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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
Novel sensory modalities for navigation and other behaviours |
Integrative biology of an embryonic respiratory behaviour in pond snails: the `embryo stir-bar hypothesis'
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
* Author for correspondence (e-mail: jeff.goldberg{at}ucalgary.ca)
Accepted 12 February 2008
Summary
Embryos of freshwater snails undergo direct development from single cell to juvenile inside egg masses that are deposited on vegetation and other substratum in pond, lake and stream habitats. Helisoma trivolvis, a member of the Planorbidae family of basommatophoran snails, has served as a model for studying the developmental and physiological roles for neurotransmitters during embryogenesis. Early studies revealed that H. trivolvis embryos from stage E15 to E30, the period between gastrulation and the trochophore–juvenile transition, display a cilia-driven behaviour consisting of slow basal rotation and transient periods of rapid rotation. The discovery of a bilateral pair of early serotonergic neurons, named ENC1, which project an apical process to the embryo surface and basal neurites to ciliated cells, prompted the hypothesis that each ENC1 is a dual-function sensory and motor neuron mediating a physiological embryonic response. This article reviews our past and present studies and addresses questions concerning this hypothesis, including the following. (1) What environmental signal regulates ENC1 activity and rotational behaviour? (2) Does ENC1 function as both a primary sensory and motor neuron underlying the rotational behaviour? (3) What are the sensory transduction mechanisms? (4) How does ENC1 regulate ciliary beating? (5) Do other basommatophoran species have similar neural–ciliary pathways and behavioural responses? (6) How is the behaviour manifest in the dynamic natural environment? In this review, we introduce the `embryo stir-bar hypothesis', which proposes that embryonic rotation is a hypoxia-sensitive respiratory behaviour responsible for mixing the egg capsule fluid, thereby enhancing delivery of environmental oxygen to the embryo.
Introduction
The phylum Mollusca includes an exceptionally wide range of organisms, with members from eight extant and two extinct classes as divergent in form and function as the octopuses, clams and snails. The vast majority of the roughly 250 000 species live in marine environments; however, two classes of mollusc, the bivalves and gastropods, have members that are adapted to a freshwater existence. Within the gastropods, freshwater species include various taxa containing snails with gills, as well as the order Pulmonata that contains snails with lungs.
Pulmonate snails lay egg masses containing encapsulated embryos whose life
history includes lecithotrophic direct development, whereby the embryos
develop directly into juveniles within the egg mass before hatching and
relying on external food sources. In contrast, most other molluscs display a
planktotrophic life history, whereby veliger larvae emerge from egg masses as
free-swimming planktotrophs, and remain in that stage until the presence of a
suitable substrate signals a settlement behaviour, followed by metamorphosis
into a benthic juvenile. While there is a rich record of studies on the marine
biology of molluscan embryos and larvae from planktotrophic species dating
back at least 60 years (Hadfield et al.,
2000
), the very nature of their life history limits their
usefulness as experimental models. On the other hand, the lecithotrophic
direct development and transparent egg mass structures of bassomatophoran
pulmonates are features that make these pond snails experimentally
tractable.
Lymnaea stagnalis and Helisoma trivolvis [also known as
Planorbella trivolvis (Brown,
1991)] are both bassomatophoran pulmonates that have served as
important model organisms in a broad range of neurobiological studies. L.
