J. Moll. Stud.

(1999), 65, 163–170 © The Malacological Society of London 1999

GAST ROINTESTIN AL TRANS IT AND DIGESTIV E RHYTHM

IN BIO MP HA LARIA GLABRA TA ( S A Y )

AN DREA FL ORSCH ÜTZ and WIL HELM BECK ER

Zoologisches Institut und Museum, Universität Hamburg, Martin-Luther-King-Platz 3 20146 Hamburg,
F.R.G.
(Received 15 July 1996; accepted 1 June 1998)

ABSTRACT for the understanding of digestion processes in

gastropods. In this context the retention time
Transit of X-ray opaque MicropaqueTM particles in of food in, and its discharge from, the digestive
the digestive tract of Biomphalaria glabrata was inves- gland are of particular interest.
tigated by X-radiography. Passage through the whole In this experimental approach X-radiography
animal lasted up to 200 min depending on the experi-
mental conditions (starvation, fed ad libitum etc.). has been applied to investigate the transport
For the first time, cyclic activity of the digestive gland of X-ray opaque diets through the gastro-
could be visualised: the gland is emptied every 60–110 intestinal tract of B. glabrata. This method
min discharging undigested particles. In the afternoon enabled us to observe individual snails over a
transport of probably predigested material from more long period without the need to sacrifice the
distally located parts of the digestive tract into animal as in comparable experiments with
the esophagus can be observed indicating resorption radioactive traces (Tur, Rial & Pomar, 1985).
processes occurring in the esophagus.

