Postprandial response of gastric ph in leopard sharks (Triakis semifasciata) and its use to study foraging ecology

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1 The Journal of Experimental Biology 7, -3 Published by The Company of Biologists doi:1.1/jeb.71 Postprandial response of gastri in leopard sharks (Triakis semifasiata) and its use to study foraging eology Yannis P. Papastamatiou* and Christopher G. Lowe Department of Biologial Sienes, California State University Long Beah, Bellflower Blvd, Long Beah, CA 9 USA *Author for orrespondene at present address: Hawaii Institute of Marine Biology, University of Hawaii at Manoa, PO Box 13, Kaneohe, HI 97, USA ( yannis@hawaii.edu) Aepted 3 Otober 3 Changes in gastri of leopard sharks Triakis semifasiata were quantified as an indiator of feeding frequeny and ration size. Continuous in situ measurements of gastri were made in aptive adult leopard sharks using an autonomous /temperature probe for periods ranging from 1 days. Instrumented sharks were fed meals of squid at different ration sizes. Gastri fluid samples were also taken from noninstrumented juvenile leopard sharks at different time intervals after feeding, and the measured to quantify effets of the probe in the stomah. Continuous in situ measurements of show that empty stomahs have a low of 1.±1. (mean ± S.D.) and that feeding auses Summary a rapid inrease in to 3.11±.71, followed by a gradual derease bak down to baseline levels. There was a positive relationship between hanges in and meal size (r =.7, P=.1). There were no signifiant differenes in between ontinuous in situ and laboratory serial sample measurements. Together these findings indiate that gastri aid seretion may be ontinuous in leopard sharks, and that hanges in gastri may be used to estimate feeding hronology, frequeny and ration size of leopard sharks in the field. Key words: digestion, feeding hronology, aid seretion, gastri evauation, leopard shark, Triakis semifasiata. Introdution Many sharks are apex predators and serve an important role in the transfer of energy from lower trophi levels in the marine eosystem (Wetherbee et al., 199; Lowe, ). However, an understanding of how sharks influene lower trophi levels requires knowledge of their feeding habits, in partiular how muh and how often they eat. Despite numerous studies on the feeding eology and behavior of sharks, little is known regarding the feeding hronology, frequeny of feeding, and daily ration for many shark speies (Medved et al., 19; Cortes, 199; Bush, ). Estimates of feeding hronology and frequeny as well as daily ration, stem from stomah ontent analysis and determination of the state of digestion of ingested prey items (Medved et al., 19; Cortes, 199; Kao, ; Bush, ). Unfortunately, the analysis of stomah ontents requires the apture, killing and extensive sampling of animals within the population. Presently, there is no established non-invasive method of quantifying feeding hronology, frequeny and daily rations of marine etotherms. In ontrast, for marine endotherms, frequeny and timing of feeding events have been made by reording assoiated hanges in stomah temperature using gastri temperature data-loggers or telemetry devies (Wilson et al., 199; Gremillet et al., ; Gunn et al., 1). Unfortunately, this tehnique is only effetive for quantifiation of feeding events for marine etotherms when the body temperature of the ingested prey differs onsiderably from the predator s own body temperature. One harateristi typial of most arnivorous vertebrates is the prodution of hydrohlori aid (HCl) within the stomah. In higher vertebrates, suh as humans, it has been shown that an inrease in HCl seretion rate is initially aused by distention of the stomah lining as food enters, and further regulated by the ativation of seretagogues (e.g. gastrin, histamine and aetylholine), produed by the stomah (Johnson, 19). The aidi fluids sereted by the stomah ause the leavage and onversion of the inative zymogen pepsinogen, into the ative protease enzyme pepsin, initiating the digestion of proteins in the stomah. Although the mehanisms of aid seretion in fishes have not been as well studied, stomah distention (Smit, 197) and seretagogues, inluding histamine and gastrin, are known to play a role in the ontrol of aid seretion after feeding (e.g. Bomgren and Jonsson, 199; Hogben, 197; Vigna, 1979). This suggests that the harateristi hanges in gastri assoiated with meal digestion ould be used to indiate when feeding has ourred. The few studies that have examined feeding-indued hanges in gastri of elasmobranhs have yielded different results. Sullivan (19) and Caira and Jolitz (199) found that

