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.

The "Mexican dancer" in Ecuador: molecular confirmation, embryology and planktotrophy in the sea slug Elysia diomedea.

Elysia diomedea, otherwise known as the "Mexican dancer", aries in adult size and color across its geographical distribution in Ecuador. Because of morphological variation and the absence of genetic information for this species in Ecuador, we analyzed mtDNA sequences in three populations (Ballenita, La Cabuya, and Mompiche) and confirmed that individuals from the three locations belonged to E. diomedea and that there was no population structure that could explain their morphological differences. Next, we analyzed general aspects about the reproductive biology and embryology of this species. Live slugs from the Ballenita population were maintained and reproduced ex situ. Egg ribbons and embryos were fixed and observed by brightfield and confocal microscopy. We observed a single embryo per capsule, 98 embryos per mm2 of egg ribbon, and compared the cleavage pattern of this species to that of other heterobranchs and spiralians. E. diomedea early development was characterized by a slight unequal first cleavage, occurrence of a 3-cell stage in the second cleavage, and the formation of an enlarged second quartet of micromeres. We observed clear yolk bodies in the egg capsules of some eggs ribbons at early stages of development. Both reproductive and embryological characteristics, such as presence of stomodeum in the larva, and ingestion of particles after hatching confirmed the planktotrophic veliger larvae of this species, consistent with the majority of sacoglossans from the Eastern and Northeast Pacific Oceans.

A serpin is required for ectomesoderm, a hallmark of spiralian development.

Among animals, diploblasts contain two germ layers, endoderm and ectoderm, while triploblasts have a distinct third germ layer called the mesoderm. Spiralians are a group of triploblast animals that have highly conserved development: they share the distinctive spiralian cleavage pattern as well as a unique source of mesoderm, the ectomesoderm. This population of mesoderm is distinct from endomesoderm and is considered a hallmark of spiralian development, but the regulatory network that drives its development is unknown. Here we identified ectomesoderm-specific genes in the mollusc Tritia (aka Ilyanassa) obsoleta through differential gene expression analyses comparing control and ectomesoderm-ablated embryos, followed by in situ hybridization of identified transcripts. We identified a Tritia serpin gene (ToSerpin1) that appears to be specifically expressed in the ectomesoderm of the posterior and head. Ablation of the 3a and 3b cells, which make most of the ectomesoderm, abolishes ToSerpin1 expression, consistent with its expression in these cells. Morpholino knockdown of ToSerpin1 causes ectomesoderm defects, most prominently in the muscle system of the larval head. This is the first gene identified that is specifically implicated in spiralian ectomesoderm development.

Quadruple domain-containing galectin from marine invertebrate disk abalone (Haliotis discus discus): Molecular perspectives in early development, immune expression, and potent antiviral responses.

Galectins are well-known ß-galactoside-binding proteins, which play vital roles in innate immune responses of both vertebrates and invertebrates. However, knowledge regarding invertebrate galectins is still in its infancy. With the intention of filling the knowledge gap, here we identified a quadruple domain-containing galectin from marine invertebrate disk abalone, Haliotis discus discus (AbGalec), and characterized it. AbGalec consisted of four distinct carbohydrate-recognition domains (CRDs) and lacked a signal peptide. Expression analysis revealed AbGalec to be ubiquitously expressed in all the examined early embryonic stages of abalone, with highest expression in the 16-cell stage, suggesting the importance of AbGalec in early developmental processes. Tissue distribution analysis revealed the highest expression of AbGalec in abalone mantle, followed by that in gills and hemocytes. Immune challenge experiments revealed significant upregulation of AbGalec at 24 h and 48 h post injection (p.i.) with bacterial and viral components. These results suggested the possible involvement of AbGalec in host defense mechanisms. Polyinosinic: polycytidylic acid (Poly I:C) and viral hemorrhagic septicemia virus (VHSV) injections were capable of inducing AbGalec transcript expression more prominently than bacterial stimulants, thus providing evidence for its role in viral infections. We determined the virus-neutralizing ability of a quadruple domain-containing galectin for the first time, by analyzing the downregulation of VHSV transcripts during the overexpression of AbGalec. Significant downregulation of VHSV transcripts was observed after 24 h and 48 h of post infection. Collectively, our findings reveal the potent antiviral responses of molluscan quadruple domain-containing galectin, AbGalec, along with its involvement in innate immunity.

