Mussel Gonad Research Articles

Fate and impact at molecular level of diatrizoic acid and iohexol contrast agents in Dreissena polymorpha mollusks.

Iodinated contrast media (ICMs) used in X-ray imaging for medical diagnostics are released into wastewater and then encountered in river water at concentrations ranging from several dozen to hundreds of µg/L, and even thousands of µg/L in hospital effluents. ICMs are considered as emerging pollutants as their occurrence and impact on ecosystems and the environment are poorly documented. Even if they are considered inert for humans, aquatic organisms are continuously exposed to ICMs, and their potential deleterious effects are therefore questioned as we have recently demonstrated that they enter into organisms such as the zebra mussels. To answer this question, Dreissena polymorpha were exposed to two ICMs of different osmolality, diatrizoic acid and iohexol, at an environmental concentration (100µg/L) for 21days before a depuration phase of 4days. The occurrence, fate, and impact of both ICMs in these organisms were studied using a metallomic approach. Thus, iodine as well as endogenous copper and zinc were quantified and analyzed in cytosolic extracts of digestive glands, gills, and gonads of mussels by size exclusion chromatography coupled to ICP MS. This work shows that a subcellular fractionation is necessary to distinguish variations in total element content. The cytosolic iodoprotein chromatographic pattern was consistent for the three organs and confirmed the presence of ICMs in cytosols. Additionally, this exploratory work tends to show a weak biological effect of ICMs with a substantial variation of the profile of Cu-binding proteins mostly in the gill cytosol and to a lesser extent, in the digestive gland cytosol.

A Metabolomic Analysis to Assess the Responses of the Male Gonads of Mytilus galloprovincialis after Heavy Metal Exposure.

In recent years, metabolomics has become a valuable new resource in environmental monitoring programs based on the use of bio-indicators such as Mytilus galloprovincialis. The reproductive system is extremely susceptible to the effects of environmental pollutants, and in a previous paper, we showed metabolomic alterations in mussel spermatozoa exposed to metal chlorides of copper, nickel, and cadmium, and the mixture with these metals. In order to obtain a better overview, in the present work, we evaluated the metabolic changes in the male gonad under the same experimental conditions used in the previous work, using a metabolomic approach based on GC-MS analysis. A total of 248 endogenous metabolites were identified in the male gonads of mussels. Statistical analyses of the data, including partial least squares discriminant analysis, enabled the identification of key metabolites through the use of variable importance in projection scores. Furthermore, a metabolite enrichment analysis revealed complex and significant interactions within different metabolic pathways and between different metabolites. Particularly significant were the results on pyruvate metabolism, glycolysis, and gluconeogenesis, and glyoxylate and dicarboxylate metabolism, which highlighted the complex and interconnected nature of these biochemical processes in mussel gonads. Overall, these results add new information to the understanding of how certain pollutants may affect specific physiological functions of mussel gonads.

Sexual dimorphism in the gonad lipidome of blue mussels (Mytilus sp.): New insights from a global lipidomics approach

