Differential intestinal effects of water and foodborne exposures of nano-TiO2 in the mussel Mytilus coruscus under elevated temperature.

With the wide use of nanomaterials in daily life, nano-titanium dioxide (nano-TiO2) presents potential ecological risks to marine ecosystems, which can be exacerbated by ocean warming (OW). However, most previous studies have only centered around waterborne exposure, while there is a scarcity of studies concentrating on the impact of trophic transfer exposure on organisms. We investigated the differences in toxic effects of 100 µg/L nano-TiO2 on mussels via two pathways (waterborne and foodborne) under normal (24 °C) and warming (28 °C) conditions. Single nano-TiO2 exposure (waterborne and foodborne) elevated the superoxide dismutase (SOD) and catalase (CAT) activities as well as the content of glutathione (GSH), indicating activated antioxidatant response in the intestine. However, depressed antioxidant enzymes and accumulated peroxide products (LPO and protein carbonyl content, PCC) demonstrated that warming in combination with nano-TiO2 broke the prooxidant-antioxidant homeostasis of mussels. Our findings also indicated that nano-TiO2 and high temperature exhibited adverse impacts on amylase (AMS), trypsin (PS), and trehalase (THL). Additionally, activated immune function (lysozyme) comes at the cost of energy expenditure of protein (decreased protein concentration). The hydrodynamic diameter of nano-TiO2 at 24 °C (1693-2261 nm) was lower than that at 28 °C (2666-3086 nm). Bioaccumulation results (range from 0.022 to 0.432 µg/g) suggested that foodborne induced higher Ti contents in intestine than waterborne. In general, the combined effects of nano-TiO2 and warming demonstrated a more pronounced extent of interactive effects and severe damage to antioxidant, digestive, and immune parameters in mussel intestine. The toxicological impact of nano-TiO2 was intensified through trophic transfer. The toxic effects of nano-TiO2 are non-negligible and can be exerted together through both water- and foodborne exposure routes, which deserves further investigation.

Low-frequency noise impairs righting reflex behavior by disrupting central nervous system in the sea slug Onchidium reevesii.

Anthropogenic noise has significantly increased due to human activities, posing a threat to the health and survival of marine organisms. However, current studies have often emphasized its effects on the physiological aspects of marine organisms, while ignored the relationship between the neuroendocrine system and behavior. This study aimed to evaluate the righting behavior and relevant physiological functions of the central nervous system (CNS) in sea slug (Onchidium reevesii) exposed to low-frequency noise and subsequent noise removal. The duration of the sea slugs' righting reflex increased with longer noise exposure time. The degree of neuronal cell damage and apoptosis were significantly increased and relevant gene expressions were affected (Glu, AChE, FMRFamide and CaMKII) (P < 0.05). After the removal of noise, the righting reflex speed gradually recovered, and the degree of neuronal cell damage, apoptosis and the expression levels of genes continued to decrease. Pearson correlation analysis showed that the righting time was positively correlated with CNS tissue and DNA damage, apoptosis rate, and negatively correlated with the expression levels of genes. Therefore, low-frequency noise exposure causes damage to the CNS of sea slugs, subsequently impairing their normal behavior. Sea slugs exhibited partial recovery within 384 h after removing noise. These findings provide valuable insights into the effects of low-frequency noise on the CNS and behavior of marine invertebrates.

Cloning and characterization of heat shock transcription factor 1 and its functional role for Hsp70 production in the sea slug Onchidium reevesii.