MATERIALS AND METHODS

INTRODUCTION
Stock cultures of B. glabrata (albino PR-strain,
In gastropods, as in other molluscs, there is a Tropeninstitut Hamburg) were kept at 26 1°C in
lack of detailed information on absorption sites 30 l aquaria in an overflow system as described by
Trede and Becker (1982). Snails were fed ad libitum
for various nutrients. Furthermore it is unknown
on Standen’s (1951) semiartificial diet. Only adult
how the activity of the different compartments specimens of 20 2 mm shell diameter were used
of the digestive trace is coordinated. Digestion and X-rayed with a Philips-Röntgenmüller type 9421
in gastropods appears to be a complex process; 172 03002 X-ray machine.
probably the digestive gland cannot be consid- Two modifications of the usual Standen’s diet were
ered the only site of nutrient absorption. The obtained by adding the X-ray opaque markers ThO2
functions of the gut include more than just or Micropaque TM. The amount of each marker giving
transport of food and waste materials. The find- optimum X-raying results had previously been deter-
ings of Heidermanns (1924) and Walker (1972) mined by testing different concentrations. Therefore
10 g of ThO2 or 30 g of MicropaqueTM was suspended
that nutrient absorption occurs also in the
in 50 ml of demineralized water. Under continuous
foregut of pulmonates were confirmed by Paine stirring, a mixture of 2 g milk powder, 2 g ground
& Crisp (1989), who described lipid absorption wheat germ and 2 g dried lettuce, plus 1 g sodium
taking place in the posterior esophagus of alginate was added. The suspension obtained was
Nassarius reticulatus (L.). The intestine, also, is poured into 500 ml 5% CaCl2 solution for polymeri-
involved in absorption processes (Bush, 1988 sation of the alginate matrix in order to render the
and El-Shaikh, Becker & Siebers, 1993). food insoluble in water. MicropaqueTM was pur-
Investigations on food transit rates can be chased from Nicholas GmbH, Sulzbach, F.R.G. and
useful for identification of possible absorption ThO 2 was purchased from Merck. ThO2-fed snails
were X-rayed using an Agfa Structurix D2-film at 80
sites: when the time profile of food transit is
kV for 4 sec whilst MicropaqueTM-fed were X-rayed
known it can be correlated with the appearance using an Agfa Structurix D4-film at 80 kV for 2 sec.
of certain hemolymph components in feeding Five different experimental approaches were per-
experiments. Investigations on the coordination formed which varied mainly in the manner of food
of the rhythmic activity of the digestive tract and administration:
the digestive gland are of crucial importance In experiment 1 snails were starved for 16 h and
164 ANDREA FLORSCHÜTZ & WILHELM BECKER
then fed on the MicropaqueTM diet for 30 min in 500 1963). As a further control, the passage of the white-
ml glass aquaria. For X-raying, snails were removed coloured MicropaqueTM food in the digestive tract
from the water and placed on the films. could be observed through the transparent shell of
In experiments 2, 3, 4 and 5 experimental condi- the albino snails.
tions were modified to imitate natural feeding condi-
tions. Snails were fed ad libitum and X-rayed in water.
During the experiments standard temperature was RESULTS
maintained as X-raying was performed on a heating
plate under a 60 W electric bulb. For X-raying, snails
Dissection of B. glabrata revealed that ana-
were placed on the films in 18 cm petri dishes contain-
ing 40 ml tap water. Under these conditions the shell tomy of the digestive tract (Fig. 1A) was in
of the submerged snails tilted in such a way that the concordance with Carriker’s (1946) detailed
digestive tract could be recorded from one side. description for Lymnea stagnalis apressa, except
After allowing the snails 20 min to adapt to experi- for the digestive gland, due to the different shell
mental conditions, X-ray opaque diets were offered forms of lymnaeids and planorbids. This part of
to the snails. This adaption period was necessary in the digestive tract resembles that of B. pfeifferi
experiments 2–5 to prevent snails from crawling as described by Meulemann (1972).
during X-raying. When the animals started feeding In X-radiographs the course of the digestive
they were X-rayed at intervals mentioned below.
tract can be seen very clearly: planorbid
In experiment 2, snails were starved for 16 h, then
fed ad libitum on the MicropaqueTM diet and X-rayed anatomy is particularly suitable for this applied
at 5 min intervals. experimental approach if shown from a lateral
In experiment 3, snails that previously had been fed view (Fig. 1, B).
ad libitum on Standen’s diet were fed on the Micro- Unlike previous X-ray experiments with D.
paque TM, or ThO2 diet and X-rayed at 5 min intervals. reticulatum investigated by Walker (1972) and
Experiment 4 was a pulse chase experiment. Snails Triebskorn & Florschütz (1993) and L. stag -
that previously had been fed ad libitum on Standen’s nalis investigated by Veldhuijzen (1974), in B.
diet were fed for 5 min on ThO2 diet and then fed glabrata marker particles were transported into
again on Standen’s diet. X-raying occurred at 10 min
the digestive gland but not phagocytozed. For
intervals.
In experiment 5 snails were fed ad libitum on the this reason, the ducts and tubes of the gland
MicropaqueTM diet 2 days before X-raying. When the could also be visualised by X-radiography.
experiment was started feeding with the labelled diet Controls revealed that X-raying did not influ-
was continuous, except for the adaption period. ence food transit.
X-raying occurred again at 10 min intervals. Food transit was fastest in starved snails
The passage of the administered markers was which were removed from the water prior to
followed by analysing positive prints of series of X-raying (experiment 1, Table 1). The time pro-
X-radiographs of each snail. Six parts of the digestive file of food transit proved to depend on feeding
tract were distinguished as shown in Figure 1. The
conditions: in starved snails food transit was
parts reached by the marker particles within a certain
period were determined. faster than in snails fed ad libitum (experiment
As a control, snails were either starved and then 2, Table 2). ThO2 reached the digestive gland
fed or feeding was continued on different coloured faster than MicropaqueTM (experiment 3, Table
diets. At different time intervals during feeding, snails 3). Both markers are considered inert (Brown
were dissected under balanced salt solution (Chernin, & Brown, 1965) but differ in particle size.

Table 1. Transit of a X-ray opaque MicropaqueTM diet in the digestive tract of previously
starved B. glabrata (experiment 1). Percentage of snails in which a given part of the digestive
tract was reached within a certain period are listed (N 20).

minutes after start of feeding

15 30 45 60 75 90 105 120

esophagus
stomach region 77.8
pro-intestine 22.2 50 50 12.5
mid-intestine I 50 50 50 30 12.5 12.5
mid-intestine II 37.5 70 50 50 25
post-intestine 37.5 37.5 75
digestive gland 100
 DIGESTION IN BIOMPHALARIA 165
TM
Table 2. Transit of a X-ray opaque Micropaque diet in the digestive tract of previously
starved and fed B. glabrata compared with snails fed ad libitum. Percentages of snails in
which the digestive gland was reached within a certain period are listed.

