Surface-Functionalized Polystyrene Nanoparticles Alter the Transmembrane Potential via Ion-Selective Pores Maintaining Global Bilayer Integrity.

Although nanoplastics have well-known toxic effects toward the environment and living organisms, their molecular toxicity mechanisms, including the nature of nanoparticle-cell membrane interactions, are still under investigation. Here, we employ dynamic light scattering, quartz crystal microbalance with dissipation monitoring, and electrophysiology to investigate the interaction between polystyrene nanoparticles (PS NPs) and phospholipid membranes. Our results show that PS NPs adsorb onto lipid bilayers creating soft inhomogeneous films that include disordered defects. PS NPs form an integral part of the generated channels so that the surface functionalization and charge of the NP determine the pore conductive properties. The large difference in size between the NP diameter and the lipid bilayer thickness (∼60 vs ∼5 nm) suggests a particular and complex lipid-NP assembly that is able to maintain overall membrane integrity. In view of this, we suggest that NP-induced toxicity in cells could operate in more subtle ways than membrane disintegration, such as inducing lipid reorganization and transmembrane ionic fluxes that disrupt the membrane potential.

Uptake and effects of different concentrations of spherical polymer microparticles on Artemia franciscana.

Artemia cysts have a huge economic importance for the aquaculture sector due to the fact that they are used as live feed for larviculture. Microplastics (MPs) are common and emergent pollutants in the aquatic environments, with unknown and potential long-term effects on planktonic species such as Artemia spp. When used as live feed, Artemia could transfer contaminants to fish along the food chain, with possible adverse effects on human health through their consumption. This study aims to assess the uptake of different concentrations of spherical polymer microparticles (FRM) (1-5 µm diameter) and their associated chronic effects on feeding, growth, mortality, and reproductive success from juvenile to adult stage of brine shrimp Artemia franciscana. Individuals were exposed for 44 days to 0.4, 0.8 and 1.6 mg.L-1 of FRM. No significant detrimental effects on growth, ingestion and mortality rates of A. franciscana were observed in all tested conditions. However, reproductive success was strongly affected by the increase of MP concentrations. The results of the present study showed that A. franciscana juveniles and adults were able to survive different experimental MP concentrations, but their reproductive success and progeny were significantly impacted by exposure to FRM particles.

Biosynthesis of Polyunsaturated Fatty Acids in Octopus vulgaris: Molecular Cloning and Functional Characterisation of a Stearoyl-CoA Desaturase and an Elongation of Very Long-Chain Fatty Acid 4 Protein.

Polyunsaturated fatty acids (PUFAs) have been acknowledged as essential nutrients for cephalopods but the specific PUFAs that satisfy the physiological requirements are unknown. To expand our previous investigations on characterisation of desaturases and elongases involved in the biosynthesis of PUFAs and hence determine the dietary PUFA requirements in cephalopods, this study aimed to investigate the roles that a stearoyl-CoA desaturase (Scd) and an elongation of very long-chain fatty acid 4 (Elovl4) protein play in the biosynthesis of essential fatty acids (FAs). Our results confirmed the Octopus vulgaris Scd is a ∆9 desaturase with relatively high affinity towards saturated FAs with ≥ C18 chain lengths. Scd was unable to desaturate 20:1n-15 (∆520:1) suggesting that its role in the biosynthesis of non-methylene interrupted FAs (NMI FAs) is limited to the introduction of the first unsaturation at ∆9 position. Interestingly, the previously characterised ∆5 fatty acyl desaturase was indeed able to convert 20:1n-9 (∆1120:1) to ∆5,1120:2, an NMI FA previously detected in octopus nephridium. Additionally, Elovl4 was able to mediate the production of 24:5n-3 and thus can contribute to docosahexaenoic acid (DHA) biosynthesis through the Sprecher pathway. Moreover, the octopus Elovl4 was confirmed to play a key role in the biosynthesis of very long-chain (>C24) PUFAs.

Characterization of type "B" esterases and hepatic CYP450 isoenzimes in Senegalese sole for their further application in monitoring studies.