stagnalis has been used primarily to study the neural mechanisms
underlying feeding and respiratory behaviours
(Vavoulis et al., 2007;
Bell et al., 2007;
Haque et al., 2006), as well
as a variety of questions on learning and memory
(Martens et al., 2007), neural
plasticity and cell biology (Dunn and
Syed, 2006; Jimenez et al.,
2006). While H. trivolvis has also contributed in some of
these areas (Murphy, 2001;
Torreano et al., 2005), it has
become a particularly good model to address a broad range of developmental
questions because of the advantageous morphological and biomechanical
properties of its egg mass. The egg mass is a flat circular structure
containing 5 to 50 sibling embryos, each housed individually in an egg capsule
(Fig. 1)
(Goldberg et al., 1988). The
organization of egg capsules on a single plane, combined with the transparency
of the egg mass and egg capsule membranes, allows for easy observation of
embryonic development and behaviour. Moreover, egg capsule membranes are
easily penetrated by relatively small compounds
(Beadle, 1969), such that
developing embryos can be subjected to in situ experimental
treatments. Finally, live embryos can be efficiently removed from their egg
capsules and isolated from the egg mass for various procedures, including
in vitro treatments, microsurgery and laser ablations, and, when
necessary, embryos can be re-implanted into egg capsules for developmental or
behavioural analyses (Kuang and Goldberg,
2001; Kuang et al.,
2002a; Kuang et al.,
2002b). L. stagnalis embryos are still accessible to many
of these same types of procedure; however, the three-dimensional arrangement
of egg capsules in an oblong egg mass, as well as the instability of egg
capsules within the egg mass, make it considerably more difficult to conduct
these kinds of experiment (Fig.
1).
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A serendipitous result from one of the earliest studies on H.
trivolvis embryos was that in situ exposure to a neurotoxin
intended to deplete embryonic serotonin and affect normal development
(Goldberg and Kater, 1989)
caused an immediate and pronounced stimulation of a rotational behaviour. The
compound 5,7-dihydroxytryptamine (5,7-DHT) is thought to deplete serotonergic
terminals by its uptake through serotonin transporters, oxidization and
production of free radicals that cause intracellular damage of the terminal
(Baumgarten et al., 1982).
Whereas 5,7-DHT produced the expected depletion and developmental
abnormalities over the long term, its immediate and unexpected effect was the
stimulation of embryonic rotation through the stimulation of serotonin
receptors (Diefenbach et al.,
1991). This one accidental finding prompted a series of studies
that continues to the present day on the integrative biology of rotational
behaviours in pond snail embryos. Earlier studies focused mainly on the
neurocircuitry, neuropharmacology and signal transduction mechanisms
underlying the cilia-driven rotational behaviour, whereas questions about
behavioural relevance, activating environmental cues and underlying sensory
pathways have been addressed more recently. With some of the advances made in
these various areas of investigation, we are now in a position to extend this
model system into the ecological and evolutionary arena, exploring both how
the behaviour is manifested under the complex dynamic environment of the
snail's natural habitat and how it is manifested in pond snails from three
different families of pulmonates.
As reviewed below, our studies have revealed that the embryonic rotational behaviour is mediated by a pair of two-cell neural circuits, each containing a serotonergic sensorimotor neuron named embryonic neuron C1 (ENC1). The cell body of ENC1 senses environmental cues by projecting an apical sensory dendrite to the anterodorsal surface of the embryo and controls the motility of the dorsolateral and pedal ciliary bands by synapsing with the ciliary cells through its neurites. Serotonin stimulates ciliary beating through multiple serotonin receptors, and signal transduction pathways involving calcium, protein kinase C and nitric oxide. One of the natural environmental cues that stimulates the behaviour is hypoxia, prompting the hypothesis that ciliary activity and embryonic rotation are respiratory behaviours that cause stirring of the egg capsule fluid and enhancement of oxygen diffusion to the embryo. Preliminary experiments on Lymnaea stagnalis and two species of snail from the family Physidae suggest that hypoxia-induced rotational responses are expressed widely throughout the pulmonates, and may confer an adaptive advantage to embryos faced with environmental fluctuations in dissolved oxygen.
Embryonic rotational behaviour and the underlying physiological machinery
H. trivolvis embryos when inside their egg capsules undergo a
constitutive rotational behaviour between the stages of E15 and E30
(Diefenbach et al., 1991),
representing the developmental period from immediately after gastrulation
until the beginning of the trochophore–juvenile transition. Embryonic
stages in H. trivolvis are expressed as a percentage of the total
development time (Goldberg et al.,
1988; Goldberg,
1995; Diefenbach et al.,
1998). Stage E0 corresponds to the formation of the zygote, E10 to
gastrulation, E25 to the partitioning of the foot primordium from the abdomen
with a morphologically distinguishable pedal furrow, and E100 to hatching.
