ABSTRACT There is growing evidence that maternal exposure to environmental stressors can alter offspring phenotype and increase fitness. Here, we investigate the relative and combined effects of maternal and developmental exposure to mild hypoxia (65 and 74% air saturation, respectively) on the growth and development of embryos of the marine gastropod Littorina littorea . Differences in embryo morphological traits were driven by the developmental environment, whereas the maternal environment and interactive effects of maternal and developmental environment were the main driver of differences in the timing of developmental events. While developmental exposure to mild hypoxia significantly increased the area of an important respiratory organ, the velum, it significantly delayed hatching of veliger larvae and reduced their size at hatching and overall survival. Maternal exposure had a significant effect on these traits, and interacted with developmental exposure to influence the time of appearance of morphological characters, suggesting that both are important in affecting developmental trajectories. A comparison between embryos that successfully hatched and those that died in mild hypoxia revealed that survivors exhibited hypertrophy in the velum and associated pre-oral cilia, suggesting that these traits are linked with survival in low-oxygen environments. We conclude that both maternal and developmental environments shape offspring phenotype in a species with a complex developmental life history, and that plasticity in embryo morphology arising from exposure to even small reductions in oxygen tensions affects the hatching success of these embryos.

ABSTRACT Hyperosmotic stress may result in osmotic volume loss from the body to the environment in animals that cannot control the water permeability of their integument. Euryhaline animals (which have a wide tolerance range of environmental salinities) have generally evolved the ability to counteract cell volume shrinkage by accumulating inorganic and organic osmolytes within their cells to balance internal and external osmolalities. Molluscs use very different combinations of amino acids and amino acid derivatives to achieve this goal. Theodoxus fluviatilis is a neritid gastropod that is distributed not only in limnic habitats in Europe but also in brackish waters (e.g. along the shoreline of the Baltic Sea). Animals from brackish sites survive better in high salinities than animals from freshwater locations. The results of the present study indicate that these differences in salinity tolerance cannot be explained by differences in the general ability to accumulate amino acids as organic osmolytes. Although there may be differences in the metabolic pathways involved in osmolyte accumulation in foot muscle tissue, the two groups of animals accumulate amino acid mixtures equally well when stepwise acclimated to their respective maximum tolerable salinity for extended periods. Among these amino acids, alanine and proline, as well as the osmolyte urea, hold a special importance for cell volume preservation in T. fluviatilis under hyperosmotic stress. It is possible that the accumulation of various amino acids during hyperosmotic stress occurs via hydrolysis of storage proteins, while alanine and proline are probably newly synthesised under conditions of hyperosmotic stress in the animals.

ABSTRACT Vertebrates, cephalopods and arthropods are equipped with eyes that have the highest spatiotemporal resolution among the animal phyla. In parallel, only animals in these three phyla have visual arrestin specialized for the termination of visual signaling triggered by opsin, in addition to ubiquitously expressed β-arrestin that serves in terminating general G protein-coupled receptor signaling. Indeed, visual arrestin in Drosophila and rodents translocates to the opsin-rich subcellular region in response to light to reduce the overall sensitivity of photoreceptors in an illuminated environment (i.e. light adaptation). We thus hypothesized that, during evolution, visual arrestin has taken over the role of β-arrestin in those animals with eyes of high spatiotemporal resolution. If this is true, it is expected that β-arrestin plays a role similar to visual arrestin in those animals with low-resolution eyes. In the present study, we focused on the terrestrial mollusk Limax valentianus , a species related to cephalopods but that has only β-arrestin, and generated antibodies against β-arrestin. We found that β-arrestin is highly expressed in photosensory neurons, and translocates into the microvilli of the rhabdomere within 30 min in response to short wavelength light (400 nm), to which the Limax eye exhibits a robust response. These observations suggest that β-arrestin functions in the visual system of those animals that do not have visual arrestin. We also exploited anti-β-arrestin antibody to visualize the optic nerve projecting to the brain, and demonstrated its usefulness for tracing a visual ascending pathway.

