Development of a duplex PCR for the simultaneous detection of EHP and IHHNV and analysis of the correlation between these two pathogens.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the linearly single-stranded DNA viruses. Ecytonucleospora hepatopenaei (EHP) is an intracellular parasitic microsporidian. IHHNV and EHP are pathogens that have been widely prevalent in shrimp farming. Both of them are associated with growth retardation of the penaeid shrimp, which causes serious economic losses to shrimp farming. Shrimp can be co-infected with IHHNV and EHP. In this study, a rapid duplex polymerase chain reaction (PCR) was developed and optimized for the simultaneous detection of EHP and IHHNV. The detection limit of the duplex PCR could reach 1.5 × 102 copies for EHP and IHHNV. A total of 578 Litopenaeus vannamei samples were detected by the established duplex PCR detection method. The results suggested that 398 samples were infected with EHP, 362 samples were infected with IHHNV, and 265 samples were co-infected with EHP and IHHNV. The case-control analysis of the detected shrimp samples showed a certain synergistic effect between EHP and IHHNV.

Study of infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in different organs of Penaeus vannamei.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured shrimp. It is generally believed that the target organs of IHHNV in shrimp include tissues of ectodermal and mesodermal origin, but do not normally include organ systems of endodermal origin, such as hepatopancreas. In this study, the feeding challenge of IHHNV in different organs (pleopods, muscles, gills, and hepatopancreas) of Penaeus vannamei was studied. The PCR results showed that hepatopancreas of P. vannamei had the strongest IHHNV positivity (100% positive, 19.4 copies/mg) in the feeding challenge experiment. Gills and pleopods had similar infectivity to IHHNV (86.7% positive, 10.6 and 10.5 copies/mg). Among the four organs tested in this study, the IHHNV positivity of muscles was the weakest (33.3% positive, 4.7 copies/mg). The IHHNV infection to hepatopancreas of P. vannamei was also histological confirmed. Our current data indicated that the shrimp tissues derived from the endoderm such as hepatopancreas could also be infected by IHHNV.

Development of a PCR assay for the effective detection of Enterocytozoon hepatopenaei (EHP) and investigation of EHP prevalence in Shandong Province, China.

Enterocytozoon hepatopenaei (EHP), a recently reported pathogen in the penaeid shrimp, is spreading widely and seriously threatening Penaeus (Litopenaeus) vannamei aquaculture. This study aimed to develop a new and more sensitive polymerase chain reaction (PCR) method for the effective detection of EHP. An EHP PCR assay with a pair of primers specifically amplifying a 358 bp EHP DNA fragment was developed, which was demonstrated to be capable of detecting as low as 2 × 101 copies of EHP and is specific for EHP without cross reaction with DNA samples prepared from five common shrimp pathogens, including white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic virus (IHHNV), hepatopancreatic parvovirus (HPV), infectious myonecrosis virus (IMNV), and yellow head virus (YHV). This new assay is more specific and more sensitive than the previously published EHP PCR methods. With the PCR assay developed in this study, we investigated the prevalence of EHP in four areas of Shandong, China by testing a total of 639 shrimp samples collected from Yantai, Binzhou, Dongying, and Weifang. The results showed that the EHP positive rate reached 51.2%, indicating that EHP is prevalent in shrimp culture in China.

[Effects of Different Land Use Practices on Nitrogen Loss from Runoff During Rainfall Events].

