JOURNAL
Wayne
/ SEASONAL
OF BIOLOGICAL
BREEDING
RHYTHMS
IN MOLLUSKS
/ August 2001
MOLLUSKS
Regulation of Seasonal Reproduction in Mollusks
Nancy L. Wayne
Department of Physiology, UCLA School of Medicine, Los Angeles, CA 90095 USA
Abstract Understanding the physiological basis of environmental regulation of
reproduction at the cellular level has been difficult or unfeasible in vertebrate
species because of the highly complex and diffuse nature of vertebrate
neuroendocrine systems. This is not the case with the simple nervous system of
mollusks in which reproductive neuroendocrine cells are often readily identifiable in living tissue. Given that there are mollusks that are seasonal breeders, that
the neuroendocrine cells controlling reproduction have been identified in several
molluskan species, that these neurons are conducive to cell physiological analysis, and that basic features of cell biology have been highly conserved between
mammals and mollusks, it seems that the mollusk would provide an excellent
model system to investigate cell-physiological events that mediate effects of
environmental signals on reproduction. The purpose of this review is to explore
this idea in three species in which the topic of the neural basis of seasonal reproduction has been studied: the giant garden slug Limax maximus, the freshwater
pond snail Lymnaea stagnalis, and the marine snail Aplysia californica.
Key words
Aplysia, Limax, Lymnaea, pheromone, photoperiod
It is widely acknowledged that for most seasonal
breeders living in temperate climates, photoperiod is
the primary environmental signal that regulates the
timing of reproduction (see Goldman, 2001 [this
issue]). Research in the area of environmental regulation of seasonal reproduction has focused on describing the effects of various environmental factors on
reproduction, the endocrine systems involved, the
role of the circadian system in photoperiodic
responses, and (in mammals) the site of action of
melatonin in the regulation of photoperiodic response
systems. This work has provided the field with a large
and rich literature that is focused largely at the system
and behavioral levels of analyses, with more recent
research at the molecular level with regard to the
melatonin receptor. There is comparatively little information on the cell physiological processes by which
environmental variables regulate reproductive functions. Why is that the case? This type of cellular work
is limited in vertebrates because the neural structures
involved in mediating effects of external signals on
JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 16 No. 4, August 2001
© 2001 Sage Publications
reproduction are diffusely located in the brain and the
neuroendocrine circuitry is highly complex. Therefore, identifying specific neurons in living tissue to
monitor their physiological activity is difficult or in
many cases unfeasible in vertebrate systems.
In comparison to the vertebrate central nervous
system, that of mollusks is relatively simple and could
provide an avenue for revealing the cell physiological
responses to environmental signals. The central nervous system of the mollusk is laid out as a series of
ganglia containing thousands, rather than billions, of
neurons. In some molluskan species, such as Aplysia
californica, specific neurons are readily identifiable
from one animal to the next. For example, the giant
neuron R15, which controls respiration in Aplysia, is
always located in the same place in the abdominal
ganglion from one animal to the next. This ability to
easily locate specific neurons in living tissue has
allowed researchers to map the role of single neurons
in controlling distinct behaviors. Molluskan neurons
are large in size (some with diameters reaching 1 mm),
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making electrophysiological measurements of membrane excitability a relatively simple task. This combination of a simple, well-defined nervous system and
large neurons has allowed researchers to describe in
great detail the ion channels and currents that underlie changes in membrane excitability, which in turn
controls physiology and behavior. In fact, Nobel Prize–
winning work by Hodgkin and Huxley (1952) used
the giant squid axon as the preferred model system to
describe ion-current changes during the action potential. Molluskan model systems have also been successfully used to understand the second-messenger
pathways activated by synaptic transmission and
modulatory peptides (Conn and Kaczmarek, 1989). In
addition, Kandel and his colleagues elevated the
lowly sea hare Aplysia to Nobel worthiness with their
work on the cellular and molecular basis of learning
and memory using this mollusk’s sensorimotor neurons as a model system for their studies (Bailey et al.,
1996).
Importantly, although the brain of the mollusk has
a gross appearance that is very different from that of
vertebrate brains, there are strong homologies at the
cellular and subcellular levels. For example, transmitters and peptide hormones acting at G-coupled receptors activate adenylate cyclase, which converts ATP
into cyclic AMP; cAMP activates cAMP-dependent
kinase, which phosphorylates specific proteins that
regulate cell functions. Rat or mollusk? Membrane
depolarization opens voltage-gated calcium channels,
allowing calcium to diffuse into the neuron, resulting
in a rise in concentrations of cytosolic calcium; this rise
in calcium activates downstream cell and molecular
events leading to release of transmitter. Rat or mollusk? These types of signal-transduction cascades
show striking homology between mammals and mollusks (Kandel, 1997). Both the structure and functions
of a wide array of neurotransmitters, receptors, second messengers, and peptide hormones have been
highly conserved—and so the cell physiology of a
molluskan neuron often looks remarkably like that of
a mammalian neuron.
