Prey Envenomation Does Not Improve Digestive

JOURNAL OF EXPERIMENTAL ZOOLOGY 307A:568–577 (2007)

Prey Envenomation Does Not Improve Digestive Performance in Western Diamondback Rattlesnakes (Crotalus atrox) MARSHALL D. MCCUE Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas

ABSTRACT

Although the toxic properties of snake venoms have been recognized throughout history, very little is known about the adaptive significance of these powerful mixtures. This study examined the popular hypothesis that prey envenomation enhances digestion by influencing the energetic costs of digestion and assimilation, gut passage time, and apparent assimilation efficiency (ASSIM) in western diamondback rattlesnakes (Crotalus atrox), a species whose venom is recognized for its comparatively high proteolytic activities. A complete randomized block design allowed repeated measures of specific dynamic action and gut passage time to be measured in eight snakes ingesting four feeding treatments (i.e., artificially envenomated live mice, artificially envenomated prekilled mice, saline injected live mice, and saline injected prekilled mice). A second experiment measured ASSIM in eight snakes ingesting a series of six artificially envenomated or six saline injected mice meals over an 8-week period. Contrary to expectations, the results of both these experiments revealed that envenomation had no significant influence on any of the measured digestive performance variables. Gut passage time averaged 6 days and ASSIM averaged 79.1%.

Twenty-one hours following ingestion, postprandial metabolic rates exhibited factorial increases that averaged 3.9-fold greater than resting metabolic rate. Specific dynamic action lasted on average 88 hr and accounted for 26% of the total ingested energy. The results of this study reinforce the need to systematically examine the potential adaptive advantages that venoms confer on the snakes that produce them. J. Exp. Zool. 307A:568–577, 2007. r 2007 Wiley-Liss, Inc. How to cite this article: McCue MD. 2007. Prey envenomation does not improve digestive performance in western diamondback rattlesnakes (Crotalus atrox). J. Exp. Zool.

307A:568–577.

For decades scientists have speculated about the various adaptive functions of snake venoms (Zeller, ’48; Gans and Elliott, ’68; Pough and Groves, ’83; Hayes et al., ’95; Daltry et al., ’96; Kardong, ’96). The most popular theories suggest that snake venoms serve chiefly predatory, defensive, and/or digestive functions. Although each of these hypotheses are supported by a long history of anecdotal evidence, because of the obvious logistical difficulties in dealing with venomous snakes, very few studies have attempted to investigate systematically the specific advantages that venoms concede to the relative fitness or life histories (sensu Dunham et al., ’89; Boggs, ’92; Zera and Harshman, 2001) of venomous snakes.

The ‘‘defensive hypothesis’’ suggests that venom functions to release snakes from predation pressures (Bogert, ’43; Ruben, ’76; Saint-Girons, r 2007 WILEY-LISS, INC.

’97), but no studies have formally examined whether venomous and nonvenomous species are subjected to differential predation pressures.

Moreover, this hypothesis does not address how snake predators may be able to ‘‘learn’’ to identify venomous from nonvenomous snake species, a lesson that is likely to be lethal to potential predators. The ‘‘predatory hypothesis’’ suggests that the primary function of snake venoms is to kill or otherwise incapacitate prey items that could somehow injure the snake (Kardong, ’75;

Grant sponsor: NSF-GRF and Walton Distinguished Doctoral Fellowship.

Correspondence to: Marshall D. McCue, Department of Biological Sciences, 601 Science Engineering, University of Arkansas, Fayetteville, AR 72701. E-mail: mmccue@uark.edu Received 10 April 2007; Revised 16 May 2007; Accepted 2 July 2007 Published online 1 August 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jez.411