2 Y. P. Papastamatiou and C. G. Lowe Table 1. Summary information for adult leopard sharks Triakis semifasiata fitted with /temperature probes, inluding residene time of probe in the shark s stomah Residene time Water Shark Total length Mass Ration size in stomah temperature number (m) (kg) Sex (%M b) (days) ( C) F 1.,.3, F 1.*, F F F., 1., F., 1.,.1,.1, 1.* M b, body mass. Ration sizes are for meals of squid exept for *, whih represents a meal of apelin (Mallotus villosus). some shark speies with food in their stomahs had aidi values ( 3.), while those with empty stomahs had neutral values ( 7), indiating that aid was only being sereted when food was in the stomah. However, studies by Williams et al. (197) and Menon and Kewalramani (199) on a number of other elasmobranh speies, revealed that the stomah remained aidi (..) regardless of the presene or absene of food, indiating that aid seretion is ontinuous. Unfortunately, none of these studies ontinuously monitored postprandial gastri. Leopard sharks Triakis semifasiata are a well-studied oastal speies of shark, ranging from Oregon to Baja California (Love, 199), and were hosen for this study beause of their abundane, large size and rapid alimation to aptive onditions. The objetives of this study were to (1) obtain ontinuous measurements of gastri, () quantify feeding-indued hanges in gastri, and (3) investigate the possible use of hanges in gastri as an indiator of feeding episodes in free-ranging leopard sharks. Materials and methods Continuous measurements To ontinuously monitor gastri, we used six adult female leopard sharks Triakis semifasiata (Girard ), total length TL 11.±11.9 m (mean ± 1 S.D.), mass 1.1±.1 kg (Table 1). Sharks were maintained in large seawater tanks, either at the Long Beah Aquarium of the Paifi (LBAOP:. 1 l) or the Sripps Institute of Oeanography Marine Laboratory (SIO: l). We ontinuously measured gastri and temperature using an autonomous stomah probe with a pressureequalizing referene eletrode (Peters, 1997a,b). The probe was initially designed to measure gastri and temperature in free-diving seabirds and onsists of a miroeletrode, a referene eletrode with free-diffusion liquid juntion and a data-logger enased within a titanium shell (earth & Oean Tehnologies, Kiel, Germany; Peters 1997a,b). The datalogging probe (11 m m, length diameter; mass in air= g) was programmed to reord gastri and temperature every 3 s. Before deployment, the probe was alibrated using three NBS standard buffers of 1.7,. and.. Further details of probe design and preparation an be found in Peters (1997a,b). Kao () fed adult leopard sharks (>1 m TL; kept at 13 1 C) meals of innkeeper worms (Urehis sp.) and sulpins (Cottidae) at 1 1.7% of the sharks body mass (M b ) and found that it took the sharks 7 3 h to empty their stomahs. Therefore before deploying the probe, we fasted all sharks for a minimum of 3 days to ensure that their stomahs were empty. The first probe deployment was arried out by onealing the probe within a herring, and then feeding the instrumented bait to an adult female shark. However, subsequent attempts to feed the probe to sharks failed beause sharks would not swallow the fish whole. Consequently, all remaining sharks were forefed the probe after the sharks were anaesthetized using MS- (7 mg l 1 ) and a 3 m diameter lubriated PVC pipe was gently inserted through the mouth into the stomah. The probe was then arefully passed down the pipe into the stomah, after whih the pipe was retrated. Sharks were revived, returned to the holding tank, and observed for min to ensure that they had reovered and did not prematurely regurgitate the probe. In all ases, the probe weighed <1.1% of the shark s body mass (M b ) in air. Every 3 days we fed sharks squid meals ranging from.1 to.1% of the shark s M b. Beause gastri evauation rates in sharks are known to vary with temperature (Shurdak and Gruber, 199, Wetherbee et al., 199), water temperatures were reorded in the tanks at the LBAOP and SIO (mean water temperature: 1.±. C). The probe an provide aurate data for periods up to 1 days, depending on eletrolyte outflow rate. Therefore, we removed the probe from the sharks after a 1 day period. Probe removal was arried out by anaesthetizing the sharks as before, reinserting the 3 m diameter PVC pipe into the stomah until the probe was felt, and then tipping the shark s head downwards, allowing the probe to fall out through the pipe. Sharks were then revived and returned to the holding tanks. Two sharks regurgitated the probe after a and 7 day period (Table 1). Immediately after reovery, the probe was realibrated in

