A systematic toxicologic study of polycyclic aromatic hydrocarbons on aquatic organisms via food-web bioaccumulation.

Pollution-induced declines in fishery resources restrict the sustainable development of fishery. As a kind of typical environmental pollutant, the mechanism of polycyclic aromatic hydrocarbons (PAHs) facilitating fishery resources declines needs to be fully illustrated. To determine how PAHs have led to declines in fishery resources, a systematic toxicologic analysis of the effects of PAHs on aquatic organisms via food-web bioaccumulation was performed in the Pearl River and its estuary. Overall, PAH bioaccumulation in aquatic organisms was correlated with the trophic levels along food-web, exhibiting as significant positive correlations were observed between PAHs concentration and the trophic levels of fishes in the Pearl River Estuary. Additionally, waterborne PAHs exerted significant direct effects on dietary organisms (P < 0.05), and diet-borne PAHs subsequently exhibited significant direct effects on fish (P < 0.05). However, an apparent block effect was found in dietary organisms (e.g., zooplankton) where 33.49 % of the total system throughput (TST) was retained at trophic level II, exhibiting as the highest PAHs concentration, bioaccumulation factor (BAF), and biomagnification factor (BMF) of ∑15PAHs in zooplankton were at least eight-fold greater than those in fishes in both the Pearl River and its estuary, thereby waterborne PAHs exerted either direct or indirect effects on fishes that ultimately led to food-web simplification. Regardless of the block effect of dietary organisms, a general toxic effect of PAHs on aquatic organisms was observed, e.g., Phe and BaP exerted lethal effects on phytoplankton Chlorella pyrenoidosa and zooplankton Daphnia magna, and decreased reproduction in fishes Danio rerio and Megalobrama hoffmanni via activating the NOD-like receptors (NLRs) signaling pathway. Consequently, an assembled aggregate exposure pathway for PAHs revealed that increases in waterborne PAHs led to bioaccumulation of PAHs in aquatic organisms along food-web, and this in turn decreased the reproductive ability of fishes, thus causing decline in fishery resources.

Potential of microplastics participate in selective bioaccumulation of low-ring polycyclic aromatic hydrocarbons depending on the biological habits of fishes.

Currently, although the cumulative effects of microplastics (MPs) and organic pollutants (OPs) in the environment and within organisms are being investigated, whether and how MPs participate in bioaccumulation of OPs based on a carrier effect is still unclear. In the present study, water and aquatic organisms were collected from the Pearl River. Polycyclic aromatic hydrocarbons (PAHs) and MPs were separated by solid phase extraction and were measured by gas chromatography mass spectrometry and Fourier transform infrared spectroscopy, respectively. Higher PAH concentrations at the river outlet and higher MPs abundance in the inner river were observed, indicating a mismatched distribution between PAHs and MPs. No correlation between MP abundance and PAH concentration in fishes was detected, implying that MPs exerted limited influence on PAH concentrations. Interestingly, bioconcentration factors of one major low-ring PAH (phenanthrene) in fishes showed a significant correlation with MPs abundance, implying that although MPs did not affect the variation in PAH concentrations, they potentially participated in selective bioaccumulation of PAHs. Moreover, significant correlations between MPs abundance and PAHs in fishes with different feeding and living habits were found, indicating that MPs' participation in PAH bioaccumulation was dependent on fish biology and life history. Furthermore, the health risk posed by PAHs in fishes at the river outlet surpassed the line of potential high risk, while the ecological risk posed by MPs at the inner river was in the danger category, indicating the ecological risks posed by PAHs and MPs are uneven along the Pearl River. These findings deepen our understanding of the underlying mechanism of MPs participating in selective bioaccumulation of low-ring PAHs in fishes based on fish biology and point out the present risks posed by these two pollutants in the Pearl River and its estuary, which contribute to aquatic environmental protection and fishery production in this region.

Seasonal variation of temperature affects HMW-PAH accumulation in fishery species by bacterially mediated LMW-PAH degradation.