In fish, the role that cholinesterases (ChEs) play in tissues other than those implicated in neural activity, as well as the involvement of carboxylesterases (CbEs) and cytochrome P450 isoenzymes (CYPs) in drug metabolism needs investigation. For that, Senegalese sole (Solea senegalensis) specimens were selected for characterization of several type B esterases and hepatic CYPs in order to further use this fish as sentinel. ChEs (acetylcholinesterase (AChE) and pseudocholinesterases (butyrylcholinesterase-BuChE and propionilcholinesterase-PrChE)) and CbEs were measured in brain, plasma, kidney, liver, gonad, muscle and gills. Moreover, seven fluorimetric substrates were selected to study CYP related activities in fish liver. The results showed that AChE was the dominant ChE form in brain whereas pseudocholinesterases were absent in most tissues, as demonstrated by low enzymatic activities using specific substrates and the lack of inhibition by iso-OMPA. Plasma exhibited trace activities of all the esterases assayed and no BuChE activity. CbEs were dominant in liver, but they were also present in kidney and brain. For CbE determination, α-naphtyl acetate (αNA) was seen as the most adequate substrate as it displayed higher enzymatic activities and showed more in vitro sensitivity to the carbamate eserine and the organophosphate pesticide dichlorvos. Alkoxyresorufin-O-dealkylase (EROD and BFCOD) activities, indicative in mammals of CYP1A and CYP3A subfamilies, respectively, were the highest microsomal CYP-related activities in liver. The results of this preliminary work allow us to select the most adequate esterase substrate, tissue and hepatic CYP substrate for further monitoring studies.

Effect of short-term exposure to fluorescent red polymer microspheres on Artemia franciscana nauplii and juveniles.

Microplastics (MPs) are ubiquitously present in the world's seas with unknown potential toxic effects on aquatic ecosystems. The aim of this study was to evaluate biochemical responses caused by 1-5 µm diameter plastic fluorescent red polymer microspheres (FRM), under short-term exposure of nauplii and juveniles of Artemia franciscana, using a set of biomarkers involved in important physiological processes such as biotransformation, neuronal transmission and oxidative stress. Two FRM concentrations (0.4 and 1.6 mg mL-1) present in the water at ecologically relevant concentrations were used to study their toxicity. No significant differences were found in growth, survival and feeding behaviour of nauplii, after 2 days of exposure to both FRM concentrations. However, in juveniles, survival decreased after 5 days of exposure to FRM1.6; but no significant differences were found in either growth or feeding behaviour. It was observed that nauplii and juveniles, under short-term exposure, had the ability to ingest and egest FRM particles, although their accumulation was higher in nauplii than in juveniles, maybe related with the capacity of the latter to empty their gut content faster, in the presence of food. Regarding biomarkers responses in nauplii, all enzymatic activities increased significantly, after short-term exposure to the higher FRM concentration tested (FRM1.6), which could be related with detoxifying MPs-triggered oxidative stress. In juveniles, the inhibition of ChE and the decrease in the activity of antioxidant enzymes, after 5 days of exposure to FRM1.6, might indicate a neurotoxic effect and oxidative damage induced by FRM. This study provides further evidences that accumulation of MPs in the gut by nauplii and juveniles of A. franciscana can induce negative effects on important physiological processes with influence on their health, highlighting the general concern about the negative effects of MPs pollution on aquatic species, as well as the need to understand the mechanism of MPs toxicity and its possible impacts on environmental safety. Graphical abstract.

De novo transcriptome reconstruction in aquacultured early life stages of the cephalopod Octopus vulgaris.

Cephalopods have been considered enigmatic animals that have attracted the attention of scientists from different areas of expertise. However, there are still many questions to elucidate the way of life of these invertebrates. The aim of this study is to construct a reference transcriptome in Octopus vulgaris early life stages to enrich existing databases and provide a new dataset that can be reused by other researchers in the field. For that, samples from different developmental stages were combined including embryos, newly-hatched paralarvae, and paralarvae of 10, 20 and 40 days post-hatching. Additionally, different dietary and rearing conditions and pathogenic infections were tested. At least three biological replicates were analysed per condition and submitted to RNA-seq analysis. All sequencing reads from experimental conditions were combined in a single dataset to generate a reference transcriptome assembly that was functionally annotated. The number of reads aligned to this reference was counted to estimate the transcript abundance in each sample. This dataset compiled a complete reference for future transcriptomic studies in O. vulgaris.

Time-dependent effects of polystyrene nanoparticles in brine shrimp Artemia franciscana at physiological, biochemical and molecular levels.

Micro- (<5 mm) and nanoplastics (<1 µm) are emerging threats for marine ecosystems worldwide. Brine shrimp Artemia is recognized as a suitable model among planktonic species for studying the impact of polystyrene nanoparticles (PS NPs) through short and long-term bioassays. Our study aims to evaluate the time-dependent effects of cationic amino-modified PS-NH2 (50 nm) in A. franciscana after short- (48 h) and long-term exposure (14 days). For this purpose, nauplii were exposed to a concentration range of PS-NH2 (0.1, 1, 3 and 10 µg/mL) in natural sea water (NSW), and physiological, biochemical and molecular responses were investigated. Short-term exposure to PS-NH2 caused a decrease in nauplii growth and affected the development in a concentration-dependent manner, long-term exposure impaired the survival, but not the growth and feeding behavior. Oxidative stress was detected after short term exposure as the decrease in the activity of antioxidant enzymes, and was fully evident in the long-term as lipid peroxidation, suggesting an accumulative effect. The decrease in Cholinesterase (ChE) activity observed indicates possible neurotoxic action of PS-NH2. Also, Carboxylesterase (CbE) inhibition by PS-NH2, described for the first time in this study, anticipates potential effects in biotransformation of exogenous and endogenous compounds, being the crustacean juvenile hormone methyl farnesoate (MF) that regulates development and molting, one candidate. Furthermore, short- and long-term exposure to PS-NH2 affect the expression of genes involved in cell protection, development and molting. Overall, our results reveal that low PS-NH2 concentrations induce physiological, biochemical and molecular (changes in gene expression) alterations in Artemia, and point at their potential risk for this model organism, supporting the general concern about nanoplastics occurrences in aquatic environments and their ability to represent an ecological threat for aquatic zooplanktonic species.