3 Postprandial gastri in leopard sharks 7 buffers (1.7,. and.). The data were then downloaded and analyzed using G. software (Jensen Software Systems, Servie earth & Oean Tehnologies) to interpolate and orret for drift of (see Peters, 1997a). The software also orrets for any orresponding hanges in stomah temperature. Measurement auray was determined by using the drift model desribed by Peters (1997a). Resolution of measurements for eah probe deployment was alulated by determining the gradient of the alibration urve before and after deployment. We pooled data for all six sharks into hourly intervals and ompared before and after feeding using a oneway analysis of variane (ANOVA). A Tukey s pairwise omparison was used to determine the loation of any hourly differenes in before and after a meal. The rate of inrease in after a meal, and the subsequent rate of derease after peak had been reahed, were alulated. The two rates were ompared using a t-test for unequal varianes. Titration time is defined as the time taken for gastri to return to baseline following a meal. Titration time was determined using the tehnique desribed by Gardner et al. (), where we alulated the perentage of time within 1 min intervals that remained below.. A of. was determined to be a baseline measurement for humans (Gardner et al., ), and appeared appropriate for leopard sharks as well, based on our examination of the gastri data. We used the 1 min intervals beginning h prior to onsumption of a meal and until returned to baseline levels. The first of two onseutive 1 min intervals, where <. for only % of the interval, was designated P 1. The first of two onseutive intervals, where <. for 9 1% of the time, was designated as P. Titration time was then alulated as P P 1. A linear regression was used to determine any relationship between titration time and meal size (expressed both in kg and %M b ). We alulated the areas under the feeding-indued profiles of gastri using Arview GIS (3.). A linear regression was used to identify the relationship between meal size (as % M b and kg) and the area under the feeding-indued urve. Time-series sampling In order to determine whether the probes themselves were triggering aid seretion in the adult leopard sharks, we quantified gastri hanges in non-instrumented juvenile sharks. For time-series sampling, 1 juvenile leopard sharks were obtained from loal aquaria and maintained in 9 l tanks ontaining reirulating seawater at California State University Long Beah Shark Laboratory. Sharks were measured, weighed, sexed, tagged and allowed a minimum of 1 week to adjust to aptive onditions before starting the experiments (seven females: five males, mean total length.±.3 m and mass.3±. kg). Sharks were fed a meal of anhovies and then fasted for a minimum of 7 h to ensure that their stomahs were empty. Sharks were then fed squid meals at 1% of the shark s M b and gastri fluid subsequently sampled at 1, 7, 1,,, 7 and 9 h following feeding. Gastri fluid samples were obtained by netting sharks and quikly inverting them, whih plaes them into toni immobility. Sharks were removed from the water, tipped head down to drain water from their mouth area, and a flexible ( mm diameter) plasti tube was then inserted through the mouth and into the stomah. We used an attahed syringe to remove. ml of gastri fluid and measured the fluid using a alibrated benh-top meter. After we obtained a fluid sample, the sharks were fed a meal of anhovies and allowed 3 days to reover before a seond sample was obtained at another sampling time, after a subsequent feeding. 3 days of fasting also allowed suffiient time for all food to leave the stomah prior to subsequent feeding events (Kao, ). In this way, eah shark was only sampled one after eah meal on any given day, and therefore eah shark was fed and sampled a total of seven times. The mean water temperature in the shark tank was 1.±. C (range:. 3. C). To asertain the effet of a seond meal on gastri, six sharks were fed a seond meal of squid (at 1% M b ) h after the first meal. 1 h after the seond meal, a sample of gastri fluid was obtained and the measured. In order to ompare probe and laboratory measurements, all laboratory measurements were orreted for temperature to a standardized value of 1 C (mean during probe trials was 1.