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Kardong, ’86; Hirabayashi et al., ’91; Daltry et al., ’96; Andrade and Abe, ’99). Although the temporal connection between prey envenomation and subsequent ingestion is clear to anyone who watches a captive venomous snake at ‘‘feeding time,’’ this hypothesis does not address the fact that constriction, which is the ancestral mode of prey immobilization (Greene and Brughardt, ’78; Kardong, ’80; Savitzsky, ’80; Greene, ’83), is still employed by a variety of extant species (Gans, ’61; Franz, ’77; Shine and Schwaner, ’85; Rochelle and Kardong, ’93; Moon and Mehta, 2007). Moreover, no studies have examined the fitness of venomous snakes that have been deprived of their ability to subdue their prey using envenomation. The ‘‘digestive hypothesis’’ suggests that the proteolytic activities of snake venoms function to increase digestive performance among venomous snakes (Gans, ’61; Pough and Groves, ’83; Andrade and Abe, ’99; Kini and Chan, ’99; Sasa, ’99), but remains unable to explain why the venoms of different species vary so widely with regard to their proteolytic activity (Zeller, ’48; Deutsch and Diniz, ’55; Oshima and Iwanaga, ’69; Passey, ’69; Mebs, ’70; Kocholaty et al., ’71; Geiger and Kortmann, ’77). Given the dearth of studies that have attempted to examine the potential function of snake venoms, this study was designed to investigate the digestive hypothesis in a snake species (i.e., Crotalus atrox), whose venom is recognized for its high proteolytic activity.

Early morphological and histological observations revealed that the venom glands of snakes were structurally homologous to mammalian parotid glands (Kellaway, ’37). This discovery, combined with observations that many snake venoms are rich in proteolytic activity (see Zeller, ’48), most likely led to the first speculations that snake venom served a functional role in prey digestion. Interestingly, the only contemporary observations that support the ‘‘digestion hypothesis’’ were not examined systematically, but rather were based on informal observations or were communicated anecdotally. For example, Reichert (’36) stated that pitvipers consuming live prey items required only 4–5 days to digest meals compared with the 12–14 days they required to process prekilled meals (cited in Thomas and Pough, ’79). A second observation was provided

by C. Stimmler–Morath who apparently noticed that the duration of digestion increased from 3 days after envenomating a meal to 5–8 days when vipers were force-fed prekilled meals (unpublished observation cited in Zeller, ’48). A limited number

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of recent studies has examined the digestion hypothesis (Thomas and Pough, ’79; RodriguezRobles and Thomas, ’92; McCue, 2002) but employed snakes belonging to the family Colubridae, which are either nonvenomous or produce comparatively small amounts of toxic secretions that lack the proteolytic activities characteristic of true-vipers and pitvipers (Weinstein and Kardong, ’94; Mackessy, 2002; Vidal, 2002; Fry and Wuster, 2004).

The only modern study to employ venomous snakes to examine the effect of prey envenomation on digestive performance consists of an abstract published by Mendes and Abe (’99). The researchers examined the metabolic costs of digestion, [i.e., specific dynamic action (SDA)] in a South American rattlesnake ingesting mice that were either artificially envenomated or freshly killed via cervical dislocation. Although they concluded that envenomation had little, if any, effect on SDA, they did not examine the possibility that prey envenomation might influence the gut passage rate or assimilation efficiency in snakes. Given the working hypotheses that envenomation increases digestive performance in C. atrox (e.g., by increasing the gut passage rate, reducing costs of digestion, or increasing assimilation efficiency), the goal of this study was to quantify the influence of prey envenomation on several digestive performance traits. The two experiments described in this study measured physiological performance variables that have clear connections to the life histories of venomous snakes.

The first experiment measures metabolic expenditure during digestion and gut passage time in C. atrox subjected to four feeding treatment levels. An understanding of the degree to which prey envenomation influences the costs associated with digestion has significant ecological importance because the costs of meal processing can account for approximately one-third of the annual energy expenditure of some pitvipers (McCue and Lillywhite, 2002). The fitness consequences of the passage rate are also ecologically important because increased gut passage rates would allow snakes to process an increased number of prey items during a limited feeding season.

The second experiment investigates the effects of assimilation efficiency in two groups of snakes consuming prekilled mice injected with either venom or saline. Although the apparent assimilation efficiency (ASSIM) of snakes is comparatively high ranging from 80 to 95% (Smith, ’76; Greenwald and Kanter, ’79; Reichenbach and Dalrymple, ’86; Bedford and Christian, 2000; J. Exp. Zool. DOI 10.1002/jez

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Canjani et al., 2003), any improvement in assimilation efficiency should translate to increased mass and energy gains from a given meal. The ultimate effects of increased passage rate, increased assimilation efficiency, and reduced costs of meal processing could provide important insight into the adaptive role that venom plays among snakes.

