Immunological assays of hemocytes in the Northern Quahog Mercenaria mercenaria.

The northern quahog Mercenaria mercenaria (commonly named hard clam) is an important aquaculture and fishery species along the Atlantic west coast. Environmental stresses, such as heat shock, fluctuating salinity, and harmful algal blooms are major challenges for clam aquaculture. In response to environmental stresses, hemocytes would change dynamically for defense and immunity. The goal of this study was to characterize basic immunological assays of hemocytes in the northern quahog by use of flow cytometry. The objectives were to: 1) develop a non-lethal method for hemolymph collection and dilution; 2) verify the capability of flow cytometry for hemocyte count and type identification through comparison with microscopic observation; 3) validate hemocyte viability assay based on plasma membrane integrity, and 4) develop hemocyte phagocytosis assay by use of fluorescein labeled microbeads. A non-lethal hemocyte collection method was developed using needle insertion through the ligament. Osmolality measurement of serum was the same as that of culture seawater. The pH measurement of serum (7.2) was significantly different from that of culture seawater (8.4). By microscopic observation, three types of hemocytes were identified with granulocytes, the dominant cell type (70 ± 16%), agranulocyte (14 ± 4%), and blast-like cell (16 ± 4%), and no differences were found from the measurements by flow cytometer on FSC/SSC plot (cell size/granularity). The viability of hemocytes based on plasma membrane integrity was 88 ± 6% ranging from 70 to 97% (n = 60, three populations), and viability protocol was further validated with the pre-set expected viability (p ≥ 0.424). Phagocytosis assay of hemocytes with fluorescence beads showed a mean capacity of 10 ± 5% (n = 60, three populations). Incubation time (up to 6 h) or bead concentrations (2:1 or 5:1 to hemocytes) did not affect the phagocytosis measurement. Overall, this study reported the basic characteristics of hemolymph (serum and hemocytes) of northern quahogs. It is expected that the assay methodologies will be applied to evaluation of hemocyte responses to environmental stresses for clam aquaculture.

Sperm Repository for a Breeding Program of the Eastern Oyster Crassostrea virginica: Sample Collection, Processing, Cryopreservation, and Data Management Plan.

The Eastern oyster Crassostrea virginica (Family Ostreidae) is one of the most important fishery and aquaculture species in the U.S. and is a keystone species for coastal reefs. A breeding program was initiated in 2019 to support the fast-growing aquaculture industry culturing this species in the Gulf of Mexico. Oysters from 17 wild populations in embayment along the U.S. Gulf of Mexico coast from southwest Florida to the Matagorda Bay, Texas were used as broodstock for the program to maximize genetic diversity in the base population. A sperm repository of the broodstock was established to support the breeding project. The goal of this study was to demonstrate the sperm sample collection, processing, cryopreservation, and the data management plan involved in the establishment of a sperm germplasm repository of base populations. The supporting objectives were to: (1) develop a data management plan for the sperm repository; (2) streamline the procedure for sample collection, processing, and cryopreservation; (3) incorporate sperm quality analysis into the procedure, and (4) archive the cryopreserved samples as a repository for future use in the breeding program. This sperm repository included a total of 102 male oysters from the 17 collection sites (six oysters per site). A data management plan was developed with six categories, including sample collection, phenotype, fresh sperm, genotype, cryopreservation, and post-thaw sperm, as guide for data collection. Sperm collection was accomplished by strip spawn, and fresh sperm production, motility, and fertility were recorded for quality analysis. Cryopreserved sperm samples were sorted, labelled, archived, and stored in liquid nitrogen for future use. Post-thaw motility (1-30%) and plasm membrane integrity (15.34-70.36%) were recorded as post-thaw quality parameters. Overall, this study demonstrated a streamlined procedure of oyster sperm collection, processing, and cryopreservation for establishing a sperm repository that can serve as a template for construction of oyster germplasm repositories for breeding programs.

Growth and nutrient uptake of Myriophyllum spicatum under different nutrient conditions and its potential ecosystem services in an enclosed sea area in the East China Sea.