Time-lapse video analyses of egg masses imaged under dissection or compound
microscopes revealed that the rate of embryo rotation is fastest at stage E25,
typically ranging from 0.6 to 1.2 rotations per minute (r.p.m.) at this stage.
These measurements represent an overall rate of rotation that combines two
underlying components, constitutive rotation at a slow basal rate and periods
of accelerated rotation called surges
(Diefenbach et al., 1991;
Cole et al., 2002).
These early behavioural observations prompted a series of hypotheses about
the underlying physiological mechanisms that were strongly supported in
subsequent studies. For example, they suggested that the slow basal rotation
was due to constitutive ciliary beating, whereas the surges resulted from the
stimulation of ciliary beating by cilio-excitatory motor neurons. Finally, the
absence of rotational arrests suggested that the cilia mediating the
rotational movement were only under excitatory control, contrary to locomotory
cilia in marine gastropods that receive both excitatory and inhibitory inputs
(Braubach et al., 2006).
Finally, the serendipitous result of the 5,7-DHT treatment described above
suggested that serotonin is the primary cilio-excitatory neurotransmitter in
H. trivolvis embryos.
Histological analyses of fixed embryos and differential interference
contrast (DIC) observation of live embryos indicated that organogenesis of the
central nervous system does not begin until after stage E20, well after the
onset of the rotational behaviour
(Goldberg, 1995;
Diefenbach et al., 1991;
Diefenbach et al., 1998).
However, immunofluorescence experiments revealed that serotonin is expressed
in a bilateral pair of large neurons as early as stage E13
(Diefenbach et al., 1998).
These neurons, named embryonic neuron C1 (ENC1) because they were originally
thought to be the metacerebral giant serotonergic neurons studied in a variety
of adult gastropods (Granzow and Rowell,
1981), projected their primary neurites ventrally to the region
adjacent to the pedal band of cilia. Thus, their morphology and
neurotransmitter phenotype were consistent with them being the
cilio-excitatory motor neurons. Interestingly, each ENC1 projects a stubby
apical dendrite dorsally that is tipped with a specialization that extends
through to the surface of the embryo (Fig.
2) (Goldberg and Kater,
1989; Diefenbach et al.,
1998). This superficial dendritic knob contains an array of
microvilli and non-motile cilia, an anatomical arrangement typical of a
sensory specialization. Thus, the early anatomical data suggested that ENC1
was a sensorimotor neuron involved in regulating ciliary activity in response
to an unknown environmental cue.
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The appearance of ENC1 prior to the formation of the central nervous
system, and the absence of any other detectable neurons between stages E13 and
E25, suggests that this neuron evolved during pulmonate evolution to play a
critical role in embryos specific to encapsulated development. However, the
relatively medial location of these neurons on the anterodorsal aspect of the
embryos, combined with their stubby apical dendrites and superficial dendritic
knobs, led to a more likely interpretation
(Diefenbach et al., 1998).
There is now general agreement that rather than being the embryonic
metacerebral giant neurons as proposed earlier, ENC1s are the evolutionary
remnants of the apical sensory organs of marine gastropods, specialized
embryonic nervous systems that control locomotory and settlement behaviours in
planktonic veligers (Voronezhskaya et al.,
1999). These are typically small ganglia containing relatively few
neurons, including three to six serotonergic neurons depending on the species
(Kempf et al., 1997). Some of
these neurons have sensory-like morphologies, including the serotonergic
para-ampulary neurons that look strikingly similar to ENC1. We hypothesize
that the apical sensory organ became drastically reduced during the evolution
of pulmonates because the absence of the planktonic larval stage eliminated
the need for the more extensive neural machinery required to control the more
complex behaviours associated with the planktonic life history. ENC1s and
their possible homologues in other pulmonates were retained to carry out a
relatively simple task specific to encapsulated development (see below).
Further comparative analyses are required to help confirm such an evolutionary
hypothesis.
