Influences of sedimentation and hydrodynamics on the spatial distribution of Alexandrium catenella/tamarense resting cysts in a shellfish farming lagoon impacted by toxic blooms

Since resting cysts are a potential seeding source for blooms, the presence of these cysts in sediments is a marker of an established population for a number of harmful algal species. The spatial patterns of cyst density in relation to sediment characteristics and hydrodynamics are still largely misunderstood. This study investigated the spatial distribution of resting cysts belonging to the Alexandrium tamarense species complex (Dinophyceae) in sediments of a Mediterranean coastal lagoon (Thau Lagoon, France). This lagoon, hosting shellfish farming, is regularly impacted by toxic Alexandrium catenella blooms. The average cyst density across the whole lagoon was rather low, <20 cysts g⁻¹ of dry sediment (DS). However, densities varied widely among sampled stations, with the highest density (∼440 cysts g⁻¹ DS) recorded in a shallow cove named Crique-de-l’Angle, which is the only area where dense blooms of A. catenella and A. tamarense have been recorded in the years preceding this survey. An analysis using spatial autoregressive models demonstrated that cyst densities were highly spatially autocorrelated (indicating that close stations tended to have more similar cyst densities) with accumulation sites. With respect to sediment characteristics (5 granulometric fractions <2 mm and biochemical components), the highest densities were found in silty sediments containing high proportions of water and organic matter. Nevertheless, the linkage between cyst density and sediment structure was not always verified; this reflected the influence of hydrodynamics on the sedimentation of cysts and sediment particles, and on the dispersal of cysts away from the bloom area by wind-induced currents, suggesting that hydrodynamics was responsible for the spatially autocorrelated distribution of cyst densities.     

Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia,Southern Mediterranean Sea)

A high spatial resolution sampling of Alexandrium pacificum cysts, along with sediment characteristics (% H₂O, % organic matter (OM), granulometry), vegetative cell abundance and environmental factors were investigated at 123 study stations in Bizerte Lagoon (Tunisia). Morphological examination and ribotyping of cells obtained from a culture called ABZ1 obtained from a cyst isolated in lagoon sediment confirmed that the species was A. pacificum. The toxin profile from the ABZ1 culture harvested during exponential growth phase was simple and composed of the N-sulfocarbamoyl toxins C1 (9.82 pg toxin cell⁻¹), the GTX6 (3.26 pg toxin cell⁻¹) and the carbamoyl toxin Neo-STX (0.38 pg toxin cell⁻¹). The latter represented only 2.8% of the total toxins in this strain.High abundance of A. pacificum cysts correlated with enhanced percentages of water and organic matter in the sediment. In addition, sediment fractions of less than 63 μm were examined as a favorable potential seedbed for initiation of future blooms and outbreaks of A. pacificum in the lagoon. A significant difference in the cyst distribution pattern was recorded among the lagoon's different zones, with the higher cyst abundance occurring in the inner waters. Also, no correlation due to the specific hydrodynamics of the lagoon was observed in the spatial distribution of A. pacificum cysts and vegetative cells.     

Role of resting cysts in Chilean Alexandrium catenella dinoflagellate blooms revisited

The detection of sparse Alexandrium catenella-resting cysts in sediments of southern Chilean fjords has cast doubts on their importance in the recurrence of massive toxic dinoflagellate blooms in the region. The role of resting cysts and the existence of different regional Chilean populations was studied by culturing and genetic approaches to define: (1) cyst production; (2) dormancy period; (3) excystment success; (4) offspring viability and (5) strain mating compatibility. This study newly revealed a short cyst dormancy (minimum 69 days), the role of key abiotic factors (in decreasing order salinity, irradiance, temperature and nutrients) controlling cyst germination (max. 60%) and germling growth rates (up to 0.36–0.52 div. day⁻¹). Amplified fragment length polymorphism (AFLP) characterization showed significant differences in genetic distances (GD) among A. catenella populations that were primarily determined by the geographical origin of isolates and most likely driven by oceanographic dispersal barriers. A complex heterothallic mating system pointed to variable reproductive compatibility (RCs) among Chilean strains that was high among northern (Los Lagos/North Aysén) and southern populations (Magallanes), but limited among the genetically differentiated central (South Aysén) populations. Field cyst surveys after a massive 2009 bloom event revealed the existence of exceptional high cyst densities in particular areas of the fjords (max. 14.627 cysts cm⁻³), which contrast with low cyst concentrations (<221.3 cysts cm⁻³) detected by previous oceanographic campaigns. In conclusion, the present study suggests that A. catenella resting cysts play a more important role in the success of this species in Chilean fjords than previously thought. Results from in vitro experiments suggest that pelagic–benthic processes can maintain year-round low vegetative cell concentrations in the water column, but also can explain the detection of high cysts aggregations after the 2009-bloom event. Regional drivers that lead to massive outbreaks, however, are still unknown but potential scenarios are discussed.     