We investigated the growth and nutrient uptake of Myriophyllum spicatum under different nutrient conditions and evaluated its implications for ecosystem services in an enclosed area of Jinshan. The specific growth rate ranged from 1.29%-4.37%/day, and the dissolved inorganic carbon and nitrogen, and phosphorus uptake rates were 1.30-1.62, 0.040-0.453, and 0.003-0.027 mg/(g∙day), respectively, under different nutrient conditions. The O2-production and carbon-sequestration efficiencies in the field were 154.30 and 1.25 mg/(g DW∙h), respectively. The average removal efficiencies of NH4+-N, NO3--N, NO2--N, and PO43--P were 43.05%, 97.03%, 64.26%, and 59.24%, respectively, in M. spicatum-cultivated areas compared with in the open sea. Harvesting of M. spicatum removed 12,936.87, 1289.97 and 114.81 kg of carbon, nitrogen, and phosphorus, respectively, from seawater in Jinshan in Nov, 2018. In conclusion, M. spicatum is a good candidate for integrated macrophyte/animal multi-trophic aquaculture in terms of nutrient extraction and economic diversification in low-salinity environments.

Annual patterns of macroalgal blooms in the Yellow Sea during 2007-2017.

The world's largest macroalgal blooms caused by Ulva prolifera have occurred in the Yellow Sea for 11 consecutive years. The area covered by blooms has been approximately 500 km2 in previous years, while in 2017, the maximum area decreased significantly to 312 km2. In this study, we concluded that species competition between Ulva and Sargassum (fast rise of the golden tides), extreme high sea surface temperature and harvest for floating Ulva macroalgae were the three critical factors influencing the sharp reduction in covered area for blooms in 2017. In addition, analysis of annual variations of Pyropia aquaculture area in the Southern Yellow Sea over the past two decades revealed that a great expansion in "Sansha" regions was mainly responsible for the initial blooms in 2007, and that this expansion supported the great biomass of the blooms in following years. Based on these findings, we suggest comprehensive utilization of the macroalgal blooms is a feasible way to control them.

Ulva prolifera green-tide outbreaks and their environmental impact in the Yellow Sea, China.

The Ulva prolifera green tides in the Yellow Sea, China, which have been occurring since 2007, are a serious environmental problem attracting worldwide attention. Despite extensive research, the outbreak mechanisms have not been fully understood. Comprehensive analysis of anthropogenic and natural biotic and abiotic factors reveals that human activities, regional physicochemical conditions and algal physiological characteristics as well as ocean warming and biological interactions (with microorganism or other macroalgae) are closely related to the occurrence of green tides. Dynamics of these factors and their interactions could explain why green tides suddenly occurred in 2007 and decreased abruptly in 2017. Moreover, the consequence of green tides is serious. The decay of macroalgal biomass could result in hypoxia and acidification, possibly induce red tide and even have a long-lasting impact on coastal carbon cycles and the ecosystem. Accordingly, corresponding countermeasures have been proposed in our study for future reference in ecosystem management strategies and sustainable development policy.

Environmental triggers of a Microcystis (Cyanophyceae) bloom in an artificial lagoon of Hangzhou Bay, China.

The relationship between Microcystis abundance and environmental variables was studied during a Microcystis bloom in the summer months of 2016 in an artificial lagoon of Hangzhou Bay, China. It was determined that Microcystis abundance increased from 0.16 × 104 cell/L to 5.8 × 107 cell/L within 17 days from 28 July to 14 August, contributing to 96.84-99.56% of the total phytoplankton abundance. Then, Microcystis gradually disappeared 57 days afterwards. The results showed that the growth of Microcystis, including the stage of recovery, outbreak, subsidence and disappearance, was significantly correlated with water temperature, salinity, soluble reactive phosphorus (PO4-P), dissolved inorganic nutrients (DIN), silicate (SiO4-Si), the ratio of DIN/SiO4-Si and zooplankton abundance, and the key environmental triggers which promoted the outbreak of Microcystis were water temperature, PO4-P concentration and zooplankton abundance in this artificial lagoon.