The synaptic connectivity between ENC1 and ciliary target cells was
confirmed through anatomical and physiological analyses. Both DIC microscopy
and serotonin immunofluorescence showed ENC1 neurite branching in close
apposition to the basal surface of ciliary cells throughout the pedal band of
cilia, as well as the most medial of the four ciliary cells that comprise each
dorsolateral band of cilia (Koss et al.,
2003). Ultrastructural experiments revealed the expected chemical
synaptic profiles between ENC1 and ciliary cells. Surprisingly, these synapses
often occurred on short projections emanating from the basal surface of the
ciliary cells (Koss et al.,
2003). Gap junction profiles between adjacent ciliary cells were
also observed, suggesting that ciliary cells not innervated by ENC1 may
respond indirectly through gap junction-mediated transfer of electrical or
chemical signals between ciliary cells.
In contrast to the circumstantial evidence of ENC1–ciliary
communication provided by the anatomical experiments, conclusive evidence
could only be obtained through experimental perturbation of ENC1 activity in
physiological experiments. Taking advantage of the ability to visualize
cellular structures in live intact embryos through DIC microscopy, laser
techniques were used to confirm functional connectivity between ENC1 and
ciliary cells. A laser stimulation protocol was developed that results in the
death of ENC1 5–6 h after laser treatment
(Kuang and Goldberg, 2001).
Not only did the bilateral laser ablation of ENC1 reduce the rate of embryo
rotation to the basal level and eliminate surges
(Kuang et al., 2002b), but
also the laser treatment caused an initial increase in cilia beat frequency
(Kuang and Goldberg, 2001).
This suggested that the initial effect of the laser treatment was an
injury-induced depolarization leading to transmitter release and postsynaptic
activation of ciliary beating. This demonstration of laser-induced stimulation
of transmitter release was further confirmed by blocking the cilioexcitatory
response with an effective serotonin antagonist
(Kuang and Goldberg,
2001).
The signal transduction mechanisms by which serotonin causes an increase in
cilia beat frequency have been examined in behavioural and cellular
experiments. Behaviourally, serotonin causes up to a fourfold increase in
rotation rate through a low affinity receptor that is preferentially blocked
by the serotonin antagonist mianserin
(Diefenbach et al., 1991;
Goldberg et al., 1994). This
response to serotonin is also blocked by treatments that interfere with nitric
oxide signalling (Cole et al.,
2002). While nitric oxide is produced and active in both ENC1 and
ciliary cells, it appears to act primarily as an intracellular messenger,
rather than in anterograde or retrograde transmission between ENC1 and ciliary
cells (Cole et al., 2002;
Doran et al., 2003) (see
below).
Our cellular studies on signal transduction pathways in ciliary cells were
recently boosted by the development of techniques to isolate identified
ciliary patches (Doran et al.,
2004), thus eliminating the problems encountered in earlier
studies on heterogeneous cell populations
(Goldberg et al., 1994;
Christopher et al., 1996;
Christopher et al., 1999).
Collectively, these studies have revealed that serotonin-induced
cilioexcitation involves a complex network of signal transduction pathways. In
the absence of serotonin, ciliary cells display constitutive ciliary beating
at low beat frequencies that underlies the basal rate of rotation observed in
intact embryos. Addition of serotonin causes an immediate increase in ciliary
beat frequency in pedal and dorsolateral ciliary cells that involves
activation of serotonin receptors, protein kinase C, nitric oxide and an
increase in intracellular calcium (see below).
Two serotonin receptor subtypes have been cloned in H. trivolvis,
one a member of the 5-HT1 family of G-protein-coupled serotonin
receptors and the other a member of the 5-HT7 family of
G-protein-coupled serotonin receptors. These receptors, named
5-HT1Hel and 5-HT7Hel, respectively, are both expressed
in embryonic ciliary cells, as well as in many neurons of the adult central
nervous system (Mapara et al.,
2008; Doran et al.,
2004). A preliminary study of these suggested that
5-HT1Hel receptors probably mediate a transient large-amplitude
cilioexcitatory response to serotonin
(Gallin et al., 2006). In
contrast, the 5-HT7Hel receptors appear to be involved in producing
long-lasting cilioexcitatory responses of lower amplitude that are sustained
even after serotonin washout. This dual control system may explain why earlier
pharmacological experiments identified very few effective blockers of the
serotonin response. Mianserin, a compound with poor selectivity for serotonin
receptor subtypes (Petrascheck et al.,
2007), is the only serotonin antagonist shown to effectively block
the cilioexcitatory or rotational responses to serotonin
(Goldberg et al., 1994).