Given that there are molluskan species that are seasonal breeders, that the neuroendocrine cells controlling reproduction have been identified in several
molluskan species, that these neurons are conducive
to cell physiological analysis, and that basic features of
cell biology have been highly conserved, it seems that
the mollusk would provide an excellent model system
to investigate the physiological basis of environmental regulation of reproduction at the cellular level. The
purpose of this review is to explore this idea.
Although there is a relatively limited literature on
physiological mechanisms underlying environmental
regulation of reproduction in mollusks, three species
in which this topic has been studied are the giant garden slug Limax maximus, the freshwater pond snail
Lymnaea stagnalis, and the marine snail Aplysia
californica. All three species are hermaphrodites, containing both male and female gonads.
LIMAX MAXIMUS
Effects of Photoperiod
Limax maximus is a common garden slug, which
undergoes reproductive maturation from mid-May
through late July and reaches full reproductive competence in August. Sokolove and colleagues demonstrated that maturation of the Limax reproductive system is dependent on photoperiodic signals (reviewed
in Sokolove et al., 1984). Slugs raised in the laboratory
on short photoperiods remain immature indefinitely.
Exposure to long day-lengths stimulates growth and
development of the gonad, penis, albumen gland, various female accessory sex organs, and maturation of
sperm. The cerebral ganglion (the “command center”
of the molluskan brain) was shown to mediate the
effects of long days on both male and female components of the reproductive system in transplant studies
(McCrone et al., 1981). Cerebral ganglia were dissected from donors either raised on long or short days
and then implanted into recipients maintained on
inhibitory short day-lengths. Slugs that received the
long-day cerebral ganglia showed normal reproductive tract development, whereas those animals that
received short-day ganglia remained immature. This
work indicates that long days stimulate the synthesis
(and perhaps secretion) of a hermaphroditic maturation factor (Fig. 1a). Further work demonstrated that
in response to long days, both the cerebral ganglion
and blood contain a male gonadotropic factor that
stimulates proliferation of spermatogonia in recipient
slugs maintained on inhibitory short days (Melrose
et al., 1983; Fig. 1a). It is likely that the male gonadotro-
Wayne / SEASONAL BREEDING IN MOLLUSKS
393
Figure 1. (a) Effects of photoperiod and brain transplants on the reproductive system of Limax maximus. Brains transplanted from donor
slugs maintained on stimulatory long days stimulated the reproductive system in recipient slugs maintained on inhibitory short days.
Brains from short-day animals had no effect on maturation of the reproductive system. These findings indicate that the brain mediates the
effects of photoperiod on maturation of the reproductive system.
(b) Effects of brain and gonad transplants on the reproductive system of Limax. Transfer of gonadectomized slugs from inhibitory short days to stimulatory long days had no effect on maturation of the accessory sex organs, indicating that secretions from the gonad
are important for mediating the effect of photoperiod on this aspect of the maturation process. Transplanting gonads from slugs maintained on long days to gonadectomized animals stimulated maturation of the accessory sex organs. Likewise, transplanting brains from
long-day slugs that were gonadectomized to gonad-intact animals kept on inhibitory short days stimulated maturation of the gonad and
accessory sex organs in the recipient animals. These experiments suggest that photoperiod activates neurons in the brain that, in turn, activate the gonad, thereby stimulating maturation of the accessory sex organs.
(c) A model for photoperiodic stimulation of the reproductive system of Limax. Long photoperiods stimulate unknown
photoreceptors, activating neurons in the cerebral ganglion to secrete a hermaphroditic maturation factor and male gonadotropic factor.
These secretions from the cerebral ganglion stimulate the gonad to release additional hormones that stimulate maturation of the female
and male accessory sex organs.
pic factor originates in the cerebral ganglion (which
contains many secretory neurons) and is secreted into
the blood, then diffuses throughout the hemolymph
where it can act at its target site(s) in the reproductive
system.
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Organization of the Reproductive
Endocrine System
There are three main types of tissues known to be
involved in the Limax photoperiodic response system:
the cerebral ganglion, which receives light input from
an unidentified photoreceptor and produces maturation/gonadotropic factors in response to long days;
the hermaphroditic gonad; and the male and female
accessory sex organs (e.g., penis, albumen gland, etc.).