Nutrient bioextraction and microalgae growth inhibition using submerged macrophyte Myriophyllum spicatum in a low salinity area of East China Sea.

Myriophyllum spicatum was cultivated in a low salinity area of Hangzhou Bay (salinity 5.8-6.5), from August to October in 2016, to evaluate the abilities of its nutrient bioextraction and microalgae growth inhibition. During the 72-day cultivation period, M. spicatum had a specific growth rate (SGR) of 6.23%day-1 and increased 20-fold in biomass (wet weight). Tissue C, N and P assimilation quantities of M. spicatum were found to be 3279.39kg, 360.61kg and 26.97kg, respectively. The concentration of NH4-N, NO3-N, NO2-N and PO4-P after M. spicatum cultivation was decreased by 47.92%, 58.28%, 36.40% and 55.57%, respectively. The phytoplankton density was decreased from 1064.60×104cellsL-1 to 12.85×104cellsL-1. These results indicated that cultivation of M. spicatum can help in nutrient bioextraction and microalgae growth inhibition in low salinity marine water bodies.

Rapid expansion of Ulva blooms in the Yellow Sea, China through sexual reproduction and vegetative growth.

Green algal blooms have occurred in the Yellow Sea for 11 consecutive years since 2007. A "seed bank" comprising micro-propagules including gametes, meiospores, and zygotes, played an important role in the rapid formation of a green tide. In the present study, germination differences among zygotes, meiospores, and gametes were examined. The growth ability and maturation period of alternating generations of sexual Ulva prolifera strains were also assessed. The zygote and meiospore germination rate was 91.67% and 80.29%, respectively, approximately three times greater than that of gametes (30%). In addition, the highest daily growth rate of sporophytes and gametophytes was 266.7% and 288.1%, respectively, and the maturation period of sporophytes and gametophytes was 35.7 and 31.3 days, respectively. These results indicate that sexual reproduction and vegetative growth are mainly responsible for the rapid expansion of macroalgal blooms in the Yellow Sea.

Variations of dominant free-floating Ulva species in the source area for the world's largest macroalgal blooms, China: Differences of ecological tolerance.

Species composition and seasonal variations of free-floating Ulva species were investigated in the source area of the world's largest macroalgal blooms during 2009-2015. Based on a combination of a morphological analysis and sequences of nuclear-encoded ITS and 5S rDNA spacer regions, the dominant species in the free-floating Ulva community at the early stage of green tides were Ulva compressa, Ulva flexuosa, and Ulva linza. The first appearance of Ulva prolifera on the sea surface was in mid-May and it dominated the floating Ulva community in June from 2009 to 2011. From 2012-2015, U. prolifera was not only the first species to appear on the sea surface but also the dominant species during the whole early stage of green tides. To explain the successional mechanism, the effects of environmental factors on the growth of four Ulva species were examined in the laboratory under different combinations of light intensity and temperature. It was found that the highest growth rate of U. prolifera was 44.9%/d, which was much higher than the other three Ulva species. The strong tolerance of U. prolifera to extreme conditions also helps it survive and bloom in the Yellow Sea.

Bioremediation using Gracilaria lemaneiformis to manage the nitrogen and phosphorous balance in an integrated multi-trophic aquaculture system in Yantian Bay, China.

To reduce negative environmental impacts from human aquaculture activities, the red alga Gracilaria lemaneiformis was co-cultured with the fish Pseudosciaena crocea in an integrated multi-trophic aquaculture (IMTA) system for 35d in Yantian Bay. The eutrophication index value decreased from 14.5 to 8.4 after seaweeds were co-cultured in cage farming areas, which indicated that the eutrophic water column in Yantian Bay could be mediated by IMTA. Total DIN and DIP of the tidal input and output were 9.23kg, 0.19kg and 11.08kg, and 0.27kg, respectively. Total 5.24kg of dissolved N and 0.81kg of dissolved P were released from IMTA system. These results indicate that G. lemaneiformis co-cultured in IMTA system could not completely remove all excess nutrients. In theory, at least 324.48kg of seaweed seedlings would be required to balance excess nutrients generated from fish cages.

