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Ott BD, Secor SM.. Adaptive regulation of digestive performance in the genus Python. J Exp Biol 210: 340-356

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The adaptive interplay between feeding habits and digestive physiology is demonstrated by the Burmese python, which in response to feeding infrequently has evolved the capacity to widely regulate gastrointestinal performance with feeding and fasting. To explore the generality of this physiological trait among pythons, we compared the postprandial responses of metabolism and both intestinal morphology and function among five members of the genus Python: P. brongersmai, P. molurus, P. regius, P. reticulatus and P. sebae. These infrequently feeding pythons inhabit Africa, southeast Asia and Indonesia and vary in body shape from short and stout (P. brongersmai) to long and slender (P. reticulatus). Following the consumption of rodent meals equaling 25% of snake body mass, metabolic rates of pythons peaked at 1.5 days at levels 9.9- to 14.5-fold of standard metabolic rates before returning to prefeeding rates by day 6-8. Specific dynamic action of these meals (317-347 kJ) did not differ among species and equaled 23-27% of the ingested energy. For each species, feeding triggered significant upregulation of intestinal nutrient transport and aminopeptidase-N activity. Concurrently, intestinal mass doubled on average for the five species, in part due to an 85% increase in mucosal thickness, itself a product of 27-59% increases in enterocyte volume. The integrative response of intestinal functional upregulation and tissue hypertrophy enables each of these five python species, regardless of body shape, to modulate intestinal performance to meet the demands of their large infrequent meals.
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340
Introduction
The integration of a species’ ecology and physiology is
exemplified in the adaptive interplay between their feeding
ecology and digestive physiology (Karasov and Diamond,
1988; 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
The adaptive interplay between feeding habits and
digestive physiology is demonstrated by the Burmese
python, which in response to feeding infrequently has
evolved the capacity to widely regulate gastrointestinal
performance with feeding and fasting. To explore the
generality of this physiological trait among pythons, we
compared the postprandial responses of metabolism and
both intestinal morphology and function among five
members of the genus Python: P. brongersmai, P. molurus,
P. regius, P. reticulatus and P. sebae. These infrequently
feeding pythons inhabit Africa, southeast Asia and
Indonesia and vary in body shape from short and stout (P.
brongersmai) to long and slender (P. reticulatus). Following
the consumption of rodent meals equaling 25% of snake
body mass, metabolic rates of pythons peaked at 1.5·days
at levels 9.9- to 14.5-fold of standard metabolic rates
before returning to prefeeding rates by day·6–8. Specific
dynamic action of these meals (317–347·kJ) did not differ
among species and equaled 23–27% of the ingested energy.
For each species, feeding triggered significant
upregulation of intestinal nutrient transport and
aminopeptidase-N activity. Concurrently, intestinal mass
doubled on average for the five species, in part due to an
85% increase in mucosal thickness, itself a product of
27–59% increases in enterocyte volume. The integrative
response of intestinal functional upregulation and tissue
hypertrophy enables each of these five python species,
regardless of body shape, to modulate intestinal
performance to meet the demands of their large infrequent
meals.
Key words: Adaptive response, digestion, intestinal enzyme, intestinal
nutrient transport, Python, reptile, specific dynamic action.
Summary
The Journal of Experimental Biology 210, 340-356
Published by The Company of Biologists 2007
doi:10.1242/jeb.02626
Adaptive regulation of digestive performance in the genus Python
Brian D. Ott* and Stephen M. Secor
Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487-0344, USA
*Author for correspondence (e-mail: brian.d.ott@ua.edu)
Accepted 31 October 2006
THE JOURNAL OF EXPERIMENTAL BIOLOGY
341Postprandial responses of Python
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
Animals and their maintenance
The five species of this study span the geographic range and
morphological diversity of the genus Python (Fig.·1). Python
brongersmai Stull 1938, the blood python, inhabits eastern
Sumatra and neighboring portions of Malaysia (Keogh et al.,
2001). 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).
The pythons used in this study were born in captivity and
purchased commercially. We housed snakes individually in
20·l plastic boxes and maintained them at 28–32°C under a
photoperiod of 14·h:10·h L:D. Snakes were fed laboratory rats
once every 2·weeks and had continuous access to water. To
reduce potential body-size effects, we used snakes of similar
mass resulting in no significant difference among the five
Python species in body mass for either the metabolic or
intestinal experiments. Prior to the start of experimentation, we
withheld food from snakes for a minimum of 30·days to ensure
that they were postabsorptive. Python molurus has been found
to complete digestion within 10–14·days after feeding (Secor
and Diamond, 1995). All individual snakes used in this study
were between 18 and 24·months old, with body masses of
THE JOURNAL OF EXPERIMENTAL BIOLOGY
342
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 (VO2) from
snakes fasted for 30·days and following feeding. Measurements
were made using closed-system respirometry as described
B. D. Ott and S. M. Secor
(Secor and Diamond, 1997; Secor, 2003). Each metabolic trial
began by measuring VO2of fasted snakes twice a day (morning
and evening) for up to 6·days and assigning the lowest
measured VO2of 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 VO2prior to feeding; (2) peak VO2, the highest
recorded VO2following feeding; (3) factorial scope of peak VO2,
calculated as peak VO2divided by SMR; (4) duration, the time
after feeding that VO2was significantly elevated above SMR;
(5) SDA, specific dynamic action: the total energy expenditure
above SMR over the duration of significantly elevated VO2; 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 VO2and 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.
A
B
E
D
C
0
3.2
6.4
9.6
M
b/
TL
(g cm–1)
c
P. brongersmai
b
P. regius
b
P. sebae
a,b
P. molurus
a
P. reticulatus
Fig.·1. Photographs and relative body shape (body mass, Mb/total
length, TL) of the five Python species used in this study. (A) P.
brongersmai, (B) P. regius, (C) P. sebae, (D) P. molurus, (E) P.
reticulatus. Note the significant variation in body shape from the short
and stout P. brongersmai to the long and slender P. reticulatus. In the
histogram, letters above bars that are different denote significant
(P<0.05) differences between means, as determined from post hoc
pairwise comparisons.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
343Postprandial responses of Python
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--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 -
naphthylamine. Enzyme activities were quantified as mol 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·m). 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 width2height). 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 mole·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 mol 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 VO2. Additionally, we used
post hoc pairwise mean comparisons (Tukey–Kramer
THE JOURNAL OF EXPERIMENTAL BIOLOGY
344
procedure) to determine when post feeding VO2was no longer
significantly different from SMR, and to identify significant
differences in VO2between 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
Metabolic response to feeding
Body mass, meal mass, and relative meal size (% of body
mass) did not differ significantly in the five species (Table·1).