Does the cerebral ganglion secrete stimulatory hormones that have wide-ranging target sites throughout
the reproductive system? Or do the hormones of cerebral origin activate gonadal secretions, which in turn
influence the accessory sex organs? Evidence favors
the latter model (Figs. 1 b,c). McCrone and Sokolove
(1986) showed that gonadectomizing short-day
immature slugs prevented photoperiodic stimulation
of the accessory sex organs. However, implanting
th ese cas t rated slugs with gonads f ro m
photoperiodically stimulated mature animals led to
development of the accessory sex organs, suggesting
that the gonad is secreting a hormone(s) that mediates
the effects of photoperiod (presumably, via input to
the cerebral ganglia) on accessory sex-organ development. Further experiments showed that the brain
becomes photoperiodically induced independent
from the gonad (McCrone and Sokolove, 1986). Immature slugs raised on short days were implanted with
brains from donor animals raised on stimulatory long
days. Compared to nonimplanted controls, the slugs
receiving a long-day brain showed stimulation of the
gonad and accessory sex organs.
To my knowledge, there was but a single laboratory
working on the physiology underlying photoperiodic
control of reproduction in the Limax, and publications
from this group ended in the mid-1980s. The work on
long-day stimulation of reproductive factors from the
cerebral ganglion was never followed up, and so the
identification of the hermaphroditic maturation factor
and the male gonadotropic factor (which may be one
and the same molecule) remains to be determined.
This is unfortunate, because without this information,
positive identification of the neurons in the cerebral
ganglion that synthesize these reproductive factors
cannot be made. And, obviously, without identification of these neurons, it is not possible to study their
physiological response to photoperiodic cues.
Because of its dramatic response to a shift between
short and long days, the Limax CNS still provides a
promising model system for investigating the detailed
cell physiology of a photoperiodic response system.
LYMNAEA STAGNALIS
The natural habitat of Lymnaea stagnalis is freshwater lakes and ditches, where they copulate and lay
eggs primarily during the summer. Studies have
shown that there are five major external signals that
regulate reproduction in this snail: photoperiod, food
consumption, temperature, water quality, and parasites (reviewed in Joosse, 1984; de Jong-Brink et al.,
1992). In general, long day-lengths and clean, oxygenated water stimulate reproductive functions, while
starvation, cold temperatures, and parasites inhibit
reproduction. Not surprisingly, there are complex
interactions between these signals that ultimately
impact whether or not the snail will reproduce. Most
studies on the Lymnaea reproductive system have
focused on the female phase of reproduction (leading
to oviposition), and that is the work that will be
reviewed here.
Organization of the Female
Reproductive Endocrine System
There are six main types of cells/organs known to
be involved in regulating egg laying in Lymnaea: the
endocrine dorsal bodies, the neuroendocrine
caudodorsal cells, the lateral lobes, the hermaphroditic gonad, the hermaphroditic duct, and the accessory sex organs (including the albumen gland)
(reviewed in Joosse, 1984; Fig. 2a). The endocrine dorsal bodies are attached to the part of the brain called
the cerebral ganglia and produce dorsal body hormone, which stimulates vitellogenesis and growth
and differentiation of the female accessory sex organs.
The caudodorsal cells are located in the cerebral ganglia and produce caudodorsal cell hormone, which
stimulates ovulation and alters behaviors associated
with egg laying. Neurons in the lateral lobes (small
ganglia attached to the cerebral ganglia) regulate maturation of the female reproductive system by activating the endocrine dorsal bodies and the caudodorsal
cells. In response to caudodorsal cell hormone, mature
oocytes located within follicles in vitellogenic areas of
the ovotestis are released into the hermaphroditic duct
where fertilization takes place. The eggs (up to 200) are
surrounded by secretions from the albumen gland
and membranes secreted by the pars contorta as they
pass along the duct. Egg-mass formation occurs in the
oothecal gland, the egg mass exits the animal’s body
and is deposited on a substrate. The behaviors triggered by secretion of caudodorsal hormone and that
Wayne / SEASONAL BREEDING IN MOLLUSKS
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Figure 2. (a) Organization of the female reproductive endocrine system of Lymnaea stagnalis. Neurons in the lateral lobes activate the
endocrine dorsal bodies and caudodorsal cells. Dorsal body hormone, secreted from the lateral and medial dorsal bodies, stimulates
vitellogenesis and growth and differentiation of the female accessory sex organs. Caudodorsal cell hormone, secreted by the caudodorsal
cells, stimulates ovulation and egg-laying behaviors. LL = lateral lobe; LDB = lateral dorsal body; MDB = medial dorsal body; DBH = dorsal
body hormone; CDC = caudodorsal cells; CDCH = caudodorsal cell hormone.
