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Organismal responses to stressful environments are influenced by numerous transcript- and protein-level mechanisms, and the relationships between expression changes at these levels are not always straightforward. Here, we used paired transcriptomic and proteomic datasets from two previous studies from gill of the California mussel, Mytilus californianus, to explore how simultaneous transcript and protein abundance patterns may diverge under different environmental scenarios. Field-acclimatized mussels were sampled from two disparate intertidal sites; individuals from one site were subjected to three further treatments (common garden, low-intertidal or high-intertidal outplant) that vary in temperature and feeding time. Assessing 1519 genes shared between the two datasets revealed that both transcript and protein expression patterns differentiated the treatments at a global level, despite numerous underlying discrepancies. There were far more instances of differential expression between treatments in transcript only (1451) or protein only (226) than of the two levels shifting expression concordantly (68 instances). Upregulated expression of cilium-associated transcripts (likely related to feeding) was associated with relatively benign field treatments. In the most stressful treatment, transcripts, but not proteins, for several molecular chaperones (including heat shock proteins and endoplasmic reticulum chaperones) were more abundant, consistent with a threshold model for induction of translation of constitutively available mRNAs. Overall, these results suggest that the relative importance of transcript- and protein-level regulation (translation and/or turnover) differs among cellular functions and across specific microhabitats or environmental contexts. Furthermore, the degree of concordance between transcript and protein expression can vary across benign versus acutely stressful environmental conditions., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Many predators fracture strong mollusk shells, requiring specialized weaponry and behaviors. The current shell fracture paradigm is based on jaw- and claw-based predators that slowly apply forces (high impulse, low peak force). However, predators also strike shells with transient intense impacts (low impulse, high peak force). Toward the goal of incorporating impact fracture strategies into the prevailing paradigm, we measured how mantis shrimp (Neogonodactylus bredini) impact snail shells, tested whether they strike shells in different locations depending on prey shape (Nerita spp., Cenchritis muricatus, Cerithium spp.) and deployed a physical model (Ninjabot) to test the effectiveness of strike locations. We found that, contrary to their formidable reputation, mantis shrimp struck shells tens to hundreds of times while targeting distinct shell locations. They consistently struck the aperture of globular shells and changed from the aperture to the apex of high-spired shells. Ninjabot tests revealed that mantis shrimp avoid strike locations that cause little damage and that reaching the threshold for eating soft tissue is increasingly difficult as fracture progresses. Their ballistic strategy requires feed-forward control, relying on extensive pre-strike set-up, unlike jaw- and clawbased strategies that can use real-time neural feedback when crushing. However, alongside this pre-processing cost to impact fracture comes the ability to circumvent gape limits and thus process larger prey. In sum, mantis shrimp target specific shell regions, alter their strategy depending on shell shape, and present a model system for studying the physics and materials of impact fracture in the context of the rich evolutionary history of predator--prey interactions. [ABSTRACT FROM AUTHOR]

Long-lived animals show a non-observable age-related decline in immune defense, which is provided by blood cells that derive from self-renewing stem cells. The oldest living animals are bivalves. Yet, the origin of hemocytes, the cells involved in innate immunity, is unknown in bivalves and current knowledge about mollusk adult somatic stem cells is scarce. Here we identify a population of adult somatic precursor cells and show their differentiation into hemocytes. Oyster gill contains an as yet unreported irregularly folded structure (IFS) with stem-like cells bathing into the hemolymph. BrdU labeling revealed that the stem-like cells in the gill epithelium and in the nearby hemolymph replicate DNA. Proliferation of this cell population was further evidenced by phosphorylated-histone H3 mitotic staining. Finally, these small cells, most abundant in the IFS epithelium, were found to be positive for the stemness marker Sox2. We provide evidence for hematopoiesis by showing that co-expression of Sox2 and Cu/Zn superoxide dismutase, a hemocyte-specific enzyme, does not occur in the gill epithelial cells but rather in the underlying tissues and vessels. We further confirm the hematopoietic features of these cells by the detection of Filamin, a protein specific for a sub-population of hemocytes, in large BrdU-labeled cells bathing into gill vessels. Altogether, our data show that progenitor cells differentiate into hemocytes in the gill, which suggests that hematopoiesis occurs in oyster gills. [ABSTRACT FROM AUTHOR]