ABSTRACT Tropical coral reef organisms are predicted to be especially sensitive to ocean warming because many already live close to their upper thermal limit, and the expected rise in ocean CO 2 is proposed to further reduce thermal tolerance. Little, however, is known about the thermal sensitivity of a diverse and abundant group of reef animals, the gastropods. The humpbacked conch ( Gibberulus gibberulus gibbosus ), inhabiting subtidal zones of the Great Barrier Reef, was chosen as a model because vigorous jumping, causing increased oxygen uptake ( Ṁ O 2 ), can be induced by exposure to odour from a predatory cone snail ( Conus marmoreus ). We investigated the effect of present-day ambient (417–454 µatm) and projected-future (955–987 µatm) P CO 2 on resting ( Ṁ O 2 , rest ) and maximum ( Ṁ O 2 , max ) Ṁ O 2 , as well as Ṁ O 2 during hypoxia and critical oxygen tension ( P O 2 , crit ), in snails kept at present-day ambient (28°C) or projected-future temperature (33°C). Ṁ O 2 , rest and Ṁ O 2 , max were measured both at the acclimation temperature and during an acute 5°C increase. Jumping caused a 4- to 6-fold increase in Ṁ O 2 , and Ṁ O 2 , max increased with temperature so that absolute aerobic scope was maintained even at 38°C, although factorial scope was reduced. The humpbacked conch has a high hypoxia tolerance with a P O 2 , crit of 2.5 kPa at 28°C and 3.5 kPa at 33°C. There was no effect of elevated CO 2 on respiratory performance at any temperature. Long-term temperature records and our field measurements suggest that habitat temperature rarely exceeds 32.6°C during the summer, indicating that these snails have aerobic capacity in excess of current and future needs.

Tritonia diomedea (synonymous with Tritonia tetraquetra ) navigates in turbulent odour plumes, crawling upstream towards prey and downstream to avoid predators. This is probably accomplished by odour-gated rheotaxis, but other possibilities have not been excluded. Our goal was to test whether T. diomedea uses odour-gated rheotaxis and to simultaneously determine which of the cephalic sensory organs (rhinophores and oral veil) are required for navigation. In a first experiment, slugs showed no coherent responses to streams of odour directed at single rhinophores. In a second experiment, navigation in prey and predator odour plumes was compared between animals with unilateral rhinophore lesions, denervated oral veils, or combined unilateral rhinophore lesions and denervated oral veils. In all treatments, animals navigated in a similar manner to that of control and sham-operated animals, indicating that a single rhinophore provides sufficient sensory input for navigation (assuming that a distributed flow measurement system would also be affected by the denervations). Amongst various potential navigational strategies, only odour-gated positive rheotaxis can produce the navigation tracks we observed in prey plumes while receiving input from a single sensor. Thus, we provide strong evidence that T. diomedea uses odour-gated rheotaxis in attractive odour plumes, with odours and flow detected by the rhinophores. In predator plumes, slugs turned downstream to varying degrees rather than orienting directly downstream for crawling, resulting in greater dispersion for negative rheotaxis in aversive plumes. These conclusions are the first explicit confirmation of odour-gated rheotaxis as a navigational strategy in gastropods and are also a foundation for exploring the neural circuits that mediate odour-gated rheotaxis.

SUMMARY Marine animals living high in the rocky intertidal zone experience long durations of aerial emersion, sometimes enduring rapid increases in temperature. To date, much of our understanding of the thermal physiology of intertidal organisms comes from studies in which organisms are exposed to increasing temperatures when immersed, with the added effect of aerial emersion rarely considered. In this study, we examined the physiological response of the finger limpet, Lottia digitalis , to increases in temperature under both immersed and emersed conditions. We investigated the thermal sensitivity and upper temperature tolerance of limpets through assessment of cardiac performance, metabolic rate, glycogen depletion and maintenance of protein integrity. Cardiac performance in response to ecologically relevant increases in temperature was similar in emersed and immersed limpets from 15 to 35°C and showed multiple break patterns in heart rate as temperature was increased. Overall, emersed limpets had a greater upper thermal limit on cardiac performance, with the ability to maintain heart rate at a temperature 3–5°C higher than that for immersed limpets. Metabolism in limpets also differed significantly between emersion and immersion, where a significant depression in aerobic metabolic rate was observed under immersion with increasing temperature. Greater levels of ubiquitin-conjugated proteins were found under emersed conditions compared with immersed limpets. Maintaining cardiac performance and aerobic metabolism to higher temperatures under emersed conditions is likely reflective of physiological adaptations to live in an aerially exposed environment. Measured field temperatures where fingered limpets were collected demonstrated that limpets have a narrow thermal safety margin for aerobic performance, and currently experience multiple days where summer temperatures might exceed their threshold limits.