(b) Organization of the female reproductive system of Aplysia californica. Signals from neurons in the cerebral ganglia are transmitted to the pleural ganglia and then to the bag cell neurons located at the junction between the abdominal ganglion and pleurovisceral
connective nerves. This electrical input from higher-order centers activates a long-lasting afterdischarge (typically 10-30 min in duration).
The afterdischarge triggers secretion of egg-laying hormone (ELH), which then stimulates ovulation and egg-laying behaviors.
(c) Temperature sensitivity of the Aplysia reproductive axis, influencing ovulation and egg-laying behavior. Studies show that
temperature has no significant effect on responsiveness of the ovotestis to ELH stimulation, has some effects on bag-cell neuron functions,
and has a robust effect on responsiveness of the head ganglia to stimulation. Collectively, this work suggests that temperature’s effects on
ovulation and egg-laying behavior is primarily through changes in excitability of the head ganglia, ultimately controlling whether or not
the bag-cell neurons will afterdischarge. The direct effects of temperature on bag cell excitability and ELH secretion are variable and less
robust, suggesting that temperature-induced changes in responsiveness of the bag cell neurons play a secondary role.
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are associated with egg laying are highly complex and
last in duration for about 2 h (Ter Maat et al., 1989).
Effects of Photoperiod on Egg Laying, and
Its Modulation by Food Consumption
and Temperature
Long day-lengths (LD 16:8) have stimulatory
effects on sexual maturation, advancing the onset of
spontaneous egg laying by 2 weeks compared with
animals raised on either short (LD 8:16) or medium
(LD 12:12) days (Bohlken and Joosse, 1982). Snails
maintained on long days are slightly more sensitive to
hormonal induction of egg laying than those animals
kept on medium days (Dogterom et al., 1983). Studies
have not revealed an effect of photoperiod on membrane excitability of the neuroendocrine caudodorsal
cells that stimulate egg laying (Joosse, 1984).
The most prominent effect of photoperiod is on the
number of eggs laid (Joosse, 1984). Snails reared in the
laboratory under artificial photoperiodic conditions
laid significantly greater numbers of eggs and laid eggs
more frequently under long days than under medium
or short days. The robustness of this photoperiodic
response was modulated by both food consumption
and temperature. Starvation completely abolished
egg laying in animals maintained on short and
medium days, but animals exposed to long days continued to lay eggs although at a significantly lower
level than prior to starvation. Similarly, cold temperatures (8 °C) completely abolished egg laying in animals maintained on short and medium days; animals
kept on long days continued to lay eggs but at a lower
level compared with when they were maintained at
room temperature. Therefore, the stimulatory effect of
long days overrides the inhibitory effects of starvation
and cold temperatures.
Effect of Photoperiod on Polysaccharide
Levels in a Female Accessory Sex Organ
Glycogen and galactogen are the primary storage
polysaccharides in pulmonate snails. Both polysaccharides are found in secretory cells of the albumen
gland, which plays an important role in the packaging
of eggs for transport out of the body. Studies showed
that short days are stimulatory to glycogen synthesis
in the albumen gland, whereas long days are inhibitory (Wijsman, 1989). On the other hand, photoperiod
had no significant effect on galactogen levels in the
albumen gland. Given that glycogen and galactogen
are synthesized in the same cells, this finding of a differential photoperiodic response suggests that
changes in day length ultimately impact different
polysaccharide signal-transduction pathways at the
cellular level. Why do long photoperiods cause a
decrease in levels of albumen-gland glycogen? Is it
due to an increase in egg-laying activity (and thus,
albumen gland activity), leading to depletion of glycogen stores? This is most likely not the case, as spontaneous egg laying has no effect on levels of glycogen in
the albumen gland (Wijsman, 1989). To my knowledge, there has been no further work on the effect of
photoperiod on polysaccharide metabolism.
Effect of Clean Water Stimulus on
Egg-Laying Behavior
Ovulation and egg-laying behavior are dependent
on secretion of the neuropeptide caudodorsal cell hormone. The immediate event that triggers release of
this hormone is a pattern of repetitive action-potential
firing called an afterdischarge. There are about 100
caudodorsal cells located in the cerebral ganglion;
these neurons form a network of electrotonically connected cells that fire in synchrony for 30 to 60 min.
Ovulation can be activated in 95% of mature snails
that have been living in filthy conditions by exposing
them to clean, aerated water (Ter Maat et al., 1983). The
time from onset of the stimulus to afterdischarge was
typically between 15 and 50 min, with ovulation
occurring within 2 h. The pattern of the caudodorsal
cell afterdischarge and behaviors associated with egg
laying were very similar regardless of how this reproductive activity was elicited: spontaneously, by clean
water stimulus, or via electrical activation of
caudodorsal cell afterdischarges (Ter Maat et al.,
1989). Unlike with photoperiod in which changes in
the animal’s reproductive state require exposure to
many weeks of the appropriate signal, the clean water
stimulus elicits a nearly immediate response to favorable conditions in which to lay eggs.