Changes in abundances of Alexandrium tamarense resting cysts after the tsunami caused by the Great East Japan Earthquake in Funka Bay,Hokkaido, Japan

The 2011 Great East Japan Earthquake and the subsequent huge tsunami greatly affected both human activity and the coastal marine ecosystem along the Pacific coast of Japan. The tsunami also reached Funka Bay in northern Japan and caused serious damage to the scallop cultures there, and this tsunami was believed to have affected the coastal environments in the bay. Therefore, we investigated the changes in the spatial abundance and distribution of the toxic dinoflagellates Alexandrium tamarense cysts before the tsunami (August 2010) and after the tsunami (May 2011, August 2011, May 2012 and August 2012) in the bay. Further, monthly sampling was conducted after the tsunami to identify seasonal changes of Alexandrium catenella/tamarense cysts and vegetative cells. Significant increases were observed in the populations of A. catenella/tamarense cysts, comparing the abundances before the tsunami (in August 2010; 70 ± 61 cysts g⁻¹ wet sediment) to those just after it (in May 2011; 108 ± 84 cysts g⁻¹ wet sediment), and both A. tamarense bloom (a maximum density was 1.3 × 10³ cells L⁻¹) and PSP (Paralytic Shellfish Poisoning) toxin contamination of scallops (9.4 mouse unit g⁻¹ was recorded) occurred in the bay. Seasonal sampling also revealed that the encystment of A. tamarense and the supply of the cysts to bottom sediments did not occur in the bay from September to April. These results strongly suggested that the mixing of the bottom sediments by the tsunami caused the accumulation of the toxic A. tamarense cysts in the surface of bottom sediment through the process of redeposition in Funka Bay. Moreover, this cyst deposition may have contributed to the toxic bloom formation as a seed population in the spring of 2011.     

Abundance and distribution of toxic Alexandrium tamarense resting cysts in the sediments of the Chukchi Sea and the eastern Bering Sea

Abundance and distribution of the toxic dinoflagellate Alexandrium tamarense species complex resting cyst were investigated in the eastern Bering Sea and the Chukchi Sea for the first time. Sediment samples (top 0–3 cm depth) were collected from the continental shelf of the eastern Bering Sea (17 stations) and the Chukchi Sea (13 stations) together with a long core sample (top 0–21 cm depth) from one station in the Chukchi Sea during 2009–2012. The cysts were enumerated using the primuline staining method. Species identification of the cysts was carried out with multiplex PCR assay and the plate morphology of vegetative cells germinated from cysts in the both areas. Alexandrium cysts were widely detected in the both areas, ranging from not detected (<1 cysts cm⁻³) to 835 cysts cm⁻³ wet sediment in the eastern Bering Sea and from not detected (<1 cysts cm⁻³) to 10,600 cysts cm⁻³ in the Chukchi Sea, and all isolated cysts were genetically and morphologically identified as the North American clade A. tamarense. Their cysts were mainly distributed in the shallow continental shelf where the water depth was less than 100 m in both areas. The cysts were detected from the deep layer (18–21 cm depth of sediment core) of the long core sample. The present study confirmed the abundant existence of A. tamarense with wide range of distribution in these areas. This fact suggests that A. tamarense vegetative cells have appeared in the water column in the both areas. Furthermore, these abundant cyst depositions indicate that this species originally distributed in the Arctic and subarctic regions and well adapted to the environments in the marginal ice zone.     

Development of the “plankton emergence trap/chamber (PET Chamber)”, a new sampling device to collect in situ germinating cells from cysts of microalgae in surface sediments of coastal waters