Radula formation in two species of Conoidea (Gastropoda).

The radula is the basic feeding structure in gastropod molluscs and exhibits great morphological diversity that reflects the exceptional anatomical and ecological diversity occurring in these animals. This uniquely molluscan structure is formed in the blind end of the radular sac by specialized cells (membranoblasts and odontoblasts). Secretion type, and the number and shape of the odontoblasts that form each tooth characterize the mode of radula formation. These characteristics vary in different groups of gastropods. Elucidation of this diversity is key to identifying the main patterns of radula formation in Gastropoda. Of particular interest would be a phylogenetically closely related group that is characterized by high variability of the radula. One such group is the large monophyletic superfamily Conoidea, the radula of which is highly variable and may consist of the radular membrane with five teeth per row, or the radular membrane with only two or three teeth per row, or even just two harpoon-like teeth per row without a radular membrane. We studied the radulae of two species of Conoidea (Clavus maestratii Kilburn, Fedosov & Kantor, 2014 [Drilliidae] and, Lophiotoma acuta (Perry, 1811) [Turridae]) using light and electron microscopy. Based on these data and previous studies, we identify the general patterns of the radula formation for all Conoidea: the dorsolateral position of two groups of odontoblasts, uniform size, and shape of odontoblasts, folding of the radula in the radular sac regardless of the radula configuration. The morphology of the subradular epithelium is most likely adaptive to the radula type.

BMP signaling plays a role in anterior-neural/head development, but not organizer activity, in the gastropod Crepidula fornicata.

BMP signaling is involved in many aspects of metazoan development, with two of the most conserved functions being to pattern the dorsal-ventral axis and to specify neural versus epidermal fates. An active area of research within developmental biology asks how BMP signaling was modified over evolution to build disparate body plans. Animals belonging to the superclade Spiralia/Lophotrochozoa are excellent experimental subjects for studying the evolution of BMP signaling because a highly conserved, stereotyped early cleavage program precedes the emergence of distinct body plans. In this study we examine the role of BMP signaling in one representative, the slipper snail Crepidula fornicata. We find that mRNAs encoding BMP pathway components (including the BMP ligand decapentaplegic, and BMP antagonists chordin and noggin-like proteins) are not asymmetrically localized along the dorsal-ventral axis in the early embryo, as they are in other species. Furthermore, when BMP signaling is perturbed by adding ectopic recombinant BMP4 protein, or by treating embryos with the selective Activin receptor-like kinase-2 (ALK-2) inhibitor Dorsomorphin Homolog 1 (DMH1), we observe no obvious effects on dorsal-ventral patterning within the posterior (post-trochal) region of the embryo. Instead, we see effects on head development and the balance between neural and epidermal fates specifically within the anterior, pre-trochal tissue derived from the 1q1 lineage. Our experiments define a window of BMP signaling sensitivity that ends at approximately 44-48 â€‹hours post fertilization, which occurs well after organizer activity has ended and after the dorsal-ventral axis has been determined. When embryos were exposed to BMP4 protein during this window, we observed morphogenetic defects leading to the separation of the anterior, 1q lineage from the rest of the embryo. The 1q-derived organoid remained largely undifferentiated and was radialized, while the post-trochal portion of the embryo developed relatively normally and exhibited clear signs of dorsal-ventral patterning. When embryos were exposed to DMH1 during the same time interval, we observed defects in the head, including protrusion of the apical plate, enlarged cerebral ganglia and ectopic ocelli, but otherwise the larvae appeared normal. No defects in shell development were noted following DMH1 treatments. The varied roles of BMP signaling in the development of several other spiralians have recently been examined. We discuss our results in this context, and highlight the diversity of developmental mechanisms within spiral-cleaving animals.