Effect of Parasitic Infection on
Egg-Laying Behavior
Infection by the schistosome parasite Trichobilharzia
inhibits reproduction in Lymnaea. Studies suggest that
some of the inhibitory effects on reproduction are
caused by release of the peptide schistosomin from the
Lymnaea CNS in response to parasitic infection
(reviewed in de Jong-Brink et al., 1992). How is this
Wayne / SEASONAL BREEDING IN MOLLUSKS
parasite-induced neuropeptide affecting reproduction? Studies have shown that schistosomin attacks
multiple components along the snail’s reproductive
axis. Schistosomin antagonizes the bioactivity of
caudodorsal cell hormone at its target sites, blocking
ovulation and many of the behaviors associated with
egg laying (Hordijk et al., 1991). In addition, both purified schistosomin and hemolymph from infected
snails (which contains schistosomin) blocked the ability of caudodorsal cells to generate afterdischarges
and thus prevented secretion of caudodorsal cell
hormone and egg laying (Hordijk et al., 1992). These
studies suggest that parasitic infection inhibits
reproduction through the actions of schistosomin at
both central (caudodorsal cells) and peripheral
(caudodorsal-cell hormone target sites) locations.
Studies done in Lymnaea have identified a number
of environmental factors that regulate reproductive
function. There is an impressive literature on the
details of the cell physiology underlying function of
the caudodorsal cells that regulate ovulation and
behaviors associated with egg laying (reviewed in
Kits et al., 1990). However, there are currently few laboratories continuing this work and even fewer investigating the effects of environmental signals on reproductive function. Given that many specific neurons
involved in the reproductive neuroendocrine pathway have been identified and are well characterized,
the Lymnaea model system offers the researcher a powerful tool to understand the neuronal basis of environmental control of reproduction.
APLYSIA CALIFORNICA
The natural habitat of Aplysia californica is the
intertidal zone of the Pacific Ocean extending from
mid-California to northern Mexico. Aplysia are reproductively active from summer through mid-autumn.
Studies have shown that temperature is the primary
environmental factor that regulates reproductive
function, while photoperiod plays a minor role
(Wayne and Block, 1992). Although each Aplysia has
the full complement of both the female and male
reproductive systems, they do not self-fertilize.
Cross-fertilization is accomplished internally, thereby
requiring that these otherwise solitary animals come
into close contact with each other during the breeding
season. Pheromones from conspecifics probably play
an important role in providing an immediate signal
397
that a potential mate is in the vicinity, and in accelerating copulation (Painter et al., 1989, 1998). As with
Lymnaea, it is the female reproductive system of
Aplysia that has gained the most experimental attention, and this review will focus on the pathway regulating ovulation and egg-laying behaviors.
Organization of the Female Reproductive
Neuroendocrine System
There are five main types of cells/organs involved
in regulating ovulation and egg laying in Aplysia: the
cerebral and pleural ganglia of the neural head-ring,
the bag cell neurons of the abdominal ganglion, the
ovotestis, the hermaphroditic duct, and the accessory
sex organs. Electrical signals initiated in the cerebral
ganglia are transmitted to the nearby pleural ganglia
and then to the bag cell neurons via the pleurovisceral
connectives (Ferguson et al., 1989; Fig. 2b). This pathway ultimately activates a pattern of repetitive, action
potential firing that is very similar to the caudodorsal
cell afterdischarge in Lymnaea. In fact, there are many
characteristics of the caudodorsal cells that are highly
homologous to those of the bag cell neurons of Aplysia.
For instance, the bag cells synthesize and secrete a
36–amino acid peptide, egg-laying hormone (ELH),
that has 40% homology with the amino-acid sequence
of caudodorsal cell hormone (Chiu et al., 1979; Newcomb and Scheller, 1990; Vreugdenhil et al., 1988).
And as with the caudodorsal cells, secretion of Aplysia
ELH is triggered by the bag cell afterdischarge
(Kupfermann, 1970). ELH is secreted into the
hemolymph where it diffuses to its target sites in the
ovotestis and central nervous system, stimulating
ovulation and altering behaviors associated with egg
laying (Coggeshall, 1970; Bernheim and Mayeri,
1995). Egg laying is dependent on secretion of ELH,
and secretion of ELH is dependent on activation of the
afterdischarge (Pinsker and Dudek, 1977; Wayne and
Wong, 1994). Once the eggs exit the gonad, they travel
along the hermaphroditic duct, where they get packaged into a long egg strand, eventually leave the animal’s body, and are deposited onto a substrate. Along
the hermaphroditic duct, there are excretory glands
(e.g., atrial gland, albumen gland) that secrete various
molecules onto the eggs and egg cordon. Some of
these molecules are attractant pheromones, which get
released into the water after the eggs are laid (Painter
et al., 1989, 1998).