Blue mussels (Mytilus sp.) are an economically important species for European aquaculture. Their importance as a food source is expected to increase in the coming net-zero society due to their low environmental footprint; however, their production is affected by anthropogenic stressors and climate change. During reproduction, lipids are key molecules for mussels as they are the main source of energy on which newly hatched embryos depend in the first days of their development. In this work, blue mussels of different origins are analysed, focusing on the differences in lipid composition between the ovary (BMO) and the testis (BMT). The lipidome of blue mussel gonads (BMG) is studied here by combining traditional lipid profiling methods, such as fatty acid and lipid class analysis, with untargeted liquid chromatography-mass spectrometry (LC-MS) lipidomics. The approach used here enabled the identification of 770 lipid molecules from 23 different lipid classes in BMG. BMT, which consists of billions of spermatocytes, had greater amounts of cell membrane and membrane lipid components such as FA18:0, C20 polyunsaturated fatty acids (PUFA), free sterols (ST), ceramide phosphoethanolamines (CerPE), ceramide aminoethylphosphonates (CAEP), cardiolipins (CL), glycerophosphocholines (PC), glycerophosphoethanolamines (PE) and glycerophosphoserines (PS). In BMO, saturated fatty acids (FA14:0 and FA16:0), monounsaturated fatty acids (MUFA) and other storage components such as C18-PUFA accumulated in triradylglycerolipids (TG) and alkyldiacylglycerols (neutral plasmalogens, TG O-), which, together with terpenes, wax esters and cholesterol esters, make up most of oocytes yolk reserves. BMO also had higher levels of ceramides (Cer) and generally alkyl/alkenyl glycerophospholipids (mainly plasmanyl/plasmenyl PC), suggesting a role for these lipids in vitellogenesis. Non-methylene interrupted dienoic fatty acids (NMID FA), typically found in plasmalogens, were the only membrane-forming PUFA predominantly detected in BMO. The results of this study are of great importance for clarifying the lipid composition of BMG and provide an important basis for future studies on the reproductive physiology of these organisms.

Effect of cadmium in the gonads of mussel (Mytilus coruscus): an ionomics and proteomics study.

The mussel Mytilus coruscus is an important and very popular seafood in China and widespread along the eastern coast of China. In this study, we investigated the molecular response of mussel gonads to cadmium accumulation at two concentrations (80 and 200µg/L) for 30days using ionomics and proteomics techniques. The shrinkage of the cells and moderate hemocytic infiltration were observed in the Cd-treated groups. The strontium, selenium (Se), and zinc contents were significantly altered, and the relationships between iron, copper, Se, manganese, calcium, sodium, and magnesium were also significantly altered. Label-free quantitative proteomics analysis revealed a total of 227 differentially expressed proteins. These proteins were associated with multiple biological processes, including the tricarboxylic acid cycle, structural reorganization of cells, biosynthesis of amino acids, inflammatory response of cells, and tumorigenesis. Nonetheless, our ionomics and proteomics analysis revealed that mussels could partly alleviate the adverse effects of Cd by altering the metal contents and correlations between minerals, thereby enhancing the biosynthesis of some amino acids and activity of antioxidant enzymes. Overall, this study provides an insight into the mechanism underlying Cd toxicity in mussel gonads from a metal and protein perspective.

Ecotoxicity of emerging contaminants in the reproductive organ of marine mussels Mytilus galloprovincialis

Contaminants of emerging concern (CECs) present a new threat to the marine environment, and it is vital to understand the interactions and possible toxicity of CEC mixtures once they reach the ocean. CECs—such as metal nanoparticles, nanoplastics, and pharmaceuticals—are groups of contaminants some of which have been individually evaluated, though their interactions as mixtures are still not fully understood. To ensure a healthy and prosperous future generation, successful reproduction is key: however, if hindered, population dynamics may be at danger leading to a negative impact on biodiversity. This study aimed to understand the effects of silver (20 nm nAg, 10 μg/L), polystyrene nanoparticles (50 nm nPS, 10 μg/L), and 5-fluorouracil (5FU, 10 ng/L) individually and as a mixture (10 μg/L of nPS + 10 μg/L of nAg +10 ng/L of 5FU) in the gonads of Mytilus galloprovincialis. A multibiomarker approach, namely the antioxidant defence system (ADS; superoxide dismutase, catalase, glutathione peroxidases, glutathione – S – transferases activities), and oxidative damage (OD; lipid peroxidation) were analysed in the gonads of mussels. All exposure treatments after 3 days led to an increase of enzymatic activity, followed by an inhibition after 14 and 21 days. Thus, ADS was overwhelmed due to the generation of ROS, resulting in OD, except for nPS exposed mussels. The OD in Mix exposed mussels increased exponentially by 57-fold. When CEC mixtures interact, they are potentially more hazardous than their individual components, posing a major threat to marine species. To understand synergistic and antagonistic interactions, a model was applied, and antagonistic interactions were observed in evaluated biomarkers at all time-points, apart from a synergistic interaction at day 3 relative to LPO. Results indicate that the effects observed in Mix-exposed mussel gonads are mainly due to the interaction of nAg and 5FU but not nPS.

