Accumulation, Depuration, and Biological Effects of Polystyrene Microplastic Spheres and Adsorbed Cadmium and Benzo(a)pyrene on the Mussel Mytilus galloprovincialis

Filter feeders are target species for microplastic (MP) pollution, as particles can accumulate in the digestive system, disturbing feeding processes and becoming internalized in tissues. MPs may also carry pathogens or pollutants present in the environment. This work assessed the influence of polystyrene (PS) MP size and concentration on accumulation and depuration time and the role of MPs as vectors for metallic (Cd) and organic (benzo(a)pyrene, BaP) pollutants. One-day exposure to pristine MPs induced a concentration-dependent accumulation in the digestive gland (in the stomach and duct lumen), and after 3-day depuration, 45 µm MPs appeared between gill filaments, while 4.5 µm MPs also occurred within gill filaments. After 3-day exposure to contaminated 4.5 µm MPs, mussels showed increased BaP levels whilst Cd accumulation did not occur. Here, PS showed higher affinity to BaP than to Cd. Three-day exposure to pristine or contaminated MPs did not provoke significant alterations in antioxidant and peroxisomal enzyme activities in the gills and digestive gland nor in lysosomal membrane stability. Exposure to dissolved contaminants and to MP-BaP caused histological alterations in the digestive gland. In conclusion, these short-term studies suggest that MPs are ingested and internalized in a size-dependent manner and act as carriers of the persistent organic pollutant BaP.

Influence of Particle Size on Ecotoxicity of Low-Density Polyethylene Microplastics, with and without Adsorbed Benzo-a-Pyrene, in Clam Scrobicularia plana

This study investigated the ecotoxicological effects of differently sized (4–6 µm and 20–25 µm) low-density polyethylene (LDPE) microplastics (MPs), with and without adsorbed benzo-a-pyrene (BaP), in clam Scrobicularia plana. Biomarkers of oxidative stress (superoxide dismutase—SOD; catalase—CAT), biotransformation (glutathione-S-transferases—GST), oxidative damage (lipid peroxidation—LPO) and neurotoxicity (acetylcholinesterase—AChE) were analysed in gills and digestive glands at different time intervals for a total of 14 days of exposure. In order to have a better impact perspective of these contaminants, an integrated biomarker response index (IBR) and Health Index were applied. Biomarker alterations are apparently more related to smaller sized (4–6 µm) MPs in gills and to virgin LDPE MPs in the digestive gland according to IBR results, while the digestive gland was more affected by these MPs according to the health index.

Tissue- and substrate-dependent mitochondrial responses to acute hypoxia-reoxygenation stress in a marine bivalve Crassostrea gigas (Thunberg, 1793)

Hypoxia is a major stressor for aquatic organisms, yet intertidal organisms like the oyster Crassostrea gigas are adapted to frequent oxygen fluctuations by metabolically adjusting to shifts in oxygen and substrate availability during hypoxia-reoxygenation (H/R). We investigated the effects of acute H/R stress (15 min at ∼0% O2, and 10 min reoxygenation) on isolated mitochondria from the gill and the digestive gland of C. gigas respiring on different substrates (pyruvate, glutamate, succinate, palmitate and their mixtures). Gill mitochondria showed better capacity for amino acid and fatty acid oxidation compared to the mitochondria from the digestive gland. Mitochondrial responses to H/R stress strongly depended on the substrate and the activity state of mitochondria. In mitochondria oxidizing NADH-linked substrates exposure to H/R stress suppressed oxygen consumption and ROS generation in the resting state, whereas in the ADP-stimulated state, ROS production increased despite little change in respiration. As a result, electron leak (measured as H2O2 to O2 ratio) increased after H/R stress in the ADP-stimulated mitochondria with NADH-linked substrates. In contrast, H/R exposure stimulated succinate-driven respiration without an increase in electron leak. Reverse electron transport (RET) did not significantly contribute to succinate-driven ROS production in oyster mitochondria except for a slight increase in the OXPHOS state during post-hypoxic recovery. A decrease in NADH-driven respiration and ROS production, enhanced capacity for succinate oxidation and resistance to RET might assist in post-hypoxic recovery of oysters mitigating oxidative stress and supporting rapid ATP re-synthesis during oxygen fluctuations such as commonly observed in estuaries and intertidal zones.