Beyond the activation of serotonin receptors, calcium, protein kinase C and
nitric oxide have all been implicated in producing the cilioexcitatory
response to serotonin (Christopher et al.,
1996; Christopher et al., 1998;
Doran et al., 2003;
Doran et al., 2004;
Doran and Goldberg, 2006). In
most ciliary systems studied to date, a rise in intracellular calcium is
directly responsible for activating the molecular ciliary machinery (Salathe
and Bookman, 1999; Lansley and Sanderson,
1999; Zagoory et al.,
2001; Doran and Goldberg,
2006). Experiments with calcium buffers, calcium-sensitive dyes
and manipulations of extracellular calcium concentration in pedal and
dorsolateral ciliary cells suggest that serotonin induces a highly localized
acute rise in intracellular calcium that stimulates beat frequency, in
addition to a slow dispersed rise in cytosolic calcium that may function in
the refilling of intracellular calcium stores
(Doran and Goldberg, 2006;
Doran et al., 2004;
Doran, 2005). Pharmacological
experiments indicated that although phospholipase C and protein kinase C
contribute partially to the cilioexcitatory response, the calcium necessary
for ciliary stimulation is released from an intracellular store through a
caffeine-sensitive release mechanism, rather than an inositol triphosphate- or
ryanodine-sensitive release mechanism
(Doran, 2005). Finally, nitric
oxide is constitutively expressed in ciliary cells and plays a permissive role
in cilioexcitation. Although serotonin only causes a moderate stimulation of
nitric oxide production in some ciliary cells, interference with ongoing
nitric oxide production or activity completely prevents the cilioexcitatory
response to serotonin (Doran et al.,
2003). This profile of signal transduction activities is
relatively unique compared with the various other vertebrate or invertebrate
systems in which cilioexcitatory responses have been examined. Whereas many of
the intracellular messengers are the same in most systems, their specific
activities, interactions and relative roles are highly variable [for example,
protein kinase C (see Doran and Goldberg,
2006; Morales et al.,
2000; Levin et al.,
1997; Mwimbi et al.,
2002)].
Identification of hypoxia as an environmental regulator of embryonic rotation
Pond snail embryos undergo encapsulated development in the relatively stable environment of the egg mass. The energy expended in producing the rotation behaviour and the precise neural circuitry underlying the rotational surges together suggest that the behaviour must provide some benefit to the embryo. The alternative interpretation that the rotational behaviour is simply a vestige of locomotory behaviours expressed by veliger larvae of ancestral planktonic species seems far less likely.
The highly specialized dendritic knob of ENC1 has an increased surface area
through sensory-like cilia and microvilli
(Diefenbach et al., 1998).
This prompted the hypothesis that the ENC1–ciliary circuits are
activated by a specific environmental cue, and the ensuing stimulation of
ciliary beating and embryo rotation comprise an adaptive response to the cue.
Candidate environmental signals include those that fluctuate independently of
embryo metabolism, such as light and temperature, and those that are affected
by embryo metabolism, such as oxygen, carbon dioxide, nutrients and metabolic
waste products. Early on in experiments evaluating this latter group of
signals, we discovered that exposure of egg masses to hypoxic pond water
induces a robust stimulation of embryo rotation similar in magnitude to that
produced by maximal concentrations of exogenous serotonin
(Kuang et al., 2002a). This
came as a great surprise since ciliary beating is highly dependent on the
availability of ATP (Woolley,
2000), and thus an adequate supply of oxygen.
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|
).
