|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online January 8, 2007
Journal of Experimental Biology 210, 340-356 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02626
Adaptive regulation of digestive performance in the genus Python
Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487-0344, USA
* Author for correspondence (e-mail: brian.d.ott{at}ua.edu)
Accepted 31 October 2006
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Key words: Adaptive response, digestion, intestinal enzyme, intestinal nutrient transport, Python, reptile, specific dynamic action
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
;
McWilliams et al., 1997;
Secor, 2005a). A well-known
example of this is the adaptive correlation between food habits (i.e.
herbivory and carnivory) and the morphology and function of the
gastrointestinal (GI) tract. The GI tract of herbivores is typically longer
than that of carnivores and possesses specialized regions for the fermentation
of plant material (Stevens and Hume,
1995; Karasov and Hume,
1997; Mackie,
2002). Equally apparent is how organisms are able to face routine
fluctuations in the amount and type of food consumed due to seasonal changes
in food availability, ontogenetic shifts in diet, reproductive status and
alternating foraging and feeding strategies
(O'Brien et al., 1989;
Stergiou and Fourtouni, 1991;
Koertner and Heldmaier, 1995;
Johnson et al., 2001). In
response to such shifts in feeding habits, and thus digestive demand, species
modulate gut performance to match pace with digestive load
(Hammond and Diamond, 1994;
Piersma and Lindström,
1997; Weiss et al.,
1998). The plasticity of GI performance is manifested in
morphological restructuring and/or functional regulation at the cellular level
(Ferraris, 1994;
Carey, 1990;
Secor, 2005a).
The adaptive capacity to regulate digestive performance in response to
changes in digestive demand is well expressed by amphibian and reptile species
that naturally experience long episodes of fasting
(Secor, 2005a). Anurans that
estivate during dry seasons and snakes that employ the sit-and-wait tactic of
foraging, and thus eat infrequently, severely downregulate GI performance upon
the completion of digestion, maintain a quiescent gut while fasting, and with
feeding, rapidly upregulate digestive performance
(Secor and Diamond, 2000;
Secor, 2005b). The benefit of
this trait is observed as a reduction in energy expenditure during the bouts
of fasting. For example, during estivation, the metabolic rates of anurans are
depressed by 70%, and the standard metabolic rates (SMR) of sit-and-wait
foraging snakes are 47% less than that of active foraging snakes that only
modestly regulate GI performance with feeding and fasting
(Guppy and Withers, 1999;
Secor and Diamond, 2000).
For sit-and-wait foraging snakes, the correlation between infrequent
feeding and wide regulation of intestinal performance has been investigated
for only four species representing three lineages [Boidae, Pythonidae and
Viperidae (Secor and Diamond,
2000)]. Given that these lineages are dominated by species that
employ the sit-and-wait tactic of foraging, and presumably eat infrequently,
it could be hypothesized that the wide regulation of digestive performance is
a conserved trait, basal for each of these lineages and expressed by all
members. Alternatively, given the species diversity within these lineages, the
capacity to modulate gut performance may be linked to species differences in
geography, morphology, habitat and feeding ecology. Hence, a recurring
question in our research on the adaptive response of the digestive system is
whether physiological responses to feeding and fasting are equivalent among
sit-and-wait foraging snakes, or if the magnitude of response varies as a
function of differences in geography, morphology and/or ecology.
To address this question, we started with a comparative study on the
physiological responses to feeding within the genus Python
(Pythonidae). We selected this genus for two reasons. First, the Burmese
python Python molurus has been the focus of a collection of studies
on physiological responses to feeding and fasting
(Secor and Diamond, 1995;
Stark and Beese, 2001; Overgaard et al.,
1999; Lignot et al.,
2005). With feeding, P. molurus experiences dramatic
increases in metabolic rate, cardiac output, gastric acid production,
intestinal nutrient transport and hypertrophy of the small intestine
(Secor and Diamond, 1995;
Secor and Diamond, 1997;
Secor et al., 2000;
Secor, 2003;
Lignot et al., 2005). Upon the
completion of digestion, these postprandial responses are reversed; metabolism
is depressed, gastric acid production ceases, intestinal nutrient transport is
downregulated, and the intestine atrophies. Second, of the nine genera within
Pythonidae, Python is considered the most derived and morphologically
diverse genus (Kluge, 1993).
The genus Python is composed of ten species, four of which inhabit
sub-Saharan Africa, whereas the other six inhabit southeastern Asia and
Indonesia (Broadley, 1984;
Kluge, 1993;
Keogh et al., 2001). Three
Python species (P. molurus, P. reticulatus and P.
sebae) are among the largest snakes in the world (>7 m in length and
100 kg in mass), whereas P. regius only reaches 2 m in length and 3
kg in mass (Obst et al., 1984;
Murphy and Henderson, 1997).
Variation in Python body shape ranges from long and slender (P.
reticulatus) to short and heavy-bodied (P. brongersmai)
(Shine et al., 1998;
Shine et al., 1999). Although
it is generally assumed from anecdotal observations that all members of
Python employ the sit-and-wait tactic of foraging and thus feed
relatively infrequently, studies on gut contents suggest a more frequent
feeding habit for several species (Pope,
1961; Murphy and Henderson,
1997; Shine et al.,
1999).
We designed this study to determine whether differences in Python geographic range, body shape and potential feeding habits impact the magnitude of postprandial metabolic responses and intestinal regulation. We selected for study five species of Python that vary in geographic range and body shape; P. brongersmai, P. molurus, P. regius, P. reticulatus and P. sebae. Our objectives were to quantify for each species and compare interspecifically: (1) the profile of postprandial metabolic response; (2) the energy expended on meal digestion and assimilation; (3) the magnitude by which intestinal function (hydrolase activity and nutrient uptake) is elevated with feeding; (4) the postprandial change in intestinal morphology and the mass of organs; and (5) the postprandial increase in intestinal performance quantified as intestinal capacity for nutrient uptake and hydrolase activity. For these five species of Python, we shall demonstrate both species-specific differences in postprandial responses and a general wide regulation of intestinal performance.