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Effect of Temperature on Egg Laying
The water temperature of the intertidal area off the
coast of southern California varies between 10 °C
(during winter/spring) and 20 °C (during late summer/early autumn). Relatively small changes in water
temperature have a dramatic influence on the frequency of egg-laying events in Aplysia, as well as on
the physiology and biochemistry of the neural pathway that regulates this behavior. Studies have shown
that Aplysia maintained in the laboratory at 15 °C lay
eggs significantly less frequently than those animals
maintained at 20 °C (Pinsker and Parsons, 1985;
Wayne and Block, 1992). This temperature-dependent
switch in reproductive activity could occur at one or
more components along the reproductive axis. Likely
tissues that could be influenced by temperature are
the head ganglia, which transmits electrical signals to
the bag cells; the bag cells, which synthesize and
secrete ELH; and the ovotestis, which responds to
ELH by extruding eggs into the hermaphroditic duct
(Fig. 2c). Aplysia acclimated to 15 °C or 20 °C showed
no significant difference in responsiveness to treatment with various doses of ELH, suggesting that the
effect of temperature on egg laying is not at the level of
the ovotestis, but at some higher-order level along the
reproductive pathway (Wayne et al., 1996).
Some characteristics of the bag cell neurons do
show significant changes in response to temperature.
Bag cell neurons from animals maintained at 15 °C
show significantly lower rates of ELH synthesis than
bag cells from animals kept at 20 °C (Berry, 1984),
potentially affecting the amount of releasable hormone. And treatment of bag cell neurons with α-bag
cell peptide (α-BCP, one of several bioactive peptides
cleaved from the ELH prohormone and co-released in
response to the afterdischarge) alters bag cell excitability and levels of cAMP in a temperature-dependent
manner (Redman and Berry, 1991). Importantly, activation of the cAMP second-messenger pathway has
stimulatory effects on bag-cell membrane excitability
(Conn and Kaczmarek, 1989), ELH synthesis (Bruehl
and Berry, 1985), and ELH secretion (Wayne et al.,
1998). Redman and Berry (1991) showed that at 15 °C
α-BCP suppressed levels of cAMP and hyperpolarized the membrane of bag cell neurons; the response at
20 °C was the opposite. Given the importance of membrane excitability and the cAMP pathway on ELH
secretion, it is possible that the effect of temperature
on egg-laying behavior is through temperaturedependent changes at the level of the bag cell neurons
and how they respond to α-BCP. However, in studies
using freely behaving Aplysia, the effects of temperature on certain bag-cell functions were inconsistent.
All animals acclimated to 20 °C showed afterdischarges and subsequent egg laying upon electrical
stimulation of the bag cell neurons; only 48% acclimated to 15 °C showed afterdischarges and subsequent egg-laying behavior (Wayne et al., 1996). These
results suggest that temperature can alter the responsiveness of bag cells to excitatory input. Studies investigating whether temperature affects the amount of
ELH released in response to afterdischarge had variable outcomes. Use of a bioassay for ELH secretion
revealed a slight but significant decrease in ELH from
cold-adapted bag cell neurons compared with
warm-adapted preparations (this difference in
bioactive ELH could not account for the dramatic difference in egg-laying behavior that is typically
observed at the two temperatures). On the other hand,
use of a specific and sensitive radioimmunoassay for
quantitative measurement of ELH showed no significant difference between bag cells from animals acclimated to 15 °C and those from animals kept at 20 °C
(Wayne et al., 1996). Overall, these results suggest that
temperature has minor or secondary effects on ELH
secretion.