SUMMARY Metal ions are present in many different biological materials, and are capable of forming strong cross-links in aqueous environments. The relative contribution of different metal-based cross-links was measured in the defensive glue produced by the terrestrial slug Arion subfuscus. This glue contains calcium, magnesium, zinc, manganese, iron and copper. These metals are essential to the integrity of the glue and to gel stiffening. Removal of all metals caused at least a 15-fold decrease in the storage modulus of the glue. Selectively disrupting cross-links involving hard Lewis acids such as calcium reduced the stiffness of the glue, while disrupting cross-links involving borderline Lewis acids such as zinc did not. Calcium is the most common cation bound to the glue (40 mmol l −1 ), and its charge is balanced primarily by sulphate at 82–84 mmol l −1 . Thus these ions probably play a primary role in bringing polymers together directly. Imine bonds formed as a result of protein oxidation also contribute substantially to the stiffness of the glue. Disrupting these bonds with hydroxylamine caused a 33% decrease in storage modulus of the glue, while stabilizing them by reduction with sodium borohydride increased the storage modulus by 40%. Thus a combination of metal-based bonds operates in this glue. Most likely, cross-links directly involving calcium play a primary role in bringing together and stabilizing the polymer network, followed by imine bond formation and possible iron coordination.

SUMMARY The invasive Pomacea canaliculata estivates during periods of drought and should cope with harmful effects of reoxygenation during arousal. We studied thiobarbituric acid reactive substances (TBARS), enzymatic (superoxide dismutase, SOD and catalase, CAT) and non-enzymatic antioxidants (uric acid and reduced glutathione), and heat shock protein expression (Hsc70, Hsp70 and Hsp90) in (1) active control snails, (2) snails after 45 days of estivation, and (3) aroused snails 20 min and (4) 24 h after water exposure, in midgut gland, kidney and foot. Both kidney and foot (but not the midgut gland) showed a TBARS increase during estivation and a decrease after arousal. Tissue SOD and CAT did not change in any experimental groups. Uric acid increased during estivation in all tissues, and it decreased after arousal in the kidney. Allantoin, the oxidation product of uric acid, remained constant in the midgut gland but it decreased in the kidney until 20 min after arousal; however, allantoin levels rose in both kidney and foot 24 h after arousal. Reduced glutathione decreased during estivation and arousal, in both midgut gland and kidney, and it remained constant in the foot. Hsc70 and Hsp70 kidney levels were stable during the activity–estivation cycle and Hsp90 expression decreases during estivation and recovers in the early arousal. In foot, the expression of Hsp70 and Hsp90 was high during activity and estivation periods and disminished after arousal. Results indicate that a panoply of antioxidant and molecular chaperone defences may be involved during the activity–estivation cycle in this freshwater gastropod.

SUMMARY Contemporary theory for thermal adaptation of ectothermic metazoans focuses on the maximization of energy gain and performance (locomotion and foraging). Little consideration is given to the selection for mechanisms that minimize resting energy loss in organisms whose energy gain is severely constrained. We tested a hypothetical framework for thermal performance of locomotor activity (a proxy for energy gain) and resting metabolism (a proxy for energy loss) in energetically compromised snails in the littoral fringe zone, comparing this with existing theory. In contrast to theory, the thermal ranges and optima for locomotor performance and metabolic performance of Echinolittorina malaccana are mismatched, and energy gain is only possible at relatively cool temperatures. To overcome thermal and temporal constraints on energy gain while experiencing high body temperatures (23–50°C), these snails depress resting metabolism between 35 and 46°C (thermally insensitive zone). The resulting bimodal relationship for metabolism against temperature contrasts with the unimodal or exponential relationships of most ectotherms. Elevation of metabolism above the breakpoint temperature for thermal insensitivity (46°C) coincides with the induction of a heat shock response, and has implications for energy expenditure and natural selection. Time-dependent mortality is initiated at this breakpoint temperature, suggesting a threshold above which the rate of energy demand exceeds the capacity for cellular energy generation (rate of ATP turnover). Mortality in a thermal range that elevates rather than limits aerobic metabolism contrasts with the hypothesis that cellular oxygen deficiency underlies temperature-related mortality. The findings of this study point to the need to incorporate aspects of resting metabolism and energy conservation into theories of thermal adaptation.