By contrast, SMR (as ml·O2·h–1 or ml·O2·g–1·h–1) varied
significantly (all P<0.0001) among species as P. reticulatus
and P. sebae had a significantly (both P<0.0013) higher SMR
than P. brongersmai and P. regius (Table·1). All species
exhibited significant (all P<0.0001) variation in VO2, both pre
B. D. Ott and S. M. Secor
and postfeeding, with VO2increasing 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 VO2, as
well as the scope of peak VO2, 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 VO2(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).
Table·1. Metabolic parameters measured in five Python species
Variable P. brongersmai P. molurus P. regius P. reticulatus P. sebae F P
N67688
Body mass (g) 763±72 719±38 715±107 730±66 706±37 0.110 0.978
Meal mass (g) 190±20 180±9 178±26 183±17 176±10 0.430 0.758
Meal size (% of body mass) 25.0±0.0 25.1±0.1 25.0±0.2 25.0±0.0 24.9±0.2 0.300 0.902
SMR (ml·h–1) 16.5±1.2a18.4±1.3a,b 16.4±3.5a24.1±1.7c22.0±1.4b,c 12.7 < 0.0001
SMR (ml·g–1·h–1) 0.021±0.001a0.026±0.001a,b 0.022±0.002a0.034±0.001c0.030±0.001b,c 14.0 <0.0001
Peak VO2(ml·h–1) 184±18a265±18b156±28a253±20b253±10b21.7 <0.0001
Peak VO2(ml·g–1·h–1) 0.241±0.011a0.374±0.034b0.216±0.012a0.347±.009b0.349±0.009b15.8 <0.0001
Scope of peak VO2(peak VO2/SMR) 11.3±0.6a14.5±1.0b9.9±0.7a10.4±0.4a11.7±0.5a7.54 <0.0003
Duration (days) 8 6 8 7 6
SDA (kJ) 322±28 317±20 326±63 340±24 347±13 2.56 0.059
SDA (kJ·kg–1) 422±18 447±37 447±22 474±20 496±16 1.43 0.248
SDA (% of ingested; kJ) 23.1±1.0 24.5±2.0 25.1±1.2 25.6±1.1 27.3±0.9 1.25 0.312
SMR, standard metabolic rate; VO2, rate of oxygen consumption; SDA, specific dynamic action.
Values are presented as means ± 1 s.e.m.
For variables with significant Pvalues, different superscript letters denote significant (P<0.05) differences between means of the five species as
determined from post hoc pairwise comparisons.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
345Postprandial responses of Python
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).
0
0.15
0.30
0.45
P. reticulatus
0
0.15
0.30
0.45
P. brongersmai
Time postfeeding (days)
P. molurus
0
0.15
0.30
0.45
P. regius
0
0.15
0.30
0.45
0246810
P. sebae
0
0.15
0.30
0.45
V
O2 (ml g–1 h–1)
.
Fig.·2. Mean rates of oxygen consumption (VO2) prior to day·0 and up
to 10·days following the consumption of rodent meals equaling 25%
of the snake body mass for Python brongersmai, P. molurus, P. regius,
P. reticulatus and P. sebae (N=6–8 for each species). In this and the
following figures, error bars indicate ± 1 s.e.m. and are omitted if the
s.e.m. is smaller than the width of the symbol used for the mean value.
Note the rapid increase in VO2following the consumption of a meal
and a slower return to fasting rates by days·6–9.
Contents (% of original meal)
25
50
75
100 B
0
25
50
75
100
cb,c
a,b,c
A
a,b
a
0
P. brongersmai
P. regius
P. sebae
P. m o lu r us
P. reticulatus
Fig.·3. Percentage of ingested meal that was recovered within the
stomach (A) and small intestine (B) of Python brongersmai, P.
molurus, P. regius, P. reticulatus and P. sebae 2·days after the
consumption of rodent meals equaling 25% of their body mass (N=3
for each species). There was significant variation in the percentage of
the ingested meal remaining within stomachs among the species, as
both P. brongersmai and P. regius had more of the meal still
remaining than P. reticulatus and P. sebae. By contrast, there was no
variation in the percentage of the ingested meal left in the small
intestine. In A, letters above bars that are different denote significant
(P<0.05) differences between means as determined from post hoc
pairwise comparisons.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
346
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).
Postprandial changes in intestinal morphology and organ
mass
There was a significant (all P<0.044) postprandial increase
in small intestinal mass in all of the five python species
B. D. Ott and S. M. Secor
(Table·2). On average, the small intestine of these pythons
doubled in mass within 2·days after feeding (Fig.·6). For P.
reticulatus only, the postprandial increase in small intestinal
mass was also accompanied by a significant (P=0.038) increase
(17%) in small intestinal length (Fig.·6). For fasted P. molurus,
P. regius and P. sebae and for all five species postfeeding, there
was significant (all P<0.046) variation in the wet mass of
intestinal segments. In each case, the proximal segment was
significantly (all P<0.045) heavier (by 100±13%) than the
distal segment.
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).
For all five species, feeding generated a significant (all
P<0.041) increase in the wet mass (and in most cases the dry
Uptake rates (nmol min–1 mg–1)
0
1
2
3
*
P. brongersmai P. molurus P. sebaeP. regius P. reticulatus
L-leucineL-prolineD-glucose
AD
0
0.5
1.0
1.5
0
0.4
0.8
1.2
0
0.7
1.4
2.1
0
0.6
1.2
1.8
0
0.7
1.4
2.1
0
0.9
1.8
2.7
0
1
2
3
0
0.15
0.30
0.45
AD AD AD AD
0
0.1
0.2
0.3
0
1.3
2.6
3.9
0
0.8
1.6
2.4
0
0.2
0.4
0.6
0
0.7
1.4
2.1
0
0.15
0.30
0.45
0
0.11
0.22
0.33
Fasted Fed
** **
**
** **
**
*
*
***
*** ** **
**
*** ***
*
**
***
*
*
*
***
*
*
Fig.·4. Uptake rates of the amino acids L-leucine and L-proline and of the sugar D-glucose by the anterior (A) and distal (D) portions of the small
intestine of fasted (following a 30-day fast, open bars) and fed (2·days postfeeding, solid bars) Python brongersmai, P. molurus, P. regius, P.
reticulatus and P. sebae. All species (with the exception of P. brongersmai for L-leucine and L-proline) showed significant postprandial increases
in nutrient uptake by the anterior small intestine and in many cases by the distal intestine. *P<0.05, **P<0.01, ***P<0.001.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
347Postprandial responses of Python
mass) of the liver and kidneys (Table·2). At 2·days postfeeding,
liver and kidney wet masses had increased by 68.3±8.7% and
62.8±10.9%, respectively, in the five species. Additionally,
postprandial changes in organ mass included a significant
decrease (P=0.005) in wet mass of the gall bladder in P.
reticulatus and increase (P=0.022) in pancreatic wet mass in P.
brongersmai (Table·2).