±. C), using the tehnique desribed by Brower et al. (199). A one-way ANOVA was used to ompare among speifi sampling times (1, 7, 1,,, 7 and 9 h after feeding). A Tukey s pairwise omparison was then used to determine the loation of any pairwise differenes. To determine any effets of the automated probe on aid seretion, we ompared values from sharks fitted with stomah probes that had onsumed squid at 1% M b, to values from juvenile sharks kept in laboratory tanks, using a t- test for equal varianes. A Kolmogorov Smirnov test was used to determine if data sets were normally distributed. Data were ompared for values at 1, 7, 1 and h after a meal. The maximum time interval between meals for sharks that had been fitted with a stomah probe and onsuming a meal of 1% M b, was h. Results Continuous measurements Shark stomah temperatures were very similar to the ambient water temperatures exept during feeding events, when ingestion of frozen squid aused a sudden derease in stomah temperature. Hene, a derease in temperature of.3 1 C was observed during these times. Resolution and auray of temperature measurements were ±.1 C. For all sharks, gastri was relatively aidi ( <.) at all sampling times. Mean gastri 1 h prior to feeding sharks with empty stomahs (i.e. sharks fasted > h) was 1.±1., although there was a large amount of variability among sharks (range:..1). Ingestion of food items indued a rapid inrease in gastri (.1±. units min 1 ) peaking approximately 1 h post-feeding, followed by a more gradual derease of.±. units min 1, until baseline

4 Y. P. Papastamatiou and C. G. Lowe 1 Leo 1 1 Leo Leo?? 1* Leo Leo 3? 1 3 Time (days) Leo 1.1 1* Time (days) 1 Fig. 1. Continuous gastri measurements from six adult female leopard sharks Triakis semifasiata (Leo 1 ) fitted with a /temperature probe. Temperature is depited in grey, in blak. Arrows point to feeding events; numbers above arrows represent meal size (perentage of the shark s body mass).? indiates the point where a meal of unknown mass was onsumed. All meals were squid exept for that marked with an asterisk, where a meal of apelin Mallotus villosus was onsumed. levels were reahed (Fig. 1). The rate of inrease was signifiantly greater than the rate of derease (t-test for unequal varianes, F=., P=.9). After a meal, inreased on average by 1.±.1 units, with the rates of inrease a funtion of meal size expressed either as %M b (r =., P=.) or in kg (r =., P=.). Pooled data for all six sharks showed a signifiant differene in from 1 h before a meal up to h after a meal (Tukey s test, F=.3, P<.1), and had returned to baseline after 7 h. We found the integrated area under the profile to be signifiantly related to meal size, expressed as a %M b (r =.3, P=.7) and in kg (r =.1, P=.). The perentage of time that the remained below. varied signifiantly with meal size. Titration time also signifiantly varied as a funtion of meal size expressed as a perentage of the shark s M b (r =.9, P=., Fig. A) and in kg (r =.73, P=.3, Fig. B). Error analysis of probe data indiated that ontinuous measurements were within a range of ±.1. units for the duration of the deployments, for the majority of sharks (Table ). Resolution of measurements for all sharks was typially. units. Time-series sampling Twelve juvenile leopard sharks were sampled over the seven sampling times; however, not all sharks ould be sampled at some of the later sampling times as insuffiient gastri fluid was obtained for measurements. There was a derease in the of gastri fluid in the hours following a feeding event, with a minimum being reahed between 1 h after the meal (Fig. 3). Leopard sharks had gastri fluids with a mean of 3.31±.3 1 h after feeding, whih had dereased to 1.7±. 9 h after the meal; however there were very small amounts of fluid left in the stomah > h after a meal. A one-way ANOVA revealed a signifiant differene in among the

5 Postprandial gastri in leopard sharks 9 Titration time (min) 1 y=71.x.7 r = Mass of food ingested (%M b ) 1 y=x+1.9 r =.73 1 A B (1) a (1) b (11) (11) (1) Time after feeding (h) Fig. 3. Change in gastri of juvenile leopard sharks following ingestion of squid at 1% body mass. Samples of gastri fluid were removed from the shark s stomah, and readings orreted to a standardized value at 1 C. Values are means ± 1 S.D.; numbers above bars are sample sizes. Bars with the same letter are statistially insignifiant from eah other (see text for details). b, () (3) Mass of food ingested (kg) Fig.. Regression orrelations between titration time and meal size in six adult leopard sharks. Sharks were fitted with /temperature probes and fed meals of squid. Meal size is expressed as both a perentage of the shark s body mass (A) and in kg (B). sampling times (F=., P<.1). A Tukey s pairwise omparison revealed a signifiant differene between the at the 1 h sampling time and the of all subsequent sampling times (P<.1, Fig. 3). In addition, gastri at 7 h postfeeding was signifiantly different from the values at 1,, 7 and 9 h (P<.1). The ingestion of a seond meal, h after the first, aused Table. Summary information of automated /temperature probe performane for eah individual adult leopard shark Triakis semifasiata Shark Resolution Drift error () number Start End Min. Max Resolution before and after deployment is inluded, as well as error analysis over a range of values. the of the gastri fluid to rise (Fig. ). 