Analysis of the Elemental Composition of Gonads, Gametes and Larvae of the Mussel <i>Mytilus galloprovincialis</i> in the Spawning Period

In this work, we studied the elemental composition of gonads, eggs, sperm and larvae of the mussel Mytilus galloprovincialis during spawning, when the contents of biologically active compounds and minerals are maximal. A comparative analysis of the contents showed that the elements are involved in the mussel gametogenesis in different pathways. There were significant differences in the contents of Li, B, Mg, Si, P, K, Ca, V, Cr, Mn, Fe, Co, Ni, Zn, Ge, As, Se, Br, Rb, Sr, Mo, Pd, Sn, I, Ba and Ce in male and female gonads before and after spawning, gametes and larvae of mussels. It is likely that some of the listed elements are not only passively accumulated in the mollusk body, but are also essential, being directly involved in the reproduction process. Most of significant differences in the element contents were found between eggs and larvae and between male gonads before spawning and sperm. By spawning, mussel gonads accumulated mainly p- and d-elements, apparently due to the ability of their ions to form complexes and, consequently, to be included in the structure of enzymes. The contents of B, Mg, Si, P, K, Ca, Cr, Mn, Fe, Cu, Zn, As, Se, Br, Sr and Ba in male and female gonads, gametes and larvae were an order or several orders of magnitude higher than those of other elements. The contents of Ca, Fe, Sr, Sn and I in mussel larvae were significantly higher than in gonads and gametes. Sn is a technological hydropollutant, and thus, larvae can serve as a bioindicator of the aquatic environment pollution with this element. The data obtained are of practical interest for improving the biotechnology of reproduction of marine hydrobionts and obtaining functional products based on them, which is important for the optimization of aquaculture management and for addressing human health-related issues.

Transcriptome analysis reveals sex-specific alterations in gonads of green mussel exposed to organophosphorus insecticide triazophos

Triazophos (TP) is a widespread pollutant in aquatic environments. A sex-specific metabolic response in green-lipped mussel Perna viridis to TP exposure was observed in our previous study, and this led us to investigate the mechanisms associated with its toxicity. P. viridis were subjected to chronic exposure (15 days) to TP at 35 μg/L to compare the sex-biased transcriptomic profiles in the gonads of male and female mussels. We identified 632 differentially expressed genes (DEGs) (348 up-regulated and 284 down-regulated) in TP-exposed males, and only 61 DEGs (9 up-regulated and 52 down-regulated) in TP-exposed females. Many DEGs were found to be involved in the nervous, reproductive endocrine, oxidative stress, and immune systems of P. viridis. Additionally, enzymatic activity analysis indicated TP induced neurotoxic effects and oxidative damage to the mussels. Our results demonstrate that the stress response and molecular mechanisms of TP toxicology are different between female and male mussels.

Morphological, Gene, and Hormonal Changes in Gonads and In-Creased Micrococcal Nuclease Accessibility of Sperm Chromatin Induced by Mercury.

Mercury is one of the most dangerous environmental pollutants. In this work, we analysed the effects of exposure of Mytilus galloprovincialis to 1, 10 and 100 pM HgCl2 for 24 h on the gonadal morphology and on the expression level of three stress genes: mt10, hsp70 and πgst. In this tissue we also evaluated the level of steroidogenic enzymes 3β-HSD and 17β-HSD and the expression of PL protein genes. Finally, we determined difference in sperm chromatin accessibility to micrococcal nuclease. We found alterations in gonadal morphology especially after exposure to 10 and 100 pM HgCl2 and hypo-expression of the three stress genes, particularly for hsp70. Furthermore, decreased labelling with both 3β-HSD and 17β-HSD antibodies was observed following exposure to 1 and 10 pM HgCl2 and complete absence at 100 pM HgCl2 exposure. Gonads of mussels exposed to all HgCl2 doses showed decreased expression of PL protein genes especially for PLIII. Finally, micrococcal nuclease digestions showed that all doses of HgCl2 exposure resulted in increased sperm chromatin accessibility to this enzyme, indicative of improper sperm chromatin structure. All of these changes provide preliminary data of the potential toxicity of mercury on the reproductive health of this mussel.

