Application of environmental DNA metabarcoding to identify fish community characteristics in subtropical river systems.

Fish are vital in river ecosystems; however, traditional investigations of fish usually cause ecological damage. Extracting DNA from aquatic environments and identifying DNA sequences offer an alternative, noninvasive approach for detecting fish species. In this study, the effects of environmental DNA (eDNA), coupled with PCR and next-generation sequencing, and electrofishing for identifying fish community composition and diversity were compared. In three subtropical rivers of southern China, fish specimens and eDNA in water were collected along the longitudinal (upstream-downstream) gradient of the rivers. Both fish population parameters, including species abundance and biomass, and eDNA OTU richness grouped 38 sampling sites into eight spatial zones with significant differences in local fish community composition. Compared with order-/family-level grouping, genus-/species-level grouping could more accurately reveal the differences between upstream zones I-III, midstream zones IV-V, and downstream zones VI-VIII. From the headwaters to the estuary, two environmental gradients significantly influenced the longitudinal distribution of the fish species, including the first gradient composed of habitat and physical water parameters and the second gradient composed of chemical water parameters. The high regression coefficient of alpha diversity between eDNA and electrofishing methods as well as the accurate identification of dominant, alien, and biomarker species in each spatial zone indicated that eDNA could characterize fish community attributes at a level similar to that of traditional approaches. Overall, our results demonstrated that eDNA metabarcoding can be used as an effective tool for revealing fish composition and diversity, which is important for using the eDNA technique in aquatic field monitoring.

Occurrence, distribution, and risk assessment of antibiotics in a typical aquaculture area around the Dongzhai Harbor mangrove forest on Hainan Island.

The trees of the Dongzhai Harbor mangrove forest suffer from antibiotic contamination from surrounding aquaculture areas. Despite this being one of the largest mangrove forests in China, few studies have focused on the antibiotic pollution status in these aquaculture areas. In the present study, the occurrence, distribution, and risk assessment of 37 antibiotics in surface water and sediment samples from aquaculture areas around Dongzhai Harbor mangrove forests were analyzed. The concentration of total antibiotics (∑antibiotics) ranged from 78.4 ng/L to 225.6 ng/L in surface water (except S14-A2) and from 19.5 ng/g dry weight (dw) to 229 ng/g dw in sediment. In the sediment, the concentrations of ∑antibiotics were relatively low (19.5-52.3 ng/g dw) at 75 % of the sampling sites, while they were high (95.7-229.0 ng/g dw) at a few sampling sites (S13-A1, S13D, S8D). The correlation analysis results showed that the Kd values of the 9 antibiotics were significantly positively correlated with molecular weight (MW), Kow, and LogKow. Risk assessment revealed that sulfamethoxazole (SMX) in surface water and SMX, enoxacin (ENX), ciprofloxacin (CFX), enrofloxacin (EFX), ofloxacin (OFX), and norfloxacin (NFX) in sediment had medium/high risk quotients (RQs) at 62.5 % and 25-100 %, respectively, of the sampling sites. The antibiotic mixture in surface water (0.06-3.36) and sediment (0.43-309) posed a high risk at 37.5 % and 66.7 %, respectively, of the sampling sites. SMX was selected as an indicator of antibiotic pollution in surface water to assist regulatory authorities in monitoring and managing antibiotic pollution in the aquaculture zone of Dongzhai Harbor. Overall, the results of the present study provide a comprehensive and detailed analysis of the characteristics of antibiotics in the aquaculture environment around the Dongzhai Harbor mangrove system and provide a theoretical basis for the source control of antibiotics in mangrove systems.

Abundance and characteristics of microplastics in the Wanquan River estuary, Hainan Island.