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.
In similar fashion, as a result of combined intestinal
hypertrophy and postfeeding increases in aminopeptidase-N
activity, all five Python species experienced significant (all
P<0.006) postfeeding increases in total intestinal
aminopeptidase-N capacity (Fig.·8). By 2·days postfeeding, P.
brongersmai, P. molurus, P. regius, P. reticulatus and P. sebae
had increased their intestinal aminopeptidase-N capacity by
10.5-, 7.3-, 8.5-, 8.2- and 5.6-fold, respectively.
Discussion
Although differing in body shape, adult size and geographic
distribution, members of the genus Python experience
significant, and in many cases dramatic, postprandial responses
in metabolism, organ mass and intestinal performance. As an
apparent adaptive feature of their infrequent feeding habits
Python brongersmai, P. molurus, P. regius, P. reticulatus and
P. sebae downregulate intestinal performance with fasting and
consequently rapidly upregulate gut performance with feeding.
In the ensuing discussion, we shall address in turn the
postprandial metabolic, functional and trophic responses of
Python, the proximate mechanisms underlying the regulation
of their intestinal performance, the adaptive significance of
their digestive physiology, and several questions that remain to
be addressed.
Metabolic responses to feeding
All five pythons of this study exhibited the characteristic
postprandial profile of metabolism, observed as a rapid
postfeeding increase in VO2that, 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 VO2, 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
Aminopeptidase-N activity
(μmol min–1 g–1 protein)
P. brongersmai P. molurus P. sebaeP. regius P. reticulatus
0
10
20
30
AD AD A D A D A D
**
*** **
***
**
**
*
Fasted Fed
Fig.·5. Aminopeptidase-N activity of the anterior (A) and distal (D) portions of the small intestine of fasted (following a 30-day fast, open bars)
and fed (2·days postfeeding, solid bars) Python brongersmai, P. molurus, P. regius, P. reticulatus and P. sebae. There were significant
postprandial increases in aminopeptidase-N activity of the anterior small intestine in all species, and the distal small intestine in three species;
*P<0.05, **P<0.01, ***P<0.001.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
348
(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, VO2peaked 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 VO2
of P. brongersmai and P. regius, the higher scope of peak VO2
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 VO21–2·days postfeeding at rates between 0.25 and
0.55·ml·g–1·h–1, a range of VO2that 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 VO2
can be explained by differences in relative meal size (20%
versus 25% of body mass), given that postprandial peaks in VO2
increase with relative meal size (Secor and Diamond, 1997). In
B. D. Ott and S. M. Secor
a study of P. regius, Starck and Wimmer (Starck and Wimmer,
2005) recorded SMR and peak VO2of 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 VO2
(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
Table·2. Body mass, snout–vent and total length, and wet and dry mass for organs removed from fasted and fed Python species
P. brongersmai P. molurus P. regius P. reticulatus P. sebae
Variable Fasted 2·d.p.f. Fasted 2·d.p.f. Fasted 2·d.p.f. Fasted 2·d.p.f. Fasted 2·d.p.f.
N3333333333
Body mass (g) 818±136 861±42 790±109 769±29 741±110 710±85 815±99 753±48 780±112 852±25
Snout–vent 89.3±4.5 89.5±1.3 120.7±8.8 122.3±1.9 98.1±9.2 97.8±6.2 154.3±7.1 145.5±4.8 119.3±5.8 117.0±1.0
length (cm)
Total length (cm) 96.2±4.8 96.5±4.8 135.0±9.5 137.0±3.1 106.7±9.6 105.7±6.9 178.2±7.8 166.7±5.2 134.7±6.6 132.7±1.3
Wet mass (g)
Heart 2.00±0.44 2.57±0.62 2.05±0.25 2.06±0.15 1.78±0.11 1.61±0.05 1.75±0.17 1.65±0.15 1.99±0.28 2.37±0.45
Lung 5.22±1.09 5.03±0.66 6.42±0.66 6.96±0.17 5.24±0.79 4.02±0.32 5.84±0.79 6.76±1.46 5.69±0.52 6.20±0.26
Liver 11.1±2.6 20.5±1.6* 8.27±0.32 14.3±0.9* 9.17±0.76 12.8±0.4* 8.50±2.16 15.8±0.8* 11.8±1.4 18.7±1.2*
Stomach 10.1±2.1 14.6±2.3 10.1±2.1 14.3±1.2 7.09±0.80 11.9±3.0 10.5±0.8 12.1±1.1 11.9±1.6 14.7±0.5
Gall bladder 1.75±0.61 1.10±0.55 2.92±0.33 2.01±0.19 1.70±0.02 1.59±0.22 3.93±0.31 2.09±0.14* 2.62±0.29 2.50±0.35
Pancreas 0.89±0.16 1.42±0.10* 0.71±0.08 0.94±0.07 0.61±0.10 0.83±0.10 0.93±0.19 0.99±0.03 0.86±0.11 1.14±0.10
Spleen 0.06±0.01 0.11±0.04 0.10±0.01 0.14±0.02 0.06±0.00 0.06±0.00 0.09±0.00 0.05±0.03 0.17±0.04 0.18±0.05
Small intestine 10.4±2.3 23.7±1.0* 10.6±0.4 25.3±4.1* 8.34±0.77 13.7±1.9* 13.9±0.7 23.0±2.6* 10.5±1.2 24.1±0.05*
Large intestine 8.17±2.16 8.89±0.95 6.51±0.97 9.14±0.90 5.07±0.75 4.91±0.66 4.86±0.52 5.34±0.37 6.03±0.80 7.42±0.33
Kidneys 3.76±0.74 7.23±0.60* 3.49±0.45 6.14±0.09* 3.32±0.64 4.74±0.35* 3.78±0.43 5.04±0.36* 4.34±0.26 7.37±0.53*
Dry mass (g)
Heart 0.30±0.13 0.34±0.08 0.33±0.05 0.32±0.02 0.27±0.05 0.28±0.01 0.33±0.01 0.26±0.03 0.38±0.07 0.38±0.05
Lung 0.79±0.22 0.74±0.10 1.19±0.14 1.20±0.04 0.83±0.07 0.74±0.10 1.26±0.13 1.22±0.26 1.08±0.17 1.20±0.04
Liver 5.05±1.22 6.19±1.02 3.01±0.05 3.75±0.26* 2.49±0.15 3.37±0.16* 3.10±0.35 4.27±0.18* 4.68±0.69 6.48±0.43*
Stomach 1.93±0.45 1.97±0.26 2.56±0.12 2.74±0.26 1.31±0.14 2.07±0.48 2.34±0.22 2.45±0.18 2.36±0.37 3.06±0.16
Gall bladder 0.32±0.21 0.31±0.24 0.56±0.08 0.48±0.10 0.17±0.08 0.21±0.09 0.47±0.17 0.32±0.06 0.48±0.10 0.43±0.06
Pancreas 0.11±0.04 0.25±0.01 0.14±0.01 0.12±0.00 0.13±0.02 0.15±0.02 0.22±0.05 0.21±0.01 0.19±0.03 0.23±0.02
Spleen 0.02±0.00 0.02±0.01 0.02±0.00 0.03±0.00 0.01±0.00 0.01±0.00 0.02±0.00 0.01±0.01 0.03±0.01 0.04±0.01
Large intestine 1.25±0.28 1.34±0.15 1.20±0.17 1.29±0.12 0.86±0.14 0.60±0.03 1.01±0.11 0.94±0.10 1.10±0.16 1.53±0.15
Kidneys 0.76±0.16 1.14±0.06 0.63±0.23 1.25±0.17* 0.64±0.13 0.75±0.06 1.03±0.11 1.04±0.13 0.80±0.08 1.40±0.10*
Organs were weighed immediately after removal from three fasted and three fed [2 days postfeeding (d.p.f.)] individuals of each Python
species.
