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Metamorphic shortening of the alimentary tract in anuran larvae (Rana catesbeiana).

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THE ANATOMICAL RECORD 242:417-423 (1995)
Metar iorphic Shortening of the Alimentary Tract in An1
Larvae ( R a m catesbeiana)
ROBERT PRETTY, TOM10 NAITOH, AND RICHARD J. WASSERSUG
Department of Anatomy and Neurobiology, Dalhousie University, Halifcu,
Nova Scotia, Canada
ABSTRACT
Background: The premetamorphic alimentary tract in
anurans can be more than 10 times a tadpole's body length but then dramatically shortens to a third or less of that length by the end of metamorphosis. Although there have been many studies on histological changes in
the anuran gut with metamorphosis, the broader question of where the
major shortening occurs has not been previously addressed. This topic is
investigated here.
Methods: We began our study by labeling intestinal coils in situ in preserved Rana catesbeiana tadpoles and then uncoiling their intestines, locating, and measuring the labeled points. This allowed us to map the coiled
gut of the tadpole, such that the distance along the oral-anal axis could be
determined by simply counting coils. We next implanted markers into the
intestinal coils of live R. catesbeiana tadpoles at five known locations along
the oral-anal axis, established from the prior mapping. The tadpoles were
then induced to metamorphose by immersion in thyroid hormone. After the
gut had shortened to a third of its premetamorphic length, the positions of
the implanted markers were determined through dissection.
Results: Relative distances between the marked points did not change
when the gut shortened.
Conclusions: The results indicate that during metamorphosis the intestine shortens uniformly along its length and not preferentially from one
region or another. Although metamorphosis was artificially induced, the
shortening that we observed matches that occurring during natural
metamorphosis. o 1995 Wiley-fiss, Inc.
Key words: Metamorphosis, Alimentary tract, Intestine, Anura, Rana
catesbeiana
The metamorphosis of a tadpole into a frog is one of
nature's most spectacular events. Part of this spectacle
includes profound changes in the alimentary tract.
During metamorphosis there are both microscopic
changes in the intestines at the histological level and
macroscopic changes in its overall length.
These histological changes are well documented.
They occur in both the epithelial and extraepithelial
layers (Dauca and Hourdry, 1985; Hourdry and Beaumont, 1985).During metamorphosis, the larval gut lining is degraded (Fox, 1984; Yoshizato, 1989). There is
increased production of acid phosphatase by lysosomes
along the alimentary tract (Kaltenbach et al., 1981)'
which helps break down the premetamorphic epithelium. Epithelial cells are autolysed and engulfed by
phagocytes. Finally, much of the degenerated tissue is
sloughed off into the intestinal lumen.
Simultaneously, the adult intestinal epithelium differentiates. Nests of undifferentiated precursor cells divide and produce a new epithelium (Marshall and
Dixon, 1978).The connective tissue and musculature of
0 1995 WILEY-LISS. INC
the gut wall also proliferate greatly (Ishizuya-Oka and
Shimozawa, 1987).
While all of these histological changes are taking
place, the intestine shortens extensively. Before metamorphosis the intestine in common frogs, such as those
of the genus Rana, can be 10 or more times the body
length of the tadpole (Rugh, 1951; this study). Intestinal shortening begins before the tail is absorbed
(Dauca and Hourdry, 1985), and by completion of metamorphosis the absolute length of the gut is usually less
than a third its premetamorphic length. Some representative values for the percentage that the gut shortens at metamorphosis are: 58.15%in Rana temporaria,
82.2% in Rana pipiens (Janes, 1934); 90% in Alytes
Received November 7, 1994; accepted January 31, 1995.
Address reprint requests to Richard Wassersug, Department of
Anatomy and Neurobiology, Sir Charles "upper Medical Building,
Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada.
Tomio Naitoh is now at the Department of Biology, Shimane University, Matsue, 690, Japan.
418
R. PRETTY ET AL.
Switchback Point
‘ G
Fig. 1. Left: Schematic ventral view of the Rana catesbeiana intestine inside the body cavity. SB = switchback point; i.e., the midpoint of
the intestinal mass, where the direction of coiling reverses itself. The
portion of the gut oral to the switchback point is shaded and individual coils numbered, with respect to distance from the switchback
point, using Roman numerals. The caudal ( = anal) portion of the gut,
with respect to the switchback point, is unshaded and coils there are
numbered with Arabic numerals. In this view, proximal coils i, iv, and
v are hidden by the overlying caudal coils. Typically in R . catesbeiana,
as one moves laterally from the switchback point, the first 2-4 coils
observed in ventral view are from the caudal portion of the alimentary tract. L = liver, P = pancreas. Right: Schematic representation of
the coiled gut showing the location of oral (light dashed line) and anal
(dark continuous line) portions of the gut. Here the coils have been
spread out and tipped en masse rostrally to help expose both superficial and deep coils.
obstetricans (Dauca and Hourdry, 1985); and 84% in R .
catesbeiana (Carver and Frieden, 1977). As these figures indicate, the shortening is both massive and well
documented. Yet, surprisingly, it is not known where
along the oral-anal axis most shortening actually occurs. That topic is addressed here.