To understand the effect of nitrogen from runoff during rainfall events for different land uses, sub-catchments A and B in the small Shipanqiu watershed in Zhong County, Chongqing-which were managed using different land use practices-were taken as research objects. Runoff flow and nitrogen levels at the outlet of the catchment were monitored. Sub-catchment A is an agroforestry-water complex and sub-catchment B is the site of traditional agriculture. EMC was used to evaluate the average concentration of runoff nitrogen during rainfall events, and the effect of this runoff nitrogen on the small watershed with different land use systems was analyzed. The results showed that the TN concentration in catchment B (1.37-15.17 mg·L-1) > catchment A (0.84-9.28 mg·L-1); the ratio of the first peak to the second peak in catchment A was 62%, which was far less than the 97% in catchment B; the average DN/TN values were 69% and 75% in catchments A and B, respectively; and the average NN/DN values were 67% and 80% in catchments A and B, respectively. The different land use practices have significant impacts on nitrogen loss. Compared with the catchment where traditional agricultural practices were followed, the agroforestry-water complex catchment effectively reduced the loss of nitrogen and decreased the first TN peak value and DN/TN and NN/DN values. This study provides a scientific basis for the prevention and control of non-point source pollution in small watersheds in the area of the Three Gorges Reservoir.

Research progress on hosts and carriers, prevalence, virulence of infectious hypodermal and hematopoietic necrosis virus (IHHNV).

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the major viral pathogens of penaeid shrimp and it has spread worldwide. IHHNV causes substantial economic loss to the shrimp farming industry and has been listed as a notifiable crustacean disease pathogen by the World Organization for Animal Health (OIE). In this paper, we reviewed studies on the hosts and carriers, prevalence, genotypes and virulence of IHHNV. The pathogenesis mechanisms of IHHNV and the viral interference between IHHNV and white spot syndrome virus (WSSV) were also discussed. The mechanism of IHHNV infection and its virulence difference in different hosts and different developmental stages have not been fully studied yet. The mechanisms underlying viral interference between IHHNV and WSSV are not yet fully understood. Further studies are needed to elucidate the precise molecular mechanisms underlying IHHNV infection and to apply the insights gained from such studies for the effective control and prevention of IHHNV disease.

[Characteristics of Nitrogen and Phosphorus Output and Loss Flux in the Shipanqiu Watershed, Three Gorges Reservoir Area].

As the source of non-point pollution in the Three Gorges Reservoir Area, small watershed is a key control object in alleviating deterioration of water quality. In the Three Gorges Reservoir Area, the Shipanqiu small watershed with various land-use types was selected as the research object, and the water quantity and quality of the outlet section of the watershed were continuously monitored. We carried out analysis of the small watershed runoff loss and nitrogen and phosphorus pollutants with concentration, analyzed the morphology change characteristics of runoff erosion, calculated the small watershed of pollutant emission flux, and analyzed the nitrogen and phosphorus nutrient loss and main human and natural factors, especially in the Three Gorges Reservoir Area of agriculture where nonpoint source pollution research has important practical significance. The results showed that the rainfall in the watershed varied significantly with the seasons, and the rainfall was mainly distributed from April to June, which was the main output period of nitrogen and phosphorus loss in the small watershed, accounting for 58.94% and 67.60% of the total nitrogen and phosphorus load, respectively, in the whole year. The total annual runoff in the Shipanqiu small watershed was 8.02×104 m3, and the annual total nitrogen loss flux was 5.04 kg·hm-2, of which nitrate nitrogen (2.54 kg·hm-2) was the main part. The total phosphorus output was 0.534 kg·hm-2, and the soluble total phosphorus (0.422kg·hm-2) accounted for 79.00% of the total phosphorus flux. The loss flux of total nitrogen was 9.51 times that of total phosphorus, and the non-point source pollution risk of nitrogen was much greater than that of phosphorus. Therefore, for the Shipanqiu small watershed, it is especially important to prevent nitrogen loss in paddy fields when fertilization and rainfall coincide.

[Effect of Optimized Fertilization and Biochar Application on Phosphorus Loss in Purple Soil Sloping Farmland].