SUMMARY Acceleration of embryonic rotation is a common response to hypoxia among pond snails. It was first characterized in Helisoma trivolvis embryos, which have a pair of sensorimotor neurons that detect hypoxia and release serotonin onto postsynaptic ciliary cells. The objective of the present study was to determine how the hypoxia response is mediated in Lymnaea stagnalis , which differ from H. trivolvis by having both serotonergic and dopaminergic neurons, and morphologically distinct ciliated structures at comparative stages of embryonic development. Time-lapse video recordings of the rotational behavior in L. stagnalis revealed similar rotational features to those previously observed in H. trivolvis , including rotational surges and rotational responses to hypoxia. Serotonin and dopamine increased the rate of rotation with similar potency. In contrast, serotonin was more potent than dopamine in stimulating the ciliary beat frequency of isolated pedal cilia. Isolated apical plate cilia displayed an irregular pattern of ciliary beating that precluded the measurement of ciliary beat frequency. A qualitative assessment of ciliary beating revealed that both serotonin and dopamine were able to stimulate apical plate cilia. The ciliary responses to dopamine were reversible in both pedal and apical plate cilia, whereas the responses to serotonin were only reversible at concentrations below 100 μmol l –1 . Mianserin, a serotonin receptor antagonist, and SKF83566, a dopamine receptor antagonist, effectively blocked the rotational responses to serotonin and dopamine, respectively. The rotational response to hypoxia was only partially blocked by mianserin, but was fully blocked by SKF83566. These data suggest that, despite the ability of serotonin to stimulate ciliary beating in L. stagnalis embryos, the rotational response to hypoxia is primarily mediated by the transient apical catecholaminergic neurons that innervate the ciliated apical plate.

SUMMARY The terrestrial slug Arion subfuscus secretes a glue that is a dilute gel with remarkable adhesive and cohesive strength. The function of this glue depends on metals, raising the possibility that metal-catalyzed oxidation plays a role. The extent and time course of protein oxidation was measured by immunoblotting to detect the resulting carbonyl groups. Several proteins, particularly one with a relative molecular mass ( M r ) of 165×10 3 , were heavily oxidized. Of the proteins known to distinguish the glue from non-adhesive mucus, only specific size variants were oxidized. The oxidation appears to occur within the first few seconds of secretion. Although carbonyls were detected by 2,4-dinitrophenylhydrazine (DNPH) in denatured proteins, they were not easily detected in the native state. The presence of reversible cross-links derived from carbonyls was tested for by treatment with sodium borohydride, which would reduce uncross-linked carbonyls to alcohols, but stabilize imine bonds formed by carbonyls and thus lead to less soluble complexes. Consistent with imine bond formation, sodium borohydride led to a 20–35% decrease in the amount of soluble protein with a M r of 40–165 (×10 3 ) without changing the carbonyl content per protein. In contrast, the nucleophile hydroxylamine, which would competitively disrupt imine bonds, increased protein solubility in the glue. Finally, the primary amine groups on a protein with a M r of 15×10 3 were not accessible to acid anhydrides. The results suggest that cross-links between aldehydes and primary amines contribute to the cohesive strength of the glue.

SUMMARY Helisoma trivolvis has served as a model system to study the functions of serotonin (5-HT) from cellular, developmental, physiological and behavioural perspectives. To further explore the serotonin system at the molecular level, and to provide experimental knockout tools for future studies, in this study we identified serotonin receptor genes from the H. trivolvis genome, and characterized the molecular structure and expression profile of the serotonin receptor gene products. Degenerate oligonucleotide primers, based on conserved regions of the Lymnaea stagnalis 5-HT 1Lym receptor, were used to amplify G protein-coupled biogenic amine receptor sequences from H. trivolvis genomic cDNA, resulting in the cloning of two putative serotonin receptors. The deduced gene products both appear to be G protein-coupled serotonin receptors, with well-conserved structure in the functional domains and high variability in the vestibule entrance of the receptor protein. Phylogenetic analysis placed these receptors in the 5-HT 1 and 5-HT 7 families of serotonin receptors. They are thus named the 5-HT 1Hel and 5-HT 7Hel receptors, respectively. In situ hybridization and immunofluorescence studies revealed that these genes and gene products are expressed most heavily in the ciliated pedal and mantle epithelia of H. trivolvis embryos. In adults, widespread expression occurred in all ganglia and connectives of the central nervous system. Expression of both receptor proteins was localized exclusively to neurites when examined in situ . In contrast, when isolated neurons were grown in culture, 5-HT 1Hel and 5-HT 7Hel immunoreactivity were located primarily in the cell body. This is the first study to reveal a 5-HT 7 receptor in a molluscan species.