To investigate the regulatory role of heat shock transcription factor 1 of sea slug Onchidium reevesii (OrHSF1) on Hsp70 expression in the sea slug under stress , the OrHSF1 gene was cloned and bioinformatics analysis was performed, then the gene and protein expressions by RNA interference (RNAi) mediated knockdown of OrHSF1 expression were measured to clarify the regulatory relationship between OrHSF1 and Hsp70 under low-frequency noise (LFN) stress. Our study was the first to clone a 1572 bp sequence of the OrHSF1 gene, with the sequence coding for amino acids (CDS) being 729 bp, encoding 243 amino acids. O. reevesii shared a close evolutionary relationship with mollusks such as the Aplysia californica. OrHSF1 gene is widely expressed in different tissues of sea slugs, with the highest expression in the intestine and the lowest in the reproductive glands. Furthermore, we used RNA interference (RNAi) as a tool to silence the OrHSF1 gene in the central nervous system (CNS) and the results indicated that gene silencing was occurring systematically in the CNS and the suppression of OrHSF1 expression by RNAi-mediated gene silencing altered the expression of Hsp70; besides, the expression trends of OrHSF1 gene and Hsp70 were consistent in the 3 and 5-day RNAi experiment. Moreover, in sea slugs injected with siHSF1 and exposed to LFN, the mRNA expression and protein expression of Hsp70 in the CNS were significantly decreased compared to the low-frequency noise group (P < 0.05). This study demonstrated that OrHSF1 regulates Hsp70 expression in marine mollusks under low-frequency noise, and HSF1-Hsp70 axis plays a key role in stress response.

Size-dependent effects of plastic particles on antioxidant and immune responses of the thick-shelled mussel Mytilus coruscus.

Micro-/nano-plastic particles (MNPs) are present in the ocean with potential detrimental impacts on marine ecosystems. Bivalves are often used as marine bioindicators and are ideal to evaluate the threat posed by various-sized MNPs. We exposed the mussel Mytilus coruscus to MNPs with different particle sizes (70 and 500 nm, 5, 10 and 100 µm) for 3, 72 h and 30 days. The antioxidant responses in digestive gland and the hemolymph were then evaluated. The time of exposure played a strong modulating role in the biological response. A 3-hour exposure had no significant impact on the digestive gland. After 72 h, an increase in oxidative stress was observed in the digestive gland, including increased hydrogen peroxide (H2O2) level, catalase (CAT), glutathione peroxidase (GPx) activities and malondialdehyde (MDA) production. After a 30-day exposure, the oxidative stress decreased while lipid peroxidation increased. A 30-day exposure increased hemocyte mortality (HM) and reactive oxygen species (ROS) levels in the hemolymph, while phagocytosis (PA), lysosome content (LC), mitochondrial number (MN) and mitochondrial membrane potential (MMP) significantly decreased. Longer-term exposure to MNPs caused oxidative stress in the digestive gland as well as impaired viability and immunity of hemocytes. Particle size also influenced the response with smaller particles having more severe effects. A depuration for 7 days was enough to reverse the negative effects observed on the digestive gland and hemolymph. This study provides new insights on the effects of small-sized MNPs, especially nanoplastic particles (NPs), on aquatic organisms, and provides a solid theoretical knowledge background for future studies on toxic effects of MNPs.

Low-frequency noise aggravates the toxicity of cadmium in sea slug Onchdium reevesii.

Industrial development not only triggers heavy metal pollution but also introduces a less easily discernible disturbance: low-frequency noise pollution. Low-frequency noise can disrupt wildlife behavior, potentially exerting complex effects through interacting with heavy metals. Nevertheless, the cumulative impacts of low-frequency noise and cadmium (Cd) pollution on marine organisms remain largely unexplored. This study aimed to evaluate the immune defense response of sea slugs (Onchdium reevesii) exposed to Cd (1.32 mg/L) and low-frequency noise (500 Hz, 1000 Hz). Our results show that Cd exposure results in Cd2+ accumulation in the sea slug's hepatopancreas, leading to a decrease in total antioxidant capacity (TAC) and a significant increase in enzyme activities, including glutathione (GSH), lipid peroxidation (LPO), and aspartate transferase (AST). Additionally, there is a substantial upregulation in the expression of genes related to tumor protein p53 (p53), Cytochrome C (CytC), Caspase 3, and Caspase 9, as well as metallothionein (MT) and heat shock protein 70 (Hsp70) genes. Concurrently, an excessive production of reactive oxygen species (ROS) occurs in the hemocytes, resulting in apoptosis and subsequent diminished cell viability, with these effects positively correlating with the exposure duration. Furthermore, when sea slugs were exposed to both Cd and low-frequency noise, there was a decrease in the hepatopancreas's antioxidant capacity and an enhancement in hemocytes immune responses, which positively correlated with low-frequency noise frequency. The comprehensive assessment of biomarker responses highlights that low-frequency noise has the potential to amplify the deleterious effects of Cd on sea slug physiology, with this negative impact positively linked to noise frequency. Consequently, our study underscores that the combined influence of low-frequency noise and Cd pollution magnifies the effects on sea slug health. This could potentially disrupt the population stability of this species within its natural habitat, providing fresh insights into the evaluation of cumulative environmental pollution risks.