Phosphorus is an essential nutrient for crop growth, but the input of excess phosphorus is a significant cause of eutrophication. This study explored the relationship between fertilization methods and phosphorus loss in actual production, providing a theoretical basis for scientific fertilization and rational reduction of fertilizer application. In the experiment, a wild-type OD flow plot was used to monitor the occurrence of multiple rainfall runoff and sediment yield in purple soil sloping farmland in 2017-2018. Four different schemes of non-fertilizer treatment, conventional fertilization treatment, optimized fertilization treatment, and reduced fertilization combined with biochar were studied. The effects of soil flow, surface runoff, and sediment phosphorus loss on purple soil sloping farmland were analyzed. The results showed that:①The total yield of each treatment was optimized (20737.23 L) > conventional (18513.17 L) > CK (18134.58 L) > biochar (13594.85 L), and the total sediment yield of each treatment was CK (1998 kg·hm-2) > biochar (1884 kg·hm-2) > optimized (1681 kg·hm-2) > conventional (910 kg·hm-2). The middle stream of soil is the main type of runoff in the rainy season, accounting for 60.14%-87.34% of the total output flow. The total amount of sediment produced by each treatment was not significantly different from that of the conventional treatment (P>0.05). ②The flux of total phosphorus loss in each treatment was characterized by sediment > surface runoff > soil middle flow. Phosphorus lost through the middle stream of soil is the least, accounting for only 2.63%-12.91% of the flux of total phosphorus loss, while the flux of sediment loss of phosphorus can reach 63.74%-78.74%, and thus is the main output route of soil phosphorus loss. ③The application of biochar can effectively reduce the abortion flow in the soil of purple soil sloping land, and the loss flux of orthophosphate in the middle stream, which are 49.94% and 56.45% lower than the conventional treatment, respectively. However, the interception effect on surface runoff is not good, and there is no significant influence on the flux loss of particulate phosphorus. At the same time, the flux of total phosphorus in surface runoff and sediment is significantly increased by 73.28% and 123.53%, respectively, compared with conventional treatment (P<0.05). Therefore, to control the loss of phosphorus in purple soil sloping farmland in southwest China, we should focus on reducing the occurrence of soil sediment loss. Bio-carbon should be further optimized in the practical application of agricultural production with the phosphorus fertilizer input ratio.

[Response of Nitrogen Loss Flux in Purple Soil Sloping Field to Reduced Fertilizer and Combining Straw].

The purple soil sloping field is considered as the main source of sediment and non-point source pollution in the Three Gorges Reservoir area. To prevent and control the non-point source pollution, it is indispensable to explore the characteristics of nitrogen loss in the overland flow and interflow of purple soil sloping field in Three Gorges Reservoir area. The purple soil sloping runoff plots, located in the Shibaozhai Experimental Station of Chengdu Institute of Chinese Academy of Sciences in Zhongxian County, Chongqing, were studied. The experiment included no fertilization treatment (CK), traditional fertilization treatment (T1), amended fertilization treatment (T2), and reduced fertilizer with straw treatment (T3). According to the data of volume of the interflow and runoff and the leach concentration and flux of nitrogen forms under rapeseed-maize rotation system, the response of nitrogen leaching flux to reduce fertilizer with straw application can be definite in purple soil sloping plots. The results show that the ratio of interflow to total runoff is 60.14%-88.56%, and the flux of nitrogen leaching in the interflow accounts for 72.88%-92.35% of total nitrogen loss flux. Ammonium was mainly leached by the overland flow. In addition, nitrate was mainly leached by the interflow and was the main form of nitrogen leaching. The fluxes of ammonium and nitrate under different treatments followed the order T1 > T2 > T3 > CK. The total nitrogen flux of T3 was 20.07 kg·(hm2·a)-1, which was 43.59% and 39.55% lower than that of T1 and T2, respectively. The reduced fertilizer with straw application significantly decreased the leaching flux of ammonium, nitrate, and total nitrogen, and weakened the effect on runoff nitrogen leaching in the purple soil sloping plots.

[Effect of Fertilizer Reduction and Biochar Application on Soil Nitrogen Loss in Purple Upland].