As the nexus where rivers and oceans meet, estuaries are vulnerable to microplastic (MP) pollution derived from rivers. However, few studies have focused on the pollution status of MPs in small estuarine areas. Here, the abundance and characteristics of MPs in surface water and sediment samples from a small estuary, the Wanquan River estuary, were studied. The average abundance of MPs was 6573 ± 2659 n/m3 in surface water and 1065 ± 696 n/kg dw in sediment samples from the Wanquan River estuary. Most of the MPs in water samples and sediments were red (92.9 % and 88.1 %) fragments (87.4 % and 95.5 %) with sizes <1.0 mm (90.8 % and 92.4 %) made up of antifouling paint particles (APPs) (83.5 % and 89.8 %), respectively. A significant positive correlation (p < 0.01) was found between the concentration of Cu2+ and the abundance of APPs in sediment samples from the Wanquan River estuary. The APPs in the sediments can act as a continuous source of toxic chemicals (e.g., Cu2+) to marine environments. The results of this study expand our knowledge about MP pollution in small estuaries, and the ecological risk of APPs in the Wanquan River estuary to aquatic organisms should not be ignored.

Perfluorooctanoic acid (PFOA)-induced alterations of biomolecules in the wetland plant Alismaorientale.

Perfluoroalkyl substances (PFASs) have been widely studied by researchers due to their environmental persistence, chemical stability and potential toxicity. Some researchers have reported the physiological and biochemical toxicity of PFASs on plants through traditional and innovative methods; however, the changes in biological macromolecules caused by PFASs are rarely studied. Here, Fourier transform infrared spectroscopy (FTIR) was used to study how exposure to perfluorooctanoic acid (PFOA) alters the structure and function of biomolecules of the wetland plant Alisma orientale. Biomass results showed that PFOA had negative effects on plant growth. FTIR results showed that PFOA could result in changes in the structures, compositions, and functions of lipids, proteins and DNA in plant cells. In the treatment groups, the ratios of CH3 to lipids and carbonyl esters to lipids increased compared with the control, while the ratios of CH2 to lipids and olefinicCH to lipids decreased, which indicated lipid peroxidation caused by PFOA exposure. Changes in the compositions and secondary structures of proteins were also found, which were indicated by the decreased ratio of amide I to amide II and the increased ratio of ß-sheet to α-helix in the treatment groups compared to the control. Moreover, PFOA affected the composition of DNA by promoting the B- to A-DNA transition. These results showed that the mechanism of PFOA toxicity toward plants at the biochemical level could be illustrated by FTIR.

Longitudinal pattern of resource utilization by aquatic consumers along a disturbed subtropical urban river: Estimating the relative contribution of resources with stable isotope analysis.

The utilization of food resources by aquatic consumers reflects the structure and functioning of river food webs. In lotic water systems, where food availability and predator-prey relationships vary with gradient changes in physical conditions, understanding diet assimilation by local communities is important for ecosystem conservation. In the subtropical Liuxi River, southern China, the relative contribution of basal resources to the diet assimilation of functional feeding groups (FFGs) was determined by stable carbon (13C) and nitrogen (15N) isotope analyses. The output of Bayesian mixing models showed that diatom-dominated periphyton (epilithic biofilm), aquatic C3 plants (submerged hydrophytes), and suspended particulate organic matter (SPOM) associated with terrestrial C3 plants contributed the most to the diet assimilation of FFGs in the upper, middle, and lower reaches, respectively. The relative contribution of consumer diet assimilation was weighted by the biomass (wet weight, g/m2) of each FFG to reflect resource utilization at the assemblage level. From the upper to the lower reaches, the spatial variation in the diet assimilation of fish and invertebrate assemblages could be summarized as a longitudinal decrease in periphyton (from 57%-76% to <3%) and an increase in SPOM (from <7% to 51%-68%) with a notable midstream increase in aquatic C3 plants (23%-48%). These results indicate that instream consumers in the Liuxi River rely more on autochthonous production (e.g., periphyton and submerged hydrophytes) than on terrestrially derived allochthonous matter (e.g., terrestrial plants). The pattern of resource utilization by consumers in the mid-upper Liuxi River is consistent with findings from other open subtropical and neotropical rivers and provides evidence for the riverine productivity model. Our study indicates that protecting inherent producers in rivers (e.g., periphyton and submerged hydrophytes) and restoring their associated habitats (e.g., riffles with cobble substrate) are conducive to aquatic ecosystem management.

Impacts of environmental factors on the food web structure, energy flows, and system attributes along a subtropical urban river in southern China.

