Japanese flounder (Paralichthys olivaceus) responsive mechanisms to hypoxia are still not fully understood. Therefore, we performed an acute hypoxic treatment (dissolved oxygen at 2.07 ± 0.08 mg/L) on Japanese flounder. It was confirmed that the hypoxic stress affected the physiological phenotype through changes in blood physiology (RBC, HGB, WBC), biochemistry (LDH, ALP, ALT, GLU, TC, TG, ALB), and hormone (cortisol) indicators. Hypoxia inducible factor-1 (HIF-1), an essential oxygen homeostasis mediator in organisms consisting of an inducible HIF-1α and a constitutive HIF-1β, and its target gene LDH-A were deeply studied. Results showed that HIF-1α and LDH-A genes were co-expressed and significantly affected by hypoxic stress. The dual-luciferase reporter assay confirmed that transcription factor HIF-1 transcriptionally regulated the LDH-A gene, and its transcription binding sequence was GGACGTGA located at -2343~-2336. The DNA methylation status of HIF-1α and LDH-A genes were detected to understand the mechanism of environmental stress on genes. It was found that hypoxia affected the HIF-1α gene and LDH-A gene methylation levels. The study uncovered HIF-1/LDH-A signaling pathway responsive mechanisms of Japanese flounder to hypoxia in epigenetic modification and transcriptional regulation. Our study is significant to further the understanding of environmental responsive mechanisms as well as providing a reference for aquaculture.

Advances in high-throughput sequencing technologies allow a more complete study of microbial plankton community composition and diversity, especially in the rare microbial biosphere. The DNA extraction of plankton is a key step for such studies; however, little is known about its influences on the abundant or rare microbial biosphere. Our aim was to quantify the influences of different DNA extraction kits on abundant and rare plankton in the surface waters of a reservoir and provide a reference for the comparisons between microbial community studies with different extraction methods. We evaluated the influence of five common commercial kits on DNA quality, microbial community diversity and composition, and the reproducibility of methods using both 16S and 18S rRNA genes amplicon sequencing. Our data showed that results of Fast DNA Spin Kit for Soil (MPF) had higher α diversity for bacteria and high DNA quality, indicating that it is the most suitable approach for bacterioplankton diversity study. However, DNeasy Blood & Tissue Kit (QD) and QIAamp DNA Mini Kit (QQ) methods could produce results that are easier to replicate for bacteria and eukaryotes, respectively, and were more comparable between studies. The use of different DNA extraction kits had larger influence on the rare taxa compared with abundant taxa. Therefore, the comparability between studies that employed different extraction methods can be improved by removing low-abundance or less-representative OTUs. Collectively, this study provides a comprehensive assessment of the biases associated with DNA extraction for plankton communities from a freshwater reservoir. Our results may guide researchers in experimental design choices for DNA-based ecological studies in aquatic ecosystem.

Aquatic environments serve as important reservoirs of antibiotic resistance genes (ARGs), but the information on the high-resolution temporal pattern of ARGs in waterbodies is extremely limited. In this study, the weekly dynamics of ARGs and their relationships with microbial taxonomic communities and environmental variables were analyzed in a subtropical urban reservoir over the period of 1 year using high-throughput approaches. In total, 197 ARGs and 10 mobile genetic elements (MGEs) were detected. The results showed that the bacterial community had a seasonal pattern, while ARGs composition did not exhibit seasonality, thereby indicating the asynchrony or decoupling of temporal patterns of microbial taxonomy and function. More importantly, bacterial abundance and community diversity were more strongly correlated with 17 measured environmental variables than ARGs (36 significant correlations for OTUs, 11 for ARGs). However, stochastic processes appeared to have a minor role in the structuring of the ARG profiles, but a more important role in the structuring of bacterial taxonomic communities. Furthermore, we found that precipitation and turbidity were significantly correlated with the richness and diversity of ARGs, suggesting that multiple environmental factors influence the composition and dynamics of ARGs in complex ways. MGEs were abundant and showed significant positive correlations with ARGs, indicating a plausible influence of MGEs on the variation of ARGs. This is the first study which provides an overview of high-resolution dynamics of ARGs in a subtropical waterbody. Our results improve the understanding of microbial processes and mechanisms of ARGs at fine temporal scale, and offer empirical data of use in the monitoring, assessment and management of the urban water environments.