METHODS

Metabolic and passage experiments Eight subadult, western diamondback rattlesnakes (C. atrox) were employed in the SDA and gut passage experiments. Before experimentation, the snakes were housed individually and allowed to acclimate to laboratory conditions of 27–301C and 12L:12D photoperiod for a minimum of 3 months and a maximum of 6 months. During the acclimation period, snakes were fed prekilled young adult mice every 10–14 days and provided water ad libitum.

A randomized block design with four treatment levels was used so that each of the eight snakes could serve as its own control. The four treatment levels [i.e., live venom, (LV), dead venom (DV), live no-venom (LNV), and dead no-venom (DNV)] involved feeding snakes similar-sized mice derived from the same feeder colony. These experimental mouse meals were offered to snakes at 14-day intervals ensuring that snakes were fully postabsorptive at the start of each feeding trial (Stevenson et al., ’85; Overgaard et al., 2002; Holmberg et al., 2003; Zaidan and Beaupre, 2003; Dorcas et al., 2004).

In the LV treatment level, a 26-g needle was used to inject 20 mg of lyophilized C. atrox venom (Kentucky Reptile Zoo) reconstituted by dissolving it in 0.2 mL of 0.9% saline warmed to 371C into the tail vein of a live adult mouse (Bolyn, ’40; Russell and Eventov, ’64; Tu et al., ’69; Ownby and Colbeg, ’88). Lyophilized venom, obtained during the year before beginning these experiments, was used because it could be conveniently reconstituted when needed and because it has been shown to retain its pharmacological properties for over 50 years (Russell and Eventov, ’64). The amount of venom used in these experiments was similar to that expended during normal predatory strikes in rattlesnakes (Hayes, ’92), but less than the total venom stores found in similar-sized rattlesnakes (Minton, ’57; Jimenez-Porras, ’61; McCue, 2006a).

Approximately 2 min after the venom injection, each mouse was also labeled with a fluorescent J. Exp. Zool. DOI 10.1002/jez

tracer (Scientific Marking Materials Inc., Seattle, WA), which allowed to track the gut passage rates of each meal. To accomplish this, a 1.0 mL saline suspension containing 50 mg of the indigestible, fluorescent tracer in one of four colors, specific to each treatment group, was injected intraperitonealy (IP) using a 16-g needle into each mouse.

The labeled mice were then placed near each snake in a manner such that the snakes did not strike at the meals, but were able to locate and consume them. In most cases snakes consumed the experimental meal within ca. 30 min.

The LNV treatment level was similar to the LV treatment except that no venom was added to the 0.2 mL intravenous (IV) saline injections. The mice were then euthanized via cervical dislocation, and injected IP with a unique color of the fluorescent tracer suspension described above.

Approximately 20–30 min before ingestion, snakes in the LNV treatment level were anesthetized using isoflurane (IsoFloTM, Abbot Laboratories, North Chicago, Il). A previous study demonstrated that metabolic rates are not altered significantly in rattlesnakes that have recently recovered from IsoFlo-induced anesthesia (McCue, 2006a). The anesthetized snakes were then placed into clear acrylic tubes appropriate for their size, and 18inch forceps were used to gently force feed the experimental meals to snakes, thus ensuring that venom was not inadvertently introduced into the meals during the normal ingestion process.

The DV treatment level utilized mice that were previously euthanized via cervical dislocation.

These mice were warmed to ca. 371C and IV injected with 0.2 mL of the reconstituted venom solution and 1.0 mLLLof the fluorescent tracer IP. The DV mice were then placed near snakes for them to consume. The DNV treatment level consisted of a frozen mouse that was warmed as described above, and injected IV with saline solution and IP with the tracer suspension. Like the LNV treatment, the DNV mice were gently force fed to anesthetized snakes.