Dietary Probiotic Pediococcus acidilactici MA18/5M Improves the Growth, Feed Performance and Antioxidant Status of Penaeid Shrimp Litopenaeus stylirostris: A Growth-Ration-Size Approach

Probiotics are increasingly documented to confer health and performance benefits across farmed animals. The aim of this study was to assess the effect of a constant daily intake of the single-strain probiotic Pedicococcus acidilactici MA18/5M (4 × 108 CFU.day−1.kg−1 shrimp) fed over fixed, restricted ration sizes (1% to 6% biomass.day−1) on the nutritional performance and metabolism of adult penaeid shrimp Litopenaeus stylirostris (initial body-weight, BWi = 10.9 ± 1.8 g). The probiotic significantly increased the relative daily growth rate (RGR) across all ration size s tested (Mean-RGR of 0.45 ± 0.08 and 0.61 ± 0.07% BWi.day−1 for the control and probiotic groups, respectively) and decreased the maintenance ration (Rm) and the optimal ration (Ropt) by 18.6% and 11.3%, respectively. Accordingly, the probiotic group exhibited a significantly higher gross (K1) and net (K2) feed conversion efficiency with average improvement of 35% and 30%, respectively. Enhanced nutritional performances in shrimps that were fed the probiotic P. acidilactici was associated with, in particular, significantly higher α-amylase specific activity (+24.8 ± 5.5% across ration sizes) and a concentration of free-glucose and glycogen in the digestive gland at fixed ration sizes of 3% and below. This suggests that the probiotic effect might reside in a better use of dietary carbohydrates. Interestingly, P. acidilactici intake was also associated with a statistically enhanced total antioxidant status of the digestive gland and haemolymph (+24.4 ± 7.8% and +21.9 ± 8.5%, respectively; p < 0.05). As supported by knowledge in other species, enhanced carbohydrate utilization as a result of P. acidilactici intake may fuel the pentose-phosphate pathway, generating NADPH or directly enhancing OH-radicals scavenging by free glucose, in turn resulting in a decreased level of cellular oxidative stress. In conclusion, the growth-ration method documented a clear contribution of P. acidilactici MA18/5M on growth and feed efficiency of on-growing L. stylirostris that were fed fixed restricted rations under ideal laboratory conditions. The effect of the probiotic on α-amylase activity and carbohydrate metabolism and its link to the shrimp’s antioxidant status raises interesting prospects to optimize dietary formulations and helping to sustain the biological and economic efficiency of the penaeid shrimp-farming industry.

Transcriptomic Profile of the Cockle Cerastoderma edule Exposed to Seasonal Diarrhetic Shellfish Toxin Contamination

Bivalves constitute an important source of proteins for human consumption, but some accumulate biotoxins such as diarrhetic shellfish toxins (DSTs), constituting a risk to human health. The cockle Cerastoderma edule is one of the most important species harvested in the Portuguese coast but also one of the most affected species due to recurrent DSTs exposure. However, little is known regarding the effects of the toxins produced by blooming dinoflagellates on C. edule. Herein, we explore the Differentially Expressed Genes (DEGs) of two tissues (gills and digestive gland) from wild cockles sampled in Portugal, through their whole transcriptomic response in two different seasons (exposed and not exposed to DSTs). The de novo transcriptome assembly returned 684,723 contigs, N50 of 1049, and 98.53% completeness. Altogether, 1098 DEGs were identified, of which 353 DEGs were exclusive for the digestive gland, 536 unique for the gills and 209 DEGs were common. Among DEGs were identified known DSTs-biomarkers including glutathione peroxidase, glutathione S-transferase, superoxide dismutase, cytochrome P450, ABC transporters, actin and tubulin-related proteins, Heat shock proteins and complement C1Q-like proteins. This study provides the first transcriptomic profile of C. edule, giving new insights about its molecular responses under different environmental conditions of DSTs exposure.