Given that there does not appear to be a robust
effect of temperature on the response of the ovotestis
to hormonal stimulation, nor on the amount of ELH
secreted in response to afterdischarge, work progressed to higher-order centers in the brain that might
account for the dramatic effect of temperature on
egg-laying behavior. Neurons in the head ganglia
(particularly, the cerebral and pleural ganglia) appear
to play a central role in the activation of bag cell
afterdischarges (Ferguson et al., 1989; Brown et al.,
1989). Studies have shown that extract from the atrial
gland contains a factor (Peptide B) that when applied
to either the cerebral or pleural ganglia activates neural signals to the bag cell neurons, which stimulates
the afterdischarge (Painter et al., 1988). Subsequent
work investigated the effect of temperature on responsiveness of the head ganglia to atrial-gland extract
stimulation of bag cell afterdischarge (Wayne et al.,
1996). That study showed a dramatic effect of temperature on the latency to afterdischarge when the head
ganglia was treated with the extract. Basically, neural
preparations maintained at 15 °C showed a significantly greater delay to afterdischarge than neural
preparations maintained at 20 °C. The outcome of this
work suggests that temperature has its primary effect
Wayne / SEASONAL BREEDING IN MOLLUSKS
on egg-laying behavior through alterations in the
responsiveness of command neurons in the head
ganglia that ultimately activate the bag cell
afterdischarge.
Effect of Photoperiod on Egg Laying
Although there does appear to be an effect of
photoperiod on egg-laying behavior, this response is
not robust compared with the effect of temperature.
One study (Wayne and Block, 1992) showed that in
animals maintained in warm temperatures, short
day-lengths (LD 8:16) enhanced the frequency of egg
laying in mature Aplysia compared with those animals
kept on long days (LD 16:8). However, this
stimulatory effect of short days was only apparent in
warm water and during the animal’s natural breeding
season (summer and autumn). The weak photoperiodic response made further investigations of the neural regulation of this phenomenon difficult. Nevertheless, a follow-up experiment showed that the eyes
play a role in mediating photoperiodic information to
the reproductive system (Wayne and Block, 1992).
Intact animals maintained on short days showed a
slightly greater frequency of egg laying than those animals kept on long days; animals that were bilaterally
enucleated showed no photoperiodic response.
Importantly, the eyes of Aplysia contain both
photoreceptors and a circadian pacemaker (Jacklet,
1969; Eskin, 1971). The circadian system is involved in
the neural pathway mediating photoperiodic
responses in most animals investigated; however, it is
not known if this is also the case with mollusks. Nevertheless, functional characteristics of the light-input
pathway, clock mechanisms, and output pathway
controlling circadian rhythmicity have been well characterized in both Aplysia and a related marine mollusk, Bulla gouldiana (reviewed in Colwell et al., 1992;
Block and Michel, 1997). In both of these mollusks, the
circadian pacemaker cells are located within the retina
but outside of the photoreceptor layer: possibly the
D-type cells in Aplysia retina (Woolum and
Strumwasser, 1980), and definitely the basal retinal
neurons (BRNs) in Bulla retina (McMahon et al., 1984).
Studies in Bulla have shown that light entrains the
ocular rhythm through direct actions on photopigments in the BRNs. This triggers a cascade of cellular
events—membrane depolarization, calcium influx
through voltage-sensitive calcium channels, and possible changes in protein synthesis—which ultimately
shifts the phase of the rhythm. Generation of the circa-
399
dian rhythm itself appears to involve gene
transcription and translational events. Finally, the output pathway of the clock mechanism involves rhythmic changes in K+ conductance, which regulates membrane potential: hyperpolarization during the day and
depolarization during the night. This rhythm in membrane potential leads to rhythmic changes in
optic-nerve impulse activity, which presumably is at
least partly responsible for rhythmic changes in the
behavior of the animal. A very similar story has
emerged with our understanding of the circadian system in the Aplysia eye, with the exception that there is
no clear evidence for transcriptional/translational
events controlling the circadian oscillator (see Block
and Michel, 1997, for details).
Notably, the eyes of Aplysia secrete melatonin in a
rhythmic pattern: elevated during the day, low during
the night—the opposite pattern expected based on
studies from most vertebrates investigated (Abran
et al., 1994). It is possible that the eyes act as both a
photoreceptor and source of a melatonin signal that
provide the link between photoperiodic input and the
reproductive axis controlling egg laying. Unfortunately, this work has not been continued. Lack of a
robust and repeatable photoperiodic response in
Aplysia has made further investigations unappealing.
Also, definitive work identifying the command neurons in the cerebral ganglia that control bag cell
afterdischarge has not been forthcoming, and so, our
ability to directly investigate the effects of photoperiod and temperature on specific neuronal functions has been limited.
Effect of Pheromones on Reproduction
The onset of the breeding season coincides with a
pronounced change in the social behavior of Aplysia.
Previously solitary creatures, these mollusks will now
prefer to congregate in rather large aggregates of copulating animals (Blankenship et al., 1983). These
aggregates contain both copulating and egg-laying
animals, with numerous masses of egg cordons in
close vicinity. How do these otherwise solitary animals find each other, and what causes this newfound
attraction? The most obvious answer is that pheromones are released into the water from reproductively
mature conspecifics.