Early embryonic exposure of freshwater gastropods to pharmaceutical 5-alpha-reductase inhibitors results in a surprising open-coiled "banana-shaped" shell.

In vertebrates, the steroidogenesis enzyme 5α-reductase converts testosterone to the more potent androgen 5α-dihydrotestosterone. Homologues of 5α-reductase genes have been identified in molluscs. However, recent findings suggest that vertebrate-type steroid androgens are not utilised in molluscan reproductive development. Genomic searches have revealed that molluscs do not possess many of the steroidogenic enzymes required to make testosterone, nor a nuclear androgen receptor. Consequently, the role of 5α-reductase in molluscs presents a mystery. Here, developmental exposures of Biomphalaria glabrata to selective pharmaceutical 5α-reductase inhibitors elicited a strong, highly reproducible phenotypic response characterised by the development of elongated "banana-shaped" shell morphology. In comparison to untreated snails, the shells are open-coiled and the whorls are unattached. Dutasteride (5α-reductase inhibitor) is approximately 10-times more potent at provoking the banana-shaped shell phenotype than finasteride, paralleling the pharmaceuticals' efficacy in humans. Other enzyme inhibitors with different modes of action were tested to investigate the specificity of the phenotype. However, only the pharmaceutical 5α-reductase inhibitors provoked the response. Dutasteride elicited the same phenotype in a second gastropod, Physella acuta. In the absence of evidence for de novo androgen steroidogenesis in molluscs, these findings suggest that novel substrates for 5α-reductase exist in gastropods, lending support to the contention that molluscan endocrinology differs from the well-characterised vertebrate endocrine system.

Trophic transfer of paralytic shellfish toxin (PST): Physiological and reproductive effects in the carnivorous gastropod Acanthina monodon (Pallas, 1774).

Harmful algal blooms can adversely affect different levels of the trophic chain, from primary consumers, such as bivalve molluscs, to higher links such as large fish, birds and mammals, including humans. Among secondary consumers, it has been described that carnivorous gastropods can accumulate these toxins when they prey on bivalves that have been exposed to toxic microalgae; these could also harm human health. In Chile, frequent events of harmful algal blooms caused by the dinoflagellate Alexandrium catenella have been described. This organism produces paralytic shellfish toxin (PST) which has been identified in some carnivorous gastropods. The objective of this research was to identify the physiological and reproductive response of the carnivorous gastropod Acanthina monodon fed on the Mytilid Perumytilus purpuratus, which had previously been maintained on a diet containing PST. Specimens of A. monodon showed a decrease in ingestion and absorption rate when they consumed PST indirectly through their diet. The oxygen consumption rate was also affected by the diet-time interaction. The variations of these parameters were reflected in the scope for growth, since the available energy was lower in gastropods exposed to toxic diet. Consumption of PST had a negative effect on the reproduction of A. monodon, since intoxicated adults presented lower egg-masses and delayed start of oviposition. We observed a delay in the development of the embryos inside the capsules, and a lower number of hatched juveniles, although these few juveniles from intoxicated parents accomplished higher growth rates during the next 6 months. We may therefore suggest that toxin transfer, from harmful microalgae through the trophic chain, can generate deleterious effects on the physiological energetics of the organisms that consume them, affecting their reproductive capacity and early ontogenetic development.

Gene Expression Does Not Support the Developmental Hourglass Model in Three Animals with Spiralian Development.

It has been proposed that animals have a pattern of developmental evolution resembling an hourglass because the most conserved development stage-often called the phylotypic stage-is always in midembryonic development. Although the topic has been debated for decades, recent studies using molecular data such as RNA-seq gene expression data sets have largely supported the existence of periods of relative evolutionary conservation in middevelopment, consistent with the phylotypic stage and the hourglass concepts. However, so far this approach has only been applied to a limited number of taxa across the tree of life. Here, using established phylotranscriptomic approaches, we found a surprising reverse hourglass pattern in two molluscs and a polychaete annelid, representatives of the Spiralia, an understudied group that contains a large fraction of metazoan body plan diversity. These results suggest that spiralians have a divergent midembryonic stage, with more conserved early and late development, which is the inverse of the pattern seen in almost all other organisms where these phylotranscriptomic approaches have been reported. We discuss our findings in light of proposed reasons for the phylotypic stage and hourglass model in other systems.

