Chemical defences indicate bold colour patterns with reduced variability in aposematic nudibranchs.

The selective factors that shape phenotypic diversity in prey communities with aposematic animals are diverse and coincide with similar diversity in the strength of underlying secondary defences. However, quantitative assessments of colour pattern variation and the strength of chemical defences in assemblages of aposematic species are lacking. We quantified colour pattern diversity using quantitative colour pattern analysis (QCPA) in 13 dorid nudibranch species (Infraorder: Doridoidei) that varied in the strength of their chemical defences. We accounted for the physiological properties of a potential predator's visual system (a triggerfish, Rhinecanthus aculeatus) and modelled the appearance of nudibranchs from multiple viewing distances (2 and 10 cm). We identified distinct colour pattern properties associated with the presence and strength of chemical defences. Specifically, increases in chemical defences indicated increases in colour pattern boldness (i.e. visual contrast elicited via either or potentially coinciding chromatic, achromatic and/or spatial contrast). Colour patterns were also less variable among species with chemical defences when compared to undefended species. Our results indicate correlations between secondary defences and diverse, bold colouration while showing that chemical defences coincide with decreased colour pattern variability among species. Our study suggests that complex spatiochromatic properties of colour patterns perceived by potential predators can be used to make inferences on the presence and strength of chemical defences.

Human-driven breakdown of predator-prey interactions in the northern Adriatic Sea.

Long-term baseline data that allow tracking how predator-prey interactions have responded to intensifying human impacts are often lacking. Here, we assess temporal changes in benthic community composition and interactions between drilling predatory gastropods and their molluscan prey using the Holocene fossil record of the shallow northern Adriatic Sea, which is characterized by a long history of human transformation. Molluscan assemblages differ between the Isonzo and Po prodelta, but both show consistent temporal trends in the abundance of dominant species. Samples of mollusc prey collected at high stratigraphic resolution indicate that drilling frequencies have drastically declined in the Po prodelta since the mid-twentieth century, while a weaker trend in the more condensed sediments of the Isonzo prodelta is not statistically significant. The decrease in drilling predation intensity and the community turnover are linked to the loss of predatory gastropods and the increased relative abundance of less-preferred prey during the most recent decades. Our results align with data showing the substantial depletion of marine resources at higher trophic levels in the region and indicate that the strong simplification of the food web initiated in the late nineteenth century accelerated further since the mid-twentieth century.

Within- and transgenerational stress legacy effects of ocean acidification on red abalone (Haliotis rufescens) growth and survival.

Understanding the mechanisms by which individual organisms respond and populations adapt to global climate change is a critical challenge. The role of plasticity and acclimation, within and across generations, may be essential given the pace of change. We investigated plasticity across generations and life stages in response to ocean acidification (OA), which poses a growing threat to both wild populations and the sustainable aquaculture of shellfish. Most studies of OA on shellfish focus on acute effects, and less is known regarding the longer term carryover effects that may manifest within or across generations. We assessed these longer term effects in red abalone (Haliotis rufescens) using a multi-generational split-brood experiment. We spawned adults raised in ambient conditions to create offspring that we then exposed to high pCO2 (1180 µatm; simulating OA) or low pCO2 (450 µatm; control or ambient conditions) during the first 3 months of life. We then allowed these animals to reach maturity in ambient common garden conditions for 4 years before returning the adults into high or low pCO2 treatments for 11 months and measuring growth and reproductive potential. Early-life exposure to OA in the F1 generation decreased adult growth rate even after 5 years especially when abalone were re-exposed to OA as adults. Adult but not early-life exposure to OA negatively impacted fecundity. We then exposed the F2 offspring to high or low pCO2 treatments for the first 3 months of life in a fully factorial, split-brood design. We found negative transgenerational effects of parental OA exposure on survival and growth of F2 offspring, in addition to significant direct effects of OA on F2 survival. These results show that the negative impacts of OA can last within and across generations, but that buffering against OA conditions at critical life-history windows can mitigate these effects.

The impact of aragonite saturation variability on shelled pteropods: An attribution study in the California Current System.