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
). They are an extremely heavy-bodied snake [body mass to
total length ratio of 8.97±0.23 (mean ± 1 s.e.m.);
Fig. 1] with a body mass
reaching 22 kg and a body length up to 2.5 m
(Shine et al., 1999;
Keogh et al., 2001).
Python molurus L. is a large snake, up to 8 m in length and 100 kg in
mass that ranges from India east into Thailand
(Murphy and Henderson, 1997).
Python regius Shaw 1802, the ball python, inhabits west-central
Africa and is the smallest of the Python species (2 m) and is stout
in body shape (Obst et al.,
1984). Python reticulatus Schneider 1801, the reticulated
python, ranges throughout southeastern Asia and Indonesia
(Pope, 1961). Considered the
longest snake in the world (reported lengths of 10 m), P. reticulatus
has the most slender body shape (body mass to total length ratio of
4.53±0.18; Fig. 1) of
the Python species used in this study. Python sebae Gmelin
1789, the northern African python, occurs throughout much of the northern
portion of sub-Saharan Africa and is also a large python (8 m in length and
100 kg in mass) with a body shape similar to that of P. molurus
(Broadley, 1984). In general,
Python species are considered to be sit-and-wait foragers that feed
relatively infrequently in the wild (Pope,
1961; Murphy and Henderson,
1997). Sit-and-wait foraging snakes lie in wait in a camouflaged
location from which they can ambush passing prey
(Pope, 1961;
Slip and Shine, 1988;
Greene, 1997).
|
). All
individual snakes used in this study were between 18 and 24 months old, with
body masses of studied P. brongersmai, P. molurus, P. regius, P.
reticulatus and P. sebae averaging 806±51 (N=9),
760±47 (N=7), 707±71 (N=10), 757±49
(N=10) and 759±47 (N=10) g, respectively. Animal care
and experimentation were conducted under protocols approved by the University
of Alabama Institutional Animal Care and Use Committee.
Measurements of postprandial metabolic response
We quantified the postprandial metabolic response of each species by
measuring rates of oxygen consumption
(
O2) from snakes
fasted for 30 days and following feeding. Measurements were made using
closed-system respirometry as described
(Secor and Diamond, 1997;
Secor, 2003). Each metabolic
trial began by measuring
O2 of fasted
snakes twice a day (morning and evening) for up to 6 days and assigning the
lowest measured
O2 of each snake
over that time period as its standard metabolic rate (SMR). Snakes were then
fed a meal consisting of one to three rats equaling 25.0±0.0% of their
body mass and metabolic measurements were resumed at 12-h intervals for 3 days
and at 24-h intervals thereafter for 11 more days. At 5-day intervals during
metabolic measurements, snakes were removed from their chambers, weighed,
provided with water, and then returned back to their chambers.
We characterized the postprandial metabolic response of meal break down,
absorption and assimilation of each snake by quantifying the following six
variables as described by Secor and Faulkner
(Secor and Faulkner, 2002):
(1) SMR, the lowest measured
O2 prior to
feeding; (2) peak
O2, the highest
recorded O2
following feeding; (3) factorial scope of peak
O2, calculated
as peak O2
divided by SMR; (4) duration, the time after feeding that
O2 was
significantly elevated above SMR; (5) SDA, specific dynamic action: the total
energy expenditure above SMR over the duration of significantly elevated
O2; and (6) SDA
coefficient, SDA quantified as a percentage of meal energy. We quantified SDA
(kJ) by summing the extra O2 consumed above SMR during the period
of significantly elevated
O2 and
multiplying that value by 19.8 J ml-1 O2 consumed
assuming that the dry matter of the catabolized rodent meal is 70% protein,
25% fat and 5% carbohydrates, and generates a respiratory quotient (RQ) of
0.73 (Gessaman and Nagy, 1988). The energy content of rodent meals was
calculated by multiplying the rodent wet mass by its energy equivalent (kJ
g-1 wet mass) determined by bomb calorimetry. Five individual rats,
each of three different size classes, were weighed (wet mass), dried,
reweighed (dry mass), ground to a fine powder, and pressed into pellets. Three
pellets from each individual rat were ignited in a bomb calorimeter (1266,
Parr Instruments Co., Moline, IL, USA) to determine energy content (kJ
g-1). For each rat, we determined wet-mass energy equivalent as the
product of dry mass energy content and rodent's dry mass percentage. The three
rodent size classes we used weighed on average 45±0.2, 65±5.0
and 150±5.0 g and had an energy equivalent of 6.5±0.3,
7.0±0.4 and 7.6±0.3 kJ g-1 wet mass,
respectively.
Tissue collection
For each species, we killed (by severing the spinal cord immediately
posterior to the head) three individuals that had been fasted for 30 days and
three individuals 2 days following the consumption of rodent meals equaling
25% of the snake's body mass. Following death, a mid-ventral incision was made
to expose the GI tract and other internal organs, which were each removed and
weighed. We emptied the contents of the stomach, small intestine and large
intestine of fed snakes and reweighed each organ. The difference between full
and empty weight of each organ was noted as the mass of the organ's content.
Organ content mass was divided by meal mass to illustrate for each species the
relative extent of digestion at 2 days postfeeding.