Invasion by exotic plant species can alter ecosystem function and reduce native plant diversity, but relatively little is known about their effects on belowground microbial communities. Here we investigated the effects of exotic cordgrass (Spartina alterniflora) invasion on the distribution of soil bacterial communities in a mangrove nature reserve of the Jiulong River Estuary, southeast China using high-throughput sequencing of 16S rRNA gene and multivariate statistical analysis. Our results showed that S. alterniflora invasion altered soil properties, and significantly increased soil bacterial taxa richness, primarily by stimulating an increase in conditionally rare or rare taxa, and changes in community composition and function. Abundant, conditionally rare and rare subcommunities exhibited similar response patterns to environment changes, with both conditionally rare and rare taxa showing a stronger response than abundant ones. Habitat generalists were detected among abundant, conditionally rare and rare taxa, whereas habitat specialists were only identified among conditionally rare taxa and rare taxa. In addition, we found that vegetation was the key factor driving these patterns. However, our comparative analysis indicated that both environmental selection, and neutral process, significantly contributed to soil bacterial community assembly. These results could improve the understanding of the microbial processes and mechanisms of cordgrass invasion, and offer empirical data of use in the restoration and management of the mangrove wetlands.

Although it is widely recognized that cyanobacterial blooms have substantial influence on the plankton community in general, their correlations with the whole community of eukaryotic plankton at longer time scales remain largely unknown. Here, we investigated the temporal dynamics of eukaryotic plankton communities in two subtropical reservoirs over a 6-year period (2010-2015) following one cyanobacterial biomass cycle-the cyanobacterial bloom (middle 2010), cyanobacteria decrease (late 2010-early 2011), non-bloom (2011-2014), cyanobacteria increase, and second bloom (late 2014-2015). The eukaryotic community succession that strongly correlated with this cyanobacterial biomass cycle was divided into four periods, and each period had distinct characteristics in cyanobacterial biomass and environments in both reservoirs. Integrated co-occurrence networks of eukaryotic plankton based on the whole study period revealed that the cyanobacterial biomass had remarkably high network centralities, and the eukaryotic OTUs that had stronger correlations with the cyanobacterial biomass exhibited higher centralities. The integrated networks were also modularly responded to different eukaryotic succession periods, and therefore correlated with the cyanobacterial biomass cycle. Moreover, sub-networks based on the different eukaryotic succession periods indicated that the eukaryotic co-occurrence patterns were not constant but varied largely associating with the cyanobacterial biomass. Based on these long-term observations, our results reveal that the cyanobacterial biomass cycle created distinct niches between persistent bloom, non-bloom, decrease and increase of cyanobacteria, and therefore associated with distinct eukaryotic plankton patterns. Our results have important implications for understanding how complex aquatic plankton communities respond to cyanobacterial blooms under the changing environments.

Plankton communities normally consist of few abundant and many rare species, yet little is known about the ecological role of rare planktonic eukaryotes. Here we used a 18S ribosomal DNA sequencing approach to investigate the dynamics of rare planktonic eukaryotes, and to explore the co-occurrence patterns of abundant and rare eukaryotic plankton in a subtropical reservoir following a cyanobacterial bloom event. Our results showed that the bloom event significantly altered the eukaryotic plankton community composition and rare plankton diversity without affecting the diversity of abundant plankton. The similarities of both abundant and rare eukaryotic plankton subcommunities significantly declined with the increase in time-lag, but stronger temporal turnover was observed in rare taxa. Further, species turnover of both subcommunities explained a higher percentage of the community variation than species richness. Both deterministic and stochastic processes significantly influenced eukaryotic plankton community assembly, and the stochastic pattern (e.g., ecological drift) was particularly pronounced for rare taxa. Co-occurrence network analysis revealed that keystone taxa mainly belonged to rare species, which may play fundamental roles in network persistence. Importantly, covariations between rare and non-rare taxa were predominantly positive, implying multispecies cooperation might contribute to the stability and resilience of the microbial community. Overall, these findings expand current understanding of the ecological mechanisms and microbial interactions underlying plankton dynamics in changing aquatic ecosystems.