Observations from the California Current System (CalCS) indicate that the long-term trend in ocean acidification (OA) and the naturally occurring corrosive conditions for the CaCO3 mineral aragonite (saturation state Ω < 1) have a damaging effect on shelled pteropods, a keystone group of calcifying organisms in the CalCS. Concern is heightened by recent findings suggesting that shell formation and developmental progress are already impacted when Ω falls below 1.5. Here, we quantify the impact of low Ω conditions on pteropods using an individual-based model (IBM) with life-stage-specific mortality, growth, and behavior in a high-resolution regional hindcast simulation of the CalCS between 1984 and 2019. Special attention is paid to attributing this impact to different processes that lead to such low Ω conditions, namely natural variability, long-term trend, and extreme events. We find that much of the observed damage in the CalCS, and specifically >70% of the shell CaCO3 loss, is due to the pteropods' exposure to naturally occurring low Ω conditions as a result of their diel vertical migration (DVM). Over the hindcast period, their exposure to damaging waters (Ω < 1.5) increases from 9% to 49%, doubling their shell CaCO3 loss, and increasing their mortality by ~40%. Most of this increased exposure is due to the shoaling of low Ω waters driven by the long-term trend in OA. Extreme OA events amplify this increase by ~40%. Our approach can quantify the health of pteropod populations under shifting environmental conditions, and attribute changes in fitness or population structure to changes in the stressor landscape across hierarchical time scales.

Egg-laying hormone expression in identified neurons across developmental stages and reproductive states of the nudibranch Berghia stephanieae.

Neuropeptides play essential roles in coordinating reproduction. Egg-laying hormone (ELH) is conserved in genetic sequence and behavioral function across molluscs, where neuronal clusters secrete ELH to modulate and induce egg-laying. Here we investigated ELH in the nudibranch mollusc, Berghia stephanieae. ELH preprohormone gene orthologs, which showed clade-specific differences at the C-terminus of the predicted bioactive peptide, were identified in brain transcriptomes across several nudipleuran species, including B. stephanieae. ELH shares deep homology with the corticotropin-releasing hormone gene family, which has roles broadly in stress response. Injection of synthesized B. stephanieae ELH peptide into mature individuals induced egg-laying. ELH gene expression in the brain and body was mapped using in-situ hybridization chain reaction. Across the adult brain, 300-400 neurons expressed ELH. Twenty-one different cell types were identified in adults, three of which were located unilaterally on the right side, which corresponds to the location of the reproductive organs. Ten cell types were present in pre-reproductive juvenile stages. An asymmetric cluster of approximately 100 small neurons appeared in the right pedal ganglion of late-stage juveniles. Additional neurons in the pleural and pedal ganglia expressed ELH only in adults that were actively laying eggs and sub-adults that were on the verge of doing so, implicating their direct role in reproduction. Outside the brain, ELH was expressed on sensory appendages, including in presumptive sensory neurons. Its widespread expression in the nudibranch B. stephanieae suggests that ELH plays a role beyond reproduction in gastropod molluscs.

Critical thermal maxima and oxygen uptake in Elysia viridis, a sea slug that steals chloroplasts to photosynthesize.

Photosynthetic animals produce oxygen, providing an ideal lens for studying how oxygen dynamics influence thermal sensitivity. The algivorous sea slug Elysia viridis can steal and retain chloroplasts from the marine alga Bryopsis sp. for months when starved, but chloroplast retention is mere weeks when they are fed another green alga, Chaetomorpha sp. To examine plasticity in thermal tolerance and changes in net oxygen exchange when fed and starving, slugs fed each alga were acclimated to 17°C (the current maximum temperature to which they are exposed in nature) and 22°C (the increase predicted for 2100) and measured at different points during starvation. We also examined increased illumination to evaluate a potential tradeoff between increased oxygen production but faster chloroplast degradation. Following acclimation, we subjected slugs to acute thermal stress to determine their thermal tolerance. We also measured net oxygen exchange before and after acute thermal stress. Thermal tolerance improved in slugs acclimated to 22°C, indicating they can acclimate to temperatures higher than they naturally experience. All slugs exhibited net oxygen uptake, and rates were highest in recently fed slugs before exposure to acute thermal stress. Oxygen uptake was suppressed following acute thermal stress. Under brighter light, slugs exhibited improved thermal tolerance, possibly because photosynthetic oxygen production alleviated oxygen limitation. Accordingly, this advantage disappeared later in starvation when photosynthesis ceased. Thus, E. viridis can cope with heatwaves by suppressing metabolism and plastically adjusting heat tolerance; however, starvation influences a slug's thermal tolerance and oxygen uptake such that continuous access to algal food for its potential nutritive and oxygenic benefits is critical when facing thermal stress.