Currently, the specific mechanism generating seasonal variation in polycyclic aromatic hydrocarbons (PAHs) via bacterial biodegradation remains unclear, and whether this alteration affects PAH bioaccumulation is unknown. Therefore, we performed a study between 2015 and 2020 to investigate the effects of seasonal variation on bacterial communities and PAH bioaccumulation in the Pearl River Estuary. Significantly high PAH concentrations in both aquatic and fishery species were determined in dry seasons (the mean ∑16PAH concentration: water, 37.24 ng/L (2015), 30.83 ng/L (2020); fish, 51.01 ng/L (2015) and 72.60 ng/L (2020)) compared to wet seasons (the mean ∑16PAH concentration: water, 22.38 ng/L (2015), 19.40 ng/L(2020); fish, 25.28 ng/L (2015) and 32.59 ng/L (2020)). Distinct differences in taxonomic and functional composition of bacterial communities related to biodegradation of low molecular weight PAHs (LMW-PAHs) were observed between seasons, and the concentrations of PAHs were negatively correlated with seasonal variation in temperature. Temperature-related specific bacterial taxa (e.g., Stenotrophomonas) directly or indirectly participated in LMW-PAH degradation via encoding PAH degradation enzymes (e.g., protocatechuate 4,5-dioxygenase) that subsequently led to bioaccumulation of high molecular weight PAHs (HMW-PAHs) in wild and fishery species due to LMW-PAHs in the water. Based on this alteration, the ecological risk posed by PAHs decreased in wet seasons, and an unbalanced spatio-temporal distribution of PAHs was observed in this estuary. These results suggest that seasonal variation of temperature affects HMW-PAH accumulation in fishery species via bacterially mediated LMW-PAH biodegradation.

Bacterioplankton community indicators for seasonal variation in a fragmented subtropical river.

In this study, spatiotemporal investigations were conducted along five cascade dams in the main channel of the North River of China during 2019 to explore bacterioplankton community indicators of water environments in a fragmented, highly regulated river. Bacterioplankton communities were good bioindicators of temporal variation in river environments, especially when considering the bacterial class level. Specifically, the most dominant bacterial classes (Gammaproteobacteria, Oxyphotobacteria, and Actinobacteria) and sub-dominant bacterial classes (Bacteroidia, Betaproteobacteria, and Acidimicrobiia) exhibited obvious temporal variation. Rainfall, water temperature (WT), water transparency (SD), and pH were all highly associated with temporal variation. In contrast, bacterioplankton indicators of spatial variation were limited to individual dominant bacterial classes for individual study periods, while rainfall, total phosphorus (TP), and pH were also associated with spatial variation. Clustering of bacterioplankton community compositions revealed that temporal differences were much stronger than spatial differences, which is consistent with most environmental parameters exhibiting obvious temporal differences, but minimal spatial differences. A possible reason for these observations could be that river fragmentation caused by cascade dams weakened spatial differences in communities, with WT, rainfall, and river runoff playing key roles in these patterns. In conclusion, bacterioplankton communities were good bioindicators of water environments in the fragmented river ecosystem of this study and their temporal variation was more apparent than their spatial variation.

Saltwater intrusion affecting NO2- accumulation in demersal fishery species by bacterially mediated N-cycling.

To investigate the underlying effects of saltwater intrusion (SWI) on bottom aquatic ecosystems, a set of environmental parameters and the bacterial community were determined and analyzed by sampling bottom water and surface sediments at the Modaomen waterway of the Pearl River Estuary. Biodiversity of fishery species and their relationship with the environment variables were analyzed together. NO3- and NO2- concentration down-regulation and NH4+ concentration up-regulation in water and sediment were observed along the resulting salinity gradient, indicating that SWI affected N-cycling. Further investigation via 16 s sequencing revealed that taxonomic and functional composition of the bacterial community in the sediment displayed greater discretization than in water, implying that SWI exerted a greater impact on the sedimentary bacterial community. Metagenomic sequencing showed that the sedimentary bacterial community was associated with NO3-, NO2-, and NH4+ transformation under SWI, and that this was driven by salinity and conductivity. Nitrogen metabolism and denitrification related genes were expressed at higher levels in high salinity than in low salinity, consistent with the increased enzymatic activities of NiR and NR. The NO2- concentration in the muscle of six selected fishery species was significantly decreased by 11.15-65.74% (P < 0.05) along the salinity gradient, indicating that SWI reduced NO2- accumulation. The results suggest that SWI alleviates NO2- accumulation in demersal fishery species via bacterial mediation of N-cycling.