By way of background, the intestines in tadpoles
form a tight double spiral, which fills most of the body
cavity. Nodzenski et al. (1989) report that in one
anuran species, the coiled intestines occupied 80% of
the body cavity, leaving little space for other viscera.
The center of the double coil is the switchback point
(SB)-the point at which the spiral reverses itself.
Hence, the anterior and posterior portions of the small
intestine concurrently spiral outward from this midpoint (Delsol and Zervudacki, 1962). The typical arrangement of the coils in premetamorphic Rana catesbezana is shown in Figure 1.
We reasoned t h a t we could determine where the
greatest amount of gut shortening took place if we
could mark sites along the oral to anal axis of the in-
testine before metamorphosis and then locate these
same sites after metamorphosis. To do this we first
needed a map of the larval gut, which would allow us to
determine the distance along the intestine of specific
coils. We constructed the map in the first part of our
study using R . catesbeiana larvae. In the second part,
the same points were surgically labeled in live R. catesbeiana and their positions relative to each other determined once the gut had shortened.
Hypothetically there are four basic topological ways
in which the intestine could shorten at metamorphosis:
(1)the gut could preferentially shorten from the oral
end, (2) the gut could preferentially shorten from the
anal end, (3) the gut could preferentially shorten from
a more central region (i.e., the switchback point), or (4)
the gut could shorten uniformly along its oral-anal
axis. These possibilities, which are not mutually exclusive, are schematically outlined in Figure 2. If the gut
shortens preferentially from one end, then, after metamorphosis the marked points should be found nearer to
each other at that end. If the gut shortens predomi-
419
Oral End
Switchback Point
Anal End
PREMETAMORPHIC POSITION OF GUT MARKERS
1
. Preferential shortening from the oral end
3. Preferential shortening from switchback point
2. Preferential shortening from the anal end 4. Uniform shortening along intestinal length
POSSIBLE POSTMETAMORPHIC POSITIONS OF GUT MARKERS
Fig. 2. Schematic drawing of the uncoiled intestine of an anuran
tadpole showing five marked positions in the larval intestine (above)
and four hypothetical ways in which the intestine could shorten with
metamorphosis and displace the points (below).The switchback point
is a permanent feature represented here by a node. The possible ways
the gut could preferentially shorten are: (1)from the oral end, (2) from
the anal end, (3) from the middle, or (4)uniformly along its total
length.
nately from the center, then, after metamorphosis the
marked points should be found closer to each other in
that region. Alternatively, if the gut shortens uniformly along its oral-anal axis, then, there will be no
change apparent in the relative distribution of the
marked points along the gut.
The alimentary tract was removed en masse from the
body cavity of each specimen by cutting the esophagus,
terminal rectum, post caval, and dorsal mesenteries.
The gut was then uncoiled by cutting away remaining
mesenteries and major vessels. The total length of the
alimentary tract and the distances from the oral end to
each marked point were recorded. The percentage distance along the oral-anal axis for each of the five
mapped points was calculated.
MATERIALS AND METHODS
Intestinal Mapping in Tadpoles
Tadpoles for this portion of the study were all colSurgical Procedures
lected in the vicinity of Halifax County, Nova Scotia.
Live R . catesbeiana tadpoles were purchased from
They were euthanized in MS-222 (Sigma, St. Louis,
MO) and preserved in 10% neutral-buffered formalin. NASCO (Fort Atkinson, WI) and kept at 15°C in a reTen specimens with conspicuous hindlimbs, but no ex- frigerated aquarium to slow natural metamorphosis
posed forelimbs (stages 37-41; Gosner, 1960) were dis- and maintained on a diet of parboiled lettuce. Ten of
sected. Prior to dissection, each specimen’s develop- the tadpoles were implanted with intestinal markers;
two were kept as controls. The control animals were
mental stage and snout-vent length were recorded.