Steroid hormones, selenium, and zinc in the gonads – gametes – larvae biological system of the mussel Mytilus galloprovincialis Lam.

Assessment of the interaction of marine farms with the environment in the industrial cultivation of the mussel Mytilus galloprovincialis is very important. In the mussel farm – environment system, biotic fluxes of chemical compounds through gonads, gametes (sperm and eggs), and larvae make a considerable contribution to this interaction. Since gonads play a key role in the mussel reproduction, it is interesting to study the budget of materials, that are directly involved in this process. Out of these materials, testosterone, estradiol, fatty acids, and some trace minerals, such as Se and Zn, are known to affect spawning, growth, and development. The molluscs absorb these materials from food and water. These materials are partly metabolically assimilated by mussels and partly excreted into the environment with gametes. The aim of this study was to estimate the components of the budget of steroid hormones, fatty acids, and two essential trace elements (Zn and Se) in mussel gonads, gametes, and larvae. The total testosterone and estradiol in gonads and gametes were quantified by enzyme-linked immunosorbent assay. The contents of the trace elements were found using inductively coupled plasma mass spectrometry. The fatty acid composition was determined by means of gas chromatography–mass spectrometry. The contents of Se and Zn in mussel gonads and gametes were found to depend on the stage of the reproductive cycle. In female gonads, Se and Zn concentrations were higher than in male ones. The highest concentration of Se was recorded in eggs: (14.7 ± 2.9) μg·g−1 dry weight (d. w.). In sperm, it was (14.4 ± 1.8) μg·g−1 d. w. Zn content in gonads before spawning was higher than in gametes. In male gonads and in sperm, its values were (27.5 ± 3.7) and (19.3 ± 6.4) μg·g−1 d. w., respectively. In female gonads and eggs, the contents of zinc were (53.6 ± 10.9) and (49.3 ± 8.2) μg·g−1 d. w., respectively. In spring, the mean values of Se and Zn assimilation degree (q) in gonads of the mussel were within 0.1–0.6. The limit values of the alimentary accumulation coefficient (Klim) of Se and Zn ranged 0.6 to 1.4. While spawning, mussels excrete polyunsaturated fatty acids (PUFA), which are probably used by other marine organisms. Up to 56.2 % of PUFA are excreted with sperm, and 48.1 %, with eggs, whereas in larvae this fraction does not exceed 10.2 %. The data obtained indicate that the molluscs assimilate sex hormones, fatty acids, selenium, and zinc to maintain vital processes: prostaglandins are synthesized from PUFA in the body, and testosterone esters are formed from testosterone. Se and Zn, when coupled with proteins, play a key role in the reproduction and formation of larval shells.

Vitellogenin gene expression in marine mussels exposed to ethinylestradiol: No induction at the transcriptional level