Intestinal nutrient uptake
In fasted and digesting snakes we measured nutrient transport rates across
the intestinal brush border membrane using the everted sleeve technique as
developed by Karasov and Diamond (Karasov
and Diamond, 1983) and modified for snakes by Secor et al.
(Secor et al., 1994) and Secor
and Diamond (Secor and Diamond,
2000). The empty small intestine was everted (turned inside out),
divided into equal-length thirds; each third was weighed and sectioned into
1-cm segments. Segments were mounted on metal rods, preincubated in reptile
Ringer's solution at 30°C for 5 min, and then incubated for 2 min at
30°C in reptile Ringer's solution containing an unlabeled and radiolabeled
nutrient and a radiolabeled adherent fluid marker (L-glucose or
polyethylene glycol). We measured, from individual intestinal segments, total
uptake (passive and carrier-mediated) of the amino acids L-leucine
and L-proline and active carrier-mediated uptake of
D-glucose. Because of the similarities between uptake rates of the
proximal and middle intestinal regions, we report the average uptake rates of
those two segments (noted hereafter as the anterior intestine) and those of
the distal segment.
A pair of studies has shown the everted sleeve technique to severely damage
the intestinal mucosa of birds, and thus question the method's ability to
accurately quantify intestinal performance for those species (Starck et al.,
2000; Stein and Williams,
2003). To determine whether the method has any damaging effects on
python intestine, we compared sets of intestinal segments removed from the
proximal region of the small intestine of fed P. molurus, P.
reticulatus and P. sebae at two stages of the everted sleeve
protocol; prior to eversion and after everted tissues were incubated at
30°C in unstirred reptile Ringers for 5 min and in stirred reptile Ringers
for 2 min. We prepared each intestinal segment for light microscopy (described
below) and examined cross sections of the intestine for damage to the mucosal
layer.
For each of these three pythons, everting, mounting and incubating
intestinal segments did not damage the mucosal layer. Between the two stages
of the procedure, we observed no significant difference (all
P>0.47) in villus length (N=20 per stage of procedure)
for these three species. In contrast to some birds, the everted sleeve can be
performed without damaging the intestinal mucosa of pythons, as well as the
mucosa of lizards and anurans (Secor,
2005b; Tracy and Diamond,
2005).
Brush border enzyme activity
From each intestinal third we measured the activity of the brush
border-bound hydrolase, aminopeptidase-N (EC 3.4.11.2) following the procedure
of Wojnarowska and Gray (Wojnarowska and
Gray, 1975). Aminopeptidase-N cleaves NH2-terminal
amino acid residues from luminal oligopeptides to produce dipeptides and amino
acids that then can be absorbed by the small intestine
(Ahnen et al., 1982). From 1-cm
segments, scraped mucosa was homogenized in PBS (1:250 dilutions) on ice.
Activity of aminopeptidase-N was measured using leucyl-ß-naphthylamide
(LNA) as the substrate and p-hydroxymercuribenzoic acid to inhibit
nonspecific cytosol peptidases. Absorbance of the product resulting from the
hydrolysis of LNA was measured spectrophometrically (DU 530, Beckman Coulter,
Inc., Fullerton, CA, USA) at 560 nm and compared to a standard curve developed
with ß-naphthylamine. Enzyme activities were quantified as µmol of
substrate hydrolyzed per minute per gram of protein. Protein content of the
homogenate was determined using the Bio-Rad Protein Assay kit based on the
method of Bradford (Bradford,
1976).
Intestinal morphology and organ masses
We quantified the effects of feeding on small intestinal morphology by
measuring intestinal mass, intestinal length, mucosa and muscularis/serosa
thickness and enterocyte dimensions from fasted and fed snakes. Immediately
following the removal and flushing of the small intestine, we measured its wet
mass and length. From the middle region of the small intestine, a 1-cm segment
was fixed in 10% neutral-buffered formalin solution, embedded in paraffin and
cross sectioned (6 µm). Several cross sections were placed on a glass slide
and stained with Hematoxylin and Eosin. We measured mucosa and
muscularis/serosa thickness and enterocyte dimensions from individual cross
sections using a light microscope and video camera linked to a computer and
image-analysis software (Motic Image Plus, Richmond, British Columbia,
Canada). We calculated the average thickness of the mucosa and
muscularis/serosa from ten measurements taken at different positions of the
cross section. Likewise, we averaged the height and width of ten enterocytes
measured at different positions of the cross section and calculated their
volume based on the formula for a cube (enterocyte width2 x
height). To assess postprandial effects on the mass of other organs, we
weighed the wet mass of the heart, lungs, liver, empty stomach, pancreas,
empty large intestine and kidneys immediately upon their removal from snakes.
Each organ was dried at 60°C for 2 weeks and then reweighed for dry
mass.
Small intestinal capacity
For each nutrient we quantified the intestine's total uptake capacity
(reported as µmole min-1) by summing together the product of
segment mass (mg) and mass-specific rates of nutrient uptake (nmole
min-1 mg-1) for the proximal, middle and distal
segments. Likewise, we quantified total small intestinal capacity for
aminopeptidase-N activity by summing the products of mucosa segment mass (mg)
times segment aminopeptidase-N activity, calculated as µmol of substrate
hydrolyzed per minute per mg of mucosa. Mucosa mass was calculated from the
mass of scraped mucosa from a 1-cm segment of intestine and multiplying that
mass by segment length.
Statistical analyses
For each metabolic trial we used repeated-measures design analysis of
variance (ANOVA) to test for significant effects of time (before and after
feeding) on O2.