15 min 30 min

previously starved ad libitum previously starved ad libitum

Percentage 95.2 0 100 20

N 21 20 23 20

Figure 1. Biomphalaria glabrata. Lateral view of the digestive tract (A) compared with two positive prints of
X-radiographs (B and C).
Abbreviations: bm, buccal mass; c, crop; dg, digestive gland; dgd, digestive gland ducts; e, esophagous; f, foot;
g, gizzard; mid-i, mid-intestine; p, pylorus; pro-e, pro-esophagus; pro-i, pro-intestine; post-e, post-esophagus;
st-r, stomach-region; t, tentacle.
166 ANDREA FLORSCHÜTZ & WILHELM BECKER
Table 3. Transit of ThO2 in the digestive tract In all ad libitum feeding experiments (2–5),
of B. glabrata compared with the transit of cyclic feeding activity could be observed: after
the other X-ray opaque MicropaqueTM ( MO). the adaptation period snails fed continuously
Percentages of snails in which the digestive for 45–60 min. These feeding periods were
gland was reached within a certain period are
interrupted by 30–60 min rest periods. In the
listed.
afternoon, pauses within feeding periods were
15 min 30 min generally longer.
Although food transit was slowest in snails
ThO2 MO ThO2 MO fed ad libitum, considerable variations occurred
between individuals. One typical example
Percentage 95.2 59.3 100 78.4 from experiment 2 is shown in Figure 2. The
N 21 27 23 28 marker initially remained in the esophagus for a
certain period (see arrow at 15 min). Then
the first marker particles appeared in the diges-
tive gland. The filling process continued

Figure 2. Biomphalaria glabrata. Experiment 4. Transit of the X-ray opaque marker ThO2 in the digestive
tract visualised by positive prints of X-radiographs taken from the same snail. Lateral view. Time is indicated
as min after feeding the marker. Abbreviations as in Fig. 1.
 DIGESTION IN BIOMPHALARIA 167

Figure 3. Biomphalaria glabrata . Experiment 5. Transit of the X-ray opaque MicropaqueTM-particles in the
digestive tract, viewed by radiographs of the same snail. In this time profile, minutes after the start of the
feeding cycle are recorded. The different stages of filling and emptying of the digestive gland can be observed
especially well in this sequence.

while marker particles progressed into the a rhythmic filling and emptying of the digestive
pro-intestine and mid-intestine (see arrows at gland. The gland is filled continuously during
85 min) forming the faecal string. Meanwhile the first 60–110 min of the experiment (Fig. 3, 6
marker particles had been completely dis- and 67 min). The highly branched tubular
charged from the digestive gland and the loop system of the gland can be observed. After the
of the midintestine had been reached (see 95 filling process the organ is emptied abruptly
min). At 165 min the marked part of the faecal within at most 10 min (Fig. 3, 77 min) Figure 4
string had been discharged via the anus. indicates the return of particles released from
In snails which were fed the MicropaqueTM the tubules of the digestive gland to the eso-
diet ad libitum throughout the whole experi- phagus. After 34 min the initially taken up
ment (experiment 3), rhythmic activity of the material has reached the distal parts of the
digestive tract could be observed, together with digestive tract, and the esophagus is empty.
168 ANDREA FLORSCHÜTZ & WILHELM BECKER

Figure 4. Biomphalaria glabrata . Experiment 5. Transit of the X-ray opaque MicropaqueTM-particles in the
digestive tract, viewed by radiographs of the same snail. In this time profile, minutes after the start of the
feeding cycle are recorded. The postesophagus and peristaltic waves are indicated by arrows.