Global change scenarios trigger carry-over effects across life stages and generations of the intertidal limpet, Siphonaria australis.

For organisms with complex life histories, carry-over effects (COEs) can manifest between life stages, when conditions experienced by one stage influence the next, as well as trans-generationally, when the parental environment affects offspring. Here we used multiple global change-associated stressors to examine both forms of COE simultaneously in an intertidal limpet with mixed development (i.e. planktonic larvae hatch from benthic egg masses). Adult Siphonaria australis were subjected to four treatments over four weeks: an ambient control, a treatment featuring elevated water temperature (25°C) and UVB (1.7 W m-2), a copper pollution treatment (5.0 µg L-1), and a treatment incorporating all three stressors. Egg masses laid by these adults were then redistributed among the same four treatments (producing 16 adult-to-egg treatment histories) and stressed until hatching. Finally, hatching larvae were reared under ambient conditions for 24 days. While adult survivorship was unaffected by treatment, embryonic viability in egg masses responded strongly to egg mass treatment, as well as parental stress exposure, therefore displaying trans-generational COEs. These trans-generational COEs interacted with COEs originating in egg masses to produce highly context-dependent hatching sizes and larval growth. This demonstrates that the performance of a given organism at a given time reflects not only conditions experienced during embryonic development, but also those of the parental generation, and suggests that COEs play an important but underestimated role in responses to global change scenarios.

Transgenerational impairments of reproduction and development of the marine invertebrate Crepidula onyx resulted from long-term dietary exposure of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47).

Polybrominated diphenyl ethers have become ubiquitous in the environment and elevated concentrations have often been found in marine organisms. Using the gastropod Crepidula onyx as a study model, this multigenerational study sets out to test the hypotheses that 1) parental dietary exposure to environmentally realistic levels of 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) would lead to transgenerational impairments on fitness traits of marine invertebrates, and 2) the organisms might develop adaptation/acclimation after exposure for one or more generations. F0 generation of C. onyx was fed with the dinoflagellate Isochrysis galbana encapsulated with two concentrations of BDE-47 (1.78 and 16.0 ng million cells-1, respectively), and half of the F1 offspring from the higher concentration treatment was returned to control condition (transgenerational group), while the other half received BDE-47 treatment continuously (continuous treatment group). Bioaccumulation and maternal transfer of BDE-47 were evident in all life stages of the F0 generation and in F1 eggs, respectively. Exposure to BDE-47 reduced fecundity, delayed sexual maturity, and impeded embryonic development in F0 to F2. In particular, developmental toxicity of F2 embryos was apparent in the transgenerational group, but not in the continuous treatment group, even when BDE-47 was not detected in the F2 embryos nor in their mothers and they have never been exposed to the chemical. This study also suggested that the offspring might have developed adaptation/acclimation to the exposure of BDE-47 within two generations of exposure, and that the physiological alterations associated with acclimation/adaptation might have hindered the normal larval development under a stress free condition. These findings highlighted the need for long-term multigenerational studies in the ecological risk assessment of chemicals alike.

Expansion of TALE homeobox genes and the evolution of spiralian development.

Spiralians, including molluscs, annelids and platyhelminths, share a unique development process that includes the typical geometry of early cleavage and early segregation of cell fate in blastomeres along the animal-vegetal axis. However, the molecular mechanisms underlying this early cell fate segregation are largely unknown. Here, we report spiralian-specific expansion of the three-amino-acid loop extension (TALE) class of homeobox genes. During early development, some of these TALE genes are expressed in staggered domains along the animal-vegetal axis in the limpet Nipponacmea fuscoviridis and the polychaete Spirobranchus kraussii. Inhibition or overexpression of these genes alters the developmental fate of blastomeres, as predicted by the gene expression patterns. These results suggest that the expansion of novel TALE genes plays a critical role in the establishment of a novel cell fate segregation mechanism in spiralians.