Upon ingesting the experimental meals, snakes were placed individually into ca. 2-L metabolic chambers where their rates of oxygen consumption were recorded hourly for 120 hr using a multiplexing flow-through respirometry system contained within an environmental chamber set to 301C. All metabolic trials began and ended at 120072 h to minimize any effects of diel variation in metabolic rate on the SDA measurements (Beck, ’95; Beaupre and Duvall, ’98; Hopkins et al., ’99; Beaupre and Zaidan, 2001). Carbon

PREY ENVENOMATION AND DIGESTIVE PERFORMANCE

dioxide-free atmospheric gas was passed through metabolic chambers at constant flow rates that were monitored daily using a mass flow meter.

The oxygen concentrations of excurrent gas from each chamber were measured using a Sable Systems FC–1TM (Las Vegas, NV) oxygen analyzer connected to a computer-controlled multiplexer that serially subsampled the excurrent gas from eight chambers each hour. During each trial, at least one respirometry chamber was left empty to monitor the baseline incurrent oxygen concentration. Flow rates through respirometry chambers averaged approximately 150 mL/min, and oxygen concentrations in the chambers never fell below 20%. At the end of each trial, rates of oxygen consumption were calculated at hourly intervals using the following equation modified from Withers (1977), Oconsumption ¼

½Oi Oe E 1 ½Oi

where Oconsumption is the aerobic metabolic rate (O2 mL/hr), E is the mass flow of gas stream passing through respirometry chambers, and O is the fractional concentration of oxygen in incurrent (i) and excurrent (e) gas streams.

Body masses and baseline resting metabolic rates (RMRs) were determined on postabsorptive snakes over a 48-hr period to get starting values, and SDA was calculated using each snake’s respective RMR. For example, the metabolic scope of SDA (i.e., SCOPE) was calculated as the highest measured metabolic rate for each postprandial snake in each treatment level divided by its mean RMR (Machida, ’81; Andrade et al., ’97; Overgaard et al., ’99; Secor and Diamond, 2000; McCue, 2006b). The time for peak SDA (i.e., PTIME) was determined as the length of time required for each snake to reach its highest postprandial metabolic rate. The duration of SDA (i.e., DURAT) was calculated as the time following a meal at which the postprandial metabolic rate of an individual returned to within the two standard deviations (i.e., ca. 95% confidence interval) of its mean RMR (Dorcas et al., 2004; McCue et al., 2005). The energetic cost of SDA (i.e., SDA-KJ) was quantified as the summation of differences between measured postprandial metabolic rates and mean postabsorptive metabolic rates each hour for 120 hr according to the following equation, " # 120 X ðSDAn RMRÞ E: n¼1

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where SDA is defined as the postprandial metabolic rate at hour n, RMR is the mean postabsorptive metabolic rate, and E is an energy conversion factor (20.13 kJ/LO2; Jobling, ’81). It should be noted that the initialism SDA is used throughout this paper to refer generally to the phenomenon of SDA, whereas the term SDA-KJ refers specifically to the energetic costs associated with SDA. The coefficient of SDA (i.e., COEFF) was calculated by dividing the SDA-KJ by the energetic content of each mouse meal assuming 7.02 kJ/g wet mass (McCue et al., 2005), and multiplying the resultant by 100 to convert it to percentage (Guinea and Fernandez, ’97; Secor and Diamond, 2000; Roe et al., 2004; McCue, 2006b).

Metabolic chambers were examined twice daily under black light to determine the gut passage time (i.e., PASS). If a snake defecated in its metabolic chamber during respirometric measurements, the chamber was cleaned and the snake was replaced into the chamber for the remainder of the measurement period. After 120 hr, the snakes were returned to their respective cages that were then checked under a black light every 24 hr over the subsequent 4 days. Response variables (i.e., SDA-KJ, COEFF, SCOPE, PTIME, DURAT, and PASS) were compared among the four treatment levels using repeated measures analysis of variance (ANOVA) ; P-values r0.05 were considered significant. All statistical tests were conducted using [StatViews (SAS), Cary, NC]. Repeated measures responses are presented in the text as mean7standard error and the results pooled among treatments are presented as mean7standard deviation.