1 h after the seond meal the was 3.±.3, and this was signifiantly greater than the at all other sampling times (7, 1,,, 7, 9 h) exept at the initial 1 h sampling time (Tukey s test, P<.1). There were no signifiant differenes observed between ontinuous probe and laboratory measurements of gastri 1 h, 7 h, 1 h and h (P>.) after a meal. Disussion Gastri hanges Gastri of leopard sharks was aidi ( <3.) at all times exept immediately following a feeding event. The postprandial inrease in gastri is undoubtedly due to the mixing of ingested food and seawater with the aidi stomah fluids. The of gastri fluid returned to baseline levels, presumably as aid seretion inreased, thereby re-aidifying stomah ontents (James, 197; Johnson, 19). Stomahs of leopard sharks remained strongly aidi ( <.) for at least days following meal ingestion. However, the average gastri evauation time estimated for adult leopard sharks is approximately h (Kao, ), indiating that leopard sharks ontinuously serete aid. Although aid seretion is ontinuous, the variation in aid seretion rates following gastri evauation is unknown. Nevertheless, larger amounts of gastri fluid were obtainable from noninstrumented sharks 1 1 h after a meal, while only very small amounts of fluid ould be obtained 9 h after feeding. Therefore, it is hypothesized that the ingestion of food auses an inrease in aid seretion triggered by the distention of the stomah (Smit, 197), and is further regulated by hemial seretagogues (Hobgen, 197; Vigna, 1979). Similarities in feeding-indued hange between

6 3 Y. P. Papastamatiou and C. G. Lowe (1) a (1) b (11) Time after feeding (h) instrumented and non-instrumented sharks indiate that it is unlikely that the physial presene of the probe within instrumented sharks stimulated long-term additional aid seretion. However, three of the sharks fitted with a probe did exhibit a slight derease in gastri followed by a gradual inrease over a 1 day period following the introdution of the probe; therefore there may be some stimulatory effets of the probe during the first day following deployment. For the smallest shark used (mass = 7.3 kg), a low stomah volume:probe volume ratio may have been responsible for the observed flutuations in gastri, whih made it diffiult to disern feeding events (see Fig. 1; Leo ). In addition to having negligible physiologial effets, the probe also appeared to have negligible behavioral effets on the leopard sharks utilized in this study. Although a majority of the sharks had to be fore-fed the probe, all sharks resumed ative feeding within 1 day of deployment of the probe and behaved similarly to non-instrumented sharks. Digestive physiology Other studies on elasmobranhs have found similar hanges in gastri to those seen in leopard sharks. Two skate speies (Raja lavata and R. naevus) were found to maintain an aidi gastri environment ( 1..), regardless of the presene or absene of food (Williams et al., 197). Babkin et al. (193) found that fasting skates sereted very small amounts of aidi fluid, with gastri ranging from. 3.. Menon and Kewalramani (199) found gastri values for three speies of elasmobranhs (Chillosyllium griseum, Dasyatis warnak, Rhinobatus halavi) of. 3. and 3.3., 1 and 7 days following feeding, respetively, indiative of ontinuous aid seretion. Dobreff (197; ited in Barrington, 197) was able (11) Fig.. Gastri hanges in juvenile leopard sharks fed a meal of squid at 1% body mass M b. Six sharks were fed a seond meal of squid (1%M b) h after the first. The of the gastri fluid was determined 1 h after the seond meal. All readings were orreted to a standardized temperature of 1 C. Values are means ± 1 S.D.; numbers above bars represent sample sizes. Bars with the same letter are statistially insignifiant from eah other (see text for details). () a to obtain small quantities of aidi fluid from the fasting stomah of an elasmobranh for up to 11 days after a meal, although there was a gradual rise in during this period. Not all elasmobranhs exhibit this pattern of gastri response to food onsumption. Sullivan (19) found that empty stomahs of eight different speies of elasmobranhs exhibited neutral values, whereas those ontaining food were aidi. Caira and Jolitz (199) found that nurse sharks Ginglymostoma irratum, olleted in the field with food in their stomahs, had a mean of.