SUMMARY Several gastropod molluscs produce glues that are interesting because they are dilute gels and yet they produce strong adhesion. Specific glue proteins have been identified that play a central role in this adhesion, possibly by crosslinking other polymers in the gel. This study investigates the role of metals in the action of these glue proteins. Atomic absorption spectrometry showed that glue from the slug Arion subfuscus contains substantial quantities of zinc (46±7 p.p.m. and 189±80 p.p.m. in two different sets of experiments) and also iron, copper and manganese (2–7 p.p.m.). Iron-specific staining demonstrates that iron is bound specifically to the 15 kDa glue protein. Several approaches were used to show that these metals have important functional effects. Adding iron or copper to dissolved glue causes the proteins to precipitate rapidly, although zinc has no effect. Removing iron and related transition metals with a chelator during secretion of the glue causes a sixfold increase in the solubility of the glue. Once the glue has set, however, removing these metals has no effect. Finally, the gel-stiffening activity of the glue proteins was measured in the presence and absence of the chelator. The chelator eliminated the gel-stiffening effect of the proteins, suggesting that transition metals were necessary for the proteins to act on the gel. Thus, the glue contains transition metals and these metals play an essential role in glue function.

SUMMARY Tritonia diomedea is a useful neuroethological model system that can contribute to our understanding of the neural control of navigation. Prior work on both sensory and locomotory systems is complemented by recent field experiments, which concluded that these animals primarily use a combination of odours and water flow as guidance cues. We corroborate these field results by showing similar navigation behaviours in a flow tank. Slugs crawled upstream towards both prey and conspecifics, and turned downstream after crawling into a section of the flow tank downstream of a predator. Controls without upstream odour sources crawled apparently randomly. We then tested whether these behaviours depend on odours detected by the rhinophores. Outflow from a header tank was used to generate prey, predator and unscented control odour plumes in the flow tank. Slugs with rhinophores crawled upstream towards a prey odour plume source, turned downstream in a predator odour plume, and showed no reaction to a control plume. Slugs without rhinophores behaved similarly to controls, regardless of odour plume type. Finally, we used extracellular recordings from the rhinophore nerve to demonstrate that isolated rhinophores are chemosensitive. Afferent activity increased significantly more after application of all three odour types than after unscented control applications. Responses were odour specific. We conclude that rhinophores mediate orientation to flow, and suggest that future work should focus on the integration of mechanosensation and chemosensation during navigation in T. diomedea .

SUMMARY Sea hares protect themselves from predatory attacks with several modes of chemical defenses. One of these is inking, which is an active release of a protective fluid upon predatory attack. In many sea hares including Aplysia californica and A. dactylomela , this fluid is a mixture of two secretions from two separate glands, usually co-released: ink,a purple fluid from the ink gland; and opaline, a white viscous secretion from the opaline gland. These two secretions are mixed in the mantle cavity and directed toward the attacking predator. Some of the chemicals in these secretions and their mechanism of action have been identified. In our study,we used western blots, immunocytochemistry, amino acid analysis, and bioassays to examine the distribution of these components: (1) an l -amino acid oxidase called escapin for A. californica and dactylomelin-P for A. dactylomela , which has antimicrobial activity but we believe its main function is in defending sea hares against predators that evoke its release; and (2) escapin's major amino acid substrates - l -lysine and l -arginine. Escapin is exclusively produced in the ink gland and is not present in any other tissues or secretions. Furthermore, escapin is only sequestered in the amber vesicles of the ink glandand not in the red-purple vesicles, which contain algal-derived chromophores that give ink its distinctive purple color. The concentration of escapin and dactylomelin-P in ink, both in the gland and after its release, is as high as 2 mg ml -1 , or 30 μmol ml -1 , which is well above its antimicrobial threshold. Lysine and arginine (and other amino acids) are packaged into vesicles in the ink and opaline glands, but arginine is present in ink and opaline at <1 mmol l -1 and lysine is present in ink at <1 mmol l -1 but in opaline at 65 mmol l -1 . Our previous results showed that both lysine and arginine mediate escapin's bacteriostatic effects, but only lysine mediates its bactericidal effects. Given that escapin's antimicrobial effects require concentrations of lysine and/or arginine >1 mmol l -1 , our data lead us to conclude that lysine in opaline is the primary natural substrate for escapin in ink. Furthermore, packaging of the enzyme escapin and its substrate lysine into two separate glands and their co-release and mixing at the time of predatory attack allows for the generation of bioactive defensive compounds from innocuous precursors at the precise time they are needed. Whether lysine and/or arginine are substrates for escapin's antipredatory functions remains to be determined.