Vitellogenin (Vtg), a large multidomain protein precursor of egg-yolk proteins, is used as an endocrine disruption biomarker in fish, and in the last decades, its use has been extended to invertebrates like mollusks. However, it remains unclear whether invertebrate endocrine system produces Vtg in response to estrogens, like it occurs in oviparous vertebrates. In a previous study, no evidence of induction of Vtg expression at protein level was found in gonads of the marine mussel Mytilus galloprovincialis after exposure to the estrogenic chemical 17α-ethinylestradiol (EE2). In the present follow-up study, it was investigated whether there is any effect of EE2 on Vtg abundance at transcriptional level in M. galloprovincialis gonads. To this aim, RT-qPCR analysis targeting three different domains of Vtg transcript was performed on gonads of mussels that were exposed either 4 or 24 days to 100 ng/L EE2. In addition, several reference genes were analysed and a selection of these for potential use in further RT-qPCR analyses on mussel male and female gonads is provided. Results showed higher expression in females than in males for the three analysed Vtg domains, and no evidence of Vtg mRNA induction due to EE2 either in females or males. The present results, together with those obtained from previous analysis at protein level, support that Vtg is not an adequate biomarker for xenoestrogenicity in marine mussels. Additionally, nucleotide sequences of Vtg transcripts of three closely-related species from Mytilus edulis complex (M. galloprovincialis, M. edulis and M. trossulus) are provided and compared with Vtg sequences from other mollusk species to assess the level of conservation and evolutionary relationships among species.

Stress responses in expressions of microRNAs in mussel Mytilus galloprovincialis exposed to cadmium

MicroRNAs (miRNAs) are known to have complicated functions in aquatic species, but little is known about the role of miRNAs in mollusk species under environmental stress. In this study, we performed small RNA sequencing to characterize the differentially expressed miRNAs in different tissues (whole tissues, digestive glands, gills, and gonads) of blue mussels (Mytilus galloprovincialis) exposed to cadmium (Cd). In summary, 107 known miRNAs and 32 novel miRNAs were significantly (p < 0.01) differentially expressed after Cd exposure. The peak size of miRNAs was 22 nucleotides. Target genes of these differentially expressions of miRNAs related to immune defense, apoptosis, lipid and xenobiotic metabolism showed significant changes under Cd stress. These findings provide the first characterization of miRNAs in mussel M. galloprovincialis and expressions of many target genes in response to Cd stress.

No evidence that vitellogenin protein expression is induced in marine mussels after exposure to an estrogenic chemical

A wide variety of endocrine disrupting chemicals reach the marine environment and can cause harmful effects in different marine organisms. Vitellogenin (Vtg), the egg-yolk precursor, is a commonly used endocrine disruption biomarker in fish and more recently in marine invertebrates under the assumption of high expected similarities in the endocrine system of vertebrates and invertebrates. However, this assumption has been recently questioned. The results from previous studies focused on bivalve molluscs showed that Vtg induction could be misleading because of the use of either non-robust or indirect techniques to measure Vtg. In this study, mussels (Mytilus galloprovincialis) were exposed to either 10 or 100 ng/L of the synthetic hormone 17α-ethinylestradiol (EE2) at different exposure times (4 and 24 days) and under different feeding regimes (representing different energy balances), and Vtg levels in both male and female mussel gonads were quantified by label free shotgun LC-MS/MS proteomic analysis. Vtg protein was not detected in male gonads. In female gonads, Vtg levels were not significantly affected by EE2 at any exposure time or EE2 concentration tested, whereas a significant correlation was found between the degree of maturation of the gonad and Vtg levels in females. Results obtained in the present study critically question the use of Vtg as a biomarker of endocrine disruption in marine mussels, and show that the degree of maturation of the gonad can be an important confounding factor in the attempts to evaluate estrogenic effects through Vtg measurement in mussel gonads.

Effects of short-term exposure of paracetamol in the gonads of blue mussels Mytilus edulis.

A growing body of literature suggests that pharmaceutical contamination poses an increasing risk to marine ecosystems. Paracetamol or acetaminophen is the most widely used medicine in the world and has recently been detected in seawater. Here, we present the results of 7 days' exposure of blue mussel adults to 40 ng/L, 250 ng/L and 100 μg/L of paracetamol. Histopathology shows that haemocytic infiltration is the most observed condition in the exposed mussels. The mRNA expression of VTG, V9, ER2, HSP70, CASP8, BCL2 and FAS in mussel gonads present different patterns of downregulation. VTG and CASP8 mRNA expression show downregulation in all exposed mussels, irrespective of sex. The V9, HSP70, BCL2 and FAS transcripts follow a concentration-dependent variation in gene expression and may therefore be considered good biomarker candidates. ER2 mRNA expression shows a downregulated trend, with a clearer dose-response relationship in males. In conclusion, this study suggests that paracetamol has the potential to alter the expression of several genes related to processes occurring in the reproductive system and may therefore impair reproduction in blue mussels.