Seasonal and inter-annual variability of bacterioplankton communities in the subtropical Pearl River Estuary, China.

It is widely recognized that environmental factors substantially influence on the seasonal and inter-annual variability of bacterioplankton communities, yet little is known about the seasonality of bacterioplankton communities in subtropical estuaries at longer-term time scales. Here, the bacterioplankton communities from the eight major outlets of the subtropical Pearl River Estuary were investigated across 3 years (2017-2019) using full-length 16S rRNA gene sequencing. Significant seasonal and inter-annual variation was observed in bacterioplankton community compositions across the 3 years (p < 0.05). In addition, the inferred functional composition of the communities varied with seasons, although not significantly, suggesting that functional redundancy existed among communities and across seasons that could help to cope with environmental changes. Five evaluated environmental parameters (temperature, salinity, pH, total dissolved solids (TDS), total phosphorus (TP)) were significantly correlated with community composition variation, while only three environmental parameters (temperature, pH, and TDS) were correlated with variation in inferred functional composition. Moreover, community composition tracked the seasonal temperature gradients, indicating that temperature was a key environmental factor that affected bacterioplankton community's variation along with seasonal succession patterns. Gammaproteobacteria and Alphaproteobacteria were the most dominant classes in the surface waters of Pearl River Estuary, and their members exhibited divergent responses to temperature changes, while several taxa within these group could be indicators of low and high temperatures that are associated with seasonal changes. These results strengthen our understanding of bacterioplankton community variation in association with temperature-dependent seasonal changes in subtropical estuarine ecosystems.

Connection between the Gut Microbiota of Largemouth Bass (Micropterus salmoides) and Microbiota of the Pond Culture Environment.

The vital role of the gut microbiota in fish growth, development, immunity, and health has been largely confirmed. However, the interaction between environmental microbiota and the gut microbiota of aquaculture species remains unclear. Therefore, we analyzed the gut microbiota of largemouth bass (Micropterus salmoides) collected from subtropical ponds in southern China, as well as the pond water and aquatic sediment microbiota, using high-throughput sequencing of the 16S rRNA gene. Our results demonstrated significant differences in the compositions of pond water, sediment, and the gut microbiota of largemouth bass. Moreover, these compositions changed throughout the culture period. Only approximately 1% of the bacterial species in the pond sediment and gut microbiota were exchanged. However, the bacterial proportion of the gut microbiota from pond water microbiota was approximately 7% in samples collected in June and August, which increased markedly to 73% in October. Similarly, the proportion of bacteria in the pond water microbiota from the gut microbiota was approximately 12% in June and August, which increased to 45% in October. The study findings provide basic information for understanding the interactions between environmental microbiota and the gut microbiota of cultured fish, which may contribute to improved pond culture practices for largemouth bass.

Occurrence, source identification, and ecological risk assessment of polycyclic aromatic hydrocarbons in sediments of the Pearl River Delta, China.

The pollution characteristics, sources, and ecological risk of polycyclic aromatic hydrocarbons (PAHs) in surface sediment samples from the river networks and outlets of the Pearl River Delta (PRD) were investigated. The total PAH concentrations were in the range of 69.1-1297 ng g-1 in river networks and 56.6-617 ng g-1 in river outlets. The results of source identification showed that PAHs in sediments were mainly derived from combined sources of coke tar and liquid fossil fuel combustion, coal/wood combustion, and petroleum contamination. The ecological risks of PAHs were evaluated based on sediment quality guidelines (SQG), mean probable effects levels quotient values (PEL-Q), and the toxicity equivalence factor (TEQBaP) method. The ecological risks of PAHs in sediments were at moderate levels in the Pearl River Delta. Although no high risk was found, regular and continuous monitoring of PAHs in sediments needs to be performed.