Additionally, we used post hoc pairwise mean comparisons
(Tukey-Kramer procedure) to determine when post feeding
O2 was no longer
significantly different from SMR, and to identify significant differences in
O2 between
sampling times. To test for species effects on metabolic variables, we used
ANOVA for mass-specific rates and analysis of covariance (ANCOVA), with body
mass as the covariate, for whole-animal measurements. Significant ANOVA and
ANCOVA results were followed by post hoc comparisons to identify
significant differences between species.
A repeated-measures design ANOVA and post hoc comparisons were employed to test for positional effects (proximal, middle and distal regions of the small intestine) on nutrient uptake rates and aminopeptidase-N activities. We used ANOVA to determine the postfeeding effects on nutrient uptake rates and aminopeptidase-N activity, and ANCOVA (body mass as the covariate) to test for postfeeding changes in total small intestinal capacity for nutrient uptake and aminopeptidase-N activity. Likewise, we used ANCOVA (body mass as the covariate) to test for postfeeding effects on intestinal mass, length and morphology, and the wet and dry masses of other organs. Species differences in intestinal morphology were also explored by ANCOVA and post hoc comparisons. We designate the level of significance as P<0.05 and report mean values as means ± 1 s.e.m.
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
O2, both pre and
postfeeding, with
O2 increasing
significantly (all P<0.0002) for each species within 12 h after
feeding (Fig. 2). Oxygen
consumption continued to increase before peaking at 1.5 days postfeeding, at
rates that ranged between 9.9- and 14.5-fold higher than SMR
(Table 1). We found peak
O2, as well as
the scope of peak
O2, to vary
significantly (all P<0.0003) among the five pythons
(Table 1). The three larger
species (P. molurus, P. reticulatus and P. sebae) showed
significantly (all P<0.0018) higher peak rates than the two
smaller species (P. brongersmai and P. regius). Python
molurus had the largest scope of peak
O2
(14.5±1.0), which was significantly (all P<0.032) greater
than the scopes exhibited by the other four species
(Table 1). For these five
pythons, the duration of significantly elevated metabolic rates lasted from 6
to 8 days (Table 1).
|
|
The summed energy expended on digestion, absorption and assimilation (SDA) did not differ among the five species when calculated either as kJ or kJ g-1 (Table 1). Given the lack of variation in SDA and meal size (and thus energy), the SDA coefficient (SDA as a percentage of meal energy) likewise did not differ significantly among the five species, averaging 25.3±0.6% (Table 1).
Digestion rates
By 2 days postfeeding, 59%, 48%, 56%, 34% and 42% of the original rodent
meals remained in the stomachs of P. brongersmai, P. molurus, P. regius,
P. reticulatus and P. sebae, respectively
(Fig. 3). The relative amount
of the meal found in the stomach differed significantly (P=0.002)
among the five Python species. Python brongersmai had a
larger percentage of its meal still within its stomach compared to P.
reticulatus and P. sebae, and P. regius retained more
of its meal than P. reticulatus. Mass of small intestinal content did
not significantly vary among species, averaging 9.8±1.0% of original
meal mass (Fig. 3).
|
Intestinal nutrient uptake
For each of the five Python species, there was no significant
difference in snout-vent length, total length, or body mass between fasted and
fed snakes. For 13 of the 30 cases (five species, two treatments, three
nutrients), intestinal position had a significant (all P<0.049)
effect on nutrient uptake rates, as uptake rates of the proximal segment were
significantly greater than rates of the distal segment. Combining all fasted
and fed pythons, uptake rates of L-leucine, L-proline
and D-glucose declined by an average of 16%, 34% and 64%,
respectively, from the proximal to distal segment.
Python molurus, P. regius, P. reticulatus and P. sebae each experienced significant (all P<0.018) postfeeding increases in L-leucine, L-proline and D-glucose uptake rates by the anterior portion of the small intestine (Fig. 4). For these four pythons, uptake rates of L-leucine increased by 6.4-, 2.9-, 5.9- and 3.4-fold, of L-proline by 4.5-, 3.5-, 5.1- and 3.1-fold, and of D-glucose by 7.7-, 27.1-, 13.6- and 16.1-fold, respectively. By contrast, P. brongersmai lacked any significant postfeeding increase in amino acid uptake by the anterior small intestine, though did significantly (P<0.0014) upregulate anterior intestinal uptake of D-glucose, by 40-fold (Fig. 4).
|
Significant postprandial upregulation of nutrient transport occurred in the distal small intestine of all five species (Fig. 4). Significant postprandial uptake of L-leucine occurred in P. brongersmai, P. molurus, P. regius, P. reticulatus and P. sebae by factors of 1.3-, 7.6-, 2.2-, 3.1- and 3.4-fold, respectively; of L-proline in P. molurus, P. regius, P. reticulatus and P. sebae by 3.7-, 2.1-, 3.0- and 3.2-fold, respectively; and of D-glucose in P. regius by 21.5-fold.
Intestinal aminopeptidase-N activity
Aminopeptidase-N activity varied significantly (all P<0.027)
depending on intestinal positions in fed P. brongersmai and P.
sebae, as activity was significantly greater in the proximal compared to
the distal region. For each species studied, aminopeptidase-N activity of the
anterior intestine was significantly (all P<0.033) greater in fed
snakes than in fasted snakes (Fig.