This study seeks to clarify the effect of biochar application on nitrogen loss patterns and flux in purple arid sloping land, so as to provide a scientific basis for improving the quality of farmland and reducing the risk of agricultural non-point source pollution in purple arid sloping land. The effects of four treatments on surface runoff and soil nitrogen loss patterns and fluxes in purple arid sloping land were studied by field experiments, including no fertilization (control), conventional fertilization, optimized fertilization, and biochar (fertilizer reduction and biochar application). The results showed that:① Of the fertilization treatments, the total runoff of conventional treatment was the highest at 16133 L·a-1, and the total runoff of biochar treatment was the lowest at 11893 L·a-1. In each fertilization treatment, soil midstream was the main mode of runoff, accounting for 61.80%-68.60% of the total loss. Compared with the control (no fertilization treatment), the sediment loss in other fertilization treatments was decreased, with conventional treatment showing the most significant effect. ② Ammonium nitrogen is mainly lost by surface runoff, accounting for 86.51%-96.58% of the total loss flux. Biochar treatment had the highest loss flux at 0.69 kg·(hm2·a)-1, and the control treatment had the lowest at 0.17 kg·(hm2·a)-1. ③ The concentration of granular nitrogen in the production flow of each fertilization treatment was higher than that of the control treatment, and the loss flux of granular nitrogen in the conventional fertilization treatment was the highest at 2.87 kg·(hm2·a)-1. ④ There was a significant positive correlation between total nitrogen concentration and nitrate nitrogen concentration in the soil midstream and surface runoff of each fertilization treatment (P<0.01). Nitrate nitrogen is the main form of total nitrogen loss, and both take soil midstream as the main way of loss. The total nitrogen loss through soil flow accounted for 72.86%-89.13%, and that of conventional fertilization was the highest at 35.58 kg·(hm2·a)-1, whereas that of biochar treatment was the lowest at 21.49 kg·(hm2·a)-1. Reducing the amount of fertilizer and applying biochar can significantly reduce the runoff and nitrogen flux, and effectively prevent and control the risk of agricultural non-point source pollution.

Transcriptome analysis of Procambarus clarkii infected with infectious hypodermal and haematopoietic necrosis virus.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured penaeid shrimp. IHHNV has many hosts, mainly including crustaceans. It has recently been reported that Procambarus clarkii can be infected by IHHNV. In the present study, we studied the hepatopancreas of P. clarkii by transcriptome high-throughput sequencing to analyze the response of P. clarkii to IHHNV infection. After de novo assembly, there were 400,340,760 clean reads. A total of 237 differentially expressed genes (DEGs) were obtained, including 77 significantly up-regulated unigenes and 160 significantly down-regulated ones. The expression levels of 12 immune-related DEGs were validated by qRT-PCR, substantiating the reliability of RNA-Seq results. The enrichment analysis of DEGs showed that the immune-related pathways were closely related to apoptosis and phagocytosis. Moreover, a large number of pathways related to metabolic function were down-regulated, suggesting that IHHNV infection might affect the growth of P. clarkii.

A novel real-time PCR approach for detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV) in the freshwater crayfish Procambarus clarkii.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) infects many crustacean hosts, including cultured penaeid shrimp. In the present study, we aimed to develop a novel sensitive SYBR Green-based real-time PCR method to specifically amplify DNA fragments of IHHNV. Our newly developed real-time PCR method with a 195-bp amplicon specifically detected IHHNV and showed no cross reaction with white spot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), infectious myonecrosis virus (IMNV) and yellow-head virus (YHV). This method could detect as low as one single copy of IHHNV plasmid DNA, more sensitive than other SYBR Green-based real-time PCR methods and less expensive and more convenient than the TaqMan probe-based real-time PCR. Moreover, our data using the newly designed method showed that 80% of IHHNV-fed Procambarus clarkii samples were IHHNV positive. Our findings further confirmed that P. clarkii can be infected by IHHNV.

[Characteristics of Deposited Sediment and Assessment of Heavy Metals in Typical Tributaries Bay Riparian Zone of the Three Gorges Reservoir].