Termination of hypoxic treatments induced a pronounced and transient
inhibition of rotation rate below normal levels. Embryos reliably responded
during repeated cycles of hypoxia and normoxia, and behavioural sensitization
of the hypoxia-induced response was observed by the third cycle. The response
was concentration dependent, with threshold, half-maximal and maximal
responses occurring at a PO2 of 60, 28 and 13
mmHg, respectively. Finally, in contrast to the potent effects of hypoxia,
embryo rotation was only weakly sensitive to hyperoxia or hypercapnia
(Kuang, 2002). Together, these
characteristics indicate that the rotation behaviour and underlying
neural–ciliary circuits function to detect and form adaptive responses
to environmental hypoxia (see below).
Whilst the physiological mechanisms underlying the motor components of the
rotational response to hypoxia are partially understood as a result of our
studies on ENC1–ciliary communication and serotonin-induced
cilioexcitation (see above), much less is known about the sensory side of the
response. Laser ablation and pharmacological experiments confirmed that the
response to hypoxia depends on intact ENC1s and serotonin release. As well,
ciliary cells isolated in cell culture had no response to hypoxia, further
suggesting that ENC1 is directly responsible for hypoxia detection
(Kuang et al., 2002a). To
confirm this electrophysiologically, the technique developed to isolate
identified patches of ciliary cells (Doran
et al., 2004) was adapted for ENC1. Whole-cell current-clamp
recordings of isolated ENC1s revealed hypoxia-induced action potential
activity in four of the four cells tested, indicating that ENC1 contains the
sensory apparatus to detect hypoxia (Fig.
3).
Mechanisms of hypoxia sensing have long been a topic of intense debate
(Buckler, 2007;
Kemp and Peers, 2007). Whilst
the hypoxia-sensing pathway in ENC1 has not yet been examined in isolated
cells, pharmacological experiments on whole embryos and isolated ciliary cells
have implicated the mitochondrial electron transport chain and potassium
channel closure (Fig. 4).
Treatment of whole embryos with either rotenone or 4-aminopyridine, inhibitors
of the electron transport chain and oxygen-sensitive potassium channels
(Lopez-Barneo et al., 2001;
Haddad and Jiang, 1997),
respectively, increased the embryonic rotation rate. In contrast, neither of
these affected the cilia beat frequency in isolated ciliary cells, suggesting
that the site of action is upstream, most likely in ENC1 cells. While these
experiments indicate possible elements of the hypoxia sensor, a more
comprehensive analysis of hypoxia sensing in isolated ENC1s is required.
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Adult pond snails, which respire both cutaneously in the aquatic
environment and through air breathing at the water surface, respond to hypoxia
through oxygen-sensing peripheral neurons that activate the air-breathing
pathway (Bell et al., 2007).
The stimulation of the embryo rotation behaviour by hypoxia suggests that this
behaviour also serves a respiratory function that helps to ensure an adequate
supply of oxygen to the developing embryo, and thus confers an adaptive
advantage to embryos. We hereby call this the `embryo stir-bar hypothesis',
whereby embryo rotation, coupled with the underlying ciliary activity, serves
to mix the egg capsule fluid (Fig.
5). In this scenario, mixing would diminish the unstirred boundary
layer underneath the capsular membrane and reduce the diffusion gradient from
the capsule surface to the oxygen-consuming embryo. Not only would this
activity increase the oxygen concentration at the embryonic surface, but it
would also steepen the diffusion gradient between the outside and inside
surfaces of the egg capsule membrane, thus enhancing the transfer of oxygen
into the egg capsule.
|
Our initial measurements of capsular oxygen using oxygen-sensitive
microelectrodes confirmed the presence of a significant oxygen gradient from
egg capsule surface to embryo under normoxic conditions
(Kuang et al., 2002a).
Furthermore, exposure of egg masses to hypoxic pond water caused rapid
reductions in egg capsule oxygen (Kuang,
2002), suggesting that the egg mass and egg capsule membranes form
weak diffusion barriers to environmental oxygen. Oxygen measurement
experiments testing the mixing effects of embryo rotation and ciliary activity
when stimulated by either serotonin or hypoxia are currently underway.