Establishment and activity of the D quadrant organizer in the marine gastropod Crepidula fornicata.

During development in metazoan embryos, the fundamental embryonic axes are established by organizing centers that influence the fates of nearby cells. Among the spiralians, a large and diverse branch of protostome metazoans, studies have shown that an organizer sets up the dorsal-ventral axis, which arises from one of the four basic cell quadrants during development (the dorsal, D quadrant). Studies in a few species have also revealed variation in terms of how and when the D quadrant and the organizer are established. In some species the D quadrant is specified conditionally, via cell-cell interactions, while in others it is specified autonomously, via asymmetric cell divisions (such as those involving the formation of polar lobes). The third quartet macromere (3D) typically serves as the spiralian organizer; however, other cells born earlier or later in the D quadrant lineage can serve as the organizer, such as the 2d micromere in the annelid Capitella teleta or the 4d micromere in the mollusc Crepidula fornicata. Here we present work carried out in the snail C. fornicata to show that establishment of a single D quadrant appears to rely on a combination of both autonomous (via inheritance of the polar lobe) and conditional mechanisms (involving induction via the progeny of the first quartet micromeres). Through systematic ablation of cells, we show that D quadrant identity is established between 5th and 6th cleavage stages, as it is in other spiralians that use conditional specification. Subsequently, following the next cell cycle, organizer activity takes place soon after the birth of the 4d micromere. Therefore, unlike the case in other spiralians that use conditional specification, the specification of the D quadrant and the activity of the dorso-ventral organizer are temporally and spatially uncoupled. We also present data on organizer function in naturally-occurring and experimentally-induced twin embryos, which possess multiple D quadrants. We show that supernumerary D quadrants can arise in C. fornicata (either spontaneously or following polar lobe removal); when multiple D quadrants are present these do not exhibit effective organizer activity. We conclude that the polar lobe is not required for D quadrant specification, though it could play a role in effective organizer activity. We also tested whether the inheritance of the small polar lobe by the D quadrant is associated with the ability to laterally inhibit neighboring quadrants by direct contact in order to normally prevent supernumerary organizers from arising. Finally, we discuss the variation of spiralian organizers in a phylogenetic context.

A set of simple cell processes is sufficient to model spiral cleavage.

During cleavage, different cellular processes cause the zygote to become partitioned into a set of cells with a specific spatial arrangement. These processes include the orientation of cell division according to: an animal-vegetal gradient; the main axis (Hertwig's rule) of the cell; and the contact areas between cells or the perpendicularity between consecutive cell divisions (Sachs' rule). Cell adhesion and cortical rotation have also been proposed to be involved in spiral cleavage. We use a computational model of cell and tissue biomechanics to account for the different existing hypotheses about how the specific spatial arrangement of cells in spiral cleavage arises during development. Cell polarization by an animal-vegetal gradient, a bias to perpendicularity between consecutive cell divisions (Sachs' rule), cortical rotation and cell adhesion, when combined, reproduce the spiral cleavage, whereas other combinations of processes cannot. Specifically, cortical rotation is necessary at the 8-cell stage to direct all micromeres in the same direction. By varying the relative strength of these processes, we reproduce the spatial arrangement of cells in the blastulae of seven different invertebrate species.

Maternal Lipid Provisioning Mirrors Evolution of Reproductive Strategies in Direct-Developing Whelks.