In order to analyze the spatial variation characteristics of grain diameter, nutrient elements and heavy metal pollution with deposition sediment in tributaries bay of the Three Gorges Reservoir, we selected 9 typical tributaries bay, 54 deposited sediment samples were collected from the riparian zone for analyzing grain diameter distribution, capacity, organic matter, nutrient elements of TN, TP and K, heavy metal elements of Cr, Cu, Ni, Pb, and Zn. The results indicated that particle size distribution from Wujiang River in Fuling to the Modao stream in Yunyang presented a trend of fluctuation, deposited sediment at 160-165 m elevation was coarser than that at 165-175 m elevation,volume percent of sand and clay presented a moderate variation at both altitudes, while silt had small variation. Independent sample t test showed that characteristics difference between the upper and lower sediments in riparian zone was not significant. The geo-accumulation index of heavy metal pollutants in the sediment from riparian zone of the Three Gorges Reservoir tributaries bay indicated that, only Zn element in Zhenxi River, Longdong River and Long River, Pb element in the Modao Stream belonged to non-moderate pollution levels, whereas there were no pollution of all other elements in tributaries.

A real-time PCR for the detection of infectious myonecrosis virus in penaeid shrimp.

Infectious myonecrosis virus (IMNV) is a recently observed shrimp virus, which threats the cultured Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry. The specific aim of this study was to develop a new sensitive real-time PCR method for the specific detection of shrimp IMNV. A real-time PCR assay with a pair of primers to specifically amplify a 101bp IMNV cDNA fragment and a corresponding TaqMan probe was developed, which shown to be specific for IMNV without cross reaction with DNA samples prepared from four other shrimp viruses including white spot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV), and infectious hypodermal and haematopoietic virus (IHHNV). The method could detect as low as one single copy of IMNV plasmid cDNA.

Assessment of the role of brine shrimp Artemia in white spot syndrome virus (WSSV) transmission.

Challenge tests with Artemia four different development stages (nauplii, metanauplii, pseudoadults and adults) to white spot syndrome virus was carried out by immersion challenge and virus-phytoplankton adhesion route in order to asses the possibility of Artemia acting as a vector of WSSV to penaeid shrimp Litopenaeus vannamei postlarvae. The WSSV succeeded in infecting four stages Artemia, and nested-PCR detection for WSSV revealed positive results to virus-phytoplankton adhesion route. No mass mortalities were observed in penaeid shrimp postlarvae fed with WSSV-positive Artemia which exposed to WSSV by virus-phytoplankton adhesion route, whereas WSSV DNA detected in penaeid shrimp postlarvae by nested-PCR. By contrary, no WSSV-positive was detected in any animal fed with WSSV-negative Artemia. These results indicated that Artemia could serve as a vector in WSSV transmission.

White spot syndrome virus (WSSV) transmission from rotifer inoculum to crayfish.

To test the possibility that shrimp pond rotifer resting eggs and hatched rotifers could transmit white spot syndrome virus (WSSV) to crayfish (Procambarus clarkii), we injected crayfish with rotifer and resting egg inocula that were WSSV-positive only by dot-blot analysis of PCR products. No crayfish became WSSV-positive after challenge with the resting egg inoculum. However, 1/15 crayfish became WSSV-positive after challenge with the rotifer inoculum. The results demonstrated that rotifers constitute a potential risk for WSSV transmission to crayfish and other cultivated crustaceans. However, the actual quantitative risk of transmission in an aquaculture setting depends on many variables that remain untested.

White spot syndrome virus (WSSV) detected by PCR in rotifers and rotifer resting eggs from shrimp pond sediments.

White spot syndrome virus (WSSV) was detected by PCR-dot blot hybridization in rotifer resting eggs from shrimp Penaeus chinensis culture-pond sediments. It was also detected in rotifers hatched from those eggs. Surface disinfection before analysis indicated that WSSV was probably present within the resting eggs. Results suggested that rotifer resting eggs may be an overwintering reservoir for WSSV in shrimp ponds.