Life history and physiological responses of native and invasive brine shrimps exposed to zinc.

Although a substantial amount of research exists on pollution and biological invasions, there is a paucity of understanding of how both factors interact. Most studies show that pollution favours the establishment of invasive species, but pollution may also promote local adaptation of native species and prevent the establishment of new incomers. However, evidence for this is extremely limited because most studies focus on successful invasions and very few on cases where an invasion has been resisted. Here we provide evidence of local adaptation of native species to pollution combining life history and physiological data. We focused on the invasion of the North American brine shrimp Artemia franciscana, which is causing a dramatic biodiversity loss in hypersaline ecosystems worldwide, and one of the last native Artemia populations in SW Europe (A. parthenogenetica from the historically polluted Odiel estuary, SW Spain). Life table response experiments were carried out in the laboratory to compare the demographic responses of A. parthenogenetica and a nearby A. franciscana population to long-term Zn exposure (0.2 mg L-1). We also evaluated oxidative stress by measuring antioxidant defences (catalase, glutathione reductase and superoxide dismutase) and lipid peroxidation (thiobarbituric acid reactive substances). A high concentration of Zn induced strong mortality in A. franciscana, which also showed high levels of lipid peroxidation, suggesting relatively poor physiological resistance to pollution compared with A. parthenogenetica. The age at maturity was shorter in A. parthenogenetica, which may be an adaptation to the naturally high mortality rate observed in the Odiel population. Exposure to Zn accelerated age at first reproduction in A. franciscana but not in A. parthenogenetica. In contrast, Zn had a stimulatory effect on offspring production in A. parthenogenetica,which also showed higher reproductive parameters (number of broods, total offspring and offspring per brood) than A. franciscana. Overall, the results of this study strongly suggest that native Artemia from Odiel estuary is locally adapted (at both, reproductive and physiological levels) to Zn contamination and that A. franciscana is highly sensitive. This is a good example of how pollution may play a role in the persistence of the last native Artemia populations in the Mediterranean.

Dietary Effect on the Proteome of the Common Octopus (Octopus vulgaris) Paralarvae.

Nowadays, the common octopus (Octopus vulgaris) culture is hampered by massive mortalities occurring during early life-cycle stages (paralarvae). Despite the causes of the high paralarvae mortality are not yet well-defined and understood, the nutritional stress caused by inadequate diets is pointed out as one of the main factors. In this study, the effects of diet on paralarvae is analyzed through a proteomic approach, to search for novel biomarkers of nutritional stress. A total of 43 proteins showing differential expression in the different conditions studied have been identified. The analysis highlights proteins related with the carbohydrate metabolism: glyceraldehyde-3-phosphate-dedydrogenase (GAPDH), triosephosphate isomerase; other ways of energetic metabolism: NADP+-specific isocitrate dehydrogenase, arginine kinase; detoxification: glutathione-S-transferase (GST); stress: heat shock proteins (HSP70); structural constituent of eye lens: S-crystallin 3; and cytoskeleton: actin, actin-beta/gamma1, beta actin. These results allow defining characteristic proteomes of paralarvae depending on the diet; as well as the use of several of these proteins as novel biomarkers to evaluate their welfare linked to nutritional stress. Notably, the changes of proteins like S-crystallin 3, arginine kinase and NAD+ specific isocitrate dehydrogenase, may be related to fed vs. starving paralarvae, particularly in the first 4 days of development.

Long-term effect of temperature on bioaccumulation of dietary metals and metallothionein induction in Sparus aurata.