Assimilation experiment Eight subadult wild-captured C. atrox were employed in assimilation experiments. Like the SDA and passage experiment, snakes were housed individually and acclimated to the laboratory conditions described above for a minimum of 3 months. After 14 days of fasting, the snakes were considered postabsorptive. Individual snakes were then assigned randomly to one of the two feeding treatment groups (i.e., envenomated and nonenvenomated) and were allowed to consume one thawed feeder mouse every week over a 42-day period. Snakes in the envenomated treatment group ingested adult mice that had been killed via CO2 asphyxiation and IV and IP injected with 0.5 mL of reconstituted C. atrox venom (1 mg dry venom per1 mL 0.9% saline). Snakes in the noneJ. Exp. Zool. DOI 10.1002/jez

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RESULTS The C. atrox used in the SDA and gut passage rate experiments had mean masses of 200747 g and RMRs of 17.477.2 mL O2/h. Meal masses averaged 20.370.3 g and did not differ significantly among the four treatment levels (P 5 0.557). The SDA-KJ and the COEFF averaged 35.8 kJ and 26%, respectively, and did not differ among treatment levels (Table 1). The SCOPE and the PTIME averaged 3.9 and 21.9 hr, respectively, and did not differ among treatment levels (Fig. 1). The duration of the SDA response was not statistically different among the four treatments and averaged 88 hr (Table 1). The PASS was not compared statistically among all snakes at all treatment levels because two snakes failed to defecate before the end of the 9-day observation period. One snake retained feces in excess of 9 days following the LV, LNV, and DV J. Exp. Zool. DOI 10.1002/jez

TABLE 1. Mean and standard errors of measurements made in the SDA and gut passage experiments LV Meal (g) SDA-KJ (kJ) COEFF (%) SCOPE PTIME (hr) DURAT (hr)

19.7 35.5 26 3.9 22.6 77.8

(0.5) (3.5) (3) (0.2) (0.7) (6.3)

LNV 20.7 35.1 24 3.9 22.1 81.5

(0.5) (2.8) (2) (0.3) (0.9) (4.6)

DV 20.3 35.3 25 3.8 19.5 92.9

DNV

(0.5) (3.1) (2) (0.3) (0.7) (7.5)

20.3 37.3 30 3.9 23.5 98.1

(0.6) (5.0) (4) (0.3) (2.5) (6.6)

Repeated-measures analysis of variances revealed no significant treatment effects.

SDA, specific dynamic action; LV, live venom; LNV, live no-venom; DV, dead venom; DNV, dead no-venom; SDA-KJ, energetic cost of SDA; COEFF, coefficient of SDA; SCOPE, metabolic scope of SDA; PTIME, time for peak SDA; DURAT, duration of SDA.

70 60

L-V L-NV

50

D-V D-NV

40 30 20 10 0 VO2(ml/h)

nvenomated treatment group ingested prekilled mice that were injected with 0.5 mL of physiological saline. All the mice were presented to the snakes in a manner such that they were unable to strike the prey before locating and ingesting the meals.

Egesta were periodically removed from each cage during the 6-week trial and for an additional 14 days after each snake’s final experimental meal to ensure their guts were cleared. All voided material was dried at 801C in an oven for approximately 72 hr (Golley, ’61; Paine, ’71).

Egested material from each snake was physically pooled and ground to a fine powder using a mortar and pestle. Six to twelve subsamples (ca. 60 mg) of the homogenized egesta from each snake were pelleted and combusted in a microbomb calorimeter (Gentry Instruments, Aiken, SC) calibrated with benzoic acid pellets of known mass.

Ash content of voided material was determined by weighing the uncombusted residue from each calorimetry sample. ASSIM was calculated by dividing the ingested energy by the energy content of each diet assuming 7.02 kJ/g wet mass. ASSIM and ash content of egesta (i.e., ASH) were compared between the two treatment levels using an unpaired t-test as well as an analysis of covariance using ‘‘energy ingested’’ as the covariate to minimize the risk of spurious autocorrelation associated with measurements of ASSIM efficiency (Packard and Boardman, ’88; Raubenheimer, ’95; Packard and Boardman, ’99); P-values r0.05 were considered significant.