Carryover effects of embryonic hypoxia exposure on adult fitness of the Pacific abalone.

The widespread and severe drop in dissolved oxygen concentration in the open ocean and coastal waters has attracted much attention, but assessments of the impacts of environmental hypoxia on aquatic organisms have focused primarily on responses to current exposure. Past stress exposure might also affect the performance of aquatic organisms through carryover effects, and whether these effects scale from positive to negative based on exposure degree is unknown. We investigated the carryover effects of varying embryonic hypoxia levels (mediate hypoxia: 3.0-3.1 mg O2/L; severe hypoxia: 2.0-2.1 mg O2/L) on the fitness traits of adult Pacific abalone (Haliotis discus hannai), including growth, hypoxia tolerance, oxygen consumption, ammonia excretion rate, and biochemical responses to acute hypoxia. Moderate embryonic hypoxia exposure significantly improved the hypoxia tolerance of adult Pacific abalone without sacrificing growth and survival. Adult abalone exposed to embryonic hypoxia exhibited physiological plasticity, including decreased oxygen consumption rates under environmental stress, increased basal methylation levels, and a more active response to acute hypoxia, which might support their higher hypoxia tolerance. Thus, moderate oxygen declines in early life have persistent effects on the fitness of abalone even two years later, further affecting population dynamics. The results suggested that incorporating the carryover effects of embryonic hypoxia exposure into genetic breeding programs would be an important step toward rapidly improving the hypoxia tolerance of aquatic animals. The study also inspires the protection of endangered wild animals and other vulnerable species under global climate change.

Characterization of myoinhibitory peptide signaling system and its implication in larval metamorphosis and spawning behavior in Pacific abalone.

Myoinhibitory peptides (MIPs) affect various physiological functions, including juvenile hormone signaling, muscle contraction, larval development, and reproduction in invertebrates. Although MIPs are ligands for MIP and/or sex peptide receptors (MIP/SPRs) in diverse arthropods and model organisms belonging to Lophotrochozoa, the MIP signaling system has not yet been fully investigated in mollusks. In this study, we identified the MIP signaling system in the Pacific abalone Haliotis discus hannai (Hdh). Similar to the invertebrate MIPs, a total of eight paracopies of MIPs (named Hdh-MIP1 to Hdh-MIP8), harboring a WX5-7Wamide motif, except for Hdh-MIP2, were found in the Hdh-MIP precursor. Furthermore, we characterized a functional Hdh-MIPR, which responded to the Hdh-MIPs, except for Hdh-MIP2, possibly linked with the PKC/Ca2+ and PKA/cAMP signaling pathways. Hdh-MIPs delayed larval metamorphosis but increased the spawning behavior. These results suggest that the Hdh-MIP signaling system provides insights into the unique function of MIP in invertebrates.

Heat hardening improves thermal tolerance in abalone, without the trade-offs associated with chronic heat exposure.

Marine animals are challenged by chronically raised temperatures alongside an increased frequency of discrete, severe warming events. Exposure to repeated heat shocks could result in heat hardening, where sub-lethal exposure to thermal stress temporarily enhances thermotolerance, and may be an important mechanism by which marine species will cope with future thermal challenges. However, we have relatively little understanding of the effects of heat hardening in comparison to chronic exposure to elevated temperatures. Therefore, we compared the effects of heat hardening from repeated exposure to acute heat shocks and chronic exposure to elevated temperatures on thermal tolerance in the European abalone, Haliotis tuberculata. Adult abalones were exposed to either control temperature (15 °C), chronic warming (20 °C) or a regime of two events of repeated acute heat shock cycles (23-25 °C) during six months, and their thermal tolerance and performance, based upon cardiac activity, compared using a dynamic ramping assay. The cost associated with each treatment was also estimated via measurements of condition index (CI). Abalone exposed to both temperature treatments had higher upper thermal limits than the control, but heat-hardened individuals had significantly higher CI values, indicating an enhancement in condition status. Differences in the shape of the thermal performance curve suggest different mechanisms may be at play under different temperature exposure treatments. We conclude that heat hardening can boost thermal tolerance in this species, without performance trade-offs associated with chronic warming.

