught sharks suggests that a tube of 30-mm diameter was insufficient to efficiently remove stomach contents from Spiny Dogfish above those size thresholds. Efficiency improved from 69.7% to 93.0% with the use of a tube diameter of 37 mm, which may have been better matched to the larger sharks. This decline in efficiency was not pronounced in the lavaging of the longline-caught sample population that included use of the 37 mm tube, which indicates that this tube size was well-matched to sharks in the size range captured during that survey. The difference between mouth width and tube diameter showed the most significant relationship with lavage efficiency, with higher differences associated with lower efficiency. Mouth width is also the most intuitive measurement to compare with tube diameter, and should be measured before choosing the lavage tube. The influence of total stomach content weight may be unaffected by the increase in tube diameter, but may potentially be overcome by flushing the stomach with greater pressure. The difference in collection methods should be addressed as a potential influence on lavage efficiency. Gear used to capture sharks can significantly affect the amount and type of food recovered during diet sampling (Wetherbee and Cortés 2004). Generally, longline sampling tends to capture sharks with relatively empty stomachs, and capture stress from any collection method can result in regurgitation. However, stomach content weight was not significantly different between trawl- and longline-caught sharks. In this study, differences in gear did not appear to significantly influence stomach content weight, but capture method should always be assessed as a possible confounding factor. During trawl sampling, time needed for the lavage procedure ranged from 2.5–4 mins per shark. This is in addition to the time taken to capture, sort, and measure the sharks, so total handling time is likely considerably higher than this approximation. Some capture methods can increase the total handling time and potential physical damage to the sharks, and gastric lavage does add another source of stress to the animal. Due to the sampling design for this survey, no measurement of post-lavage survival was possible. The Spiny Dogfish is an especially hardy species with regard to physiological stress, and individuals are likely to survive capture and handling unless fatally injured (Mandelman and Farrington 2009). However, stress tolerance varies by species and thus should be considered when using this method on any shark. This method of stomach content sampling is inexpensive and easy to use. The tubes used in this study were constructed from available acrylic tubing, but other materials such as PVC should be just as effective. The low amount of equipment C.W. Bangley, R.A. Rulifson, and A.S. Overton 2013 Southeastern Naturalist Vol. 12, No. 3 532 required makes the stomach-tube method appropriate aboard crowded research and fishing vessels where space and time may be limited. This method was easiest with a two-person team: one researcher handled the shark, inserted the tube, and performed the flushes while the other held the bag open and recorded data. However, it is feasible with one person. There was some concern over possible injury from the shark’s teeth, but the tubes used for this survey were of sufficient length to keep hands a safe distance from the mouth during insertion and retrieval. Because this method involves directly handling the sharks, it is best used on juveniles or species with a maximum total length of 1–1.2 m, such as those in the dogfish and small coastal shark fishery complexes. It is difficult for any nonlethal method of collecting stomach contents to be as effective as sacrifice and dissection. However, increased sensitivity towards shark conservation is already strongly encouraging the use of nonlethal methods. The results of this assessment suggest that flushed tubes may be an inexpensive and effective means of sampling the diets of dogfish in the field, but the efficiency of the method is dependent on selecting the appropriate tube width for the mouth width of the shark. Pre-measuring the shark’s mouth width and selecting a tube width within 10–20 mm of it will likely provide the highest possible lavage efficiency, and should be standard procedure whenever this method is performed. Acknowledgments The authors acknowledge East Carolina University for financial support, the scientific crew of the NOAA/NMFS R/V Henry B. Bigelow and Mike Pratt and Max Carpman aboard the F/V Perfect C’s for providing vessels and field assistance, Dr. Brad Wetherbee at the University of Rhode Island and Dr. Patrick Harris at East Carolina University for providing guidance on the sample design, and Evan Knight, Daniel Zapf, and Sara Addis for assistance with field sampling, statistical analysis, and editing. Thanks are also due to two anonymous reviewers who were instrumental in improving this manuscript. Literature Cited Barnett, A., K.S. Redd, S.D. Frusher, J.D. Stevens, and J.M. Semmens. 2010. 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