0

24

48

72

96

120

80 70

L-V L-NV

60

D-V

50

D-NV

40 30 20 10 0 0

24

48 72 Time (h)

96

120

Fig. 1. Postprandial metabolic responses measured in two rattlesnakes ingesting four experimental mouse meals. The horizontal line represents mean resting metabolic rate for each snake and the gray region represents the 95% confidence interval of postabsorptive metabolic rates.LV, live venom; LNV, live no-venom; DV, dead venom; DNV, dead no-venom.

treatments, and another snake retained feces for Z9 days following the DV and the DNV treatments. When those trials were excluded from

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analyses, the average passage time was 6 days (n 5 27); analysis of variance revealed no significant difference in PASS among the four treatment levels (P 5 0.834).

The masses of the two C. atrox assimilation groups did not differ significantly (P 5 0.405) and averaged 233717 g (n 5 8). Moreover, the total energy ingested by each group did not differ significantly (P 5 0.234) and averaged 1098 kJ (Table 2). According to an unpaired t-test, neither ASSIM nor ash content of egesta differed significantly between envenomation treatments (P 5 0.814 and P 5 0.646, respectively) averaging 79.174.8% and 16.872.5%, respectively. The results of the analysis of covariance also revealed that the ASSIM of the two envenomation treatments did not differ significantly (Pslope 5 0.913, Pintercept 5 0.617).

DISCUSSION The western diamondback rattlesnake (C. atrox) was selected as a model organism in which to investigate the influence of prey envenomation on digestive performance because its venom is among the best pharmacologically characterized (Minton and Weinstein, ’86; Baramova et al., ’91; Hirabayashi et al., ’91; Hung and Chiou, ’94; Fox and Bjarnason, ’95) and possesses some of the greatest enzymatic activities found among snake venoms (Oshima and Iwanaga, ’69; Passey, ’69; Mebs, ’70; Kocholaty et al., ’71; Russell, ’72; Geiger and Kortmann, ’77). Given the well-known proteolytic and necrotizing properties of rattlesnake venoms (Michaelis and Russell, ’63; Homma and Tu, ’71; Pereira et al., ’71; Huang and Perez, ’82; Nakada et al., ’84; Ownby and Colbeg, ’88; Rodriguez-Acosta et al., ’99; Salvini et al., 2001), the finding that envenomation did not influence any of the measured digestive performance variables was surprising, although not unprecedented (see Mendes and Abe, 1999).

Several factors might help explain the results of the experiment examining the effect of envenomation on the cost of digestion and gut passage rates.

First, it is possible that the digestion temperature of 301C was too great to detect any significant influence of envenomation on digestive performance. In their study examining qualitative rates of digestion in artificially envenomated prey items fed to nonvenomous snakes, Thomas and Pough (‘79) found that the digestive advantages conceded by prey envenomation were more dramatic at 151C compared with 251C. They subsequently speculated that prey envenomation might allow

TABLE 2. Mean and standard deviation of responses measured in snakes digesting envenomated and nonenvenomated prey Envenomated Snake (g) Ingest (kJ) ASSIM (%) ASH (%)

228 1048 80 16

(20) (98) (4) (3)

Nonenvenomated 239 1148 79 17

(14) (115) (6) (3)

Envenomation had no significant effect on meal assimilation efficiency.

ASSIM, apparent assimilation efficiency; ASH, ash content of egesta.

pitvipers to digest meals at cooler temperatures, and thus inhabit more extreme elevations and latitudes compared with most other snake taxa; however, this hypothesis has yet to be examined formally. Second, it is possible that increased digestive performance was not detected because the meal sizes used in these experiments were too small. Although the snakes in this study were allowed to consume adult mice, pitvipers are known to be able to ingest prey items in excess of 50% of their own body mass (Beavers, ’76; Greene, ’83; Pough and Groves, ’83; Forsman and Lindell, ’93; Andrade et al., ’97). It has been suggested that venom may only confer digestive advantages when exceptionally large meals are consumed and the low ratio of surface area to volume necessitates additional digestive surfaces (Pough and Groves, ’83), but again, this possibility remains unexamined.