Tracing the origin of green macroalgal blooms based on the large scale spatio-temporal distribution of Ulva microscopic propagules and settled mature Ulva vegetative thalli in coastal regions of the Yellow Sea, China.

From 2008 to 2016, massive floating green macroalgal blooms occurred annually during the summer months in the Yellow Sea. The original source of these blooms was traced based on the spatio-temporal distribution and species composition of Ulva microscopic propagules and settled Ulva vegetative thalli monthly from December 2012 to May 2013 in the Yellow Sea. High quantities of Ulva microscopic propagules in both the water column and sediments were found in the Pyropia aquaculture area along the Jiangsu coast before a green macroalgal bloom appeared in the Yellow Sea. The abundance of Ulva microscopic propagules was significantly lower in outer areas compared to in Pyropia aquaculture areas. A molecular phylogenetic analysis suggested that Ulva prolifera microscopic propagules were the dominant microscopic propagules present during the study period. The extremely low biomass of settled Ulva vegetative thalli along the coast indicated that somatic cells of settled Ulva vegetative thalli did not provide a propagule bank for the green macroalgal blooms in the Yellow Sea. The results of this study provide further supporting evidence that the floating green macroalgal blooms originate from green macroalgae attached to Pyropia aquaculture rafts along the Jiangsu coastline of the southern Yellow Sea.

Eutrophication assessment and bioremediation strategy using seaweeds co-cultured with aquatic animals in an enclosed bay in China.

Intensive mariculture results in a rise in nutrient concentrations, then leads to serious eutrophication in coastal waters. Based on the sampling data obtained between August 2012 and July 2013, the eutrophication status in Yantian Bay was assessed, and the proportion of marine animals co-cultured with seaweeds was evaluated. The nutritional quality index (NQI) ranged from 4.37 to 13.20, indicating serious eutrophication conditions. The annual average ratio of nitrogen/phosphorus (N/P) was 25.19, indicating a nitrogen surplus in this system. DIN was selected as the best parameter to balance seaweed absorption and marine animal DIN production. Gracilaria lemaneiformis and Laminaria japonica were selected as co-cultured seaweeds. The optimal proportion of G. lemaneiformis production was assessed as 20074.14 tonnes. The optimal proportion of L. japonica production was evaluated as 15890.68 tonnes. High-temperature adapted seaweeds should be introduced for removing nutrients releasing by farmed aquatic animals in the summer in Yantian Bay.

Bioremediation using Gracilaria chouae co-cultured with Sparus macrocephalus to manage the nitrogen and phosphorous balance in an IMTA system in Xiangshan Bay, China.

A cage experiment using the red alga Gracilaria chouae co-cultured with the black seabream Sparus macrocephalus in Xiangshan Bay, China was conducted to measure the nutrient flux of the integrated multi-trophic aquaculture (IMTA) system. Results showed that trash fish were the main nutrient input contributor and adult fish were the main nutrient output contributor in the system. Contents of N and P in adult fish accounted for 54.45% and 59.48% of N and P in trash fish and fry, which suggests that 45.55% of N and 40.52% of P generated by fish farming were released into to the water. G. chouae proved to be an efficient bioremediation species in this IMTA system. To balance the excess nutrients generated by the system, 231.09 kg of seedlings should be cultured and 5315.07 kg of adult seaweed should be harvested.

Adaptability of free-floating green tide algae in the Yellow Sea to variable temperature and light intensity.