Ocean acidification due to increasing atmospheric CO2 concentrations results in a decrease in seawater pH and shifts in the carbonate chemistry that can negatively affect marine organisms. Marine bivalves such as the hard-shell clam, Mercenaria mercenaria, serve as ecosystem engineers in estuaries and coastal zones of the western Atlantic and, as for many marine calcifiers, are sensitive to the impacts of ocean acidification. In estuaries, the effects of ocean acidification can be exacerbated by low buffering capacity of brackish waters, acidic inputs from freshwaters and land, and/or the negative effects of salinity on the physiology of organisms. We determined the interactive effects of 21 weeks of exposure to different levels of C02 (-395, 800 and 1500 patm corresponding to pH of 8.2, 8.1 and 7.7, respectively) and salinity (32 versus 16) on biomineralization, shell properties and energy metabolism of juvenile hard-shell clams. Low salinity had profound effects on survival, energy metabolism and biomineralization of hard-shell clams and modulated their responses to elevated Pco2- Negative effects of low salinity in juvenile clams were mostly due to the strongly elevated basal energy demand, indicating energy deficiency, that led to reduced growth, elevated mortality and impaired shell maintenance (evidenced by the extensive damage to the periostracum). The effects of elevated Pco2 on physiology and biomineralization of hard-shell clams were more complex. Elevated Pco2 (-800-1500 patm) had no significant effects on standard metabolic rates (indicative of the basal energy demand), but affected growth and shell mechanical properties in juvenile clams. Moderate hypercapnia (-800 patm Pco2) increased shell and tissue growth and reduced mortality of juvenile clams in high salinity exposures; however, these effects were abolished under the low salinity conditions or at high Pco2 (-1500 patm). Mechanical properties of the shell (measured as microhardness and fracture toughness of the shells) were negatively affected by elevated C02 alone or in combination with low salinity, which may have important implications for protection against predators or environmental stressors. Our data indicate that environmental salinity can strongly modulate responses to ocean acidification in hard-shell clams and thus should be taken into account when predicting the effects of ocean acidification on estuarine bivalves. [ABSTRACT FROM AUTHOR]

What are the mechanisms of multifunctionality, i.e. the use of the same peripheral structures for multiple behaviors? We studied this question using the multifunctional feeding apparatus of the marine mollusk Aplysia californica, in which the same muscles mediate biting (an attempt to grasp food) and swallowing (ingestion of food). Biting and swallowing responses were compared using magnetic resonance imaging of intact, behaving animals and a three-dimensional kinematic model. Biting is associated with larger amplitude protractions of the grasper (radula/odontophore) than swallowing, and smaller retractions. Larger biting protractions than in swallowing appear to be due to a more anterior position of the grasper as the behavior begins, a larger amplitude contraction of protractor muscle 12, and contraction of the posterior portion of the I1/I3/jaw complex. The posterior I1/I3/jaw complex may be context-dependent, i.e. its mechanical context changes the direction of the force it exerts. Thus, the posterior of I1/I3 may aid protraction near the peak of biting, whereas the entire I1/I3/jaw complex acts as a retractor during swallowing. In addition, larger amplitude closure of the grasper during swallowing allows an animal to exert more force as it ingests food. These results demonstrate that differential deployment of the periphery can mediate multifunctionality. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the neural basis of operant conditioning in a semi-intact preparation of the pond snail, Lymnaea stagnalis. Lymnaea learns, via operant conditioning, to reduce its aerial respiratory behaviour in response to an aversive tactile stimulus to its open pneumostome. Here we report the successful conditioning of naive semi-intact preparations to show ‘learning in the dish’ and long-term memory that persists for at least 18 h. The neurons that generate this behaviour are readily identifiable and, for the first time, we have recorded from a neuron during a training paradigm that leads to long-term memory formation in the same preparation. Specifically, we recorded from the respiratory neuron Right Pedal Dorsal 1 (RPeD1), which is part of the respiratory central pattern generator and initiates the aerial respiratory behaviour. Previous studies have shown that long-term memory of this behaviour results in reduced RPeD1 activity. In the present study, we demonstrate that preventing RPeD1 impulse activity between training sessions reduces the number of sessions needed to produce long-term memory in our semi-intact preparation. [ABSTRACT FROM AUTHOR]