Comparative morphology and ph of the alimentary tract in Archachatina marginata and Achatina achatina

A study was carried out to investigate the comparative morphology and pH of the gut in the Giant African Land Snails, in Archachatina marginata and Achatina achatina respectively. The crop is long, distended and less fleshy with reddish brown coloured juice (5.8 ml volume) in A. marginata, while it is short, compact and fleshy with greenish brown coloured juice (3.2 ml volume) in A. achatina. The digestive gland was heavier (17.36 vs. 15.74 g) in the latter than the former. The absolute weight of the crop in A. marginata was significantly (p<0.05) higher than in A. Achatina, however, the absolute and relative weights of the digestive gland in A. achatina was significantly (p<0.05) higher than in A. marginata. There were significant (p<0.05) differences in linear measurements of the salivary gland and crop for absolute values and the salivary gland, crop, stomach and digestive gland for relative values. Archachatina marginata had significantly (p<0.05) longer salivary gland and crop than Achatina achatina for absolute length values and significantly (p<0.05) longer salivary gland, crop and digestive gland for relative length values. However, the relative stomach length in A. achatina was significantly (p<0.05) longer than in A. marginata. The gut pH was more acidic in the crop (5.38 and 4.83) than in the mouth (6.93 and 6.82) where it tended towards neutrality in A. marginata and A. achatina respectively. The pH profiles in the two species, decreased by 1.65 and 1.99 from the mouth to the crop while it increased by 1.46 and 1.97 from the crop to the distal digestive gland in A. marginata and A. achatina respectively. In the herbivorous snails, the pH along the gut regions remained acidic all through (5.38 – 6.93 and 4.83 – 6.82) in A. marginata and A. achatina respectively, confirming the herbivorous habits in these snail species. The longer gut and distended crop with higher volume of crop juice in A. marginata, implies its ability to store, utilize and digest more food materials than A. achatina. These results provide the basis for a better understanding of the physiology of the digestive processes in these snails.

Ascorbic Acid Regulates the Immunity, Anti-Oxidation and Apoptosis in Abalone Haliotis discus hannai Ino

The present study was conducted to investigate the roles of ascorbic acid (AA) in immune response, anti-oxidation and apoptosis in abalone (Haliotis discus hannai Ino). Seven semi-purified diets with graded levels of AA (0, 50, 100, 200, 500, 1000 and 5000 mg/kg) were fed to abalone (initial weight: 12.01 ± 0.001 g, initial shell length: 48.44 ± 0.069 mm) for 100 days. The survival, weight gain rate and daily increment in shell length were not affected by dietary AA. The AA content in the gill, muscle and digestive glands of abalone was significantly increased by dietary AA. In terms of immunity, dietary AA significantly improved the total hemocyte count, respiratory burst and phagocytic activity in hemolymph, and lysozyme activity in cell-free hemolymph (CFH). In the digestive gland, the TLR-MyD88-dependent and TLR-MyD88-independent signaling pathways were suppressed by dietary AA supplementation. The mRNA levels of β-defensin and arginase-I in the digestive gland were significantly increased by dietary AA. In the gill, only the TLR-MyD88-dependent signaling pathway was depressed by dietary AA to reduce inflammation in abalone. The level of mytimacin 6 in the gill was significantly upregulated by dietary AA. After Vibrio parahaemolyticus infection, the TLR signaling pathway in the digestive gland was suppressed by dietary AA, which reduced inflammation in the abalone. In terms of anti-oxidation, superoxide dismutase, glutathione peroxidase and catalase activities, as well as total anti-oxidative capacity and reduced glutathione content in CFH, were all significantly upregulated. The malondialdehyde content was significantly downregulated by dietary AA. The anti-oxidative capacity was improved by triggering the Keap1-Nrf2 pathway in abalone. In terms of apoptosis, dietary AA could enhance the anti-apoptosis ability via the JNK-Bcl-2/Bax signaling cascade in abalone. To conclude, dietary AA was involved in regulating immunity, anti-oxidation and apoptosis in abalone.