The skin and musculature of the anterior abdominal not subjected to surgery but were otherwise treated the
wall were cut away to reveal the coiled gut. Five points same as the specimens with the intestinal markers. All
along the alimentary tract were marked with a loop of these specimens were closely matched to those used in
6 - 0 monofilament polypropylene suture (SurgileneB, the premetamorphic mapping study (above) in size and
Davis & Geck, Baie d’Urf6, PQ) tightly knotted around metamorphic stage.
The animals selected for surgery were anesthetized
the gut. In each specimen the marked points were: the
third and fifth anterior intestinal coils, the third and by immersion in a 0.15% solution of Benzocaine (Sigfifth posterior intestinal coils, plus the “central switch- ma) for -6.5 min (following Vanable, 1985). Surgery
was performed under a dissecting microscope with fiber
back” point (Fig. 1).
420
R. PRETTY ET AL.
optic illumination. The tadpoles were placed on a bed of
ice to slow metabolism during surgery. Throughout
surgery the tadpoles were bathed in a cold tadpole
Ringer's solution (NASCO), and a n antibiotic antimycotic solution (Sigma) of penicillin, streptomycin, and
amphotericin was applied to minimize the possibility of
infection.
A longitudinal ventral incision was made slightly to
the righi of the midline, to avoid cutting sagittal blood
vessels. The incision measured no more than 25 mm in
any specimen. Using a wet spatula, the intestinal coils
were gently lifted out of the surgical wound and placed
directly on the abdominal wall. Care was taken not to
tear the mesentery.
In preliminary experiments a variety of in vivo
marking techniques was tried, such as histological dyeing and branding of points along the gut with a hot
needle. None of these labels proved to be permanent
enough. Ultimately we settled on inserting short
lengths of nonabsorbable 4-0 monofilament polypropylene suture (SurgileneB)directly through the gut wall.
Alternating black and blue sutures were used to distinguish between neighboring points. At each of the
five previously mapped points, a length of suture was
passed completely through the gut wall into the lumen
of the alimentary tract and back out again. The ends of
the sutures were flared with a hot spatula to hold them
in place. After all five sutures were in place, the intestines were tucked back into the body cavity and the
incision closed using 4-6 absorbable 6-0 sutures (Ethicone, Ethicon Suture, Peterborough, ON). The operation typically lasted between 35-60 min, depending on
the size of the tadpole and the accessibility of the individual gut coils. After surgery, the tadpoles were kept
individually in aerated aquarium water, with a small
amount of tetracycline to avoid wound infection. Total
behavioral recovery from anesthesia was observed
within 2 hr.
Metamorphic Induction
Postoperatively the animals were kept at room temperature (c. 20-22°C). Initially we planned to let animals metamorphose spontaneously, but our R. catesbeiana tadpoles did not spontaneously metamorphose
after such major intestinal surgery. Therefore metamorphosis was induced with the hormone thyroxine.
Thyroxine is the endogenous biochemical promoter of
metamorphosis, and thus it emulates natural metamorphosis of the gut at physiologically realistic dosages (Etkin, 1981; Shi and Brown, 1993).
Thyroid induction was performed with two groups of
six tadpoles, each consisting of five tadpoles with the
implanted markers and one control tadpole. Generic
SynthroidO (L-thyroxine sodium, Boots Pharmaceuticals, Etobicoke, ON) was dissolved in water to give
concentrations of 7.5 x lo-' M and 1.5 x l o p 7 M. Postsurgical tadpoles were kept individually in 1.0 1 of the
thyroid solution. The molarities were maintained by
replacing 62.5 ml of the thyroid solutions daily. Once a
week the containers were cleaned and new solutions
were started.
For the first group of six tadpoles we used a thyroxine concentration of 1.5x lop7 M and thyroxine treatment continued until metamorphic climax began (defined as the appearance of front limb bulges and/or
shortening of the tail). However, because of high mortality among the metamorphosing froglets, the dosage
was reduced in the second group of tadpoles. In that
group we used a thyroxine concentration of 7.5 x lop8
M and stopped treatment when we saw the first signs of
transformation (e.g., forelimbs about to emerge). Treatment periods were 15-18 days (1721 day; TikSE) and
11-16 days of treatment (13+2 days), respectively.
When animals died, they were promptly preserved in
neutral-buffered formalin.
Postmetamorphic Observation of Implanted Markers
The procedures for determining the position of the
gut markers in the hormonally treated specimens were
identical to those used to establish the premetamorphic
intestinal map. Prior to dissection, the size and developmental stage of each specimen were recorded. Gut
lengths and distances from the oral end to each marker
were taken. These measurements were then converted
to percent distance of each marker along the oral-anal
axis.