Gonadal Histopathological Disorders in Mytilus galloprovincialis Male Exposed to Tars Used in Mussel Farms

Polycyclic aromatic hydrocarbons (PAHs) and trace metals are pollutants widely distributed in the marine environment with toxic effects on live organisms, but few laboratory studies have proved their causal relationship with histopathologic alterations from invertebrates. We analyse the histopathologic effects of a tar mixture used in mussel farms on the gonad of male mussels. Mussels were exposed to water-accommodated fractions from different sub-lethal concentrations of that tar mixture during different times. The accumulation of PAHs and metals in mussels was quantified in each one tar exposure condition, and a comparative histological and morphometric analysis of the male gonad was performed, as well as of the gametogenic disorders and the cellular pathologies induced. Spermatogenesis disruption and alterations of somatic and germinal cells were observed as a direct effect of treatment. The pathologies observed followed an increasing malignity sequence depending on dose and exposure time: spawning induction; arrest of spermatogenesis and spermiogenesis; suppression of immune response; dysplasia of Sertoli and connective cells and finally, severe disorders of germinal cells. We demonstrate for the first time that tar used in waterproofing of floating rafts from the mussel farms causes severe effects on male mussel gonad, constituting a direct risk to the health of these organisms, affecting gametogenesis, quality of gametes and, ultimately, the sustainability of this resource and their quality for human consumption. Moreover, we propose the pathologies described in mussel as biomarkers of marine pollution and the mussel gonad as study model of testicular toxicity mechanisms in male reproduction.

Development in culture of larval spotted spiny lobster Panulirus guttatus (Latreille, 1804) (Decapoda: Achelata: Palinuridae)

AbstractThere is little information on the early life history of the spotted spiny lobster Panulirus guttatus (Latreille, 1804), an obligate reef resident, despite its growing importance as a fishery resource in the Caribbean and as a significant predator. We cultured newly-hatched P. guttatus larvae (phyllosomata) in the laboratory for the first time, and the growth, survival, and morphological descriptions are reported through 324 days after hatch (DAH). Phyllosomata were cultured at 25 °C in a flow-through seawater system within a series of custom 80 l plankton-kreisel tanks and provided with ongrown Artemia and mussel gonad. Mean body length (BL) of phyllosomata was 1.70 mm (N = 10) at hatch and increased linearly to 22.20 mm at 226 DAH (N = 3). Morphological characters from a total of 164 sampled phyllosomata were ascribed to nine distinct developmental stages (stages I-IX), and described and illustrated. Although no final stage phyllosomata (stage X) were obtained, the BL in the final stage was extrapolated at 39.6 mm using a Gompertz function, expressing the relationship between phyllosoma stages and BL. The total duration of phyllosomata for P. guttatus was estimated at 410 d, with the 5th and 95th percentiles at 334 and 526 d, respectively. Our data suggest that P. guttatus has a larger body size in the final larval stage and a substantial pelagic larval duration compared with other related panulirid lobsters in its geographic range. The described morphological and biological attributes associated with the early-life history for this species can inform future studies, and add value to models of distribution and population connectivity.