5). On average, among the five species, aminopeptidase-N activity
of the anterior small intestine increased by 4.4-fold with feeding. Three
species, P. brongersmai, P. molurus and P. reticulatus, also
experienced significant (all P<0.0095) upregulation of
aminopeptidase-N activity in the distal intestine
(Fig. 5).
|
|
|
For each Python species, the thickness of the combined muscularis and serosa layers did not differ significantly between fasted and fed snakes (Fig. 7). By contrast, the mucosal layer increased significantly (all P<0.017) in thickness postfeeding in all five species, increasing on average by 85±10% (Fig. 7). The thickening of the mucosa reflects the postprandial lengthening of the villi, which was largely due to the hypertrophy of the epithelial cells, the enterocytes. For all species, enterocyte height did not change with feeding, whereas enterocyte width did increase significantly (all P<0.036) by 27%, to 59% (Fig. 7). Applying the equation for a cube, we calculated enterocyte volume for fasted and fed snakes, and observed a 37%, 27%, 43%, 42% and 59% postprandial increase in enterocyte volume for P. brongersmai, P. molurus, P. regius, P. reticulatus and P. sebae, respectively (Fig. 7).
|
Intestinal digestive capacity
The combined postprandial increase in small intestinal mass and
mass-specific rates of brushborder function underlie the dramatic upregulation
of intestinal performance that each of these pythons experience with feeding.
When summed for the full length of the small intestine, each species' capacity
to transport nutrients increased significantly (all P<0.036) with
feeding (Fig. 8). When averaged
across the three measured nutrients, total intestinal uptake capacity
increased with feeding by factors of 13-, 15-, 20-, 12- and 15-fold for P.
brongersmai, P. molurus, P. regius, P. reticulatus and P. sebae,
respectively. When averaged across the five species, we found
L-leucine and L-proline uptake capacities to increase by
similar magnitudes, 7.6-fold and 6.5-fold, respectively, with feeding. More
dramatic is the concurrent upregulation of D-glucose uptake
capacity, averaging 31.2-fold among the five species.
|
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Metabolic responses to feeding
All five pythons of this study exhibited the characteristic postprandial
profile of metabolism, observed as a rapid postfeeding increase in
O2 that, upon
peaking, declined more gradually to prefeeding rates
(Fig. 2). Similar profiles of
postprandial metabolism have been observed for invertebrates, fishes,
amphibians, other reptiles, birds and mammals
(Jobling, 1981;
LeBlanc and Diamond, 1986;
Carefoot, 1990;
Janes and Chappell, 1995;
Secor and Phillips, 1997;
Hailey, 1998;
Secor, 2005a). For pythons we
can imagine that the large postprandial increases in their metabolic rates
stem from the elevated activity of gastrointestinal and associated organs
(heart, lung, kidneys, etc), and the transport and assimilation of the
absorbed nutrients from their large meals. Generating the SDA response is the
gastric breakdown of the intact rodent meal, the intestinal absorption of
approximately 91% of ingested nutrients, and the synthesis of new body tissues
equivalent to approximately 40% of ingested meal energy
(Secor, 2003;
Cox and Secor, 2005). For
P. molurus, it has been estimated that gastric performance and
postabsorptive protein synthesis accounts for 55% and 26.3% of SDA,
respectively (Secor,
2003).
For pythons, as well as for other reptiles and amphibians, the magnitude of
peak O2, the
duration of the metabolic response, and overall SDA are affected by meal type,
meal size, body temperature and body size
(Secor and Diamond, 1997;
Hailey, 1998;
Toledo et al., 2003;
Wang et al., 2003;
McCue et al., 2005;
Pan et al., 2005;
Secor and Boehm, 2006).
Therefore, interspecific comparisons of the SDA response are best made when
meal type, relative meal size, body temperature and body size are
standardized. To a common meal type (rats), meal size (25% of body mass), body
temperature (30°C) and body size (mean=706-763 g), the five pythons of our
study showed similar SDA responses. For each,
O2 peaked 1.5
days after feeding at 9.9- to 14.5-times SMR before declining back to
prefeeding values after an additional 5-8 days
(Fig. 2). Subtle interspecific
differences included the lower SMR and peak
O2 of P.
brongersmai and P. regius, the higher scope of peak
O2 of P.
molurus, and the shorter duration for P. molurus and P.
sebae. These differences essentially cancelled each other out in
generating similar SDAs (422-496 kJ kg-1) in the five species
(Table 1).
In previous studies in which P. molurus consumed rodent meals
equaling 20-25% of their body mass, snakes achieved peaks in
O2 1-2 days
postfeeding at rates between 0.25 and 0.55 ml g-1 h-1, a
range of O2 that
encompasses our peak rates for P. molurus, P. reticulatus and P.
sebae (Secor and Diamond,
1997; Secor et al.,
2000; Overgaard et al.,
2002; Wang et al.,
2003). Some of the variation in reported peak
O2 can be
explained by differences in relative meal size (20% versus 25% of
body mass), given that postprandial peaks in
O2 increase with
relative meal size (Secor and Diamond,
1997). In a study of P. regius, Starck and Wimmer
(Starck and Wimmer, 2005)
recorded SMR and peak
O2 of 0.021 and
0.08 ml g-1 h-1, respectively, and a duration of the SDA
response of approximately 10 days. The P. regius of our study had
similar SMR (0.022 ml g-1 h-1) and response duration (8
days), however, our P. regius attained a higher peak
O2 (0.21 ml
g-1 h-1).
For other infrequently feeding snakes, including the boa constrictor
Boa constrictor, sidewinder Crotalus cerastes, timber
rattlesnake Crotalus horridus, water python Liasis fuscus,
rosy boa Lichanura (=Charina) trivirgata, and
carpet python Morelia spilota, the consumption of rodent meals of 25%
of their body mass likewise generated 6- to 18.5-fold increases in metabolic
rate, which remained elevated for 6-8 days
(Thompson and Withers, 1999;
Secor and Diamond, 2000;
Bedford and Christian, 2001;
Zaidan and Beaupre, 2003). For
B. constrictor, C. cerastes and L. trivirgata, SDA ranged
between 357 and 670 kJ kg-1, and together with the pythons of the
present study, SDA coefficients vary between 18 and 33%
(Secor and Diamond, 2000). By
contrast, snake species that feed more frequently in the wild have more modest
SDA responses to similar size meals (20-25% of body mass), as noted by 5- to
8-fold increases in metabolism, metabolic rates that remain elevated for 3.5-5
days, SDAs of 258-309 kJ kg-1, and SDA coefficients of 13-15%
(Secor and Diamond, 2000;
Zaidan and Beaupre, 2003;
Hopkins et al., 2004;
Roe et al., 2004).