±.3, whereas those with empty stomahs had a gastri of 7.19±., indiating that aid seretion may not be ontinuous for this speies. There are also different patterns in pre- and postprandial gastri hanges for teleosts. A similar pattern in feedingindued gastri hanges to those seen in leopard sharks has also been observed for a number of oral reef fishes (e.g. Caranx ignobolis, Aanthurus nigrofusus), where those with empty stomahs had lower gastri values than those ontaining food (Lobel, 191; Montgomery and Pollak, 19). However, some oral reef fishes have been shown to exhibit the opposite trend, with lower gastri when food was present in the stomah (e.g. Aanthurus triostegus; Lobel, 191). A great deal more information exists regarding postprandial gastri for terrestrial vertebrates. Gastri has been measured ontinuously for snakes (Seor, 3), birds (Peters 1997a,b; Gremillet et al., ), ruminants (Enemark et al., 3) and humans (e.g. Evans et al., 19; Gardner et al., ). As with leopard sharks, a rise in gastri with meal ingestion, followed by a derease to baseline levels, has been observed for birds (Duke et al., 197; Peters, 1997a; Gremillet et al., ) and humans (James, 197; Gardner et al., ). Humans ontinuously serete aid, with an unstimulated (i.e. empty) stomah possessing an aid seretion rate 1 % that of a stimulated stomah (i.e. food present). Thus, in the absene of food the human stomah has a of 1.. (Johnson, 19; Evans et al., 19). While the postpandrial pattern of gastri for leopard sharks is similar to that of humans, it differs from that seen in some snakes. The empty stomah of Burmese pythons Python molurus maintains a neutral and beomes aidified upon the ingestion of food items (Seor, 3). These studies suggest that there are differenes in the pattern and mehanisms of gastri aid seretion among vertebrates. The variation in gastri hanges between speies may be due to differenes in methodology, suh as measurement tehnique, sampling interval, meal size and meal omposition. Nevertheless, digestive physiology is likely to be strongly influened by a speies feeding eology. Feeding frequeny has been shown to be responsible for inter-speifi differenes in the digestive physiology for a number of reptile speies (Seor et al., 199; Seor and Diamond, 199) and it is possible that this may be true for fish as well. Continuous seretion of aidi fluid is likely to be energetially expensive, as suggested by the high number of mitohondria found within oxynti and oxyntopepti ells (Seor, 3; Rebolledo and Vial, 1979), and the reasons for

7 Postprandial gastri in leopard sharks 31 maintaining a ontinuously aidi gastri environment are unresolved. It has been suggested that humans ontinuously serete gastri aid to prevent growth of pathogeni baterial flora on the empty stomah muosa (Seor, 3), although there have been few quantitative studies to test this hypothesis. We therefore propose two additional hypotheses to explain the ontinual seretion of gastri aid in leopard sharks. Elasmobranhs are the earliest known vertebrates to possess a stomah apable of sereting aid (Smolka et al., 199), and ontinual maintenane of a low gastri in leopard sharks may be a primitive mehanism to inrease gastri evauation rate, ausing a more rapid return of appetite (Wetherbee et al., 199; Sims et al., 199). This may be important, as many sharks are known to be opportunisti in their feeding habits (Wetherbee et al., 199), and maintaining low gastri would failitate rapid digestion of a subsequent meal. 1 h after a meal, the in the leopard shark stomah ranges from. to., and this may be an optimum range to ause both the onversion of pepsinogen into pepsin (ours at <.; Johnson, 19) and an inrease in aid seretion rates through the ation of aid-stimulating seretagogues suh as gastrin. In mammals, gastrin is sereted when gastri rises above 3. (Johnson, 19). Although there is limited information on gastrin seretion and its regulation in fish, od have been shown to exhibit a higher gastri aid seretion rate when stomah ontent is inreased (Bomgren and Jonsson, 199). In addition, the exogenous introdution of gastrin has been shown to stimulate