SUMMARY A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica was cloned and sequenced, and is the first sea hare antimicrobial protein to be functionally expressed in E. coli . Sequence analysis suggested that this protein is a flavin-containing l -amino acid oxidase (LAAO), with one predicted potential glycosylation site, although the glycosylation could not be experimentally confirmed. This protein, which we call `escapin', has high sequence similarity to several other gastropod proteins. Escapin was verified by NMR, mass spectroscopy and HPLC to have FAD as its flavin cofactor. Escapin's antimicrobial effects, bacteriostasis and bactericidal, were determined using a combination of two assays: (1) incubation of bacteria on solid media followed by assessment of inhibition by direct observation of zones of inhibition or by turbidity measurements; and (2) incubation of bacteria in liquid media followed by counting viable colonies after growing on agar plates. Native escapin inhibited the growth of Gram-positive and Gram-negative bacteria, including marine bacteria ( Vibrio harveyii and Staphylococcus aureus ) and pathogenic bacteria( Staphylococcus aureus , Streptococcus pyogenes and Pseudomonas aeruginosa ). Escapin also inhibited the growth of yeast and fungi, with different efficacies. Escapin's antimicrobial activity was concentration dependent and did not decrease when stored for more than 5 months at room temperature. Escapin was bacteriostatic and not bactericidal in minimal media (e.g. salt media) with glucose, yeast extract, and a mixture of 20 amino acids each at 50 μmol l -1 , but was bactericidal in media enriched with Tryptone Peptone. Escapin was also strongly bactericidal in media with l -lysine at concentrations as low as 3 mmol l -1 and slightly bactericidal in 50 mmol l -1 l -arginine, but not in most other amino acids even at 50 mmol l -1 . Escapin had high oxidase activity (producing hydrogen peroxide) with either l -arginine or l -lysine as a substrate and little to no oxidase activity with other l -amino acids. Hydrogen peroxide alone (without escapin or amino acids) was strongly bacteriostatic but poorly bactericidal, similar in this respect to l -arginine but different from l -lysine in the presence of escapin. Together these results suggest that there are multiple mechanisms to escapin's antimicrobial effects, with bacteriostasis resulting largely or entirely from the effects of hydrogen peroxide produced by escapin's LAAO activity, but bactericidal effects resulting from lysine-dependent mechanisms not directly involving hydrogen peroxide. Recombinant escapin expressed in bacteria was also active against Gram-positive and Gram-negative bacteria,suggesting that glycosylation is not essential for antimicrobial activity.

SUMMARY Differential screening of a Littorina littorea (the common periwinkle) cDNA library identified ferritin heavy chain as an anoxia-induced gene in hepatopancreas. Northern blots showed that ferritin heavy chain transcript levels were elevated twofold during anoxia exposure, although nuclear run-off assays demonstrated that ferritin heavy chain mRNAs were not transcriptionally upregulated during anoxia. Polysome analysis indicated that existing ferritin transcripts were actively translated during the anoxic period. This result was confirmed via western blotting, which demonstrated a twofold increase in ferritin heavy chain protein levels during anoxia, with a subsequent decrease to control levels during normoxic recovery. Organ culture experiments using hepatopancreas slices demonstrated a >50%increase in ferritin heavy chain transcript levels in vitro under conditions of anoxia and freezing, as well as after incubation with the second messenger cGMP. Taken together, these results suggest that ferritin heavy chain is actively regulated during anoxia exposure in the marine snail, L. littorea .