Plasticity of intestinal function
There is a distinct gradient in function of the python intestine, as
aminopeptidase-N activity and nutrient transport rates decline distally. A
proximal to distal gradient of intestinal hydrolase activities has also been
observed for amphibians, birds and mammals
(McCarthy et al., 1980:
Martinez del Rio, 1990;
Hernandez and Martinez del Rio,
1992; Sabat et al.,
2005). Similar decreases with position in mass-specific and
length-specific rates of nutrient uptake have been documented for fishes,
amphibians, reptiles, birds and mammals
(Karasov et al., 1985;
Karasov et al., 1986;
Buddington and Hilton, 1987;
Buddington et al., 1991;
Secor and Diamond, 2000;
Secor, 2005a). This
phenomenon, especially evident for the active uptake of D-glucose,
may best be explained by the reduction distally in functional surface area of
the small intestine, a product of decreases in villus and microvillus surface
area (Ferraris et al., 1989).
In addition, the density of glucose transporters on the surface of the
microvilli of mice (Mus musculus) and woodrats (Neotoma
lepida) decreases step-wise from proximal to middle to distal regions
(Ferraris et al., 1989). For
pythons and other species, the positional decline in intestinal function
undoubtedly reflects a response to the distal decrease in the concentration of
luminal nutrients.
For fasted and fed pythons, as for most carnivores studied, intestinal
uptake rates of amino acids are significantly greater than uptake rates of
D-glucose, usually by an order of magnitude
(Buddington et al., 1991;
Secor and Diamond, 2000;
Secor, 2005a). This difference
is explained by the predominance of protein within the snake's diet and the
relatively small amount of dietary carbohydrates. Additionally, this
difference may, in part, be due to the combined measurement of passive and
active uptake of the amino acids and/or measurement of only the active
transport of D-glucose. With feeding, pythons rapidly increase
intestinal uptake of amino acids (with the exception of P.
brongersmai) and D-glucose. In four of the python species,
L-leucine and L-proline uptake rates increased with
feeding by 2.9- to 6.4-fold, a magnitude similar to the postfeeding increases
in amino acid uptake observed for B. constrictor, C. cerastes and
L. trivirgata (Secor and Diamond,
2000). Whereas P. brongersmai lacked significant
postfeeding increases in amino acid transport, this species, along with the
other four pythons, dramatically upregulated the active transport of
D-glucose by an average of 21-fold in the anterior small intestine.
Likewise, significant postprandial increases in D-glucose active
transport have also been documented for B. constrictor (5-fold),
C. cerastes (6.8-fold) and L. trivirgata (4.3-fold)
(Secor and Diamond, 2000).
In the anterior portion of the small intestine and in some cases in the distal portion, aminopeptidase-N activity increased significantly with feeding for each of the five pythons. This increase in peptidase activity is expected given both the large protein content of their meals and that the overall upregulation of intestinal function would also include increases in brush border hydrolase activity. A matched response of hydrolase activity and nutrient transport was observed in this study by the average 4.46-fold and 4.36-fold postprandial increases in anterior aminopeptidase-N activity and amino acid uptake, respectively, for four of the pythons (excluding P. brongersmai).
Studies on the postprandial responses of intestinal hydrolases have
generated mixed results. In rats, fasting results in an increase in intestinal
peptidase activity that is reversed when the rats feed
(Kim et al., 1973;
Ihara et al., 2000). The
Andean toad, Bufo spinulosus, shows no change in either intestinal
aminopeptidase-N or maltase activity between fasting and feeding
(Naya et al., 2005). By
contrast, the pythons of this study had large postfeeding increases in the
activity of intestinal aminopeptidase-N. Explanations for this continuum of
regulatory responses include the increased activity of cellular peptidases in
fasting rats in order to hydrolyze cellular proteins as a fuel source and for
gluconeogenesis, the lack of response in the Andean toad because they may feed
frequently and, like other frequently feeding anurans they do not widely
regulate intestinal function, and the large postfeeding increase in pythons,
because as infrequent feeders they widely regulate intestinal function with
each meal.
Trophic responses of the intestine and other organs
An apparent universal response to fasting is the reduction in mass
(independent of changes in body mass) of the small intestine, manifested as
atrophy of the intestinal epithelium
(Bogé et al., 1981;
Carey, 1990;
Secor, 2005a). In blackcaps
Sylvia atricapilla, small intestinal mass and villus height are
reduced by 45% and 18%, respectively, after a 2-day fast, in rats by 42% and
30% after a 5-day fast, and in garter snakes Thamnophis sirtalis by
38% and 50% after a 4-week fast (Dunel-Erb
et al., 2001; Starck and
Beese, 2002; Karasov et al.,
2004). Feeding rapidly reverses intestinal atrophy by triggering
the hypertrophy of enterocytes, which quickly restores intestinal mass to
prefeeding levels (Dunel-Erb et al.,
2001; Karasov et al.,
2004).