Earlier work showed that animals in the process of
laying eggs are more attractive to other Aplysia than
are nonlaying animals; latency to copulate was significantly shorter between an egg layer and conspecific
400
JOURNAL OF BIOLOGICAL RHYTHMS / August 2001
than between a nonlaying animal and conspecific
(Painter et al., 1989). T-maze studies showed that
freshly laid egg cordons (with or without the egglaying animal) are more attractive to conspecifics than
sham egg cordons (Painter et al., 1991). This finding
suggests that the attractant pheromone is being
released from the egg cordon and not from some other
part of the egg-laying animal. Given that there are several excretory glands that deposit secretions onto the
eggs or egg cordon as they move through the hermaphroditic duct, it was hypothesized that the secretions contain pheromones and that these molecules
get released from the egg cordon into the surrounding
water, making an egg-laying animal highly attractive
to conspecifics. Two sources of attractant pheromones
have been revealed: the atrial gland and albumen
gland. Both glands deposit secretions onto the egg cordon. Experiments in which homogenates of the atrial
gland were placed into water containing pairs of animals showed that this treatment led to a significant
decrease in the latency to copulate compared with animals exposed to just seawater or homogenates of the
large hermaphroditic duct (Painter et al., 1989). This
response was very similar to what was observed when
animals were exposed to a freshly laid egg cordon.
A partial sequence of a potential peptide pheromone from freshly laid egg cordons was obtained and
used to design a polymerase chain reaction (PCR)
primer for identification of the attractant pheromone
(Fan et al., 1997). Given that earlier work had identified an attractant pheromone in the atrial gland, it was
assumed that the peptide pheromone from the egg
cordon would be the same as that from the atrial
gland. Surprisingly, total RNA isolated from the atrial
gland did not yield a PCR band of the expected size,
but RNA from another exocrine gland—the albumen
gland—yielded a 160-base pair PCR band that was
consistent with the attractant peptide pheromone isolated from fresh egg cordons. This albumen gland
molecule was cloned and the amino-acid sequence
determined. The sequence of the cDNA clone contained a single open reading frame encoding a
76–amino acid precursor protein. Northern blot analysis of mRNA from a variety of Aplysia tissues (CNS,
ovotestis, small hermaphroditic duct, atrial gland,
albumen gland) confirmed that the attractant
pheromone is synthesized only in the albumen gland.
Subsequent work had purified the attractant
pheromone (called attractin) as a 58-residue peptide
and used it to test behavioral responses in a T-maze
(Painter et al., 1998). The number of animals attracted
to a non-egg-laying animal was greatly increased
when 10 pmol of purified attractin was placed just
next to it in the seawater. The response was very similar to what was observed in response to eluate from
freshly laid egg cordons. This is one of the few examples of a water-borne peptide pheromone and the first
to be characterized in an invertebrate. Release of
attractant pheromones provides the final step toward
copulation (and cross-fertilization) between reproductively mature animals during the breeding season.
CONCLUSION
It is glaringly obvious that the literature on seasonal
regulation of reproduction in mollusks is relatively
old. Few studies have been published in the last
decade (see reference list from this article). The use of
molluskan model systems for understanding the neural basis of behavior has fallen out of favor over the
past two decades. Advances in electrophysiological
t e ch n iq u e s ( e .g., w h o le - ce ll pat ch c l a m p
electrophysiology) have allowed researchers to monitor membrane excitability in small mammalian neurons, which was unfeasible until relatively recently.
Research that previously could only be performed in
giant neurons (of which molluskan neurons reigned
queen) can now be done in the typical, small mammalian neuron. And so, there are fewer laboratories still
using mollusks as a model system for exploring the
neural basis of behavior. Young investigators in the
training phase of their careers, who might be attracted
to working on problems associated with seasonal
reproduction, do not have a core of laboratories using
molluskan model systems in this area of research.
Nevertheless, the experiments discussed in this
review are waiting to be continued. Interesting and
viable questions pertaining to molluskan seasonal
breeding can still lead to findings with broad impact in
the fields of neuroscience and reproduction. For
example, what effect does temperature have on
caudodorsal cell membrane potential and synthesis
and secretion of caudodorsal cell hormone of
Lymnaea? What effect does schistosomin have on ionchannel activity and signal-transduction pathways in
the caudodorsal cells? What is the command neuron(s)
in the Aplysia cerebral ganglion that controls bag cell
afterdischarge, and what is its physiological response
to changes in water temperature and photoperiod?
The relatively large and detailed literature regarding
Lymnaea and Aplysia neurophysiology makes these
Wayne / SEASONAL BREEDING IN MOLLUSKS
two model systems especially attractive for future
work.
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