In this study, the influence of temperature and light intensity on the growth of seedlings and adults of four species of green tide algae (Ulvaprolifera, Ulvacompressa, Ulva flexuosa and Ulvalinza) from the Yellow Sea was evaluated. The results indicated that the specific growth rate (SGR) of seedlings was much higher than that of adults for the four species. The adaptability of U. prolifera is much wider: Adult daily SGRs were the highest among the four species at 15-20 °C with 10-600 µmol · m(-2) · s(-1) and 25-30 °C with 200-600 µmol · m(-2) · s(-1). SGRs were 1.5-3.5 times greater than the other three species at 15-25 °C with 200-600 µmol · m(-2) · s(-1). These results indicate that U. prolifera has better tolerance to high temperature and light intensity than the other three species, which may in part explain why only U. prolifera undergoes large-scale outbreaks and floats to the Qingdao coast while the other three species decline and disappear at the early stage of blooming.

Quantitative distribution and functional groups of intertidal macrofaunal assemblages in Fildes Peninsula, King George Island, South Shetland Islands, Southern Ocean.

To evaluate spatial distribution pattern of intertidal macrofauna, quantitative investigation was performed in January to February, 2013 around Fildes Peninsula, King George Island, South Shetland Islands. A total of 34 species were identified, which were dominated by Mollusca, Annelida and Arthropoda. CLUSTER analysis showed that macrofaunal assemblages at sand-bottom sites belonged to one group, which was dominated by Lumbricillus sp. and Kidderia subquadrata. Macrofaunal assemblages at gravel-bottom sites were divided into three groups while Nacella concinna was the dominant species at most sites. The highest values of biomass and Shannon-Wiener diversity index were found in gravel sediment and the highest value of abundance was in sand sediment of eastern coast. In terms of functional group, detritivorous and planktophagous groups had the highest values of abundance and biomass, respectively. Correlation analysis showed that macrofaunal abundance and biomass had significant positive correlations with contents of sediment chlorophyll a, phaeophorbide and organic matter.

Bioremediation efficiency of the largest scale artificial Porphyra yezoensis cultivation in the open sea in China.

The bioremediation efficiency of China's largest scale Porphyra yezoensis cultivation for removing dissolved nutrients and controlling harmful algae was studied in the radial sandbanks waters of Jiangsu Province in the year 2012-2013. Mean nutrient concentration values in the P. yezoensis cultivation area were significantly lower than those in the non-cultivation area, especially during the cultivation season (p<0.05). Tissue nitrogen and phosphorus contents of seaweeds were 5.99-0.80% (dry weight (DW)) and 0.16-0.19% (DW), respectively. Production of P. yezoensis was 58950.87tons DW. Based on these values, 3688.15tons of tissue nitrogen and 105.61tons of tissue phosphorus were removed by harvesting P. yezoensis. The richness index of the red tide species Skeleton emacostatum declined from 0.32 to 0.05 during the P. yezoensis cultivation season. These results indicate that large-scale cultivation of P. yezoensis can be used to efficiently alleviate eutrophication and control harmful algae blooms in open sea.

Changes to the biomass and species composition of Ulva sp. on Porphyra aquaculture rafts, along the coastal radial sandbank of the Southern Yellow Sea.

Compositions, changes and biomass of attached Ulva species on Porphyra rafts along the radial sandbank in the Yellow Sea were investigated, and potential contributions to green tides was analyzed. Ulva prolifera, Ulva flexuosa and Ulva linza were all appeared throughout the investigated period. U. prolifera and U. flexuosa dominated attached Ulva population on Porphyra rafts. Attached Ulva species biomass showed obviously spatial and temporal variations. Temperature, Ulva microscopic propagules and human activities were main factors to influence attached Ulva species biomass. The total attached Ulva species biomass was more than 20,000 fresh weight tons in April, and the green tide causative species U. prolifera accounted 51.03% in April 2013 before green tides occurred. The high biomass of attached Ulva species would contribute most to green tides in the Yellow Sea. But how attached Ulva species on Porphyra rafts contributing to green tides in the Yellow Sea should be further studied.

The origin of the Ulva macroalgal blooms in the Yellow Sea in 2013.