*Significant differences in organ mass between fasted and fed snakes, determined by ANCOVA (P<0.05).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
349Postprandial responses of Python
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
Fig.·6. Small intestinal mass and length of fasted (following a 30-day fast, open bars) and fed (2·days postfeeding, solid bars) Python brongersmai,
P. molurus, P. regius, P. reticulatus and P. sebae. All five species had significant postfeeding increases in small intestinal mass, whereas only
P. reticulatus showed an increase in intestinal length with feeding. The photographs of small intestines of fasted and fed P. molurus illustrate
the postprandial trophic response. Asterisks indicate significant differences between the two states; *P<0.05, **P<0.01.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
350
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
B. D. Ott and S. M. Secor
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
Fig.·7. Width of the intestinal muscularis/serosa and mucosa layers, and height, width and volume of intestinal enterocytes and micrographs of
the intestinal epithelium of fasted (following a 30-day fast, open bars) and fed (2·days postfeeding, solid bars) Python brongersmai, P. molurus,
P. regius, P. reticulatus and P. sebae. Note that after feeding there is a lack of change in muscularis/serosa layer thickness and enterocyte height,
and the significant increase in thickness of the mucosal layer and enterocyte width and volume. Asterisks indicate significant differences between
the two states; *P<0.05, **P<0.01, ***P<0.001.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
351Postprandial responses of Python
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
D-glucose
0
12
24
36
0
11
22
33
0
17
34
51
0
15
30
45
0
13
26
39
0
4
8
12
Uptake capacity (μmol min–1)
0
3
6
9
0
1.1
2.2
3.3
0
20
40
60
0
10
20
30
0
8
16
24
0
15
30
45
0
8
16
24
P. brongersmai P. molurus P. sebaeP. regius P. reticulatus
Aminopeptidase-N
capacity (μmol min–1)
**
0
15
30
45 ***
***
*
**
**
**
*
**
*
*
**
***
0
20
40
60 **
0
2
4
6**
0
8
16
24 **
0
22
44
66 ***
0
16
32
48 **
0
3
6
9*
**
Fasted Fed
L-leucine
L-proline
Fig.·8. Small intestine uptake capacities of L-leucine, L-proline and D-glucose. and intestinal aminopeptidase-N activity of fasted (following a
30-day fast, open bars) and fed (2·days postfeeding, solid bars) Python brongersmai, P. molurus, P. regius, P. reticulatus and P. sebae. After
feeding, all five Python species significantly increased uptake of each nutrient and aminopeptidase-N activity; *P<0.05, **P<0.01, ***P<0.001.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
352
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
B. D. Ott and S. M. Secor
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·m) 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
THE JOURNAL OF EXPERIMENTAL BIOLOGY
353Postprandial responses of Python
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).
The conclusions we draw from this study are: (1) members of
the genus Python respond to each meal with large increases in
metabolic rate, intestinal hypertrophy, and the elevation of
intestinal function; (2) the subtle interspecific variation in
physiological and morphological responses among the five
species of Python are not associated with either geography
(Africa vs Asia) or body shape (stout vs elongated body shape);
and (3) as previously described for P. molurus, these five species
share the adaptive capacity to widely regulate gastrointestinal
performance with feeding and fasting. Although not studied, we
predict that the other five species of Python (P. anchietae, P.
breitensteini, P. curtus, P. natalensis and P. timoriensis) likewise
up- and downregulate gastrointestinal performance with each
meal.
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,
99
Body mass (g)
SMR (ml O2 h–1)
9
Frequently feeding
SMR=0.075Mb0.95
r2=0.90
1
10
100
100 100050
1
2
3
4
56
7
8
13
12
10 11
9
14
16
1517
18
Infrequently feeding
SMR=0.047Mb0.92
r2=0.97
Fig.·9. Standard metabolic rate (SMR) at 30°C plotted against body
mass Mbfor seven species of frequently feeding snakes and 11 species
of infrequently feeding snakes. Interspecific allometric equations were
generated from least-squares regression analysis of data from Secor
and Diamond (Secor and Diamond, 2000), this study, and our
unpublished observations. Numbers signify the following species: 1,
Thamnophis marcianus; 2, Thamnophis sirtalis; 3, Lampropeltis
getula; 4, Coluber constrictor; 5, Masticophis flagellum; 6, Nerodia
rhombifer; 7, Pituophis melanoleucus; 8, Morelia spilota; 9, Crotalus
cerastes; 10, Lichanura trivirgata; 11, Acrantophis dumerili; 12, Boa
constrictor; 13, Python sebae; 14, P. regius; 15, P. molurus; 16, P.
reticulatus; 17, P. brongersmai; 18, Eunectes murinus. Note that
across the range of body masses compared, infrequently feeding
snakes have SMR that are almost 50% less than those of frequently
feeding species.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
354
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 include arboreal species of the genus Morelia
(Pythonidae) and Corallus (Boidae) that are extremely long and
slender and may feed more frequently in the wild (Henderson,
2002). Studies on these genera, and others, may reveal the
independent evolution of the narrow regulation of
gastrointestinal performance within lineages of snakes that
largely regulate gut performance. A further expansion of this
inquiry would include studies on members of adjoining basal
lineages (Loxocemidae and Xenopeltidae) and those
(Acrochordidae, Bolyeriidae, Tropdophiidae and
Xenophidiidae) positioned phylogenetically between pythons
and boas and the family Viperidae, which includes the wide
regulating Crotalus cerastes (Secor et al., 1994; Pough et al.,
2004). These studies would be the next step in elucidating the
evolutionary pattern of digestive response among snakes.