Measurement, quantification, and potential risk of microplastics in the mainstream of the Pearl River (Xijiang River) and its estuary, Southern China.

The goals of this study were to investigate the distribution profiles, sources, and inventory of microplastics in the surface water of the mainstream of the Pearl River (Xijiang River) and its estuary, China, and to assess the potential ecological risks of the microplastics in this subtropical riverine habitat. The results showed that the microplastic abundances of the Humen (HUM, 16.33 ± 0.88 items/L), Zhaoqing (ZQ, 15.33 ± 0.67 items/L), and Hutiaomen (HTM, 14.67 ± 1.33 items/L) sites were significantly higher than those of the other sampling sites, indicating that the microplastics in Xijiang River and its estuary exhibited an unbalanced spatial distribution. The most common microplastic color was transparent and the major shape of the microplastics was fragments. Microplastics with a size of 0.01-0.1 mm were dominant throughout the 16 sampling sites. Polyolefin elastomer (POE, 33.33%), polyethylene terephthalate (PET, 23.81%), and polyurethane acrylate (PUA, 14.29%) were the dominant microplastic polymers. The microplastic abundance co-varied with most of the selected socio-economic indicators, including the population density, urban land area, gross domestic product (GDP), freshwater aquatic products, and freshwater cultivated area, but without significant differences, indicating that there are additional factors affecting the microplastic abundance. The potential risk (i.e., the potential ecological risk, RI; polymer risk index, H; and pollution load index, PLI) posed by the microplastics was higher in HUM, followed by ZQ and HTM, and it was lower for the other sites, suggesting that the main outlets and the biggest city on the Xijiang River contributed more to the high risks of microplastic pollution. Consistent with microplastic abundance distribution profiles, the average potential risk index values of the western river outlets were higher than those of the eastern river outlets and the Xijiang River, implying that the western river outlets had non-negligible potential ecological risks. Our findings deepen the understanding of the risks posed by microplastics and further contribute to microplastic risk management of riverine ecosystems.

NOD-like receptor signaling pathway activation: A potential mechanism underlying negative effects of benzo(α)pyrene on zebrafish.

Benzo(α)pyrene (BaP) is one of typical polycyclic aromatic hydrocarbons (PAHs) in aquatic environments and has been shown to cause toxic effects to aquatic animals. Although the negative effects of BaP have been investigated, the potential toxic mechanisms remain uncharacterized. To explore the potential mechanisms mediating the toxic effects of BaP, zebrafish (Danio rerio) were exposed to BaP for 15 days and the toxic effects of BaP in zebrafish liver were investigated using physiological and transcriptomic analyses. After 15-day BaP exposure, zebrafish liver exhibited abnormalities including increased cytoplasmic vacuolation, inflammatory cell infiltration, swelled nuclei and irregular pigmentation. BaP exposure also induced oxidative stress to the liver of zebrafish. Transcriptomic profiles revealed 5129 differentially expressed genes (DEGs) after 15-days of BaP exposure, and the vast majority of DEGs were up-regulated under BaP treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggest that genes related to immune response were significantly dysregulated. Furthermore, the nucleotide-binding, oligomerization domain (NOD)-like receptor signaling pathway was significantly enriched and most of the genes in this pathway exhibited enhanced expression after BaP exposure. These results partially explained the mechanisms underlying the toxic effects of BaP on zebrafish liver. In conclusion, BaP has the potential to induce physiological responses in zebrafish liver through altering associated genes.

Histological, biochemical and transcriptomic analyses reveal liver damage in zebrafish (Danio rerio) exposed to phenanthrene.