In another direct test of the embryo stir-bar hypothesis, assessing the
effects of hypoxia in rotation-compromised embryos would determine whether
rotational responses are required for normal development or viability, and
thus address the adaptiveness of the behavioural response. Embryos are able to
survive long-term exposure to strong hypoxia (<10% normal oxygen
concentration) pond water for 10h (Shartau
and Goldberg, 2007). We are now testing how this survival period
might be affected if the rotation behaviour was experimentally attenuated.
De-ciliation treatments such as chloral hydrate or hypertonicity
(Quarmby, 2004) were partially
effective in causing de-ciliation, but not specific enough to clearly
determine whether the loss or slowing of rotation has negative consequences on
embryo development and viability (Shartau
and Goldberg, 2007). In preliminary experiments, pharmacological
perturbation of embryo rotation using the serotonin antagonist mianserin
affected the progression of embryonic development. Whilst this result helps
support the hypothesis, approaches that more specifically attenuate the
rotation behaviour, such as molecular knockout of the ciliary serotonin
receptors or other ciliary proteins, or double laser ablation of ENC1
(Kuang et al., 2002b), will
produce more definitive tests. These experiments may also reveal whether the
basal-unstimulated rotation behaviour also plays an adaptive role under
normoxic conditions.
A comparative analysis of the embryonic rotation behaviour can also shed
light on whether the hypoxia-induced rotational response is a functional
adaptation in pond snails. It would be expected that if this is an important
survival strategy, it would be expressed in related gastropod species that
have encapsulated, directly developing embryos found in diverse fresh water
environments. Indeed, in recent experiments on representative species from the
three prominent families of freshwater pond snails, the Planorbidae,
Lymnaeidae and Physidae, hypoxia-induced rotational responses were observed in
all species tested (Fig. 6).
Interestingly, the ENC1 homologues in Lymnaea stagnalis are the
transient apical catecholaminergic (TAC) neurons
(Voronezhskaya et al., 1999),
in which dopamine has replaced serotonin as the primary cilioexcitatory
neurotransmitter in the rotation response
(Voronezhskaya et al., 1999;
Kuang, 2002). Since physids
are thought to be more closely related to lymnaeids and planorbids than these
latter two groups are to each other
(Jorgensen et al., 2004), it
will be interesting to determine whether the ENC1 homologues in physid embryos
use serotonin or dopamine as a cilio-excitatory neurotransmitter. Furthermore,
testing whether hypoxia is an important cue in more distantly related marine
snail species with encapsulated direct embryonic development will reveal
whether this is an ancient adaptation, or one that evolved more recently as
pulmonates inhabited fresh water environments
(Moran and Woods, 2007;
Lee and Strathmann, 1998).
A critical final element in validating the embryo stir-bar hypothesis is understanding how the embryonic rotation behaviour functions in nature. Snails reside in diverse freshwater environments, ranging from pristine oligotrophic waters that undergo only limited fluctuations in oxygen concentration to productive eutrophic waters that undergo large seasonal and diurnal fluctuations in oxygen concentration. Our initial field experiments on H. trivolvis embryos in a small spring-fed oligotrophic pond revealed that embryonic rotation rates varied according to the daily cycle in temperature, whereas oxygen concentrations were relatively stable throughout the daily temperature and light cycles (R.B.S. and J.I.G., unpublished observations). With the feasibility of these field experiments and a preliminary baseline data set now established, we look forward to testing how the embryo rotation behaviour helps embryos in eutrophic or stagnant habitats weather periods of severe hypoxia induced by algal blooms and heat. Furthermore, studying hypoxia responses under a variety of natural conditions may reveal how embryos balance the advantages gained through active responses that enhance oxygen availability over the short term with the metabolic cost of expending energy during sustained periods of hypoxia. The instigation of these ecological experiments highlights how the rotational respiratory behaviour in pond snail embryos is an ideal model system for integrative biology in the broadest sense, incorporating analyses at molecular, physiological, behavioural, ecological and evolutionary levels.
Acknowledgments
This work was supported by NSERC Canada Discovery Grants to J.I.G. and D.W.A. The authors thank Siva Muruganathan for editing the manuscript.
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