inreased aid prodution by the isolated dogfish muosa (Vigna, 193). Maintenane of low gastri may also be attributed to the energeti ost of regulating aid sereting oxynto-pepti ells. Based on the gastri evauation time for adult leopard sharks and the perentage of sharks aught with empty stomahs, it appears that leopard sharks feed approximately one every 3 h (Talent, 197; Kao, ). Therefore, for leopard sharks, the time interval between gastri emptying and the introdution of a new meal is relatively short, hene it may be energetially more expensive to ompletely downregulate and then upregulate aid seretion, than to merely redue the aid seretion rate. Similarly, snakes that feed frequently are found to maintain their intestinal trat in a state of physiologial readiness (Seor et al., 199), in antiipation of the next meal. Continuous gastri aid seretions may plae onsiderable strain on the gastri muosa. The mehanisms by whih the gastri muosa in leopard sharks resists damage from the aid are unknown. Quigley and Turnberg (197) found that in humans there was a steep gradient in ranging from the stomah lumen aross the muus membrane. While the lumen ontents were always aidi (.1±.17), the mean juxtamuosal was.±.37 and was often lose to neutral. Although the of the leopard shark gastri muosa was not measured in this study, a muus layer ould play a similar role in proteting the gastri muosa for the leopard shark. Appliation for studying foraging eology In addition to furthering the urrent knowledge of gastri aid seretion for an elasmobranh fish, this study also demonstrates the potential use of ontinuous gastri measurements as a tool in the study of leopard shark foraging eology. Presently the only tehnique used to diretly quantify feeding events and meal size in marine endotherms is in situ measurement of stomah temperature. Stomah temperature is relatively easy to measure, and hanges markedly as food enters the stomah (Wilson et al., 199; Gremillet et al., ; Gunn et al., 1; Klimley et al., 1). However, hanges in stomah temperature an only be observed if the predator has a different body temperature than that of its prey. This obviously limits this tehnique, given the lak of a temperature differene between etothermi fishes and their etothermi prey. In ontrast, gastri hanges when food items are ingested, independent of prey temperature. The observed feeding-indued hanges in gastri for leopard sharks made it easy to identify individual feeding events and to distinguish them from bakground flutuations in gastri. Disrete feeding events ould be resolved within h of eah other (Fig. 1; Leo 3), whih is important beause some speies of sharks may eat multiple meals over a short period before fasting for a number of days (Cortes and Gruber, 199; Wetherbee et al., 199). Another finding of this study was the orrelation between gastri hange and meal size. The larger the meal, the greater the volume of aid that needs to be sereted and the longer the duration before gastri returns to baseline values. However, titration time and hanges are likely to be strongly affeted by hanges in gastri evauation times. Gastri evauation rates in sharks are temperature dependent (Shurdak and Gruber, 199; Wetherbee et al., 199; Bush, ), and also sensitive to food type (Jakson et al., 197), prey energy ontent (Wetherbee and Gruber, 199), and prey fat ontent (Shurdak and Gruber, 199). Changes in titration times may be partiularly suseptible to rustaean prey items ontaining exoskeletons and hard parts (see Jakson et al., 197) and further studies are required to determine how different food types an effet gastri hanges. This study demonstrates that the monitoring of gastri an be used to quantify feeding hronology, frequeny and daily ration of leopard sharks in the field. Inorporation of the probe with a telemetry transmitter (aousti or radio) would allow researhers to ontinuously monitor the gastri of free-ranging animals and identify those hanges in assoiated with feeding (see Nelson, 197, 199; Lowe and Goldman, 1). We would like to thank E. Forsman and the Long Beah Aquarium of the Paifi (LBAOP) and E. Kisfaludy with the Sripps Institute of Oeanography (SIO) for use of their aptive adult leopard sharks, and the Roundhouse Aquarium (Manhattan Beah) for supplying juvenile sharks for this study. Thanks go to G. Peters of earth & Oean Tehnologies for tehnial support and advie. We would also like to aknowledge L. Berg, D. Cartamil, D. Topping, G. Moss, G. Hoisington, M. Neilson, M. Marotte, K. Forsgren, J. Vaudo

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