RESULTS
Intestinal Mapping
Figure 3 (top) shows the relative distribution of the
five mapped points along the oral-anal gut axis in 10
premetamorphic but mature (i.e., Gosner stage 2-37)R.
catesbeiana tadpoles. The switchback point was a n easily recognized permanent feature in both the coiled and
uncoiled gut (Figs. l A , 2); in our R. catesbeiana tadpoles, it was 6210.9% (Z+SE) along the gut axis. Because of its location toward the caudal end of the gut,
the remaining mapped points are unevenly distributed.
The data reveal that the mapped points represent
completely independent regions; there was no overlap
in the ranges except for two marked points anal to the
switchback point, and the mean location for those
two points differed significantly (Mann-Whitney U,
P<.0002).
Metamorphic Induction
All tadpoles submerged in thyroid hormone solution
began to transform within 3 weeks of surgery, Unfortunately, all died during the 10 days following forelimbs emerging (Gosner stage 42). Control tadpoles,
however, did not differ in metamorphosis from experimental animals.
The experimental group that experienced the lower
dose of exogenous thyroxine lived for 22-27 days (2322
days) after thyroid treatment started, whereas survival
times for the group receiving the higher thyroxine dosage were 16-30 days (23k5 days). Survival times did
not differ between the two groups (Mann-Whitney U,
P = 0.52).
Gut Shortening
Gut lengths of the transformed froglets were compared with those of the mature tadpoles measured during the mapping experiment. The mean gut length in
mature tadpoles (i.e., Gosner stages 37-41) was 1522.9
times their mean body length. In contrast, the mean
gut length of the partially transformed froglets (of
stages 42-44) was only 5.141.6 times their mean body
length. Since the tadpoles in our premetamorphic sample were at metamorphic size, i.e., essentially the same
421
GUT METAMORPHOSIS I S X S U R A K S
PREMETAMORPHIC
Oral End
25%
47%
t
?
Coil v
62%
T
Coll Ili
52%
26%
f
Switchback
point
Coil 3
Coil 5
74%
62%
86%
POSTMETAMOR PHlC
I
0
I
10
I
I
I
20
30
I
Anal End
84%
73%
m k l S D
I
I
I
I
I
I
40
50
60
70
80
90
I
I
100
PERCENTAGE DISTANCE ALONG ALIMENTARY TRACT
Fig. 3. Above: Distribution of mapped points along the alimentary
tract in R . catesbeiana tadpoles. Below: The same marked points in
metamorphic R . catesbeiana after the gut has shortened to a third of
its length. The intestines are shown here scaled to the same length.
The ruler below shows the 410 distance of the marked points from the
oral end. Arrows indicate the mean values obtained from 10 specimens, whereas the banded regions indicate i one standard deviation.
The switchback point is a permanent feature represented here by a
node. Roman numerals are assigned to points anterior to the switchback point, whereas Arabic numerals refer to points posterior. There
is no overlap in the mapped regions in either the tadpole or metamorphic sample. The similar distribution above and below confirms that
shortening is uniform along the length of the intestine.
body length a s the froglets, this means that the intestinal tract in our R. catesbeiana had shortened both
relatively and absolutely to a third of its premetamorphic length (i.e,, shortened by 66%). As expected, we
found that the length of the thyroid treatment (in days)
and the relative amount of gut shortening were correlated (Spearman Rank, 0.1 > P>.05).
Not all gut implants were recovered from the partially transformed froglets; recovery ranged from a single implant in one specimen to all five implants in
another (Table 2). However, all implants that were recovered properly fit the color scheme used a t the time of
implantation and fell within the region (as % of gut
length) described by the larval intestinal map (Table 1,
Fig. 3). Most significantly, the relative distances of the
markers along the oral-anal axis did not differ from the
those in the premetamorphic specimens (Mann-Whitney U, P>O.lO).
just the spiral portion of the small bowel. If the foregut
(stomach and duodenum) were excluded from our measurement of gut length, the switchback point would be
located much closer to the midpoint of the intestine.
A concern to us was the fact that our surgically
treated animals did not metamorphose spontaneously
and that all of the hormonally treated specimens died
before completing metamorphosis. The high mortality
in surgical specimens is one reason the experiment was
terminated and our sample sizes are small. An overdose of thyroxine may have been a factor, but our dosages were not exceptionally high compared to previous
studies on metamorphosis where this hormone has
been used. The possibility that the animals died because of either blockage of the digestive tract or starvation can be ruled out because we used large, latestage tadpoles about to metamorphosis and tadpoles
normally stop feeding during metamorphosis (Duellman and Trueb, 1986).