Playing it safe; risk-induced trait responses increase survival in the face of predation.

Predation risk effects are impacts on prey caused by predators that do not include consumption. These can include changes in prey behaviour, physiology, and morphology (i.e. risk-induced trait responses), which can have consequences to individual fitness and population dynamics (i.e. non-consumptive effects). While these risk-induced trait responses (RITRs) can lower individual fitness as compared to prey not exposed to risk, they are assumed to increase fitness in the presence of predators. While much work has been built upon this assumption, most evidence occurs in consumptive experiments where the trait values of consumed prey are unknown. We have little evidence showing individuals with a greater magnitude of RITR have greater survival. Here, we tested the hypothesis that RITRs increase survival in the presence of predators, but come at a cost to growth. We tested this hypothesis using Nucella lapillus as prey and Carcinus maenas as a predator and including mussels as a basal resource in a two-phase mesocosm experimental set-up. In phase 1, Nucella were placed into either a control or risk treatment (exposure to non-lethal Carcinus) for 28 days and their behaviour and growth measured. In phase 2, a lethal Carcinus was added to all mesocosms (non-lethal crabs were removed), and survival was recorded for 15 days. At the treatment (group) level, we found that Nucella exposed to predation risk in phase 1 had significantly greater risk aversion behaviour (summed score of risky vs. safe behaviour) and significantly lower growth. In phase 2, we found that Nucella exposed to predation risk had greater survival. At the individual level (regardless of treatment), we found that Nucella with greater risk aversion scores in phase 1 had significantly higher survival in phase 2 when exposed to a lethal predator, but this came at a cost to their growth. This study provides some of the first empirical evidence, at both the group and individual level, testing a long-held assumption that predation risk-induced behavioural responses increase survival in the face of direct predation, but that these responses come at a cost to the prey. These results add to our growing understanding of the benefits of RITRs to individual fitness and non-consumptive effects generally.

The origin and diversification of pteropods precede past perturbations in the Earth's carbon cycle.

Pteropods are a group of planktonic gastropods that are widely regarded as biological indicators for assessing the impacts of ocean acidification. Their aragonitic shells are highly sensitive to acute changes in ocean chemistry. However, to gain insight into their potential to adapt to current climate change, we need to accurately reconstruct their evolutionary history and assess their responses to past changes in the Earth's carbon cycle. Here, we resolve the phylogeny and timing of pteropod evolution with a phylogenomic dataset (2,654 genes) incorporating new data for 21 pteropod species and revised fossil evidence. In agreement with traditional taxonomy, we recovered molecular support for a division between "sea butterflies" (Thecosomata; mucus-web feeders) and "sea angels" (Gymnosomata; active predators). Molecular dating demonstrated that these two lineages diverged in the early Cretaceous, and that all main pteropod clades, including shelled, partially-shelled, and unshelled groups, diverged in the mid- to late Cretaceous. Hence, these clades originated prior to and subsequently survived major global change events, including the Paleocene-Eocene Thermal Maximum (PETM), the closest analog to modern-day ocean acidification and warming. Our findings indicate that planktonic aragonitic calcifiers have shown resilience to perturbations in the Earth's carbon cycle over evolutionary timescales.

Evolved differences in energy metabolism and growth dictate the impacts of ocean acidification on abalone aquaculture.

Ocean acidification (OA) poses a major threat to marine ecosystems and shellfish aquaculture. A promising mitigation strategy is the identification and breeding of shellfish varieties exhibiting resilience to acidification stress. We experimentally compared the effects of OA on two populations of red abalone (Haliotis rufescens), a marine mollusc important to fisheries and global aquaculture. Results from our experiments simulating captive aquaculture conditions demonstrated that abalone sourced from a strong upwelling region were tolerant of ongoing OA, whereas a captive-raised population sourced from a region of weaker upwelling exhibited significant mortality and vulnerability to OA. This difference was linked to population-specific variation in the maternal provisioning of lipids to offspring, with a positive correlation between lipid concentrations and survival under OA. This relationship also persisted in experiments on second-generation animals, and larval lipid consumption rates varied among paternal crosses, which is consistent with the presence of genetic variation for physiological traits relevant for OA survival. Across experimental trials, growth rates differed among family lineages, and the highest mortality under OA occurred in the fastest growing crosses. Identifying traits that convey resilience to OA is critical to the continued success of abalone and other shellfish production, and these mitigation efforts should be incorporated into breeding programs for commercial and restoration aquaculture.