A third possibility is that the enzymatic and proteolytic activities of C. atrox venom did function to break down prey items more rapidly, congruent with the previous observations of Thomas and Pough (’79). Although a trend of reduced SDA duration was observed in snakes consuming envenomated prey, this trend remained statistically insignificant. Interestingly, there was no apparent energetic cost reduction associated with prey envenomation. A recent study by McCue et al. (2005) revealed that the SDA-KJ in pythons resulting from the digestion of a whole mouse meal was not significantly reduced when an equal-sized meal was pureed and force fed to snakes; in fact, the SDA-KJ was slightly increased in the pythons digesting and assimilating the pureed meals. The same study concluded that the majority of metabolic expenditure associated with SDA resulted from the energetic costs of protein synthesis; if this was also the case with the rattlesnakes, the influence that prey J. Exp. Zool. DOI 10.1002/jez

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envenomation might have on net metabolic cost of digestion would, therefore, be minimal.

Even if SDA and assimilation efficiency were not influenced by envenomation, it would still be reasonable to expect to see an increase in gut passage rate in snakes consuming envenomated meals; such a result was also not observed.

Although the fluorescent tracer was used to track the ingested food bolus, previous studies have reported that egestion events are not always predictably correlated with ingestion events. For example, some snakes are noted for their tendency to retain a large bolus of digested materials in their distal large intestine for periods lasting as long as several months (Lillywhite et al., 2002).

A previous study investigating the SDA in postprandial pythons also reported that egestion events occurred irregularly despite a regular feeding regime (Overgaard et al., 2002). Although the fluorescent tracer proved to be an effective tool for identifying which egested bolus was associated with which particular meal, it was less suited to interpret results of irregular egestion events. One solution to this problem might be to label food with a radioopaque tracer and conduct periodic X-ray measurements on postprandial snakes to determine when undigested matter first enters the distal large intestine (Stevenson et al., ’85; Dorcas et al., ’97).

The inability to detect increased assimilation efficiency in envenomated prey could be related to the fact that venom was administered to prekilled mice, and was therefore unable to circulate, if only for a few seconds, throughout the bloodstream of the prey items. Although necrosis is often observed at local sites of envenomation, venom hyaluronidase activity and circulatory transport are known to be critical in distributing venom components throughout distal prey tissues (Russell, ’72; Schiff et al., ’84; Kasturi and Gowda, ’89). Although the snakes in the assimilation experiment were not allowed to strike and envenomate prey items, a second possibility is that a significant amount of venom was introduced into the prey items during the ingestion process.

The role of the surrounding musculature on the mechanics of venom delivery has been studied in defensive and predatory strikes (Young and Zahn, 2001; Young et al., 2003), but nothing is known about the amount of venom that is released by snakes that are using the same muscle groups to ingest prekilled prey items.

This study failed to find any support for the popular long-standing paradigm that snake J. Exp. Zool. DOI 10.1002/jez

venoms facilitate digestion, by increasing metabolic, assimilatory, and passage rates in a single pitviper species. Given this outcome, it may be prudent to further examine the generality of these findings by investigating the influence that prey envenomation may have on the digestive performance of other venomous snakes that are distantly related to rattlesnakes (e.g., true-vipers and members of the family Elapidae). A promising species to conduct similar studies on is the king cobra (Ophiophagus hannah), the venom of which possesses the greatest enzyme activities found among elapid snakes (McCue, 2005). If forthcoming studies confirm that venom concedes limited or no digestive advantages to other snakes, it could lead to an important paradigm shift regarding the adaptive significance of snake venoms. Such a shift would thus underscore the need for controlled studies that characterize the defensive and predatory functions of snake venoms.

ACKNOWLEDGMENTS Experiments were conducted in accordance with the University of Arkansas Institutional Animal Care and Use Committee protocol 05011. Thanks also to J. Agugliaro, Dr. K.E. Cano-McCue, and two anonymous reviewers for helpful comments on this manuscript, as well as Prof. S. Beaupre for providing the laboratory space required for this study.Funding was provided by a NSF-Graduate Research Fellowship and a Walton Distinguished Doctoral Fellowship awarded to MDM. LITERATURE CITED Andrade DV, Abe AS. 1999. Relationship of venom ontogeny and diet in Bothrops. Herpetologica 55:200–204.

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