Effects of diel-cycling hypoxia and salinity on lipid metabolism and fatty acid composition of the oyster Crassostrea hongkongensis.

For marine animals living in estuarine, coastal, and intertidal areas, salinity changes and periodic hypoxia are typical stressors; however, how the varying salinity and dissolved oxygen affect the quality and nutrition of marine aquaculture species, such as oysters remains unknown. In this study, we evaluated the diel-cycling hypoxia under different salinities on fatty acid composition and lipid metabolism in oyster Crassostrea hongkongensis digestive glands. After 28 days of exposure, both hypoxia and elevated salinity caused a decrease in the saturated fatty acid (SFA)/polyunsaturated fatty acid (PUFA) ratio of C. hongkongensis, salinity mainly causes changes in C17:0, C17:1, C18:1n9, C20:1n9, C20:4n6, C21:5n3, C22:5n3, with high salinity being more damaging to the fatty acid fractions. Also, Hypoxia accelerates the synthesis of C18:1n9 and C20:4n6. Fatty acid synthase (FAS) synthesis is increased by reduced salinity or hypoxia, but Acetyl CoA carboxylase (ACC) only weakly promotes fatty acid synthesis. Under hypoxic conditions, the activity of both hepatic lipase (HL) and lipoprotein lipase activity (LPL) decreases, which is contrary to the results for dissolved oxygen. The increase in salinity under dissolved oxygen leads to a decrease in LPL activity and an increase in HL activity. Our findings highlighted that exposure to a combination of salinity and hypoxia stressors, can disrupt the protective mechanisms of the oyster and affect the function of its lipid metabolism. Therefore, long-term exposure to periodic hypoxia with salinity changes poses a risk to the nutritional quality of C. hongkongensis, affecting oyster aquaculture and the coastal ecosystem.

Combined effect of salinity and hypoxia on digestive enzymes and intestinal microbiota in the oyster Crassostrea hongkongensis.

Anthropologic activities caused frequent eutrophication in coastal and estuarine waters, resulting in diel-cycling hypoxia. Given global climate change, extreme weather events often occur, thus salinity fluctuation frequently breaks out in these waters. This study aimed to evaluate the combined effects of salinity and hypoxia on intestinal microbiota and digestive enzymes of Crassostrea hongkongensis. Specifically, we sequenced 16 S rRNA of intestinal microbiota and measured the digestive enzymes trypsin (TRS), lipase (LPS) and amylase (AMY) in oysters exposed for 28 days to three salinities (10, 25 and 35) and two dissolved oxygen conditions, normoxia (6 mg/L) and hypoxia (6 mg/L for 12 h, 2 mg/L for 12 h). Oysters in normoxia and salinity of 25 were treated as control. After 28-day exposure, for microbial components, Fusobacteriota, Firmicutes, Bacteroidota, Proteobacteria and Actinobacteriota comprised the majority for all experimental groups. Compared with the control group, the diversity and structure of intestinal microbiota tended to change in all treated groups. The species richness in C. hongkongensis intestine also changed. It was the most significant that high salinity increased Proteobacteria proportion while low salinity and hypoxia increased Fusobacteriota but decreased Proteobacteria, respectively. Additionally, Actinobacteriota was sensitive and changed under environmental stressor (P < 0.01). The prediction results on intestinal microbiota showed that, all functions of oysters were up-regulated to distinct degrees under low/high salinity with hypoxia. According to the KEGG prediction, cellular processes were more active and energy metabolism upregulated, indicating the adaptation of C. hongkongensis to environmental change. Periodical hypoxia and low/high salinity had complex effect on the digestive enzymes, in which the activity of TRS and LPS decreased while AMY increased. High/low salinity and periodical hypoxia can change the secretion of digestive enzymes and influence intestinal microbial diversity and species richness of C. hongkongensis, deducing the chronic adverse effects on the digestive physiology in long-term exposure.