Bursting emerges from the complementary roles of neurons in a four-cell network.

Reciprocally inhibitory modules that form half-center oscillators require mechanisms for escaping or being released from inhibition. The central pattern generator underlying swimming by the nudibranch mollusc, Dendronotus iris, is composed of only four neurons that are organized into two competing modules of a half-center oscillator. In this system, bursting activity in left-right alternation is an emergent property of the network as a whole; none of the neurons produces bursts on its own. We found that the unique synaptic actions and membrane properties of the two neurons in each module (Si2 and the contralateral Si3) play complementary roles in generating stable bursting in this network oscillator. Although Si2 and Si3 each inhibits its contralateral counterpart, Si2 plays a dominant role in evoking fast and strong inhibition of the other module, the termination of which initiates postinhibitory rebound in the Si3 of that module by activating a hyperpolarization-activated inward current. Within each module, the synaptic actions and membrane properties of the two neurons complement each other: Si3 excites Si2, which then feeds back slow inhibition to Si3, terminating the burst. Using dynamic clamp, we showed that the magnitude of the slow inhibition sets the period of the oscillator. Thus, the synaptic actions of Si2 provide the hyperpolarization needed for the other module to rebound stably, whereas the membrane properties of Si3 in each module cause it to rebound first and excite Si2 to maintain the burst until terminated by the slow inhibition from Si2, which releases the other module to become active.NEW & NOTEWORTHY Half-center oscillators composed of reciprocally inhibitory neurons have been posited for over a century to underlie the production of rhythmic movements. The Dendronotus swim central pattern generator may be the simplest such circuit with only two pairs of bilaterally represented neurons. This study completes the description of the mechanism by which this network oscillator functions, showing how stable rhythmic activity arises from the complementary membrane and synaptic properties of the two neurons in the competing modules.

Weapons or deterrents? Nudibranch molluscs use distinct ecological modes of chemical defence against predators.

Defensive chemicals are used by plants and animals to reduce the risk of predation through different mechanisms, including toxins that cause injury and harm (weapons) and unpalatable or odiferous compounds that prevent attacks (deterrents). However, whether effective defences are both toxins and deterrents, or work in just one modality is often unclear. In this study, our primary aim was to determine whether defensive compounds stored by nudibranch molluscs acted as weapons (in terms of being toxic), deterrents (in terms of being distasteful) or both. Our secondary aim was to investigate the response of different taxa to these defensive compounds. To do this, we identified secondary metabolites in 30 species of nudibranch molluscs and investigated their deterrent properties using antifeedant assays with three taxa: rock pool shrimp, Palaemon serenus, and two fish species: triggerfish Rhinecanthus aculeatus and toadfish Tetractenos hamiltoni. We compared these results to toxicity assays using brine shrimp Artemia sp. and previously published toxicity data with a damselfish Chromis viridis. Overall, we found no clear relationship between palatability and toxicity, but instead classified defensive compounds into the following categories: Class I & II-highly unpalatable and highly toxic; Class I-weakly unpalatable and highly toxic; Class II-highly unpalatable but weakly toxic; WR (weak response)-weakly unpalatable and weakly toxic. We also found eight extracts from six species that did not display activity in any assays indicating they may have very limited chemical defensive mechanisms (NR, no response). We found that the different classes of secondary metabolites were similarly unpalatable to fish and shrimp, except extracts from Phyllidiidae nudibranchs (isonitriles) that were highly unpalatable to shrimp but weakly unpalatable to fish. Our results pave the way towards better understanding how animal chemical defences work against a variety of predators. We highlight the need to disentangle weapons and deterrents in future work on anti-predator defences to better understand the foraging decisions faced by predators, the resultant selection pressures imposed on prey and the evolution of different anti-predator strategies.

Crystal growth kinetics as an architectural constraint on the evolution of molluscan shells.