However, 10 min later an increased number of living on algae and detritus (Thomas, Nuanko
particles and strong peristaltic and antiperi- & Sterry, 1985). This habit is reflected in the
staltic activity was observed. This must be the anatomy of the digestive tract (Fig. 1) and the
result of a retransport from more distal parts as dynamics of the digestive process are discussed
no fresh food particles or faecal materials from below.
previous meals were ingested. The return of Although long starvation intervals between
labelled particles from the digestive tract to the feeding periods cannot be considered a normal
esophagus occurred only in the afternoon. physiological state for B. glabrata, using starved
snails for measuring food transit rates can be
useful. Experiments on metabolism are often
DISCUSSION conducted under starvation (Horst, Becker &
Kemper, 1986) to make sure that the tested
Under natural conditions B. glabrata, like other diets were consumed by the snails in a sufficient
basommatophoran snails is a continuous feeder amount.
 DIGESTION IN BIOMPHALARIA 169
The fact that food transit in ad libitum-fed as already described by Krikgsmann (1925)
snails is slower than in starved ones cannot be and Heidermanns (1926) for the stylom-
attributed solely to the fact that the marker matophoran snail Helix pomatia, by Morton
proceeds faster in the digestive tract because (1979) for the slug Deroceras caruanae and by
the organs are depleted of food. After a 16 h Morgan & Last (1972) for the freshwater proso-
starvation period food particles can still be branch Melanoides tuberculata. Also Becker’s
found in the whole digestive tract with the (1983) microcalorimetric investigations on B.
exception of the esophagus. Rather it is likely glabrata support the idea of a rhythmic cellular
that feeding conditions such as starvation influ- activity. Nevertheless no information from
ence speed of food transit as a compensatory histological investigations on basommatophor-
effect: after good deprivation the amount of ans has been obtained. Meulemann (1972), for
food taken up is increased. This conclusion is in instance, excluded the possibility of a cyclic cel-
accordance with the findings of Calow (1975) lular activity in B. pfeifferi. Therefore, further
that in Planorbis contortus food absorption investigations with more sophisticated tech-
efficiency depends on feeding conditions. niques should help to decide whether a periodic
The comparison of the different results from activity is really present in basommatophorans.
experiments 1, 2 and 3 show that the food tran- A transport of particles into the esophagus
sit rates given might only be indicative values from the stomach region has not been de-
for other workers because the special experi- scribed previously. The pronounced peristaltic
mental conditions have a very important influ- and antiperistaltic movements of the esopha-
ence. gus support the theory of the foregut as one
In the present experimental approach we site of food resorption in B. glabrata. This
succeeded for the first time in visualising the is performed mainly in the afternoon when
1–2 h cycle of the digestive gland’s rhythmic snails interrupt their feeding activity for longer
activity. These results could be obtained be- periods and no newly ingested particles have
cause the marker substance MicropaqueTM was been transported into the foregut. This phe-
distributed throughout the lumina of the gland. nomenon is in concordance with the situation
Other authors, such as Walker (1972) and Veld- in the stylommatophoran D. reticulatum where
huijzen (1974) reported that the markers were Walker (1972) found resorption processes in
phagocytoxed (as ThorotrastTM) or did not the foregut, too.
enter the gland (various BaSO4-containing Despite these similarities the dynamics of
substances, such as MicropaqueTM). In both food transit in the investigated species differ
cases the filling and emptying of the gland could from those in D. reticulatum. This may reflect
not be visualised. When comparing Veld- the distinct habitats of the two pulmonates:
huijzen’s results with ours the question arises: in B. glabrata food transit through the digestive
why did the marker enter B. glabrata digestive tract is faster (max. 2 h) when compared to
gland but not that of the other basom- D. reticulatum (9–12 h) (Walker, 1972 and
matophoran snail L. stagnalis? Particle size and Triebskorn & Florschütz, 1993) Even retention
formulation of the X-ray opaque diets are not time of food particles in the foregut is shorter
likely to be crucial, because these factors are to in the former indicating more continuous feed-
be considered similar. Rather it seems probable ing behaviour by the freshwater pulmonate.
that preadaptation to the diets in B. glabrata On the other hand D. reticulatum, a terrestrial
might be of some significance: our snails were species, usually interrupts feeding during the
reared on the same type of Standen’s diet less favourable dry day time and spends these
before and during the experiments. The L. hours in the enzymatic digestion of plant
stagnalis, however, were fed on lettuce in the material in its large crop.
pre-experimental period, which is a completely Obviously the course of digestion in gastro-
different type of diet for the snail. pods can differ in a very pronounced manner
Although food transit in B. glabrata is as it is influenced considerably by ecological
slightly faster than in L. stagnalis the 1–2 h aspects. This fact must be taken into account in
rhythm of the digestive gland is in good accor- all investigations performed in this field.
dance with the one described for L. stagnalis by
Veldhuijzen, deducing a 2 h rhythm from the
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