The energetic input that offspring receive from their mothers is a well-studied maternal effect that can influence the evolution of life histories. Using the offspring of three sympatric whelks: Cominella virgata (one embryo per capsule); Cominella maculosa (multiple embryos per capsule); and Haustrum scobina (multiple embryos per capsule and nurse-embryo consumption), we examined how contrasting reproductive strategies mediate inter- and intraspecific differences in hatchling provisioning. Total lipid content (as measured in µg hatchling(-1) ± SE) was unrelated to size among the 3 species; the hatchlings of H. scobina were the smallest but had the highest lipid content (33.8 ± 8.1 µg hatchling(-1)). In offspring of C. maculosa, lipid content was 6.6 ± 0.4 µg hatchling(-1), and in offspring of C. virgata, it was 21.7 ± 3.2 µg hatchling(-1) The multi-encapsulated hatchlings of C. maculosa and H. scobina were the only species that contained the energetic lipids, wax ester (WE) and methyl ester (ME). However, the overall composition of energetic lipid between hatchlings of the two Cominella species reflected strong affinities of taxonomy, suggesting a phylogenetic evolution of the non-adelphophagic development strategy. Inter- and intracapsular variability in sibling provisioning was highest in H. scobina, a finding that implies less control of allocation to individual hatchlings in this adelphophagic developer. We suggest that interspecific variability of lipids offers a useful approach to understanding the evolution of maternal provisioning in direct-developing species.

Are embryonic developing modes determinant in the acquisition and levels of photoprotective compounds in slipper limpets of the Crepipatella genus?

The type of embryonic development (mixed and direct) and its influence on the accumulation and translocation of photoprotective compounds from the mother to the encapsulated embryo was studied in the intertidal gastropods Crepipatella peruviana and Crepipatella dilatata during their reproductive peak. HPLC/MS was used to determine type and levels of sunscreen compounds (total carotenoids; TC/and mycosporine-like amino acid; MAA) in brooding females, capsule walls and early and pre-hatching embryos of both species. Photoprotective compounds were only quantified in nurse eggs of C. dilatata. Our results indicate that females of both species can accumulate TC and MAA at different levels, and they are able to transfer them selectively to capsule walls, embryos and nurse eggs. Palythine-serine (MW=244Da; λmax=320nm) and MAA-330 (MW=234Da; λmax=330nm) constitute total MAA pool in brooding females, whereas brooded embryos incorporate palythine (MW=244Da; λmax=320nm) to the MAA pool. Although TC was transferred from the mother to the embryo through the yolk in both species, MAA trespass showed differences. Females of C. peruviana transfer MAA to their embryos through the embryonic yolk; C. dilatata can transfer MAA only through their nurse eggs, which are consumed by embryos during the terminal stages of intracapsular development. Differences between mixed and direct embryonic development, as well as environmental UV-R levels, which the recently hatched larvae and juveniles of C. peruviana and C. dilatata are exposed to, would determine levels of sunscreen compounds in each species. Higher TC and MAA levels in pre-hatching larvae of C. peruviana compared to C. dilatata, indicate a necessity of C. peruviana for protection against UV-R radiation during approximately 15days when their veliger larvae remain in the water column before metamorphosis is complete. Conversely, low photoprotective levels in pre-hatching juveniles of C. dilatata could be related to low UV-R exposure levels due to the direct incorporation to the benthos and the presence of a protective shell.

Ecosystem Alterations and Species Range Shifts: An Atlantic-Mediterranean Cephalaspidean Gastropod in an Inland Egyptian Lake.