Tropical and subtropical rivers are being subjected to multiple stressors from human disturbance (e.g., water pollution and habitat degradation). Understanding the relationship between environmental conditions and the river ecosystem is important for improving river management. We built 14 Ecopath models composed of 28 functional groups (trophic levels [TLs] of 1.0-3.8) along a subtropical urban river to explore the influence of environmental changes on system attributes. From headwaters to downstream, the model outputs showed that the transfer efficiency (TE), energy flow parameters, and ecosystem theory indices exhibited significant (P < 0.05) differences across a longitudinal gradient of disturbance, indicating heterogeneous attributes of local river segments. The high TE values of TLs I, II, and III separated the upper, middle, and lower reaches, respectively, which could be attributed to the shift in dominant consumption flows from upstream 'periphyton - aquatic insects - insectivorous fish' to midstream 'detritus - shrimp - crustaceavorous fish' and to downstream 'phytoplankton - filter-feeding invertebrates/fish'. Structural equation modelling was used to test the causal relationships among environmental variables and demonstrated that abiotic factors directly influenced biomass composition and indirectly influenced trophic networks. Water quality, including dissolved oxygen and flow velocity; habitat characteristics, such as riffles, cobble-gravel substrate, and seasonal floodplain; and biological indicators, including the relative contributions (%) of decapods, insectivorous fish, and insect scrapers to biomass composition, had significant (P < 0.05) positive impacts on system maturity (evaluated by omnivory, connectance, and cycling indices). In the future, it will be possible to evaluate the health of river ecosystems by monitoring representative environmental factors, which could be a cost-effective approach to system-level improvement.

Fish isotopic niches associated with environmental indicators and human disturbance along a disturbed large subtropical river in China.

Stable isotopes are increasingly used to detect and understand the impacts of environmental changes on riverine ecological properties. The δ13C and δ15N signatures of fish with different feeding habits were measured in a large subtropical river to evaluate how fish isotopic niches respond to environmental gradients and human disturbance. From basal resources to fish consumers, the high values of epilithic periphyton (biofilm) δ13C and suspended particulate organic matter δ15N concurrently determined the niche ranges and space (e.g., convex hull area) of fish communities. Along a longitudinal gradient (except in the industrial zone), the number of fish trophic guilds identified by Bayesian ellipses continuously increased; meanwhile, higher trophic diversity and less redundancy were observed near the lower reaches and estuary. Variance inflation factors were estimated to detect the multicollinearity of 40 environmental variables, 14 of which were selected as indicators. Relative importance (RI) analysis was used to evaluate the explanatory power of these indicators for the spatial variation in isotopic niche metrics; the results showed that riffle habitat area, water nitrate concentration, gravel-cobble substrate, and riparian buffer width were the 4 key environmental indicators (average RI > 12%) that determined the longitudinal pattern of fish isotopic niches. These findings suggested that community-level δ13C signatures are more responsive to changes in habitats (e.g., riffle) and substrates (e.g., gravel-cobble) supporting the productivity of autochthonous diatoms while δ15N signatures respond to water quality altered by nitrogen pollution from manure-fertilized farming and poultry livestock effluent. Furthermore, δ15N may be more robust and interpretable than δ13C as an isotopic indicator of ecosystem change in rivers exposed to multiple or complex anthropogenic stressors.

The feeding of dietary Codonopsis pilosula polysaccharide enhances the immune responses, the expression of immune-related genes and the growth performance of red swamp crayfish (Procambarus clarkii).