Even though mollusks' capacity to repair shell damage is usually studied in response to a single event, their shells have to defend them against predatory and environmental threats throughout their potentially multi-decadal life. We measured whether and how mollusks respond to chronic mechanical stress. Once a week for 7 months, we compressed whole live California mussels (Mytilus californianus) for 15 cycles at ∼55% of their predicted one-time breaking force, a treatment known to cause fatigue damage in shells. We found mussels repaired their shells. Shells of experimentally stressed mussels were just as strong at the end of the experiment as those of control mussels that had not been experimentally loaded, and they were more heavily patched internally. Additionally, stressed shells differed in morphology; they were heavier and thicker at the end of the experiment than control shells but they had increased less in width, resulting in a flatter, less domed shape. Finally, the chronic mechanical stress and repair came at a cost, with stressed mussels having higher mortality and less soft tissue than the control group. Although associated with significant cost, mussels' ability to maintain repair in response to ongoing mechanical stress may be vital to their survival in harsh and predator-filled environments., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Hard external armors have to defend against a lifetime of threats yet are traditionally understood by their ability to withstand a single attack. Survival of bivalve mollusks thus can depend on the ability to repair shell damage between encounters. We studied the capacity for repair in the intertidal mussel Mytilus californianus by compressing live mussels for 15 cycles at ∼79% of their predicted strength (critically fracturing 46% of shells), then allowing the survivors 0, 1, 2 or 4 weeks to repair. Immediately after fatigue loading, mussel shells were 20% weaker than control shells that had not experienced repetitive loading. However, mussels restored full shell strength within 1 week, and after 4 weeks shells that had experienced greater fatiguing forces were stronger than those repetitively loaded at lower forces. Microscopy supported the hypothesis that crack propagation is a mechanism of fatigue-caused weakening. However, the mechanism of repair was only partially explained, as epifluorescence microscopy of calcein staining for shell deposition showed that only half of the mussels that experienced repetitive loading had initiated direct repair via shell growth around fractures. Our findings document repair weeks to months faster than demonstrated in other mollusks. This rapid repair may be important for the mussels' success contending with predatory and environmental threats in the harsh environment of wave-swept rocky coasts, allowing them to address non-critical but weakening damage and to initiate plastic changes to shell strength. We highlight the significant insight gained by studying biological armors not as static structures but, instead, as dynamic systems that accumulate, repair and respond to damage., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Sleep is essential for memory consolidation after learning as shown in mammals and invertebrates such as bees and flies. Aplysia californica displays sleep, and sleep in this mollusk was also found to support memory for an operant conditioning task. Here, we investigated whether sleep in Aplysia is also required for memory consolidation in a simpler type of learning, i.e. the conditioning of the siphon withdrawal reflex. Two groups of animals (Wake, Sleep, each n=11) were conditioned on the siphon withdrawal reflex, with the training following a classical conditioning procedure where an electrical tail shock served as the unconditioned stimulus (US) and a tactile stimulus to the siphon as the conditioned stimulus (CS). Responses to the CS were tested before (pre-test), and 24 and 48 h after training. While Wake animals remained awake for 6 h after training, Sleep animals had undisturbed sleep. The 24 h test in both groups was combined with extinction training, i.e. the extended presentation of the CS alone over two blocks. At the 24 h test, siphon withdrawal duration in response to the CS was distinctly enhanced in both Sleep and Wake groups with no significant difference between groups, consistent with the view that consolidation of a simple conditioned reflex response does not require post-training sleep. Surprisingly, extinction training did not reverse the enhancement of responses to the CS. On the contrary, at the 48 h test, withdrawal duration in response to the CS was even further enhanced across both groups. This suggests that processes of sensitization, an even simpler non-associative type of learning, contributed to the withdrawal responses. Our study provides evidence for the hypothesis that sleep preferentially benefits consolidation of more complex learning paradigms than conditioning of simple reflexes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The crustacean cardioactive peptide (CCAP) is an important neuropeptide involved in the regulation of a variety of physiological processes in arthropods. Although this family of peptides has an ancestral origin, its function remains poorly understood among protostome species - apart from arthropods. We functionally characterized three G protein-coupled receptors (GPCRs) in the oyster Crassostrea gigas, phylogenetically related to ecdysozoan CCAP receptors (CCAPRs) and to chordate neuropeptide S receptors (NPSRs). Cragi-CCAPR1 and Cragi-CCAPR2 were specifically activated by the Cragi-CCAP1 and Cragi-CCAP2 peptides, respectively, both derived from the same CCAP precursor. In contrast, Cragi-CCAPR3 was only partially activated by CCAP1 and CCAP2 at high concentrations. The Cragi-CCAPR1 and Cragi-CCAPR2 genes were expressed in various adult tissues. They are both most expressed in the gills, while Cragi-CCAPR3 is mainly expressed in the visceral ganglia (VG). Cragi-CCAP precursor transcripts are higher in the VG, the labial palps and the gills. Receptor and ligand-encoding transcripts are more abundantly expressed in the gonads in the first stages of gametogenesis, while the Cragi-CCAP precursor is upregulated in the VG in the last stages of gametogenesis. This suggests a role of the CCAP signaling system in the regulation of reproductive processes. A role