We wish to thank M. Addington, K. Asbill, J. Bagley, S.
Boback, M. Boehm, S. Cover, C. Cox, B. Gandolfi, L. Kirby, E.
Newsom, J. Phillips, K. Picard, E. Roth, K. Stubblefield and J.
Wooten for their assistance in this project. Funding for this
study was provided in part by the National Science Foundation
(IOB-0466139 to S. Secor) and the University of Alabama.
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B. D. Ott and S. M. Secor356
THE JOURNAL OF EXPERIMENTAL BIOLOGY
... The energy expenditure required to process a meal is termed specific dynamic action (SDA) (Rubner 1902;McCue 2006). The SDA for Burmese pythons eating meals equal to 25% of their body mass was measured at ~ 450 kJ/kg, approximately 25% of the ingested energy, though SDA increases with larger meals (Secor and Diamond 1997b;Ott and Secor 2007;Cox and Secor 2007). By comparison, the average SDA for frequently feeding species like mammals is ~ 10% (McCue 2006). ...
... (D) Structural and functional changes in the heart throughout digestion (Secor 2008;Secor and White 2010) By comparison, humans and other frequently feeding species display little or no change in microvillus morphology during or between meals (Secor 2005). This increased surface area paired with an up to 10-fold increase in intestinal enzyme activity (including maltase and aminopeptidase-N) synergize to maximize nutrient uptake (Ott and Secor 2007;Cox and Secor 2008;Secor 2008). For example, intestinal glucose and amino acid uptake increase up to 15-fold by 1DPF (Secor et al. 2000b). ...
... Once the feeding starts, the stomach is flooded with hydrochloric acid, and the pH quickly drops below 2, where it remains for over a week before climbing to neutral again by 15DPF (Secor 2003;Bessler and Secor 2012) (Fig. 1 C). The intestines undergo the greatest magnitude of remodeling of any organ during digestion, with the small intestine more than doubling in mass (Holmberg et al. 2002;Ott and Secor 2007). This organ growth is specifically driven by changes in the mucosal layer (Cox and Secor 2008). ...
Article
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Non-traditional animal models present an opportunity to discover novel biology that has evolved to allow such animals to survive in extreme environments. One striking example is the Burmese python (Python molurus bivittatus), which exhibits extreme physiological adaptation in various metabolic organs after consuming a large meal following long periods of fasting. The response to such a large meal in pythons involves a dramatic surge in metabolic rate, lipid overload in plasma, and massive but reversible organ growth through the course of digestion. Multiple studies have reported the physiological responses in post-prandial pythons, while the specific molecular control of these processes is less well-studied. Investigating the mechanisms that coordinate organ growth and adaptive responses offers the opportunity to gain novel insight that may be able to treat various pathologies in humans. Here, we summarize past research on the post-prandial physiological changes in the Burmese python with a focus on the gastrointestinal tract, heart, and liver. Specifically, we address our recent molecular discoveries in the post-prandial python liver which demonstrate transient adaptations that may reveal new therapeutic targets. Lastly, we explore new biology of the aquaporin 7 gene that is potently upregulated in mammalian cardiac myocytes by circulating factors in post-prandial python plasma.
... Ambush-foraging snakes are characterized by infrequent feeding bouts, low standard metabolic rates (SMR), and the capacity to widely regulate digestive performance (Secor and Nagy, 1994;Secor and Diamond, 2000;Wang, 2001;Secor, 2007a, 2007b;Stuginski et al., 2018). With the completion of meal digestion and absorption, these snakes terminate gastric acid production, downregulate pancreatic and intestinal performance, and undergo atrophy of the liver, kidneys, pancreas, and small intestine (Secor and Diamond, 2000;Secor, 2003;Ott and Secor, 2007a;Cox and Secor, 2008;Wang and Rindom, 2021). Like other vertebrates, carbohydrates and lipids reserves in snakes sustain energy homeostasis during regular fasting, with proteins being used as the last energy resource in prolonged fasting (McCue, 2010;McCue et al., 2012;Secor and Carey, 2016). ...
... Moreover, ambush-foraging snakes can reverse these conditions rapidly and upregulate gastrointestinal performance after ingesting large meals, undergoing organ hypertrophy necessary for digestion, absorption, and the incorporation of the nutrients Secor and Diamond, 2000;Ott and Secor, 2007a;Cox and Secor, 2008;Henriksen et al., 2015;Enok et al., 2016). ...
... Consequently, a dramatic metabolic increment follows the ingestion of large preys, representing the sum of several physiological and morphological processes (Andrade et al., 1997;Secor and Diamond, 1997;Secor and Diamond, 2000;Overgaard et al., 2002;Ott and Secor, 2007a). Thus, when such processes require energy the ensure of food processing and nutrients absorption, representing the remarkable transition from a state of energy conservation (fasting) to a condition in which this energy is widely used (postprandial; Secor and Diamond, 1995;McCue, 2006;Secor, 2009). ...
Article
Ambush-foraging snakes that ingest large meals might undergo several months without eating when they use the internal reserves to support the energetic costs of living. Then, morphological and physiological processes might be orchestrated during the transition from fasting to the postprandial period to rapidly use the energetic stores while the metabolic rate is elevated in response to food intake. To understand the patterns of substrates deposition after feeding, we accessed the morphological and biochemical response in Boa constrictor snakes after two months of fasting and six days after feeding. We followed the plasma levels of glucose, total proteins, and total lipids, and we performed the stereological ultrastructural analysis of the liver and the proximal region of the intestine to quantify glycogen granules and lipid droplets. In the same tissues and stomach, we measured the activity of the enzyme fructose-1,6-biphosphatase (FBPase1) involved in the gluconeogenic pathway, and we measured pyruvate kinase (PK) and lactate dehydrogenase (LDH) enzymatic activities involved in the anaerobic pathway in the liver. Briefly, our results indicated an increase in boas' plasma glucose one day after meal intake compared to unfed snakes. The hepatic glycogen reserves were continuously restored within days after feeding. Also, the enzymes involved in the energetic pathways increased activity six days after feeding in the liver. These findings suggest a quick restoring pattern of energetic stores during the postprandial period.