Polysaccharides have many functions in aquatic animals and are widely used as immunopotentiators. However, despite the emergence of serious diseases, few studies have explored the effects of Codonopsis pilosula polysaccharide (CPP) on crustaceans. We studied the effects of CPP on the growth performance, nonspecific immunity, antioxidant activity and disease resistance of red swamp crayfish (Procambarus clarkii). Healthy crayfish (5.80 ± 0.1 g) were fed diets supplemented with 0% (control), 0.05%, 0.1%, 0.15%, 0.20%, and 0.30% CPP for 8 weeks. At the end of the 8-week feeding trial, the optimal final body weight (FBW), weight gain (WG), specific growth rate (SGR), and feed conversion ratio (FCR) were observed in the crayfish fed the diets with 0.15% and 0.20% CPP, followed by those fed the diet with 0.30% CPP and then those fed the diet with 0.10% CPP, whereas the values of these parameters were obtained with the control crayfish (P < 0.05). The crayfish fed the diets with 0.15% and 0.20% CPP exhibited a significantly higher total hemocyte count (THC) and significantly increased phenoloxidase (PO), lysozyme (LZM), hemocyte (Hc), acid phosphatase (ACP) and alkaline phosphatase (AKP) compared with those belonging to the other groups (P < 0.05). The crayfish fed the diets with 0.15% and 0.2% CPP exhibited significantly higher total superoxide dismutase (T-SOD) and glutathione peroxidase (GPx) activities, a significantly increased total antioxidant capacity (T-AOC) and a significantly lower malondialdehyde (MDA) content compared with the other groups (P < 0.05), which indicated that antioxidant capacity was significantly induced by the CPP-supplemented diets. Significantly upregulated expression of immune-related genes (anti-lipopolysaccharide factors (alf), peroxiredoxin (prx5), cathepsin B (ctsb), mitochondrial manganese superoxide dismutase (mtMnsod), cyclophilin A (cypa), glutathione peroxidase (gpx), Toll-like receptor 3 (tlr3), and heat shock protein 70 (hsp70)) was detected in the crayfish fed the diets supplemented with 0.15% and 0.20% CPP diet compared with the levels observed in the control crayfish. These results showed that dietary CPP supplementation greatly improved the growth, immunity and antioxidant capacities of crayfish, and according to the observed results, 0.15%-0.2% is the recommended optimal level of CPP dietary supplementation for crayfish.

Application of mass-balance modelling to assess the effects of ecological restoration on energy flows in a subtropical reservoir, China.

Eutrophication is a leading cause of impairment of lentic water bodies throughout the world. To inhibit algal blooms and remove excess nutrients, a 10,000 m2 restoration project consisting of vegetation establishment and fish manipulation was conducted in the eutrophic bay of the Yantian Reservoir, southern China. Three Ecopath models were constructed to assess the recovery effects at an ecosystem level, and time series data were simulated to propose a fishery policy. During the restoration, 1) the redundant primary production flowing back to detritus decreased due to the increased predation of four stocked fish with different feeding habits; 2) the transfer efficiencies (TEs) through trophic levels increased due to the reinforced energy flows along the planktivorous, herbivorous, and molluscivorous food chains; 3) the groups that had the highest keystoneness shifted from carnivorous fish to invertivorous fish and omnivorous shrimp, indicating the shift of mixed trophic impacts from top-down to wasp-waist control; and 4) the changing indices of path length, flow fluxes, matter cycling, and network information showed that the restored system was more mature, developed, and organized than before. To sustain the long-term energy balance and functioning of the ecosystem, the maximum fishing yields (0.37-8.53 g/m2/year) were determined to maintain the relative biomass (close to 1) of stocked fish and wild tilapia by harvesting their annual production.

[Study on comprehensive monitoring of mouse and effect of hemorrhagic fever with renal syndrome vaccine in high prevalence areas of natural focus infectious disease of Zhejiang province in 1994--2010].

OBJECTIVE: To study the comprehensive monitoring mechanism of mouse and the effect of hemorrhagic fever with renal syndrome (HFRS) vaccine in the high prevalence areas of natural focus infectious disease of Zhejiang province in 1994 - 2010. METHODS: The night trapping method was used to monitor the population proportion, density and the rate of hantavirus (HV) carriers in mice in Xikou township Longyou county in August and September from 1994 to 2010. The healthy residents in Xikou township aged 16 to 60 years were recruited. The subjects were randomly selected as vaccination group and control group according to age, sex, occupational distribution (10 178 in intervention group and 16 159 in control group). Intervention group was given purified and inactivated vaccine from suckling mouse brain, while the control group received no intervention. The prevention effect was evaluated by protective rate of vaccine. RESULTS: The mouse population was stable in the sixteen years and the apodemus agrarius was the main type (76.5% (564/737)). The average density of mouse was 4.73% (1170/24 727). The average rate of virus carrier of mouse was 3.87% (41/1033). In 1994 - 1995, the density of mouse was 22.82% (186/815) and the rate of virus carrier was 7.0% (10/143). In 2009 - 2010, the density of mouse decreased to 2.75% (119/4330) and the rate of virus carrier was 5.5% (13/237). The average antibody positive rate of mouse from 2005 to 2010 was 4.8% (35/728) and the rate was 4.4% (6/138), 0.0% (0/113), 11.8% (16/136), 1.0% (1/104), 3.7% (4/109) and 6.3% (8/128) in each year (P < 0.01). The protective rate of HFRS vaccine was 96.2% (1 case in intervention group and 41 cases in control group). CONCLUSION: The density of mouse decreased significantly in Zhejiang province. The rate of virus carrier of mouse is stable. The vaccine is effective.