in water and ionic regulation is also supported considering the differential expression of the CCAP signaling components in oysters exposed to brackish water., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Climate change is increasing the temperature variability animals face, and thermal acclimatization allows animals to adjust adaptively to this variability. Although the rate of heat acclimatization has received some study, little is known about how long these adaptive changes remain without continuing exposure to heat stress. This study explored the rate at which field acclimatization states are lost when temperature variability is minimized during constant submersion. California mussels ( Mytilus californianus ) with different acclimatization states were collected from high- and low-zone sites (∼12 versus ∼5°C daily temperature ranges, respectively) and then kept submerged at 15°C for 8 weeks. Each week, the cardiac thermal performance of mussels was measured as a metric of acclimatization state: critical ( T crit ) and flatline ( T flat ) temperatures were recorded. Over 8 weeks of constant submersion, the mean T crit of high-zone mussels decreased by 1.07°C from baseline, but low-zone mussels' mean T crit was unchanged. High- and low-zone mussels' mean maximum heart rate (HR) and resting HR decreased ∼12 and 35%, respectively. T flat was unchanged in both groups. These data suggest that T crit and HR are more physiologically plastic in response to the narrowing of an animal's daily temperature range than T flat is, and that an animal's prior acclimatization state (high versus low) influences the acclimatory capacity of T crit Approximately 2 months were required for the cardiac thermal performance of the high-zone mussels to reach that of the low-zone mussels, suggesting that acclimatization to high and variable temperatures may persist long enough to enable these animals to cope with intermittent bouts of heat stress., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Mollusk shells protect against diverse environmental and predatory physical threats, from one-time impacts to chronic, low-magnitude stresses. The effectiveness of shells as armor is often quantified with a test of shell strength: increasing force is applied until catastrophic fracture. This test does not capture the potential role of fatigue, a process by which chronic or repeated, low-magnitude forces weaken and break a structure. We quantified the strength and fatigue resistance of California mussel ( Mytilus californianus ) shells. Shells were fatigue tested until catastrophic failure by either loading a valve repeatedly to a set force (cyclic) or loading a valve under constant force (static). Valves fatigued under both cyclic and static loading, i.e. subcritical forces broke valves when applied repeatedly or for long durations. Stronger and more fatigue-resistant valves tended to be more massive, relatively wider and the right-hand valve. Furthermore, after accounting for the valves' predicted strength, fatigue resistance curves for cyclic and static loading did not differ, suggesting that fatigue fracture of mussels is more dependent on force duration than number of cycles. Contextualizing fatigue resistance with the forces mussels typically experience clarifies the range of threats for which fatigue becomes relevant. Some predators could rely on fatigue, and episodic events like large wave impacts or failed predation attempts could weaken shells across long time scales. Quantifying shell fatigue resistance when considering the ecology of shelled organisms or the evolution of shell form offers a perspective that accounts for the accumulating damage of a lifetime of threats, large and small., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Many predators fracture strong mollusk shells, requiring specialized weaponry and behaviors. The current shell fracture paradigm is based on jaw- and claw-based predators that slowly apply forces (high impulse, low peak force). However, predators also strike shells with transient intense impacts (low impulse, high peak force). Toward the goal of incorporating impact fracture strategies into the prevailing paradigm, we measured how mantis shrimp ( Neogonodactylus bredini ) impact snail shells, tested whether they strike shells in different locations depending on prey shape ( Nerita spp., Cenchritis muricatus , Cerithium spp.) and deployed a physical model (Ninjabot) to test the effectiveness of strike locations. We found that, contrary to their formidable reputation, mantis shrimp struck shells tens to hundreds of times while targeting distinct shell locations. They consistently struck the aperture of globular shells and changed from the aperture to the apex of high-spired shells. Ninjabot tests revealed that mantis shrimp avoid strike locations that cause little damage and that reaching the threshold for eating soft tissue is increasingly difficult as fracture progresses. Their ballistic strategy requires feed-forward control, relying on extensive pre-strike set-up, unlike jaw- and claw-based strategies that can use real-time neural feedback when crushing. However, alongside this pre-processing cost to impact fracture comes the ability to circumvent gape limits and thus process larger prey. In sum, mantis shrimp target specific shell regions, alter their strategy depending on shell shape, and present a model system for studying the physics and materials of impact fracture in the context of the rich evolutionary history of predator-prey interactions., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)