The postprandial increase in small intestinal mass observed for the five
Python species is similar in magnitude to that previously noted for
B. constrictor, C. cerastes and L. trivirgata, as well as
for several species of estivating anurans
(Secor and Diamond, 2000;
Secor, 2005b). For each of
these organisms, the increase in small intestinal mass is largely attributed
to the thickening of the intestinal mucosa, which results from villus
lengthening, itself a product of enterocyte hypertrophy. For pythons and
estivating anurans, enterocyte width and volume increase with feeding by
40-90% and 50-440%, respectively (Cramp
and Franklin, 2005; Lignot et
al., 2005; Secor,
2005b). In addition to enterocyte hypertrophy, cellular
hyperplasia (replication) may also contribute to the postprandial increase in
intestinal mass. However, for P. molurus, the postprandial increases
in enterocyte replication are matched by a concurrent increase in apoptosis
(Lignot and Secor, 2003;
Lignot et al., 2005). Hence,
the postprandial increase in python intestinal mass appears largely to be a
product of cellular hypertrophy rather than hyperplasia.
Postprandial increases in the mass of organs, other than the small
intestine, have also been observed for B. constrictor, C. cerastes
and L. trivirgata (Secor and
Diamond, 2000). For these snakes, together with pythons, feeding
generates increases in liver and kidney wet masses of 
59% and 70%,
respectively. These tissues may also be experiencing cellular hypertrophy,
contributed, in part, by the accumulation of material absorbed through the gut
and filtered from circulation. Other organs involved in digestion that also
increase in mass with feeding, though not consistently among infrequently
feeding species, include the pancreas (P. brongersmai and B.
constrictor) and stomach (C. cerastes, L. trivirgata and P.
molurus) (Secor and Diamond,
2000) (this study). We did not observe a significant postfeeding
increase in heart mass in any of the pythons of this study as previously
documented for P. molurus (Secor
and Diamond, 1995; Andersen et
al., 2005).
Regulatory mechanisms of intestinal performance
Each of the five Python species in this study exhibited the
ability to widely modulate the capacity of the intestine for nutrient uptake
and aminopeptidase-N activity, with feeding and fasting. For pythons, the
underlying mechanisms for the regulation of intestinal performance are split
between those responsible for the trophic responses and those for the
functional responses of the intestinal epithelium. Within 2 days after
feeding, intestinal nutrient uptake and aminopeptidase-N capacities have
increased in the five pythons by 2- to 49-fold. On average, the increase in
small intestinal mass and the increase in mass-specific function accounts for
21.6% and 78.4%, respectively, of the postprandial increase in intestinal
capacity.
As noted earlier, the increase in small intestinal mass is due largely to
hypertrophy of the epithelium enterocytes. Plausible mechanisms for enterocyte
hypertrophy include the mobilization of amino acids from protein sources for
enterocyte rebuilding and the absorption of luminal nutrients. Although there
is no current evidence to support the former explanation, the latter
explanation is well supported from observations of enterocytes of P.
molurus filled with lipid droplets originating from the meal
(Starck and Beese, 2001;
Lignot et al., 2005). Our
histological examinations revealed the presence of lipid droplets within
enterocytes of fed snakes in all of the five python species.
There are several specific and nonspecific mechanisms by which intestinal
function, independent of mass, can be regulated. First, by increasing or
decreasing the specific activity of membrane transporters and enzymes. Second,
by modulating the rate of synthesis and thus the density of brushborder
transporters and enzymes. And third, by altering the functional surface areas
of the luminal membrane without changing transporter or enzyme activity or
density. The former two mechanisms have been proposed to explain shifts in
nutrient transporter function with changes in diet
(Buddington and Diamond, 1989;
Ferraris et al., 1992). The
third mechanism, involving the movement to and from the brush-border membrane
of intracellular stores of membrane proteins, explains the compensatory
restoration of lost function following the surgical removal of a portion of
the small intestine, as the remnant intestine responds by increasing villus
length (Fenyö et al.,
1976; Hanson et al.,
1977).
For pythons, although there is support for the second mechanism from the
findings of a postprandial increase in protein and mRNA expression of the
Na+/glucose co-transporter (SGLT1) for P. molurus, we
propose that it is the third mechanism that is largely responsible for the
regulation of intestinal function (Secor,
2005a). Pythons, like other organisms, experience a
fasting-to-feeding increase in villus length, but this would only be
responsible for about an 85% increase in surface area. Unlike other organisms,
pythons experience a postprandial increase in microvillus length
(Secor, 2005a). All five
python species in this study possessed stunted microvilli (0.5 µm)
while fasting, which increased 5-fold within 2 days after feeding (S. Secor
and J-H. Lignot, unpublished data). Given that the microvilli are minute
compared to the rest of the enterocyte, their increase in length contributes
insignificantly to the postprandial increase in small intestinal mass. If for
pythons transporter and enzymes activities and densities on the microvilli are
stable from fasting to feeding, the resulting increase in microvillus surface
area, resulting from the mobilization and insertion of membrane proteins from
within the cell, would account for much of the upregulation of intestinal
function (Secor, 2005a). This
certainly would be the case for amino acid uptake and aminopeptidase-N
activity, but would only account for a portion of the increase in the
carrier-mediated uptake of D-glucose. We suspect that the remainder
of the upregulation of D-glucose uptake is provided by the
aforementioned increase in the expression and thus the density of SGLT1.
Adaptive correlates of Python digestive physiology
Our original question asked whether Python species possess unique
differences in their digestive response that reflects species differences in
biogeography, body shape and/or feeding habits, or if they exhibit, in common,
the wide regulation of digestive response indicative of their infrequent
feeding habits. We will first comment on species-specific differences before
addressing the generality of the python digestive response. The pythons of
this study are split geographically between subSaharan Africa (P.
regius and P. sebae) and southeast Asia and Indonesia (P.
brongersmai, P. molurus and P. reticulatus). A comparison of
these two sets of snakes revealed no significant differences in metabolic,
morphological, or functional responses to fasting or feeding with respect to
geography. Interestingly, P. molurus from southeast Asia and P.
sebae from Africa have numerous similarities in body morphology and in
physiological responses, whereas P. brongersmai from Indonesia and
P. regius from Africa likewise share similar morphologies
(short-bodied) and a relatively low rate of standard metabolism.