Despite these technical problems there is strong evidence that the intestines of the marked animals shortened in a natural fashion. First, the shortening occurred a t a constant rate. This conclusion is based on
data of gut length versus length of time that the tadpoles were exposed to thyroid hormone. Also, the mean
amount of shortening observed was consistent with the
amount that has been reported in the literature for R .
catesbeiana. We found a mean shortening of 66% between stages 41 and 43. Carver and Frieden (1977)
DISCUSSION
The coiling pattern of the R. catesbeiana larval gut
varies little among individuals. Thus one can identify
individual gut coils and determine how far along the
oral-anal axis particular coils lie (Fig. 4).The most
obvious feature in both the coiled and uncoiled gut is
the switchback point, located 62?.91% from the oral
end of the gut. This point is not halfway along the total
length of the alimentary tract because we included in
our measurements the whole alimentary tract and not
422
R. PRETTY ET AL.
TABLE 1. Position of marker along alimentary tract
as % distance from oral end (%+SE)
Implant
location
Coil v
At implantation
in larvae (n = 10)
25 0.7
Coil iii
47 t 1.5
Switchback point
62 2 0.9
Coil 3
73 2 1.0
Coil 5
84
*
-t
1.2
After
metamorphosis
26 t 2.3
(n = 3)
52 -t 0.5
(n = 2)
62 2 0.5
(n = 8)
74 -t 1.1
(n = 5)
86 -t 0.9
(n = 8 )
TABLE 2. Implants recovered from surgery specimens
after thyroxine-inducedmetamorphosis
Specimen
number
1
2
3
4
5
6
7
8
9
Oral end
Coil v Coil iii
(blue) (black)
SB Point
(blue)
\’
\
\
\
\
\
\’
\
\
\
\
\
\
\
\
Anal end
Coil 3
Coil 5
(black) (blue)
\.
\
\
\
\
---Anal End
-c--
reported a total shortening of 83% by stage 46 in this
species.
Our data indicate that intestinal shortening in R.
catesbeiana occurs uniformly along the length of the
gut. This conclusion is supported indirectly by Kaltenbach et al. (1981),who found that in natural metamorphosis, thyroid hormone was located in the gut epithelium along its entire length.
Curiously, the surgical implants were not recovered
uniformly along the intestine. Sutures placed at the
switchback point and farther down the alimentary canal were recovered most often (Table 2). In contrast,
those in the anterior 60% of the gut were more often
lost. Although only 26/50 of the implants were recovered, there was little positional variation among the
recovered implants (Fig. 3), consistent with our primary conclusion that gut shortening occurs uniformly
along the intestine. The low recovery rate of implants
from the surgical specimens can be explained by one or
more of the following: (1)because of the delicate nature
of the surgery, some implants may not have been properly secured in the first place; loose implants could
have become dislodged and fallen either into the body
cavity or into the gut lumen, then been passed by the
tadpole; (2) during metamorphosis, the epithelium of
the gut degenerates and sloughs off into the lumen of
the gut (Marshall and Dixon, 1978); this could have
loosened some implants and caused them to be carried
into the lumen of the intestine; (3) the circumference of
the intestine changes during metamorphosis, and as
this happens, implants could have been excluded from
Fig. 4. Intestine of R . catesbeiana larva shown coiled and uncoiled.
Shaded areas refer to the anterior portion of the gut while white areas
refer to the posterior portion of the gut. See Figures 1 and 3 for an
explanation of other labels. Coil v is shown in this view but would
more commonly be hidden by overlying coils in ventral view.
the gut wall. The possibility, however, that some implants were digested by the tadpoles can be ruled out
because, if this were the case, the implants that were
recovered would have borne some sign of degradation,
and that was never observed.
In summary, this experiment shows that as the intestine of R. catesbeiana shortens to a third of its
premetamorphic length, it does so uniformly along its
oral-anal axis. Although metamorphosis was artificially induced, the shortening we observed closely
matched the regression in intestinal length that occurs
during natural metamorphosis.
ACKNOWLEDGMENTS
This study was part of the joint JapaneseICanadian
program on “Visceral Function in Amphibians.” We
thank Scott Pronych, Edward Hitchcock, and Ruth
Waldick for help with animal collection and general
laboratory assistance. Brian Hall, Jane Kaltenbach,
and Monika Fejtek critically reviewed manuscript
drafts. We are particularly grateful to Monika Fejtek
who helped with computer graphics, statistical analysis, and manuscript formatting. The research was sup-
GUT METAMORPHOSIS IN ANURANS
ported by the Natural Sciences and Engineering Research Council of Canada, Pacific 2000 Program of the
Canadian Department of External Affairs, and the International Scientific Research Program of Monbusho,
Japan.
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