Molluscan shells are a classic model system to study formation-structure-function relationships in biological materials and the process of biomineralized tissue morphogenesis. Typically, each shell consists of a number of highly mineralized ultrastructures, each characterized by a specific 3D mineral-organic architecture. Surprisingly, in some cases, despite the lack of a mutual biochemical toolkit for biomineralization or evidence of homology, shells from different independently evolved species contain similar ultrastructural motifs. In the present study, using a recently developed physical framework, which is based on an analogy to the process of directional solidification and simulated by phase-field modeling, we compare the process of ultrastructural morphogenesis of shells from 3 major molluscan classes: A bivalve Unio pictorum, a cephalopod Nautilus pompilius, and a gastropod Haliotis asinina We demonstrate that the fabrication of these tissues is guided by the organisms by regulating the chemical and physical boundary conditions that control the growth kinetics of the mineral phase. This biomineralization concept is postulated to act as an architectural constraint on the evolution of molluscan shells by defining a morphospace of possible shell ultrastructures that is bounded by the thermodynamics and kinetics of crystal growth.

Becoming a limpet: An 'intermittent limpetization' process driven by host features in the kleptoparasitic gastropod family Capulidae.

A coiled shell is the most evident feature of the typical Bauplan of a gastropod mollusc. However, at least 54 families independently evolved an apparently simplified shell morphology: the limpet. Species with this largely uncoiled, depressed shell morphology occur in almost every aquatic habitat and are associated to a number of different lifestyles and diets. The marine gastropod family Capulidae includes 18 recognised genera, the large majority of which are coiled, but with a number of limpet-like species. Capulid shell plasticity is also associated to a broad range of feeding ecologies, from obligate suspension feeders to kleptoparasites. To investigate the evolution of the limpet-like shell in the family Capulidae we performed an ancestral state reconstruction analysis on a time-calibrated phylogenetic tree (COI, 16S, and ITS2) including 16 species representing a good deal of its morphological diversity. Our results identified at least three capulid lineages that independently evolved limpet-like shells, suggesting that a recurrent limpetization process characterizes this family. One of the limpet-like genera was undescribed and was here named Cryocapulus n. gen. We suggest that capulids evolved from a coiled suspension feeder lineage and that the shift to kleptoparasitism, which occurred in the family ancestor, may have represented a strategy to save energy through the exploitation of the water current produced by the host. Probably the major drivers of shell evolution in capulids are related to their ecology, most of them being kleptoparasites, include the shape and the kind of host substrate, and lead to the repeated acquisition of a limpet-like shape.

Demographics of the semi-slug Parmarion martensi, an intermediate host for Angiostrongylus cantonensis in Hawai'i, during laboratory rearing.

The semi-slug, Parmarion martensi, is an intermediate host of the zoonotic nematode, Angiostrongylus cantonensis, the aetiological agent of neuroangiostrongyliasis or rat lungworm disease in humans. Rearing methods were developed for P. martensi to facilitate studies on nematode transmission and control. Parmarion martensi exhibited high survivorship when reared on a diet of dog food and fresh fruits and vegetables in temperature-controlled cabinets at 21.4°C, 98% relative humidity and 12:12 L:D cycle. Rearing containers were lined with moist paper towels for substrate and plastic pots were provided for hiding/resting and egg-laying. Under these conditions, time to first reproduction was 165.3 ± 12.3 days, fecundity was approximately 34.5 ± 7.8 eggs per adult, and hatch rate was 52.7 ± 3.2%. Survivorship post egg hatch was 86.2 ± 2.9% at 30 days (neonates had a mortality rate of about 14%) and 99% thereafter for up to a year. The demographics of laboratory-reared and wild-caught P. martensi were similar except for the weight of reproductive adults, which was significantly higher in laboratory-reared adults (4.0 ± 0.2 g) than in field-collected adults (1.5 ± 0.1 g).

Induction of Phlorotannins and Gene Expression in the Brown Macroalga Fucus vesiculosus in Response to the Herbivore Littorina littorea.