As an index of body shape, the ratio of body mass to body length ranged from 4.53±0.18 for P. reticulatus to 8.45±0.56 for P. brongersmai (Fig. 1). Along this continuum of body shape index, we did not find any significant correlation between this ratio and metabolic responses, intestinal responses or organ masses. In looking at the two extremes of Python body shape, we note that the elongated P. reticulatus possessed the highest SMR and largest SDA, whereas the stout P. brongersmai exhibited the smallest upregulation of amino acid uptake capacity and the largest increase in aminopeptidase-N capacity. It is interesting that despite having the shortest SVL, P. brongersmai small intestines are similar in length to those of P. molurus, P. reticulatus and P. sebae. Snake small intestines are arranged in a serpentine fashion and therefore are much longer than the length of body cavity that they occupy. For P. brongersmai, the ratio of small intestinal length to body cavity length occupied by the small intestine (13.1±1.6) was significantly greater that that of the other four species (6.1±0.4).
Although data on feeding habits for these five species is scant, the
existing anecdotal and scientific reports suggest that Python species
utilize an ambush foraging strategy to feed chiefly upon birds and mammals
(Pope, 1961;
Murphy and Henderson, 1997).
In southern Sumatra, P. reticulatus consume mostly rats as juveniles,
graduating to monkeys, wild pigs and small deer as adults
(Shine et al., 1998). On oil
palm plantations in northeastern Sumatra, juvenile and adult P.
brongersmai feed almost exclusively on rats
(Shine et al., 1999). In these
two previous studies, it was found that 50% of collected P.
reticulatus had the remains of a meal within their gut (stomach, small
and large intestines), whereas 78% of collected P. brongersmai had
food items in their guts. This difference in the occurrence of gut contents
may suggest that P. brongersmai feeds more frequently than P.
reticulatus, but the presence of food in the gut may not always be a good
predictor of feeding frequency for pythons. Observed in captivity,
Python species vary tremendously in the duration that they retain
fecal matter within their large intestine, from 1-2 weeks for P.
reticulatus to 2-4 months for P. brongersmai (B. Ott, personal
observations). Apparently, retention of fecal matter for extended lengths of
time has also been observed for other short and stout, sit-and-wait foraging
snakes (Lillywhite et al.,
2002). Therefore, these five species may possess similar feeding
frequencies, and hence similar magnitudes of postprandial responses.
Stepping back from the interspecific variation in Python
postprandial physiological responses, each of the five Python species
was observed to significantly regulate intestinal performance with each meal.
The downregulation of intestinal performance with fasting is proposed to be an
adaptation for organisms that predictably experience long intervals between
meals (Secor, 2001;
Secor, 2005a). For fasting
animals relying solely upon stored energy to meet metabolic demands, any trait
that reduces daily energy expenditure would be favored by natural selection.
Given the high maintenance cost of the gastrointestinal epithelium, due in
part to its high rate of cell turnover
(Johnson, 1987), its
downregulation in structure and function during fasting would therefore reduce
overall energy expenditure. For pythons and other snakes that naturally
experience long fasts between meals, this reduction in gut maintenance is
manifested in part as a lowering of their SMR. On average, SMR of the pythons
of this study and other infrequently feeding snakes is 48% lower than the SMR
of frequently feeding snakes that do not significantly downregulate intestinal
performance with fasting (Fig.
9).
|
Further inquiry in snake digestive physiology
Seldom is a study undertaken that does not generate new questions,
alternative hypotheses, and further explorations. While examining the
metabolism, morphology and postfeeding responses of these five python species,
we identified two further areas warranting further investigation.
(1) What is the significance of interspecific differences in postfeeding responses and morphology among python species? Although we have downplayed the importance of those differences in the overall regulation of digestive performance, they are worthy of further attention. Consider P. brongersmai; why did this species not downregulate amino acid uptake while fasting as it did D-glucose uptake and aminopeptidase-N activity, and why does it possess such a long intestine for its body length? Additional information on the feeding habits of this species, repeating the study, and studying the postprandial responses of sister taxa (P. breitensteini and P. curtus) may explain (or refute) this species' lack of amino acid transport regulation. The small intestine of P. brongersmai is similar in length to that of the three longest pythons (P. molurus, P. reticulatus and P. sebae), although relative to body length, it is twice as long. Is this trait unique for P. brongersmai, or is intestinal length conserved with respect to body mass, and it is P. regius that possess the uniquely short small intestine (averaging 66% the length of the small intestine of the other four species)?
(2) Is the wide regulation of gastrointestinal performance an inherent
plesiomorphic character of lineages of infrequently feeding snakes? In
the present study we were not surprised to find that Python species
widely regulate intestinal performance, given their infrequent feeding habits
and the large postprandial responses known for P. molurus. In the
family Pythonidae there are approximately 27 species within eight genera, and
in the sister family Boidae there are approximately 40 species within 11
genera. Members of these two families are generalized as sit-and-wait foragers
that feed relatively infrequently (Greene,
1997), and therefore hypothetically they all possess the
plesiomorphic trait of widely regulating digestive performance with each meal.
Alternatively, given the much broader variation in biogeography, body shape,
ecology and feeding habits among all pythons and boas (compared to just
Python), there may be species that lack this trait, and, like
frequently feeding colubrid snakes, only modestly regulate intestinal function
between meals. Candidate species for the modest regulation of digestive
function could