Expression Analyses of Genes Related to Multixenobiotic Resistance in Mytilus galloprovincialis after Exposure to Okadaic Acid-Producing Dinophysis acuminate

The mussel Mytilus galloprovincialis is one of the most important aquaculture species in Europe. Its main production problem is the accumulation of toxins during coastal blooms, which prevents mussel commercialization. P-glycoprotein (ABCB1/MDR1/P-gp) is part of the multixenobiotic resistance system in aquatic organisms, and okadaic acid, the main DSP toxin, is probably a substrate of the P-gp-mediated efflux. In this study, the presence and possible role of P-gp in the okadaic acid detoxification process was studied in M. galloprovincialis. We identified, cloned, and characterized two complete cDNAs of mdr1 and mdr2 genes. MgMDR1 and MgMDR2 predicted proteins had the structure organization of ABCB full transporters, and were identified as P-gp/MDR/ABCB proteins. Furthermore, the expression of mdr genes was monitored in gills, digestive gland, and mantle during a cycle of accumulation-elimination of okadaic acid. Mdr1 significantly increased its expression in the digestive gland and gills, supporting the idea of an important role of the MDR1 protein in okadaic acid efflux out of cells in these tissues. The expression of M. galloprovincialismrp2, a multidrug associated protein (MRP/ABCC), was also monitored. As in the case of mdr1, there was a significant induction in the expression of mrp2 in the digestive gland, as the content of okadaic acid increased. Thus, P-gp and MRP might constitute a functional defense network against xenobiotics, and might be involved in the resistance mechanisms to DSP toxins.

Inhibition of Diarrheal Shellfish Toxins Accumulation in the Mussel Perna viridis by Curcumin and Underlying Mechanisms

Diarrheal shellfish toxins (DSTs) are among the most widely distributed phytotoxins, and are associated with diarrheal shellfish poisoning (DSP) events in human beings all over the world. Therefore, it is urgent and necessary to identify an effective method for toxin removal in bivalves. In this paper, we found that curcumin (CUR), a phytopolylphenol pigment, can inhibit the accumulation of DSTs (okadaic acid-eq) in the digestive gland of Perna viridis after Prorocentrum lima exposure. qPCR results demonstrated that CUR inhibited the induction of DSTs on the aryl hydrocarbon receptor (AhR), hormone receptor 96 (HR96) and CYP3A4 mRNA, indicating that the CUR-induced reduction in DSTs may be correlated with the inhibition of transcriptional induction of AhR, HR96 and CYP3A4. The histological examination showed that P. lima cells caused severe damage to the digestive gland of P. viridis, and the addition of curcumin effectively alleviated the damage induced by P. lima. In conclusion, our findings provide a potential method for the effective removal of toxins from DST-contaminated shellfish.

Microscopic Anatomy of the Lining of Hemal Spaces in the Penaeid Shrimp, Sicyonia ingentis

The purpose of this paper is to present a morphological description of three different types of acellular material lining hemal spaces in a shrimp, providing a background for addressing future questions. The vasculature of the penaeid shrimp, Sicyonia ingentis, includes vessels leading from the heart into arteries which branch and expand into sinuses before returning hemolymph back to the heart. Early work showed that an endothelium was absent, and a basement membrane (BM) separated tissues from the hemolymph. Therefore, it was suggested that hemocytes could identify anything other than the BM as a “foreign” entity. This study demonstrates three major types of acellular material lining the hemal spaces of S. ingentis. Cardiomyocytes, digestive gland tubules, and abdominal muscle fibers are covered by BMs. Major arteries are lined by a fibrillin-like fibrous material. Finally, sheaths of collagenous connective tissues cover the heart and digestive gland as well as the outer surface of arteries, the gut, and gonad. Our understanding of hemocyte receptors and extracellular matrices in general have greatly expanded but the biochemical composition of the matrices lining crustacean hemal spaces, their role in regulating nutrient uptake, and the cells responsible for their deposition deserve further attention.