... Although pancreatic enzyme activity was unaltered with feeding, acinar cell size was increased and returned to fasting levels by 72 h. Increases in pancreas wet mass have been previously observed in the southern catfish, Atlantic cod (Gadus morhua), and in multiple snakes and anurans (Blier et al. 2007;Cox and Secor 2008;Ott and Secor 2007;Secor 2005;Secor and Diamond 2000;Zeng et al. 2012). This pancreatic hypertrophy is attributed to acinar cells responding to an increased need for digestive enzymes and neutralizing bicarbonate and has been linked to increases in RNA, protein, and water content in mammals (Webster et al. 1972). ...
Article
Full-text available
Secretions of the exocrine pancreas contain digestive enzymes integral to the digestive process. The Pacific spiny dogfish (Squalus suckleyi) has a discrete pancreas, divided into two lobes termed the dorsal and ventral lobes. These lobes drain into the anterior intestine via a common duct to enable digestion. Previous studies have identified that the exocrine pancreas produces (co)lipases, chymotrypsin, carboxypeptidase, and low levels of chitinases; however, investigations into other digestive enzymes are limited. We detect the presence of lipase, trypsin, and carbohydrase and show that activities are equivalent between both lobes of the pancreas. Additionally, we sought to investigate the influence of a single feeding event (2% body weight ration of herring by gavage) on enzyme activities over an extended time course (0, 20, 48, 72, 168 h) post-feeding. The results indicate that there are no differences in pancreatic tissue digestive enzyme activities between fed or fasted states. Analysis of acinar cell circumference post-feeding demonstrates a significant increase at 20 and 48 h, that returns to fasting levels by 72 h. No significant changes were observed regarding whole-tissue insulin or glucagon mRNA abundance or with glucose transporter (glut) 1 or 3. Yet, a significant and transient decrease in glut4 and sodium glucose-linked transporter mRNA abundance was found at 48 h post-feeding. We propose that the constant enzyme activity across this relatively large organ, in combination with a relatively slow rate of digestion leads to an evenly distributed, sustained release of digestive enzymes regardless of digestive state.
... Multiple heavily bodied lineages of snakes (including boas, pythons, and rattlesnakes) that experience monthslong fasts between meals have evolved the ability to downregulate and atrophy energetically costly digestive organs during fasting, only to subsequently regenerate organ tissue and function at unequaled rates and magnitudes after feeding. For example, within 24 h of consuming a meal, the Burmese python (Python bivittatus), increases its small intestine wet mass two-fold, intestinal mucosa thickness three-fold, and intestinal microvillus length five-fold [19][20][21][22][23]. Physiological activity simultaneously increases, including a 44-fold increase in metabolic rate and 20-fold increase in intestinal nutrient transport [15,16,[22][23][24][25]. ...
Article
Full-text available
Background Snakes exhibit extreme intestinal regeneration following months-long fasts that involves unparalleled increases in metabolism, function, and tissue growth, but the specific molecular control of this process is unknown. Understanding the mechanisms that coordinate these regenerative phenotypes provides valuable opportunities to understand critical pathways that may control vertebrate regeneration and novel perspectives on vertebrate regenerative capacities. Results Here, we integrate a comprehensive set of phenotypic, transcriptomic, proteomic, and phosphoproteomic data from boa constrictors to identify the mechanisms that orchestrate shifts in metabolism, nutrient uptake, and cellular stress to direct phases of the regenerative response. We identify specific temporal patterns of metabolic, stress response, and growth pathway activation that direct regeneration and provide evidence for multiple key central regulatory molecules kinases that integrate these signals, including major conserved pathways like mTOR signaling and the unfolded protein response. Conclusion Collectively, our results identify a novel switch-like role of stress responses in intestinal regeneration that forms a primary regulatory hub facilitating organ regeneration and could point to potential pathways to understand regenerative capacity in vertebrates.
... Moreover, the absence of a response of MEL PI to feeding in boas is in agreement with previous studies, since MEL PI showed a modest increase in mice and did not increase in anurans during fasting (Bubenik et al., 1992;Figueiredo et al., in press). The intestines of extremely infrequent feeders, such as boas, experience the most marked phenotypic modulation during fasting, including mucosa atrophy and a reduction in enterocyte volume (Ott and Secor, 2007;Secor and Carey, 2016), contributing to reduced energy expenditure and possibly to the protection of the gut in this period. The production and function of MEL on the GIT during fasting periods remains to be explored in ectothermic animals. ...
Article
Feeding upregulates immune function and the systemic and local (gastrointestinal tract) concentrations of some immunoregulatory hormones, as corticosterone (CORT) and melatonin (MEL), in mammals and anurans. However, little is known about the immune and hormonal regulation in response to feeding in other ectothermic vertebrates, especially snakes, in which the postprandial metabolic changes are pronounced. Here, we investigated the effects feeding have on hormonal and innate immune responses in the snake, Boa constrictor. We divided juvenile males into two groups: fasting and fed with mice (30% of body mass). We measured the rates of oxygen consumption, plasma CORT levels, heterophil/lymphocyte ratio (HL ratio), plasma bacterial killing ability (BKA), and stomach and intestine MEL in fasting snakes and 48 h after meal intake. We observed increased rates of oxygen consumption, plasma CORT levels, and HL ratio, along with a tendency of decreased stomach and intestine MEL in fed snakes compared to fasting ones. BKA was not affected by feeding. Overall, we found that feeding modulates metabolic rates, CORT levels, and immune cell distribution in boas. Increased baseline CORT may be important to mobilize energy to support the metabolic increment during the postprandial period. Increased HL ratio might be an immunoregulatory effect of increased CORT, which has been shown in different physiological situations such as in response to immune challenge. Our results suggest that feeding activates the hypothalamic-pituitary-adrenal axis and modulates immune cell redistribution, possibly contributing to fighting potential injuries and infections derived from predation and from pathogens present in ingested food.