Polybrominated diphenyl ether-47 and food shortage impair the byssal attachment and health of marine mussels.

Polybrominated diphenyl ethers (PBDEs) are one of the most used halogenated flame retardants worldwide, and exert neurotoxicity, reproductive toxicity, endocrine interference, and carcinogenic effects on organisms. However, there are insufficient studies on the physical and immune defense at the individual level of mussels under different food conditions. To explore the defense strategy and individual health status, the thick-shelled mussels Mytilus coruscus were exposed to different BDE-47 concentrations (0, 0.1 and 10 µg/L) and nutritional conditions (feeding and starvation) for 21 days. The results showed that BDE-47 exposure and starvation significantly decreased the number of byssus threads (NBT), adhesion, and condition index (CI) of mussels, whereas increased the reactive oxygen species (ROS) production and the combined stress further declined the CI. BDE-47 exposure and starvation induced decreased adhesive capability and healthy state along with oxidative lesions in mussels. The downregulation gene expression of foot adhesion proteins (mfp-2/3/4/5/6) under starvation or combined exposure also proved the reduced adhesion of mussels. However, up-regulated mfp-1 and pre-collagens proteins (preCOL-D/P/NG) indicated mussels would adjust energy allocation to enhance the strength and extensibility of byssal threads for compensating reduced adhesion and CI. As global climate change and organic pollution have dramatically impacted the ocean, hazardous substances and the fluctuated primary productivity have frequently co-occurred, which will affect the structure of coastal biomes and fishery production.

Multi-omics reveals response mechanism of liver metabolism of hybrid sturgeon under ship noise stress.

Underwater noise from ship engines can affect the metabolism and immune system of various fish species. Meanwhile, changes in the metabolic pathways in liver are important for fish to adapt to adverse environments. We used a combined multi-omics analysis to investigate the response mechanism of hybrid sturgeon to continuously played ship noise. A control group and a noise group (simulated ship noise: 12 h) were set up, and liver tissues were extracted for high-throughput transcriptome and metabolome sequencing. The results show that a total of 588 differentially expressed genes (DEGs) and 58 DEGs metabolites were detected. The joint analysis of transcriptome and metabolome showed that under noise stress, apoptosis and cell motility were intensified, DNA replication, RNA transcription and translation, and protein synthesis were inhibited, and lipid metabolism, nucleotide metabolism, and vitamin D3 metabolic pathways were also inhibited. Interestingly, the initiation of a partial immune responses ensured their normal immunity abilities. Moreover, material and energy requirements of the organism under noise stress were guaranteed by upregulation of carbohydrate and amino acid metabolic pathways.

Effect of low-frequency noise on the survival rate and immunity of infected Vibrio parahaemolyticus sea slug (Onchidium reevesii).

Anthropogenic noise in the marine environment has become a global environmental pollutant that affects the behavior, physiology and immunity of marine animals. However, the resistance of marine animals to pathogens while under the influence of noise is a topic that has received little attention. To assess the immune defense response of sea slugs against pathogens when exposed to low frequency noise, we performed 120 h exposure experiments on sea slugs after a Vibrio parahaemolyticus application in low frequency noise at 500 Hz and 1000 Hz. We found that after the infection with V. parahaemolyticus, the survival rate of the sea slugs decreased, the apoptosis rate and reactive oxygen species (ROS) production of hemocytes increased significantly (P < 0.05), the proliferation of hemocytes accelerated, the activities of enzymes such as superoxide dismutase (SOD), catalase (CAT), alkaline phosphatase (AKP), alanine transaminase (ALT) and lysozyme (LZM) in the hepatopancreas increased significantly, and the expression of TNF signaling pathway-related genes (TNF-α, FADD, Caspase 8, Caspase 3) and Hsp70 genes were generally upregulated. In addition, exposure of sea slug after infected with V. parahaemolyticus to low frequency noise resulted in a significant increase in both antioxidant and immune parameters, which were positively correlated with frequency. The results showed that noise frequency and exposure time had an interactive effect on the above indicators. In summary, low-frequency noise exposure increases the risk of pathogenic infections in sea slugs and exacerbates the negative effects on the antioxidant capacity and immune metabolism of the organism.