The effect of conspecific interaction on survival, growth and feeding behaviour of early juvenile tropical spiny lobster Panulirus ornatus

Behaviour underpins many facets of the performance of animals in aquaculture. By manipulating culture systems to segregate or allow particular aspects of conspecific interaction, we found physical interactions between P. ornatus individuals to be essential for better culture performance. Three culture types were used to control conspecific interactions: isolated culture (individual vessels) excluded all conspecific interactions, separated culture (lobsters cultured in adjacent cages) excluded physical interactions, and communal culture allowed for all interactions. Two water exchange rates were introduced to investigate the influence of chemical cue intensity on growth and survival. Time-series photography was used to determine the feeding behaviour and preference for different feeds including mussel gonad, commercial prawn feed and moist feed. The experiment showed improved growth and moulting frequency in communally cultured lobsters. These results suggest that direct physical contact between conspecifics is required to optimise growth of lobsters, which may be related to the complex social structures of this gregarious species. Behavioural observations of two juvenile instars (2 and 4), revealed circadian rhythm of interactions with feeds, feed preferences and intake. Observations revealed differing behaviours between the different culture types, where lobsters reared in separation displayed higher level of interactions with feeds; however this was not associated with higher feed intake. Observations of two juvenile instars (2 and 4) exhibited increase of daylight activity (interactions with feeds and feed intake) in older lobsters (instar 4).

Catostylus tagi: survival and maintenance trials of planula and polyps

Introduction: Besides the knowledge of life cycles in nature, the sustained use of aquatic biological resources also requires the development of know-how to reproduce and maintain them in captivity [1]. The scyphozoan Catostylus tagi, native to the Tagus and Sado estuaries, has already shown applicability in the areas of gastronomy and drugs for humans and has potential to be used as food for aquaculture organisms [2]. In this work we started to investigate the benthic life stages of C. tagi and their maintenance in laboratory. Materials and methods: In September 2017, gonads of 20 specimens of C. tagi (12 males and 8 females) were collected in the Tagus estuary. Pairwise samples of male and female gonads were placed in containers with 80 mL of Tagus water and different substrate for in vitro fertilization. The settlement, growth and survival of the polyps were monitored daily by microscope. The survival rate was calculated as the daily percentage of live polyps and planulae, relative to the total number of initial planulae (Table 1). Polyps were cultured under constant conditions of temperature (19 ± 1 °C) and salinity (34 ± 1 ‰) and kept in the dark to minimize the algae growth. When the polyps developed 6 tentacles, they were fed every 2 days, alternating between macerated female mussel gonads and rotifers supplied ad libitum. Waste particles and food remnants were removed daily from the containers. Between 20-50% of the water in each container was replaced with filtered Tagus water after the polyps had been fed. Statistical approach was made by Mann-Whitney U test, considering p <0.05. Results: Polyp survival rate was high on the second day (∼81%), but with a marked fall on the day after, and a trend for stability with some decrease on the following days (∼50%) (Figure 1). The highest survival rate was observed in containers that had substrate, specifically wood and plastic, while the lowest rate was observed in containers without substrate. Sand and shell were the substrates with the lowest results, the latter having a survival rate below 50% in the last days. Figure 1. Rate of C. tagi polyps survival. Discussion and conclusions: At the conditions of temperature and salinity used in this trial, statistical comparison confirmed that the survival rate of the polyps was significantly different in environments with and without substrate. Other authors observed similar rates of growth and survival in related species [3].

Surf-zone hydrodynamics alter phytoplankton subsidies affecting reproductive output and growth of tidal filter feeders.