... After meal intake, the switch from fasting to the postprandial period is marked by a set of alterations in morphology and physiology of the gastrointestinal tract (GIT), as well as by local (GIT) and systemic immune and endocrine modulation (Hansen et al., 1997;Bubenik, 2001;Secor, 2003;Crespi et al., 2004;Lignot et al., 2005;Luoma et al., 2016). All these processes are accompanied by an energy investment (specific dynamic action or SDA; Kleiber, 1961) that demands increased aerobic metabolism (Secor and Diamond, 2000;Secor, 2005Secor, , 2009Ott and Secor, 2007). In ectothermic animals, SDA modulation is especially pronounced as a result of the low basal metabolism cost and ingestion of large preys (Andrade et al., 2005). ...
Article
Mammals show immune up-regulation and increased plasma and local (gastrointestinal tract) concentrations of some immunoregulatory hormones, such as corticosterone and melatonin, after feeding. However, little is known about the endocrine and immune modulation in the postprandial period of ectothermic animals. This study investigated the effects of feeding on endocrine and immune responses in the bullfrog (Lithobates catesbeianus). Frogs were fasted for 10 days and divided into two groups: fasted and fed with fish feed (5% of body mass). Blood and gastrointestinal tract tissues (stomach and intestine) were collected at 6, 24, 48, 96, and 168 h to measure neutrophil/lymphocyte ratio, plasma bacterial killing ability, phagocytosis of blood leukocytes, plasma corticosterone and melatonin; and stomach and intestine melatonin. Feeding increased plasma corticosterone at 24 h and decreased at 168 h; and increased neutrophil/lymphocyte ratio at 6, 24, and 96 h. We also observed decreased bacterial killing ability 48 h after feeding. Stomach melatonin increased after 17-days fasting. We show that feeding activates the hypothalamic-pituitary-interrenal axis and promotes transient immunosuppression, without stimulating an inflammatory response. Increased CORT may mobilize energy to support the digestive processes and melatonin may protect the stomach during fasting. We conclude feeding modulate secretion of immunoregulatory hormones, increasing plasma CORT levels in the beginning followed by a decrease in the end of meal digestion; and systemic immune cell redistribution, increasing NL ratio during almost all meal digestion in bullfrogs. Also, fasting modulate secretion of melatonin in the stomach.
... Based on the concept of geometric gut surface scaling, a possible reason for the lower scaling in snakes could lie in the very intermittent feeding pattern in the snakes [41,42] with a particular dependence on gastric dilatation, but little dependence on a long intestine with a high throughput capacity. ...
Article
Full-text available
Although relationships between intestinal morphology between trophic groups in reptiles are widely assumed and represent a cornerstone of ecomorphological narratives, few comparative approaches actually tested this hypothesis on a larger scale. We collected data on lengths of intestinal sections of 205 reptile species for which either body mass (BM), snout-vent-length (SVL) or carapax length (CL) was recorded, transforming SVL or CL into BM if the latter was not given, and analyzed scaling patterns with BM and SVL, accounting for phylogeny, comparing three trophic guilds (faunivores, omnivores, herbivores), and comparing with a mammal dataset. Length-BM relationships in reptiles were stronger for the small than the large intestine, suggesting that for the latter, additional factors might be relevant. Adding trophic level did not consistently improve model fit; only when controlling for phylogeny, models indicated a longer large intestine in herbivores, due to a corresponding pattern in lizards. Trophic level effects were highly susceptible to sample sizes, and not considered strong. Models that linked BM to intestine length had better support than models using SVL, due to the deviating body shape of snakes. At comparable BM, reptiles had shorter intestines than mammals. While the latter finding corresponds to findings of lower tissue masses for the digestive tract and other organs in reptiles as well as our understanding of differences in energetic requirements between the classes, they raise the hitherto unanswered question what it is that reptiles of similar BM have more than mammals. A lesser effect of trophic level on intestine lengths in reptiles compared to mammals may stem from lesser selective pressures on differentiation between trophic guilds, related to the generally lower food intake and different movement patterns of reptiles, which may not similarly escalate evolutionary arms races tuned to optimal agility as between mammalian predators and prey.
... Research programs in integrative biology, which focus on assimilating techniques and data from diverse fields of study, have long engaged in severe tests of hypotheses (Brodie and Brodie 1990;Karasov and Levey 1990;Hutchinson et al. 2007;Ott and Secor 2007). However, the value of this approach is sometimes appreciated only for single-species studies, while studies comparing two or a few species can be considered problematic. ...
Article
Full-text available
Phylogenetic comparative methods represent a major advance in integrative and comparative biology and have allowed researchers to rigorously test for adaptation in a macroevolutionary framework. However, phylogenetic comparative methods require trait data for many species, which is impractical for certain taxonomic groups and trait types. We propose that the philosophical principle of severity can be implemented in an integrative framework to generate strong inference of adaptation in studies that compare only a few populations or species. This approach requires (1) ensuring that the study system contains species that are relatively closely related; (2) formulating a specific, clear, overarching hypothesis that can be subjected to integrative testing across levels of biological organization (e.g., ecology, behavior, morphology, physiology, and genetics); (3) collecting data that avoid statistical underdetermination and thus allow severe tests of hypotheses; and (4) systematically refining and refuting alternative hypotheses. Although difficult to collect for more than a few species, detailed, integrative data can be used to differentiate among several potential agents of selection. In this way, integrative studies of small numbers of closely related species can complement and even improve on broadscale phylogenetic comparative studies by revealing the specific drivers of adaptation.
Animals which feed infrequently and on large prey, like many snake species, are characterized by a high magnitude of gut upregulation upon ingesting a meal. The intensity of intestinal upregulation was hypothesized to be proportional to the time and energy required for food processing (Specific-Dynamic-Action; SDA); hence, a positive correlation between the scope of intestinal growth and SDA response can be deduced. Such a correlation would support the so far not well established link between the intestinal and metabolic consequences of digestion. In this study I tested this prediction using an interspecific dataset on snakes gleaned from published sources. I found that SDAduration and SDAscope were positively correlated with post-feeding factorial increase in small intestine mass, but not with microvillar elongation. This indicates that a wide range of whole intestine remodelling (up- but potentially also downregulation) may temporarily prolong meal processing and that a greater magnitude of intestinal growth requires a stronger metabolic elevation. However, these effects do not seem large enough to drive the variation in the entire energetic costs of digestion, because SDAexpenditure was not affected either by intestinal or microvillar growth. I therefore propose that intestinal upregulation elicits non-negligible costs, but that these costs are a fairly small component of the whole SDAexpenditure.