[Study on the integrated monitoring program regarding mouse and main mouse-borne disease in Zhejiang province].

OBJECTIVE: To study the integrated monitoring program regarding mouse and plague, hemorrhagic fever of renal syndrome (HFRS) and leptospirosis. METHODS: Integrated monitoring plan was used. A designated office coordinated 5 departments' actions within the Zhejiang Provincial Center for Disease Control and Prevention (CDC). Cage-trapping method was conducted to monitor the density of mice from June to October, respectively. RESULTS: Lishui municipal CDC had finished the integrated monitoring program on mouse and mouse-borne disease while the Longyou CDC had finished the field investigation, using the integrated monitoring program. Specimens were sent to provincial CDC. The integrated monitoring program needed more number of personnel and better coordination.Lishui reported 3 leptospirosis cases and 58 HFRS cases in 2009, with the incidence rates as 0.13 and 2.44 per 100 000, respectively. Longyou reported 2 leptospirosis case and 1 HFRS cases in 2009, with the incidence rates as 0.49 and 0.25 per 100 000, respectively. Lishui and Longyou had no plague case. Lishui caught 91 mice in 2009 and the density was 4.17%. Longyou caught 37 mice in 2009, with the density as 1.18 percent. Most mice caught from Lishui were Apodemus agrarius and the next was Mus musculus. In Longyou the Rattus tanezumi ranked the first, followed by Apodemus agrarius. The positive rate of HFRS antigen in Lishui and Longyou were 10.42% and 4.59% respectively. The positive rate of HFRS antibody in Longyou was 3.70%. The culture positive rate of leptospirosis in mouse renal of Lishui and Longyou were 0 and 0.98% respectively. The culture positive rate of leptospirosis in pig renal, duck renal, frog renal and cattle urine of Longyou was 0. The culture positive rate of leptospirosis in duck blood of Longyou was 80%. CONCLUSION: The integrated monitoring program on mouse and mouse-borne disease seemed to be feasible and could promote the integrated surveillance and control program on mouse and mouse-borne diseases in China.

[Study on a 10-year protective effects of vaccination against hemorrhagic fever with renal syndrome].

OBJECTIVE: To evaluate the epidemiological and serological efficacy after 10 years of vaccination against hemorrhagic fever with renal syndrome (HFRS) vaccines in Zhejiang province. METHODS: One county was randomly chosen as the research unit with all the healthy people between 16 and 60 years old were equally divided into study and control groups. The study group was vaccinated. Immunofluorescent antibody assay was used to test specific IgG antibody and Mcro-CPE method was used to test the titer of neutralizing antibody. RESULTS: Two weeks after the full-course immunization, the seroconversion rate became 100% (67/67, with 95% CI as 96.3%-100%) by immunofluorescent antibody test (IgG) and 44.4% (8/18 with 95% CI as 22.0%-69.0%) by neutralization test with GMT titers as 72.1 and 4.6 respectively. Booster immunization was provided one year later. Time span as two weeks prior to, one year, one and half years, two years, three years and five years after booster immunization, the rates of seroconversion on immunofluorescent antibody using IFAT method, were 28.6%, 83.3%, 75.0%, 53.1%, 22.6%, 10.0% and 55.0% respectively, and rates of seroconversion of neutralizing antibody by Mcro-CPE method were 14.8%, 55.6%, 35.0%, 31.3%, 26.0%, 10.0% and 50.0% respectively. Nine years after the reinforcement, the rates of seroconversion of immunofluorescent antibody by IFAT method was only 7.1%. The vaccinated group had no patient seen but the control group appeared 34 patients including 3 deaths. According to the ten-year observation, the vaccine seemed effective with the protection rate in population reached 100%. CONCLUSION: HFRS vaccine was effective on epidemiological, social and economical efficacy.