The eastern Atlantic and Mediterranean marine Cephalaspidea gastropod Haminoea orbignyana was collected from Lake Qarun (Fayoum, Egypt), a landlocked lake that has undergone a shift from freshwater to estuarine conditions in the past 100 years. Species identity was confirmed by both morphological (anatomical dissection and scanning electron microscopy) and molecular methods (COI gene phylogeny). Observations suggested a robust population of H. orbignyana in the lake with a density of ca. 64 individuals/m2 and ca. 105 egg masses/m2 during surveys conducted in the summer of 2013. The vast majority of snails and egg masses were found under rocks. Observations of egg masses in the lab showed a gradual change from whitish to yellow-green as the eggs matured and the release of veliger larvae alone after about a week. Although adult cephalaspideans readily consumed filamentous red and green algae, and cyanobacteria, laboratory trials showed that they consumed significantly more of the red alga Ceramium sp., than of the green alga Cladophora glomerata, with consumption of Oscillatoria margaritifera being similar to those on the two algae. When grown on these resources for 16 days, H. orbignyana maintained their mass on the rhodophyte and cyanobacterium, but not in starvation controls. No cephalaspideans grew over the course of this experiment. Lake Qarun has been periodically restocked with Mediterranean fishes and prawns since the 1920s to maintain local fisheries, which represents a possible route of colonization for H. orbignyana. Yet, based on literature records, it seems more likely that invasion of the lake by this gastropod species has occurred only within the last 20 years. As human activities redistribute species through direct and indirect means, the structure of the community of this inland lake has become unpredictable and the long-term effects of these recent introductions are unknown.

Isolation-hypoxia and re-oxygenation of the pallial cavity of female Crepipatella dilatata during estuarine salinity changes requires increased glyoxylase activity and antioxidant metabolism to avoid oxidative damage to female tissues and developing embryos.

The estuarine slipper limpet Crepipatella dilatata is a gastropod that can survive prolonged periods of low salinities (< 24 PSU) caused by tidal changes and/or prolonged periods of rain. During low salinity events, C. dilatata can isolate its body from the outside environment, by sealing its shell against the substrate on which it grows. Prolonged isolation periods from the surrounding environment can greatly lower available oxygen levels inside of the pallial cavity, impacting on the physiology of both females and their incubated encapsulated embryos. When salinity levels return to normal, isolation is terminated and the inflow of seawater results in re-oxygenation. In this study we show that when re-oxygenation of the pallial cavity takes place, oxidative damage, in the form of increased levels of lipid peroxides and protein carbonyls, occurs in both maternal tissues and in incubated embryos. To avoid terminal oxidative damage both females and their embryos increase their levels of the glyoxalase pathway enzymes (GLX-I and GLX-II) and general antioxidant metabolism (SOD, CAT, GR, GPOX and GST). As a result the levels of oxidative damage decline to basal levels within 24 h of reoxygenation. Thus the combination of isolation, a behavioural strategy, combined with encapsulation of embryos and a capacity to up regulate relatively rapidly the glyoxylase pathway and general antioxidant metabolism, play major roles in facilitating the survival of C. dilatata in the small estuaries of Southern Chile.

Standardization of a Patella spp. (Mollusca, Gastropoda) embryo-larval bioassay and advantages of its use in marine ecotoxicology.

The use of three limpet species, Patella vulgata Linnaeus, 1758, Patella depressa Pennant, 1777 and Patella ulyssiponensis Gmelin, 1791 as model organisms in marine ecotoxicology has been evaluated. Initial laboratory experiments were aimed to standardize a biological test with embryos and larvae of Patella spp, establishing the percentage of normal trochophore larvae as endpoint. Before conducting in vitro fertilization, oocytes must be maturated artificially by incubation in an alkaline solution; therefore, alkalinizing agent, pH and time of eggs alkalinization were evaluated. Moreover, time of sperm activation, optimum sperm and oocytes concentration during fertilization, gamete contact time, use of stirring during the fertilization, egg concentration and incubation temperature were examined. Minimum sample size per treatment was also estimated. Exposure of oocytes for 10min to FSW alkalinized with NH4OH at pH 9.0, the use of undiluted sperm pre-activated during 45min and a concentration of 200 oocytesmL(-1), a gamete-contact time of 180min and egg incubation at 18°C during 24h at a concentration of 80 eggsmL(-1) were the conditions allowing maximal embryo-larval development success. With an error of 0.05, a sampling size ≥320 allows a 95% confidence in the estimate. This Patella spp. acute bioassay fulfills a number of important a priori requirements to be used in ecotoxicological studies. Nevertheless, in vitro fertilization requires considerable handling, which may lead to failure in fecundation. Such difficulties are also addressed, in order to facilitate the routine use of this protocol by other laboratories.