SUMMARY Over the last 30 years, many laboratories have examined, in parallel, the feeding behaviour of gastropod molluscs and the properties of the nervous system that give rise to this behaviour. Equal attention to both behavioural and neurobiological issues has provided deep insight into the functioning of the nervous system in generating and controlling behaviour. The conclusions derived from studies on gastropod feeding are generally consistent with those from other systems, but often provide more detailed information on the behavioural function of a particular property of the nervous system. A review of the literature on gastropod feeding illustrates a number of important messages. (i) Many of the herbivorous gastropods display similarities in behaviour that are reflected in corresponding similarities in neural anatomy,pharmacology and physiology. By contrast, the same aspects of the behaviour of different carnivorous species are quite variable, possibly because of their specialised prey-capture techniques. Nonetheless, some aspects of the neural control of feeding are preserved. (ii) Feeding in all species is flexible,with the behaviour and the physiology adapting to changes in the current environment and internal state and as a result of past experience. Flexibility arises via processes that may take place at many neural sites, and much of the modulation underlying behavioural flexibility is understood at a systems and at a cellular level. (iii) Neurones seem to have specific functions that are consistent with their endogenous properties and their synaptic connections, suggesting that individual neurones code specific pieces of information (i.e. they are `grandmother cells'). However, the properties of a neurone can be extremely complex and can be understood only in the context of the complete neural circuit and the behaviour that it controls. In systems that are orders of magnitude more complex, it would be impossible to understand the functional properties of an individual neurone, even if it also coded specific information. (iv) Systems such as gastropod feeding may provide a model for understanding the functional properties of more complex systems.

SUMMARY Nitric oxide (NO) is a gaseous intercellular messenger produced by the enzyme nitric oxide synthase. It has been implicated as a neuromodulator in several groups of animals, including gastropods, crustaceans and mammals. In this study, we investigated the effects of NO on the swim motor program produced by isolated brains and by semi-intact preparations of the nudibranch Melibe leonina . The NO donors sodium nitroprusside (SNP, 1 mmol l –1 ) and S -nitroso- N -acetylpenicillamine (SNAP, 1 mmol l –1 ) both had a marked effect on the swim motor program expressed in isolated brains, causing an increase in the period of the swim cycle and a more erratic swim rhythm. In semi-intact preparations, the effect of NO donors was manifested as a significant decrease in the rate of actual swimming. An NO scavenger, reduced oxyhemoglobin, eliminated the effects of NO donors on isolated brains, supporting the assumption that the changes in swimming induced by donors were actually due to NO. The cGMP analogue 8-bromoguanosine 3′,5′-cyclic monophosphate (1 mmol l –1 ) produced effects that mimicked those of NO donors, suggesting that NO is working via a cGMP-dependent mechanism. These results, in combination with previous histological studies indicating the endogenous presence of nitric oxide synthase, suggest that NO is used in the central nervous system of Melibe leonina to modulate swimming.

SUMMARY We investigated divalent cation and anaerobic end-product concentrations and the interactive effects of these substances and pH on haemocyanin oxygen-binding (Hc-O 2 ) in the New Zealand abalone Haliotis iris . During 24 h of environmental hypoxia (emersion), d -lactate and tauropine accumulated in the foot and shell adductor muscles and in the haemolymph of the aorta, the pedal sinus and adductor muscle lacunae, whereas l -lactate was not detected. Intramuscular and haemolymph d -lactate concentrations were similar, but tauropine accumulated to much higher levels in muscle tissues. Repeated disturbance and short-term exposure to air over 3 h induced no accumulation of d - or l -lactate and no change in [Ca 2+ ], [Mg 2+ ], pH and O 2 -binding properties of the native haemolymph. The haemolymph showed a low Hc-O 2 affinity, a large reverse Bohr effect and marked cooperativity. Dialysis increased Hc-O 2 affinity, obliterated cooperativity and decreased the pH-sensitivity of O 2 binding. Replacing Mg 2+ and Ca 2+ restored the native O 2 -binding properties and the reverse Bohr shift. l - and d -lactate exerted minor modulatory effects on O 2 -affinity. At in vivo concentrations of Mg 2+ and Ca 2+ , the cooperativity is dependent largely on Mg 2+ , which modulates the O 2 association equilibrium constants of both the high-affinity ( K R ) and the low-affinity ( K T ) states (increasing and decreasing, respectively). This allosteric mechanism contrasts with that encountered in other haemocyanins and haemoglobins. The functional properties of H. iris haemocyanin suggest that high rates of O 2 delivery to the tissues are not a priority but are consistent with the provision of a large O 2 reserve for facultatively anaerobic tissues during internal hypoxia associated with clamping to the substratum.