Phenanthrene (PHE) is a common polycyclic aromatic hydrocarbon (PAH) in aquatic environments, and this contaminant can cause adverse effects on teleostean performance. In this study, we exposed the model freshwater fish (zebrafish; Danio rerio) to 300 µg/L PHE for 15 days. Histological analysis demonstrated that liver morphology deteriorated in PHE-exposed zebrafish, and cellular damage in the liver increased. Biological analysis revealed that exposure to PHE elicited significant changes in glutathione S-transferases (GST) and superoxide dismutase (SOD) activities. 476 differentially expressed genes (DEGs) were identified in liver between control and PHE treated groups through the transcriptomic analysis. Gene Ontology enrichment analysis (GO) suggested that PHE exposure induced changes in the expression of genes associated with "lipid transporter activity", "catalytic activity", "metal ion binding", "lipid transport" and "transmembrane transport". Furthermore, the "vitamin digestion and absorption" and "fat digestion and absorption" pathways enriched in Kyoto Encyclopedia of Genes and Genomes analysis (KEGG). Additionally, five candidate biomarkers associated with the PHE response in zebrafish were identified. In conclusion, our results elucidate the physiological and molecular responses to PHE exposure in the liver of zebrafish, and provide a framework for further studies of the mechanisms underlying the toxic effects of polycyclic aromatic hydrocarbons (PAHs) on aquatic organisms.

Structural and functional shifts of bacterioplanktonic communities associated with spatiotemporal gradients in river outlets of the subtropical Pearl River Estuary, South China.

In this study, we used high-throughput sequencing of 16S rRNA gene amplicons, to investigate the spatio-temporal variation in bacterial communities in surface-waters collected from eight major outlets of the Pearl River Estuary, South China. Betaproteobacteria were the most abundant class among the communities, followed by Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, and Acidimicrobiia. Generally, alpha-diversity increased in winter communities and the taxonomic diversity of bacterial communities differed with seasonal and spatial differences. Temperature, conductivity, salinity, pH and nutrients were the crucial environmental factors associated with shifts in the bacterial community composition. Furthermore, inferred community functions that were associated with amino acid, carbohydrate and energy metabolisms were lower in winter, whereas the relative abundance of inferred functions associated with membrane transport, bacterial motility proteins, and xenobiotics biodegradation and metabolism, were enriched in winter. These results provide new insights into the dynamics of bacterial communities within estuarine ecosystems.

Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans.

Cryptocaryoniasis is a severe disease of farmed marine fish caused by the parasitic ciliate Cryptocaryon irritans. This disease can lead to considerable economic loss, but studies on proteins linked to disease development and antigenic proteins for vaccine development have been relatively scarce to date. In this study, 53 protein spots with differential abundance, representing 12 proteins, were identified based on a pair-wise comparison among theronts, trophonts, and tomonts. Meanwhile, 33 protein spots that elicited serological responses in rabbits were identified, representing 9 proteins. In addition, 27 common antigenic protein spots reacted with grouper anti-sera, representing 10 proteins. Most of the identified proteins were involved in cytoskeletal and metabolic pathways. Among these proteins, actin and α-tubulin appeared in all three developmental stages with differences in molecular weights and isoelectric points; 4 proteins (vacuolar ATP synthase catalytic subunit α, mcm2-3-5 family protein, 26S proteasome subunit P45 family protein and dnaK protein) were highly expressed only in theronts; while protein kinase domain containing protein and heat shock protein 70 showed high levels of expression only in trophonts and tomonts, respectively. Moreover, actin was co-detected with 3 rabbit anti-sera while ß-tubulin, V-type ATPase α subunit family protein, heat shock protein 70, mitochondrial-type hsp70, and dnaK proteins showed immunoreactivity with corresponding rabbit anti-sera in theronts, trophonts, and tomonts. Furthermore, ß-tubulin, the metabolic-related protein enolase, NADH-ubiquinone oxidoreductase 75 kDa subunit, malate dehydrogenase, as well as polypyrimidine tract-binding protein, glutamine synthetase, protein kinase domain containing protein, TNFR/NGFR cysteine-rich region family protein, and vacuolar ATP synthase catalytic subunit α, were commonly detected by grouper anti-sera. Therefore, these findings could contribute to an understanding of the differences in gene expression and phenotypes among the different stages of parasitic infection, and might be considered as a source of candidate proteins for disease diagnosis and vaccine development.