Green algal blooms have occurred in the Yellow Sea for seven consecutive years from 2007 to 2013. In this study, satellite image analysis and field shipboard observations indicated that the Ulva blooms in 2013 originated in the Rudong coast. The spatial distribution of Ulva microscopic propagules in the Southern Yellow Sea also supported that the blooms originated in the Rudong coast. In addition, multi-source satellite data were used to evaluate the biomass of green algae on the Pyropia aquaculture rafts. The results showed that approximately 2784 tons of Ulva prolifera were attached to the rafts and possessed the same internal transcribed spacer and 5S rDNA sequence as the dominant species in the 2013 blooms. We conclude that the significant biomass of Ulva species on the Pyropia rafts during the harvesting season in radial tidal sand ridges played an important role in the rapid development of blooms in the Yellow Sea.

[Bioremediation of river water quality by consecutively adjustable submerged vegetation net].

A series of consecutively adjustable submerged vegetation nets were constructed in a polluted shallow river with a length of about 200 m and nearby the water resource protection area of Taihu Lake in East China, forming an aquatic vegetation consisted of submerged plant species Cabomba caroliniana, Vallisneria natans, Elodea nuttallii, Hydrilla verticillata, and Potamogeton crispus. The water quality indices including total nitrogen (TN), ammonium nitrogen (NH4(+)-N), nitrite nitrogen (NO2(-)-N), nitrate nitrogen (NO3(-)-N), total phosphorus (TP), and phosphate (PO4(3-)-P) were monitored, and the bioremediation effect of the vegetation nets was evaluated. After setting up the vegetation nets, the Secchi depth (SD) of the river changed from 0.5 m to 1.7-1.8 m, and the TN and TP concentrations 15 and 20 days after the nets constructed decreased by 35.6% and 66.3%, and 29.4% and 63.2%, respectively. After five months, the concentrations of NH4(+)-N, NO2(-)-N, NO3(-)-N, TN, TP, and PO4(3-)-P decreased by 92.4%, 76.8%, 72.7%, 73.9%, 90.5%, and 92.0%, respectively. This study showed that consecutively adjustable submerged vegetation net could be a potential approach for treating polluted river waters, particularly for the bioremediation of polluted small landscape shallow water bodies.

[Bioremediation efficiency of applying Daphnia magna and submerged plants: a case study in Dishui Lake of Shanghai, China].

From April 2007 to January 2008, a bioremediation experiment was conducted in a diversion channel of D-port pilot area of Dishui Lake (the channel length is 950 m, and its water volume is 10000 m3). Daphnia magna was first introduced to filter the high biomass of phytoplankton and other particulate organic matter, and then, five submerged plant species Elodea canadensis, Vallisneria spiralis, Hydrilla verticillata, Potamogeton lucens, and Potamogeton crispus were transplanted. Water samples were collected monthly to monitor the water quality and to investigate the bioremediation efficiency. Ten months monitoring data showed that in the remediation area, the water body's total nitrogen (TN), ammonium nitrogen (NH4(+)-N), nitrate nitrogen (NO3(-)-N), nitrite nitrogen (NO2(-)-N), total phosphorus (TP), and reactive phosphate (PO4(3-)-P) concentrations and chemical oxygen demand (COD) were significantly lower (P < 0.01), dissolved oxygen (DO) was increased by 50.4%, and the Secchi depth (SD) reached to an average of 3.4-3.7 m. Overall, the water quality was up to grades II or III of state water quality standards for surface water. In March 2008, the established submerged plant community was used to test its effectiveness in improving the eutrophicated water body from Dishui Lake, and the results showed that after 7-day treatment, except biological oxygen demand (BOD), the TN, TP, NO3(-)-N, NO2(-)-N, NH4(+)-N, and PO4(3-)-P concentrations and COD of the eutrophicated water were all decreased significantly, the DO was increased by 17.98%, and the SD was increased by 30 cm. The present study demonstrated the effectiveness of introducing D. magna and transplanting submerged plants in improving the water quality of Dishui Lake.