Freshwater ecosystems are important sources of drinking water and provide natural settings for the proliferation and dissemination of bacteria and antibiotic resistance genes (ARGs). However, the biogeographical patterns of ARGs in natural freshwaters and their relationships with the bacterial community at large scales are largely understudied. This is of specific importance because data on ARGs in environments with low anthropogenic impact is still very limited. We characterized the biogeographical patterns of bacterial communities and their ARG profiles in 24 reservoirs across southeast China using 16S rRNA gene high-throughput sequencing and high-throughput-quantitative PCR, respectively. We found that the composition of both bacterial communities and ARG profiles exhibited a significant distance-decay pattern. However, ARG profiles displayed larger differences among different water bodies than bacterial communities, and the relationship between bacterial communities and ARG profiles was weak. The biogeographical patterns of bacterial communities were simultaneously driven by stochastic and deterministic processes, while ARG profiles were not explained by stochastic processes, indicating a decoupling of bacterial community composition and ARG profiles in inland waters under relatively low-human-impact at a large scale. Overall, this study provides an overview of the biogeographical patterns and driving mechanisms of bacterial community and ARG profiles and could offer guidance and reference for the control of ARGs in drinking water sources. IMPORTANCE Antibiotic resistance has been a serious global threat to environmental and human health. The "One Health" concept further emphasizes the importance of monitoring the large-scale dissemination of ARGs. However, knowledge about the geographical patterns and driving mechanisms of bacterial communities and ARGs in natural freshwater environments is limited. This study uncovered the distinct biogeographical patterns of bacterial communities and ARG profiles in inland waters of southeast China under low-anthropogenic impact at a large scale. This study improved our understanding of ARG distribution in inland waters with emphasis on drinking water supply reservoirs, therefore providing the much-needed baseline information for future monitoring and risk assessment of ARGs in drinking water resources.

Unraveling the relative importance of ecological processes regulating microbial community structure is a central goal in microbial ecology. Here, we used high-throughput sequencing to examine the relative contribution of selective and neutral processes in the assembly of abundant and rare subcommunities from three subtropical bays of China. We found that abundant and rare bacterial taxa were distinctly different in diversity, despite the similar biogeographic patterns and strong distance-decay relationships, but the dispersal of rare bacterial taxa was more limited than that of abundant taxa. Furthermore, the environmental (selective processes) and spatial (neutral processes) factors seemed to govern the assembly and biogeography of abundant and rare bacterial subcommunities, although both factors explained only a small fraction of variation within the rare subcommunity. More importantly, variation partitioning (based on adjusted R2 in redundancy analysis) showed that spatial factors exhibited a slightly greater influence on both abundant and rare subcommunities compared to environmental selection; however, the abundant subcommunity had a much stronger response to spatial factors (17.3% of pure variance was explained) than that shown by the rare bacteria (3.5%). These results demonstrate that environmental selection and neutral processes explained the similar biogeographic patterns of abundant and rare subcommunities, but a large proportion of unexplained variation in the rare taxa (91.1%) implies that more complex assembly mechanisms may exist to shape the rare bacterial assemblages in the three subtropical bays.

Microeukaryotes play key roles in the structure and functioning of marine ecosystems. Little is known about the relative importance of the processes that drive planktonic and benthic microeukaryotic biogeography in subtropical offshore areas. This study compares the microeukaryotic community compositions (MCCs) from offshore waters (n = 12) and intertidal sediments (n = 12) around Xiamen Island, southern China, using high-throughput sequencing of 18S rDNA. This work further quantifies the relative contributions of spatial and environmental variables on the distribution of marine MCCs (including total, dominant, rare and conditionally rare taxa). Our results showed that planktonic and benthic MCCs were significantly different, and the benthic richness (6627 OTUs) was much higher than that for plankton (4044 OTUs) with the same sequencing effort. Further, we found that benthic MCCs exhibited a significant distance-decay relationship, whereas the planktonic communities did not. After removing two unique sites (N2 and N3), however, 72% variation in planktonic community was explained well by stochastic processes. More importantly, both the environmental and spatial factors played significant roles in influencing the biogeography of total and dominant planktonic and benthic microeukaryotic communities, although their relative effects on these community variations were different. However, a high proportion of unexplained variation in the rare taxa (78.1-97.4%) and conditionally rare taxa (49.0-81.0%) indicated that more complex mechanisms may influence the assembly of the rare subcommunity. These results demonstrate that patterns and processes in marine microeukaryotic community assembly differ among the different habitats (coastal water vs. intertidal sediment) and different communities (total, dominant, rare and conditionally rare microeukaryotes), and provide novel insight on the microeukaryotic biogeography and ecological mechanisms in coastal waters and intertidal sediments at local scale.