First published online May 19, 2008
Journal of Experimental Biology 211, 1729-1736 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.016014
Integrative biology of an embryonic respiratory behaviour in pond snails: the `embryo stir-bar hypothesis'
Jeffrey I. Goldberg1,*,
Shandra A. Doran1,
Ryan B. Shartau1,
Julia R. Pon1,
Declan W. Ali2,
Rose Tam1 and
Shihuan Kuang3
1 Department of Biological Sciences, University of Calgary, Calgary, Alberta,
Canada, T2N 1N4
2 Department of Biological Sciences, University of Alberta, Edmonton, Alberta,
Canada, T6G 2E9
3 Department of Animal Sciences, Purdue University, West Lafayette, IN
47907-2054, USA
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Fig. 1. Morphological comparison of egg masses and embryos from H.
trivolvis (stage E25, top) and L. stagnalis (stage E39, bottom).
Whole egg masses and decapsulated embryos (inset) were imaged 3 days after egg
masses were laid by young adult snails. The characteristic tight planar
arrangement of egg capsules (open arrow) inside the egg mass (arrowhead) and
the flatter translucent embryo (arrow, also see inset) facilitate imaging of
early development and behaviour in H. trivolvis. The L.
stagnalis egg mass shown is considerably smaller than egg masses
typically laid by older snails, whereas the H. trivolvis egg mass is
more representative of the typical size. In the inset, embryos are shown in
left-side view under differential interference contrast (DIC) optics. The
stages shown represent the period of robust rotational behaviour for each
species. The thin scale bar represents 500 µm and the thick scale bar in
the inset represents 50 µm.
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Fig. 2. ENC1 and post-synaptic ciliary cells viewed under DIC optics. (A) ENC1 soma
(horizontal arrow) and its primary neurite (vertical arrowheads) projecting
towards the dorsolateral (DLB) and pedal ciliary (PB) bands. (B) ENC1 viewed
through a series of descending focal planes. The sensory-like dendritic knob
(horizontal arrowhead) is located on the embryo surface (left panel), leading
to a stubby apical process containing numerous prominent vesicles (vertical
arrows) just below the surface (middle panel). Below that sits the soma, with
its characteristic large nucleus (horizontal open arrow) and prominent
nucleolus (vertical open arrow, right panel). Scale bar, 10 µm.
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Fig. 3. Electrophysiological responses of ENC1 to hypoxia in an explant culture
recorded under whole-cell current clamp. (A) Under normoxic conditions, the
membrane potential of ENC1 was around –70 mV. Hypoxia (approximately 90%
reduction in oxygen concentration) elicited membrane depolarization and a
burst of action potentials, followed by sustained membrane depolarization.
Restoration of normoxia partially repolarized the membrane potential. A second
cycle of hypoxia again elicited membrane depolarization and an action
potential. (B,C) Action potentials elicited by the first and second cycle of
hypoxia viewed on an expanded time scale.
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Fig. 4. Involvement of the mitochondrial electron transport chain and potassium
channels in embryonic rotation. (A) Rotenone (10 µmol
l–1), an inhibitor of the mitochondrial electron transport
chain, stimulated embryonic rotation (r.p.m., rotations per minute) after 10
min of incubation (left panel, N=15 embryos), but had no direct
effect on the ciliary beat frequency (CBF) of isolated ciliary cells (right
panel, N=5 cells). (B) 4-Aminopyridine (4-AP, 5 mmol
l–1), a potassium channel inhibitor, induced prolonged
increases in embryonic rotation (left panel, N=15 embryos), but had
no direct effect on the CBF of isolated ciliary cells (right panel,
N=5 cells). *P<0.05 compared with zero time
point, ANOVA followed by Fisher's PLSD.
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Fig. 5. The embryo stir-bar hypothesis. (A) In the absence of rotation, a large
oxygen concentration gradient will form due to the metabolic consumption of
oxygen by the embryo (depicted as stationary stir bar) and the unstirred
boundary layer of high oxygen concentration below the capsule surface. (B)
Embryonic rotation and ciliary activity (depicted as rotating stir bar)
function to mix the capsular fluid, causing a reduction in the size of the
oxygen gradient inside the capsule, a higher concentration of oxygen at the
embryo surface, and enhanced transfer of oxygen into the egg capsule. The
arrow size in A and B represents the relative size of the oxygen gradient
across the egg capsule membrane. Stir bar length is not representative of
embryo size (see Fig. 1).
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Fig. 6. Hypoxia stimulates embryonic rotation behaviour in three families of
freshwater pond snails. H. trivolvis (A), L. stagnalis (B)
and Physa gyrina (C), members of the Planorbidae, Lymnaeidae and
Physidae families of basommatophoran snails, respectively, demonstrated
similar profiles of increased embryonic rotation rates upon prolonged exposure
to hypoxia (open bars). Upon return to normoxic conditions (filled bars), an
initial inhibition of rotation followed by a return to baseline levels was
observed in all three species. Each bar represents mean r.p.m. ± s.e.m.
from 22–26 embryos from two stage E25 egg masses (A), 13–17
embryos from one stage E39 egg mass (B) and 23–27 embryos from two stage
E35 egg masses (C). Asterisk in B represents loss of a data point due to a
corrupted digital file.
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© The Company of Biologists Ltd 2008