Mechanisms related to the induction of phlorotannin biosynthesis in marine brown algae remain poorly known. Several studies undertaken on fucoid species have shown that phlorotannins accumulate in the algae for several days or weeks after being exposed to grazing, and this is measured by direct quantification of soluble phenolic compounds. In order to investigate earlier inducible responses involved in phlorotannin metabolism, Fucus vesiculosus was studied between 6 and 72 h of grazing by the sea snail Littorina littorea. In this study, the quantification of soluble phenolic compounds was complemented by a Quantitative real-time PCR (qRT-PCR) approach applied on genes that are potentially involved in either the phlorotannin metabolism or stress responses. Soluble phlorotannin levels remained stable during the kinetics and increased significantly only after 12 h in the presence of grazers, compared to the control, before decreasing to the initial steady state for the rest of the kinetics. Under grazing conditions, the expression of vbpo, cyp450 and ast6 genes was upregulated, respectively, at 6 h, 12 h and 24 h, and cyp450 gene was downregulated after 72 h. Interestingly, the pksIII gene involved in the synthesis of phloroglucinol was overexpressed under grazing conditions after 24 h and 72 h. This study supports the hypothesis that phlorotannins are able to provide an inducible chemical defense under grazing activity, which is regulated at different stages of the stress response.

Effect of temperature on embryonic development of the marine gastropod Charonia seguenzae (Aradas & Benoit, 1870).

The Mediterranean Triton Charonia seguenzae (Aradas & Benoit, 1870) is an endangered marine gastropod. Re-establishment of C. seguenzae populations in the depleted habitats requires knowledge of its biology and breeding in captivity. Temperature has been recorded to affect the development in marine gastropods. The present study aims to describe the encapsulated development and study the effect of three temperature regimes, that the embryos are exposed to in the wild (17, 20, 23οC), on it. At the stage of morula (7th Day After Deposition - DAD) 180 egg capsules were separated in three groups of 60 capsules. One group remained at 23 °C and the other two were acclimated at 20 and 17 °C. Two capsules were sampled randomly from each temperature setup (every day during the first 5 days, D1-D5, every other day from D7 to D17 and every other two days from D20 to eclosion), opened and the eggs, embryos or larvae were photographed under stereoscope. Stages of development and shape were assessed and dimensions were measured from microphotographs. All developmental stages were described in relation to temperature and time. At 23οC eclosion of free swimming veligers occurred 49 DAD, 17 days faster than the embryonic development at 20οC. Eclosion at 17 οC was not observed up until the 74th DAD when the last sampling was conducted. An increased amount of larval deformities was observed at 17οC reaching 94% during the last sampling (D74), while at eclosion only 4 and 3% of the hatching larvae were deformed at 20 and 23 οC respectively. In this study temperature appears as a key factor during the development of the marine gastropod Charonia seguenzae, affecting the survival and developmental rate. Although temperature affected the size of intermediate stages, the size of free swimming veligers at 20 and 23 οC did not differ.

Effect of temperature on the development of deformities during the embryonic stages of Charonia seguenzae (Aradas & Benoit, 1870).

The Mediterranean Triton Charonia seguenzae (Aradas and Benoit, 1870) is an endangered marine gastropod. Re-establishment of C. seguenzae populations in the depleted habitats requires knowledge of its biology and breeding in captivity. Deformities have a huge impact on offspring survival and quality. Temperature has been recorded to affect the development of deformities in marine gastropods. The present study aims to identify the stage of development at which deformities occur, under four temperature regimes (17, 23, 26 and 29 °C). At the stages of trochophore, veliger and free veliger larvae, three capsules that were acclimated at the examined temperatures at the stage of morula were collected, opened and 50 larvae per capsule sampled. Deformities were observed at every examined developmental stage under all tested temperatures. The lower rate of deformities at every stage occurred at 23 °C. The higher tested temperature (29 °C) was lethal and at the lower tested temperature (17 °C) almost every specimen was deformed (96.66%) at eclosion. The effect of acclimation at four developmental stages (morula, trochophore, shell formation and veliger) on the development of deformities at the free veligers of Charonia seguenzae, was studied under three temperature conditions (17, 26 and 29 °C). At eclosion, three capsules were collected, opened and 50 larvae per capsule were sampled. The acclimation at morula and trochophore larva stages led to the higher rates of deformities at eclosion. The size of the free veliger larvae was also affected by temperature with maximum size at eclosion observed at 23 °C. Charonia seguenzae's embryos tolerate elevated temperatures within environmental limits (26 °C) but near future global warming will probably pose a threat to their survival. The free veliger larvae survival at the environmental minimum is related to the time window of the acclimation, since Triton's embryos are more vulnerable to temperature alterations during the early developmental stages.