Previous studies have demonstrated that the commercial feed of aquacultured fish contains trace amounts of toxic and essential metals which can accumulate in tissues and finally be ingested by consumers. Recently rising temperatures, associated to the global warming phenomenon, have been reported as a factor to be taken into consideration in ecotoxicology, since temperature-dependent alterations in bioavailability, toxicokinetics and biotransformation rates can be expected. Sparus aurata were kept at 22°C, 27°C and 30°C for 3 months in order to determine the temperature effect on metallothionein induction and metal bioaccumulation from a non-experimentally contaminated commercial feed. A significant temperature-dependent accumulation of cadmium (Cd), copper (Cu), mercury (Hg), zinc (Zn), lead (Pb) and iron (Fe) was found in liver, together with that of manganese (Mn), Fe and Zn in muscle. Hg presented the highest bioaccumulation factor, and essential metal homeostasis was disturbed in both tissues at warm temperatures. An enhancement of hepatic metallothionein induction was found in fish exposed to the highest temperature.

A revision of Artemia biodiversity in Macaronesia.

In a biogeographical context, the term Macaronesia broadly embraces the North Atlantic archipelagos of the Azores, Madeira, Selvagens, the Canary Islands, and Cape Verde. The peculiar arid climatic conditions in some of these places have led to the development of marine salt exploitations, which can be counted among the hypersaline habitats of the brine shrimp Artemia (Branchiopoda, Anostraca). Parthenogenetic populations of this anostracan were described in the Canary Islands during the last decades of the 20th century, while the American Artemia franciscana species was recently found in the Cape Verde archipelago. Following an invasive pattern, this exotic species has recently reached the Canary Islands, too. This paper reports information dealing with biotope loss (solar saltworks) in this biogeographical region, together with possible consequences concerning the arrival of invasive species, two factors that frequently promote dramatic biodiversity losses. The discussion of this threat focuses mainly on the Canary Islands archipelago where native species of Artemia still exist.

Characterization of the organic contamination pattern of a hyper-saline ecosystem by rapid screening using gas chromatography coupled to high-resolution time-of-flight mass spectrometry.

In this paper, gas chromatography coupled to high-resolution time-of-flight mass spectrometry (GC-TOF MS) has been applied to evaluate organic pollution in a hyper-saline aquatic environment. Firstly, a target screening was made for a list of 150 GC-amenable organic micro-contaminants, including PAHs, octyl/nonyl phenols, PCBs, PBDEs, and a notable number of pesticides, such us insecticides (organochlorines, organophosphorus, carbamates and pyrethroids), herbicides (triazines and chloroacetanilides), fungicides and several transformation products. This methodology was applied to brine samples, with a salt content from 112 g/L to saturation, and to samples from Artemia populations (crustacean Anostraca) collected during 1 year from three sampling stations in saltworks bodies sited in the Ebro river delta. Around 50 target contaminants, belong to chemical families included in the list of priority substances within the framework on European water policy. Additionally, a non-target analysis was performed in both types of samples with the objective of investigating the presence of other non-selected organic compounds taking advantage of the potential of GC-TOF MS (high sensitivity in full-spectrum acquisition mode, accurate mass measurements) for searching unknowns. Organophosphorus pesticides were the contaminants more frequently detected in brine samples. Other compounds usually present in urban and industrial wastewaters, like caffeine, methylparaben, butylated-hydroxytoluene and N-butylbenzenesulfonamide were also detected in brines. The herbicide simazine and the insecticide chlorpyrifos were among the contaminants detected in Artemia samples. Results of this work reveal a potential threat to vulnerable populations inhabiting the hyper-saline ecosystem. The valuable contribution of GC-TOF MS in environmental analysis, allowing the rapid screening of a large number of organic contaminants, is also demonstrated in this paper.

Biomagnification study on organochlorine compounds in marine aquaculture: the sea bass (Dicentrarchus labrax) as a model.

Biomagnification of organochlorine compounds (pesticides and polychlorinated biphenyls) through the marine aquaculture food chain is investigated. From first-feeding, specimens of sea bass were exposed to commercial fish feed (that contained DDTs and PCBs residues) for ca. 24 months, and selected tissues (white and red muscle, liver, and visceral fat) were analyzed after 6 and 24 months of diet exposure. Data obtained showed that experimental fish tissues presented a similar contamination pattern to that of fish feed, and biomagnification processes of these compounds were proved. Additionally, commercial sea bass cultured in farms from the western Mediterranean were analyzed, their organochlorine concentrations being significantly lower than those of the 24 month old experimental fish. Thus, the exposition of human population to OCs through consumption of cultured fish would be lower than expected from experimental biomagnification studies, although red muscle presented similar OC levels in both cases, which were much higher than those of white muscle. Although levels of organochlorine compounds were found to be low, the persistence, ubiquity, and toxicity of these compounds, together with their presence in fish feed, make it necessary to monitor OC residues in the routine quality assurance programs of aquaculture activities, as this food chain is a source of these toxic compounds for human consumers. The development of sensitive analytical methodology based on GC-MS/MS has allowed for the reaching of low detection limits required to carry out the present study.