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ABSTRACT
Allhough the toxic properties of snake venoms have been recognized throughout
history, very little is known about the adaptive significance of these powerful mixtures. Thill 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
(ASSlM) in western diamondback rattlesnakes (Crotalus otrox), 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 injeeted prekilled mice). A second experiment
measured ASSTM 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 lhan 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
2007 Wiley-Liss, Inc.
produce them. J. Exp. 7..001. 307A-568-577. 2007.
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.
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M.D.MCCUE
Canjani et aI., 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 rattle
snakes (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 27-30"C
and 12L: 120 photoperiod for a minimum of 3
months and 8 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 li.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 aI., 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 37C into the
tail vein of a live adult mouse (Bolyn, '40; Russell
and Eventov, '64; Tu et aI., '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, 2oo6a).
Approximately 2 min after the venom injection,
each mouse was also labeled with a fluorescent
J. Exp. Zool. DOl 1O.1002Jjez
[0, - O,IE
Ooonsumption
1 - rOd
~ (SDA" - RMR) E.
571
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 (20.13kJIL0 2 ; 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 (Le., 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 a1., 2004; McCue, 200Gb).
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., SDAKJ, COEFF, SCOPE, PTIME,
DURAT, and PASS) were compared among the
four treatment levels using repeated measures
analysis of variance (ANOVA) ; P-values ::;0.05
were considered significant. All statistical tests
were conducted using [StatView<E (SAS), Cary,
NC]. Repeated measures responses are presented
in the text as mean standard error and the
results pooled among treatments are presented
as mean standard 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 (I mg dry
venom perl mL 0.9% saline). Snakes in the noneJ. Exp. Zool. DOl 1O.IO02/jez
572
M. D. MCCUE
LV
Meal (g)
SDA-KJ Ow)
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)
LNY
20.7
35.1
24
3.9
22.1
81.5
<0.5)
(2.8)
(2)
(D.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)
70
60
-L-V
oL-NV
'0
aO-V
~O-NV
40
30
20
'0
0
...
80
70
0>
24
48
72
96
120
-L-V
oL-NV
aO-V
60
~D-NV
'0
40
30
20
10
24
46
72
96
120
Time (h)
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.
573
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 pharmaoologically 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 aI., '84; Ownby and
eoH>eg, '88; Rodriguez-Acosta et aI., '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 30"C 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 15C
compared with 25"C. They subsequently specu
lated that prey envenomation might allow
Envenomated
Snake (g)
Ingest (kJ)
228 (20)
1048 (98)
ASSIM ('.II)
ASH ('.II)
80 (4)
15 (3)
Nonenvenomated
239 (14)
1148 11151
i9,51
1 i (3)
Dor
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574
M. D. MCCUE
envenomation might have on net metabolic cost venoms facilitate digestion, by increasing metaof digestion would, therefore, be minimal.
bolic, assimilatory, and passage rates in a single
Even if SDA and assimilation efficiency were not pitviper species. Given this outcome, it may be
influenced by envenomation, it would still be prudent to further examine the generality of these
reasonable to expect to see an increase in gut findings by investigating the influence that prey
passage rate in snakes consuming envenomated envenomation may have on the digestive performeals; such a result was also not observed. mance of other venomous snakes that are disAlthough the fluorescent tracer was used to track tantly related to rattlesnakes (e.g., true-vipers and
the ingested food bolus, previous studies have members of the family Elapidae). A promising
reported that egestion events are not always species to conduct similar studies on is the king
predictably correlated with ingestion events. For cobra (Ophiophagus hannah), the venom of which
example, some snakes are noted for their tendency possesses the greatest enzyme activities found
to retain 8 large bolus of digested materials among elapid snakes (McCue, 2005). If forthin their distal large intestine for periods lasting coming studies confirm that venom concedes
as long as several months (Lillywhite et aI., 2002). limited or no digestive advantages to other snakes,
A previous study investigating the SDA in post- it could lead to an important paradigm shift
prandial pythons also reported that egestion regarding the adaptive significance of snake
events occurred irregularly despite a regular venoms. Such a shift would thus underscore the
feeding regime (Overgaard et a1., 2002). Although need for controlled studies that characterize
the fluorescent tracer proved to be an effective tool the defensive and predatory functions of snake
for identifying which egested bolus was associated venoms.
with which particular meal, it was less suited to
ACKNOWLEDGMENTS
interpret results of irregular egestion events. One
solution to this problem might be to label food
Experiments were conducted in accordance with
with a radioopaque tracer and conduct periodic
X-ray measurements on postprandial snakes to the University of Arkansas Institutional Animal
determine when undigested matter first enters the Care and Use Committee protocol 05011. Thanks
distal large intestine (Stevenson et al., '85; Dorcas also to J. Agugliaro, Dr. K.E. Cano-McCue, and
two anonymous reviewers for helpful comments
et a\., '971.
The inability to detect increased assimilation on this manuscript, as well as Prof. S. Beaupre for
efficiency in envenomated prey could be related to providing the laboratory space required for this
the fact that venom was administered to prekilled study.Funding was provided by a NSF-Graduate
mice, and was therefore unable to circulate, if only Research Fellowship and a Walton Distinguished
for a few seconds, throughout the bloodstream Doctoral Fellowship awarded to MDM.
of the prey items. Although necrosis is often
LITERATURE CITED
observed at local sites of envenomation, venom
hyaluronidase activity and circulatory transport Andrade nv, Abe AS. 1999. Relationship of venom ontogeny
are known to be critical in distributing venom
and diet in Bothrops. Herpetologica 55:200-204.
components throughout distal prey tissues Andrade nv, CruzNeto AP, Abe AS. 1997. Meal size and
specific dynamic action in the rattlesnake Crotalus durissus
(Russell, '72; Schiff et al., '84; Kasturi and Gowda,
(Serpentes: Viperidae). Herpetologica 53:485-493.
'89). Although the snakes in the assimilation
Baramova EN, Shannon JD, Fox JW, Bjarnason JB. 1991.
experiment were not allowed to strike and
Proteolytic digestion of non-collagenous basement memenvenomate prey items, a second possibility is
brane proteins by the hemorrhagic metalloproteinase Ht-e
that a significant amount of venom was introduced
from Crotalus alrox venom. Biomed Biochim Acta 50:
763-768.
into the prey items during the ingestion process.
The role of the surrounding musculature on the Beaupre SJ, Duvall OJ. 1998. Variation in oxygen consumption of the western diamondback rattlesnake (Crotalus
mechanics of venom delivery has been studied in
atrox): implications for sexual size dimorphism. J Comp
defensive and predatory strikes (Young and Zahn,
Physiol 1688:497-506.
2001; Young et aI., 2003), but nothing is known Beaupre SJ, Zaidan F. 2001. Scaling of CO 2 production in the
Timber rattlesnake (Crolalus horridus), with comments on
about the amount of venom that is released by
cost of growth in neonates and comparative patterns.
snakes that are using the same muscle groups to
Physiol Biochem Zoo] 74:757-768.
ingest prekilled prey items.
Beavers RA. 1976. Food habits of the western diamondback
This study failed to find any support for the
rattlesnake, Crotalus atrox, in Texas (viperidae). Southwest
Nat 20:503-515.
popular long-standing paradigm that snake
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