In utero Exposure to Excessive Estrogen Impairs Homologous Recombination and Oogenesis via Estrogen Receptor 2 in Mice.

The association between the accumulation of synthetic chemicals with estrogenic activity and risks to oogenesis has become a growing concern. This study indicates that in utero estrogen exposure can affect homologous recombination in early oogenesis and influence the reproductive potential and lifespan of female offspring. We conducted this study in developing mouse ovaries using two different models: oral doses administered to the mother, and fetal ovary cultures. Our analyses of meiotic fetal oocytes suggest that 17-ß-estradiol induces gross aberrations in prophase I events, including delayed meiotic progression, increased unrepaired DNA damage, and altered homologous recombination levels. These effects were mainly mediated by estrogen receptor 2 (ESR2) activation. Mid-gestation exposure to estrogen also led to delayed primordial folliculogenesis after birth, impaired follicle development after prepuberty, and ultimately reduced the total litter size of the offspring. This raises the concern that maternal exposures to substances activating ESR2 may compromise the fertility of the exposed female fetus.

Cloning, molecular characterization, and tissue differential expression of connective tissue growth factor (ctgf) of grass carp.

Connective tissue growth factor (ctgf) is involved in the proliferation, migration, adhesion of cell, and the constituent of extracellular matrix, which plays an important role in embryogenesis, angiogenesis, wound repair, and fibrosis diseases. In this study, the cDNA sequence of grass carp ctgf gene was cloned by rapid amplification of cDNA ends (RACE) method; then, the characteristics of this gene and the predicted protein sequence were analyzed by bioinformatics methods, and the tissue differential expression pattern was detected by the quantitative real-time PCR. The results showed that the grass carp ctgf gene has a full-length of 2223 bp, encoding 343 amino acids. The deduced CTGF protein is a hydrophilic and secretary protein with a molecular mass of 37,978.2 Da and an isoelectric point of 8.22. The signal peptide locates between residue positions 1 and 22 of the polypeptide chain. The protein contains α-helix, ß-strand, and loops. The CTGF protein of grass carp shows a homology of 98%, 96%, 91%, and 91% with Wuchang bream (Megalobrama amblycephala), zebrafish (Danio rerio), common carp (Cyprinus carpio), and Mexican tetra (Astyanax mexicanus). The grass carp ctgf gene expressed significantly higher in blood and spleen than that in other tissues (P < 0.05). The low expression tissues included the heart, gill, skin, muscle, kidney, brain, and intestinal, and the lowest expression tissue was the liver. The results are consistent with the function of this gene.

Dibutyl phthalate exposure disrupts the progression of meiotic prophase I by interfering with homologous recombination in fetal mouse oocytes.

Dibutyl phthalate (DBP), one of the most widely used plasticizers, is a known environmental endocrine disruptor that impairs male and female fertility. In this study, oral administration of DBP was given to pregnant mice on 14.5 days post coitus (dpc) for 3 days; and additionally, DBP was added into the culture of 14.5 dpc fetal ovaries for 3 days. DBP exposure during gestation disturbed the progression of meiotic prophase I of mouse oocytes, specifically from the zygotene to pachytene stages. Meanwhile, the DBP-exposed pachytene oocytes showed increased homologous recombination sites and unrepaired DNA damage. Furthermore, DBP caused DNA damage by increasing oxidative stress, decreased the expression of multiple critical meiotic regulators, and consequently induced oocyte apoptosis. Moreover, the effect of DBP on meiosis I prophase involved estrogen receptors α and ß. Collectively, these results demonstrated a set of meiotic defects in DBP-exposed fetal oocytes. As aberrations in homologous recombination can result in aneuploid gametes and embryos, this study provides new support for the deleterious effects of phthalates.