Surf zones, classified from reflective to dissipative, separate the ocean from shore and subsidies from the coastal ocean must pass through surf zones to reach the shore. We have observed that variations in phytoplankton concentrations in the water over the intertidal zone varied with surf-zone hydrodynamics and we hypothesized that this variation would alter growth rates, population structure, and reproductive output of Mytilus californianus and Balanus glandula. From May 2016 to April 2017, along 7km of Cape Arago, Oregon, USA surf-zone phytoplankton concentrations were determined weekly at nine sites with varying surf-zone hydrodynamics as indicated by surf-zone widths. Throughout the year, concentrations of phytoplankton in wider, more dissipative surf zones were, on average, 16× higher than in narrow, more reflective surf zones. Similar to previous observations, surf-zone width explained >90% of the variability in phytoplankton concentrations in the surf-zone. On average, ~83% of B. glandula had egg lamellae at more dissipative shores compared to only 8% at more reflective. An index of potential reproductive output by barnacle populations as measured by ash free dry mass (AFDM) of egg lamellae was ~243× larger at more dissipative than at reflective shores and surf-zone phytoplankton concentrations and surf-zone width explained ~96 and 92% of the variability in this index. On average, density of M.californianus was ~2× higher at more reflective shores, but 60% of these individuals were smaller and non-reproductive compared to only 24% at the more dissipative sites. The gonad tissue mass/m2 of medium sized mussels was ~5× greater at more dissipative than reflective shores. Surf-zone phytoplankton concentrations and surf-zone width explained ~80% and 65% of the variability in individual mussel gonad mass and ~69% and 56% of the variability in mussel population gonad mass, respectively. M.californianus were out-planted to assess growth rates and, after 5months, average body mass was ~3× greater at more dissipative than reflective shores. Surf-zone phytoplankton concentrations and width explained ~85% and 92% of the mass increase, respectively. Phytoplankton subsidies varied with surf-zone hydrodynamics altering the growth and reproductive output of two ecologically important intertidal filter feeders.

Molecular characterization and expression of the feminization-1c (fem-1c) in the freshwater mussel (Hyriopsis cumingii)

The feminization-1c (fem-1c) gene has been shown to be associated with sex differentiation and determination in many metazoan species. It belongs to the fem-1 family which is a member of the ANK superfamily. In this study, the full-length cDNA of the fem-1c (Hcfem-1c) gene was isolated from the freshwater mussel (Hypriopsis cumingii). The isolated Hcfem-1c cDNA was 2196 bp in length and encoded a putative protein of 621 amino acids that contains seven ANK domains. Phylogenetic analysis of the deduced HcFEM-1C protein showed that it clustered with the other invertebrates homologues, indicating that the sequence of HcFEM-1C was conserved during evolution. Quantitative real-time PCR (qPCR) expression revealed that the Hcfem-1c gene was expressed in the adductor muscle, foot, liver, gill, kidney, mantle, and gonads of male and female adult mussels (two years old). In the gonads Hcfem-1c was much less abundant in males than that in females. During early development of the gonads, Hcfem-1c transcripts were significantly increased in the primordial germ cell differentiation stage (5 months old). We hypothesized that Hcfem-1c probably regulates female gonad differentiation. In situ hybridization showed that a strong and specific signal concentrated in the female oocyte cell membrane and male follicular wall, indicating that Hcfem-1c gene may not only be involved in female gonad differentiation, but also participates in egg development. This study laid the foundations for a better understanding of gender differentiation mechanism in H. cumingii.

Shotgun Proteomics Analysis Discards Alkali Labile Phosphate as a Reliable Method To Assess Vitellogenin Levels in Mytilus galloprovincialis.

Vitellogenin, the egg yolk precursor, is a well-known biomarker of endocrine disruption in oviparous vertebrates. In invertebrates, such as bivalves, it has been used in the last 10 years for the same purpose, despite the limited knowledge of invertebrate endocrinology. In bivalves, vitellogenin levels are usually estimated using an indirect technique, alkali labile phosphate (ALP), that assumes that vitellogenin is the most abundant phosphorylated protein in the analyzed tissue. In this study, we applied shotgun proteomics for the identification and quantification of vitellogenin in marine mussel gonads and compared the results with those obtained with the ALP method. The proteomic analysis revealed that vitellogenin is only detected in female gonads with expression levels that are rather variable among female mussels at different stages of gonad development. ALP analysis, on the contrary, detected similar amounts of phosphorylated proteins regardless of sex or gonad development stage. These results show evidence that the ALP method is not providing reliable information about Vtg levels, at least in marine mussel gonads. ALP is not a good proxy to assess Vtg levels in marine mussels, and careful verification of the adequacy of the procedure should be done before ALP is further assumed as a proxy of Vtg in other bivalve mollusks.