Article
The postprandial period is characterized by a modification of the gastrointestinal activity after food intake, accompanied by an increase in metabolic rate, secretion of acids, and absorption of nutrients. For ectothermic vertebrates, those changes are particularly prominent given the relatively low metabolic cost and the low frequency of food uptake. However, prolonged fasting periods decrease energy reserves and may compromise the upregulation of costly processes, such as the increase in metabolic rate after resuming the meal intake. Assuming that the main source of energy needed to support such events is provided from the animal's own body reserves, our aim with this study is to test the hypothesis that the longer the period of fasting, the smaller the metabolic rate increase during the postprandial period, since lesser energy reserves trigger these increases. For this, we measured the oxygen consumption rates (V̇O2) of red‐eared slider turtles, Trachemys scripta elegans, submitted to different periods of fasting (47 and 102 days), before and after the ingestion of meals equivalent to 5% of their body masses. Despite the longer fasting period, which led to a reduction of 10.77% in the body mass of the turtles, there were no differences between the two experimental groups regarding maximum V̇O2 values after food intake (V̇O2 peak), postprandial metabolic scope, mean time to V̇O2 peak, and postprandial duration. Results indicate that 102 fasting days does not compromise aerobic metabolic increase during postprandial period and does not impair digestive process of the turtles, even with a loss of body mass. Highlights • 102 Days of fasting reduced body mass of the turtles Trachemys scripta elegans but not impair the metabolic increase after a new meal intake. The turtles use their energy reserves to sustain the postprandial metabolism.
Conference Paper
Among snakes a correlation exists between feeding habits (frequent or infrequent) and the magnitude by which digestive performance is regulated (modest or large). This paper investigates whether the observed regulation of digestive performance is an adaptation to feeding habits and therefore, a product of natural selection. Using data on metabolic and intestinal responses to feeding for amphibians and reptiles, it is attempted to show the selective advantage and independent origin of either modestly or widely regulating gut performance. In an energetic model, snakes that naturally feed frequently on small meals benefit (from lower energy output) from modestly regulating gut performance as opposed to widely regulating gut performance. Likewise, the model suggests an energetic benefit for infrequently-feeding snakes secondary to the wide regulation of gut performance. This benefit is a function of long spans of fasting with a down-regulated gut (thereby incurring a lower standard metabolic rate) and the occasionally incursion of a costly up-regulation of the gut. In a comparison across several distantly-related lineages of amphibians and reptiles, frequently-feeding species all exhibit small postprandial responses in metabolism and intestinal nutrient transport capacities. In contrast, frogs and snakes that routinely fast for long periods independently experience five- to 30-fold increases in metabolism and intestinal performance with feeding. Among amphibians and reptiles the evidence presented supports the hypothesis that the extent by which the gut is regulated is an adaptive trait that evolved with divergence in feeding habits and energy budgets. In finishing, the foundations, caveats, and suggested future tests of this adaptive hypothesis are presented.
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We studied adaptations of digestive physiology that permit Rufous (Selasphorus rufus) and Anna's hummingbirds (Calypte anna) to absorb sugar-water meals rapidly and efficiently. As measured with soluble markers, transit times (<15 min) and mean retention times (ca. 48 min) of meals in the hummingbird digestive tract are brief compared with values for most other vertebrates. Glucose is extracted with an efficiency of 97%. We describe a new method, employing double isotope dilution, for measuring crop-emptying kinetics. Based on this method, the crop empties half of a meal in ca. 4 min and all of the meal in 15-20 min. Rufous and Anna's hummingbirds may be energy maximizers limited by crop emptying times, rather than foraging-time minimizers. This would explain why hummingbirds spend a majority of each hour sitting rather than feeding. The intestine's passive permeability to glucose is the lowest of any vertebrate studied to date. This may be an adaptation to prevent solute loss from the blood in the face of high fluid transit rates through the intestine. Active transport accounts for essentially all intestinal glucose absorption. Compared with intestines of other vertebrates, the glucose absorption sites of hummingbird intestines have normal binding constants but are present at extremely high densities. Comparisons of hummingbirds, chickens, and shrikes suggest that intestinal absorption rates for amino acids are independent of trophic habits in birds as in other vertebrate classes, but that sugar absorption decreases in the sequence herbivore > omnivore > carnivore.
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
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Examination of specimens collected for the international leather trade provided data on two species of large, heavy-bodied snakes: blood pythons (Python brongersmai) from northeastern Sumatra and short-tailed pythons (P. curtus) from northwestern Sumatra. Measurement and dissection of 2063 P. brongersmai and 181 P. curtus revealed broad interspecific similarities in morphology (size, shape, sexual dimorphism), food habits (feeding frequencies, dietary composition) and reproductive output (reproductive frequencies, egg sizes, and clutch sizes). Females of both species attain larger sizes than males, mature at larger sizes, and contain larger abdominal fatbodies. Python curtus is more heavy-bodied and longer-tailed than P. brongersmai, and more heavily infested with gut parasites. Both species feed almost exclusively on commensal rodents. Feeding rates increase with body size, and vary seasonally. Reproduction is highly seasonal. Adult females reproduce biennially, producing an average clutch of 12 to 16 large (mean = 83 to 90 g) eggs. The data also enable us to comment on the sustainability of the existing commercial trade, which is based mainly on adult males, and adult plus juvenile females. Anthropogenic habitat modification (especially, the establishment of oil-palm plantations) has increased the abundance of these taxa. Although neither species is likely to be extirpated by current levels of offtake, we need additional information to evaluate long-term sustainability of the commercial industry based on these snakes.
Article
When an animal fasts throughout the period of measurement of CO₂ production but is presumed to be eating, the error of estimating energy metabolism can be as high as 23% for herbivores; the error is small (≤3.2%) for uricotelic carnivores, and it increases steadily with the protein content of the diet for ureotelic carnivores. Errors in converting CO₂ production measurements to energy metabolism are associated with small errors (for animals that are eating daily) when a respiratory quotient of 0.83 for all herbivores, 0.80-0.83 for mammalian carnivores, and 0.72 for uricotelic carnivores is used. Similar conversions of oxygen consumption measurements into energy metabolism are small (<±3%) when urinary nitrogen excretion is not measured and RQ is assumed to be 0.8, regardless of the actual protein, fat, and carbohydrate mixture being catabolized by fed carnivores, omnivores, or herbivores. The error is even smaller in nearly all cases if the respiratory quotient is measured and is used to derive energy conversion factors (<±0.6% for uricotelic herbivores and carnivores).