Molecular cloning of NCCRP-1 gene from orange-spotted grouper (Epinephelus coioides) and characterization of NCCRP-1(+) cells post Cryptocaryon irritans infection.

Nonspecific cytotoxic cells (NCCs) are an important cytotoxic cell population in the innate teleost immune system. The receptor designated "NCC receptor protein 1" (NCCRP-1) has been reported to be involved in the recognition and activation of NCCs. In this study, the full-length cDNA of Epinephelus coioides NCCRP-1 (ecnccrp-1) was cloned. The open reading frame (ORF) of ecnccrp-1 is 699 bp, encoding a 232 amino acid protein that includes proline-rich motifs at the N-terminus and is related to the F-box associated family. Although a bioinformatics analysis showed that EcNCCRP-1 had no signal peptide or transmembrane helices, a polyclonal antibody directed against recombinant EcNCCRP-1 efficiently labeled a membrane protein in the head kidney, detected with Western blot analysis, which indicated that the protein localized to the cell surface. RT-PCR showed that the constitutive expression of ecnccrp-1 was higher in the lymphoid organs, such as the trunk kidney, spleen, head kidney, and thymus, and lower in brain, heart, fat, liver, muscle, and skin. After infection with Cryptocaryon irritans, the transcription of ecnccrp-1 was analyzed at the infected sites (skin and gills) and in the systemic immune organs (head kidney and spleen). At the infected sites, especially the skin, ecnccrp-1 expression was upregulated at 6h post infection, reaching peak expression on day 3 post the primary infection. However, the expression patterns differed in the systemic immune organs. In the spleen, ecnccrp-1 was gradually increased in the early infection period and decreased sharply on day 3 post the primary infection, whereas in the head kidney, the transcription of ecnccrp-1 was depressed during almost the whole course of infection. An immunohistochemical analysis showed that EcNCCRP-1(+) cells accumulated at the sites of infection with C. irritans. These results suggested that NCCs were involved in the process of C. irritans infection in E. coioides.

Comparative proteome analysis of two Streptococcus agalactiae strains from cultured tilapia with different virulence.

Streptococcus agalactiae is a major piscine pathogen, which causes significant morbidity and mortality among numerous fish species, and results in huge economic losses to aquaculture. Many S. agalactiae strains showing different virulence characteristics have been isolated from infected tilapia in different geographical regions throughout South China in the recent years, including natural attenuated S. agalactiae strain TFJ0901 and virulent S. agalactiae strain THN0901. In the present study, survival of tilapia challenged with S. agalactiae strain TFJ0901 and THN0901 (10(7)CFU/fish) were 93.3% and 13.3%, respectively. Moreover, there are severe lesions of the examined tissues in tilapia infected with strain THN0901, but no significant histopathological changes were observed in tilapia infected with the strain TFJ0901. In order to elucidate the factors responsible for the invasive potential of S. agalactiae between two strains TFJ0901 and THN0901, a comparative proteome analysis was applied to identify the different protein expression profiles between the two strains. 506 and 508 cellular protein spots of S. agalactiae TFJ0901 and THN0901 were separated by two dimensional electrophoresis, respectively. And 34 strain-specific spots, corresponding to 27 proteins, were identified successfully by MALDI-TOF mass spectrometry. Among them, 23 proteins presented exclusively in S. agalactiae TFJ0901 or THN0901, and the other 4 proteins presented in different isomeric forms between TFJ0901 and THN0901. Most of the strain-specific proteins were just involved in metabolic pathways, while 7 of them were presumed to be responsible for the virulence differences of S. agalactiae strain TFJ0901 and THN0901, including molecular chaperone DnaJ, dihydrolipoamide dehydrogenase, thioredoxin, manganese-dependent inorganic pyrophosphatase, elongation factor Tu, bleomycin resistance protein and cell division protein DivIVA. These virulence-associated proteins may contribute to identify new diagnostic markers and help to understand the pathogenesis of S. agalactiae.