A simple device was developed to collect germinating cells from settled cysts of microalgae on the sea bottom. This new device – the plankton emergence trap chamber (PET Chamber) – made of a clear acrylic plastic, consists of a top cylinder and a base plate attached to a bottom cylinder. After plugging the bottom cylinder with sediment collected from in situ sea bottom and filling the top cylinder with filtered seawater, the PET Chamber attached to a platform specially made for submerging the chambers is placed on the seafloor. An adequate area of the lateral side of the top cylinder is opened and covered by a plankton net (10 μm mesh) to allow a water exchange between the inside and outside the cylinder. Thus, the PET Chamber can replicate in situ environments, such as temperature, irradiance and dissolved oxygen. Using the PET Chamber, we have succeeded in collecting germinating cells and estimating the germination flux (cells m⁻² day⁻¹) of two dinoflagellates, Alexandrium catenella and Scrippsiella spp., in Ago Bay, Japan. Mircoscopic observations on the samples collected from July to October 2003 revealed fluxes of 124–2022 and 622–3732 cells m⁻² day⁻¹ in A. catenella and Scrippsiella spp., respectively. The data indicate that the new device can detect variations in the number of germinating cells of dinoflagellates. Its sampling ability, coupled with simplicity in deployment and retrieval procedures, can allow one to monitor the in situ emergence from/on the surface sediments of various organisms, that possess resting stages during their life histories, in various coastal waters.     

Germination fluctuation of toxic Alexandrium fundyense and A. pacificum cysts and the relationship with bloom occurrences in Kesennuma Bay,Japan

While cyst germination may be an important factor for the initiation of harmful/toxic blooms, assessments of the fluctuation in phytoplankton cyst germination, from bottom sediments to water columns, are rare in situ due to lack of technology that can detect germinated cells in natural bottom sediments. This study introduces a simple mesocosm method, modeled after previous in situ methods, to measure the germination of plankton resting stage cells. Using this method, seasonal changes in germination fluxes of toxic dinoflagellates resting cysts, specifically Alexandrium fundyense (A. tamarense species complex Group I) and A. pacificum (A. tamarense species complex Group IV), were investigated at a fixed station in Kesennuma Bay, northeast Japan, from April 2014 to April 2015. This investigation was conducted in addition to the typical samplings of seawater and bottom sediments to detect the dinoflagellates vegetative cells and resting cysts. Bloom occurrences of A. fundyense were observed June 2014 and February 2015 with maximum cell densities reaching 3.6 × 10⁶ cells m⁻² and 1.4 × 10⁷ cells m⁻², respectively. The maximum germination fluxes of A. fundyense cysts occurred in April 2014 and December 2014 and were 9.3 × 10³ cells m⁻² day⁻¹ and 1.4 × 10⁴ cells m⁻² day⁻¹, respectively. For A. pacificum, the highest cell density was 7.3 × 10⁷ cells m⁻² during the month of August, and the maximum germination fluxes occurred in July and August, reaching 5.8 × 10² cells m⁻² day⁻¹. Thus, this study revealed the seasonal dynamics of A. fundyense and A. pacificum cyst germination and their bloom occurrences in the water column. Blooms occurred one to two months after peak germination, which strongly suggests that both the formation of the initial population by cyst germination and its continuous growth in the water column most likely contributed to toxic bloom occurrences of A. fundyense and A. pacificum in the bay.     

The influence of bloom intensity on the encystment rate and persistence of Alexandrium minutum in Cork Harbor,Ireland

Toxic Alexandrium minutum blooms recur annually in Cork Harbor, Ireland where they initiate in an inlet known as the North Channel. The dynamics of these blooms have been studied since 2003, and a high degree of inter-annual variability in the cell densities has been observed. Two intense blooms, with maximum cell densities >500,000 cells L⁻¹, were observed in the summers of 2004 and 2011. Annual cyst surveys during winter found that cyst densities decreased after the 2004 bloom, and by 2010 an average of ca. 40 cysts g dry wt sediment⁻¹ was recorded. The intensity of blooms was found to be independent of the cyst density measured the previous winter. The cyst input to the sediment during both intense and low density blooms was measured directly through the deployment of sediment traps in the North Channel. The data allowed an estimate of the proportion of the A. minutum vegetative cells that underwent successful encystment, which averaged at 2.5% across a range of cell densities spanning three orders of magnitude. Maturation times of fresh cysts were determined at 5, 10 and 15 °C. The maturation time at 15 °C was found to be approximately 5 months, a value which increased by two months for a 5° decrease in temperature. A cyst dynamics model was constructed based on the field data to simulate the temporal variation of A. minutum cysts in the oxic layer of sediment. It revealed that a degree of resuspension is required to prevent cyst stocks from becoming exhausted in the thin oxic layer at the surface of the sediment. The model also demonstrated that the cysts supplied by periodic intense blooms, which occur with a frequency of every 7–8 years, are not in themselves enough to allow the population to persist over long time scales (decades). The cyst input from interim blooms of lower density is however enough to ensure the annual inoculation of the water column with A. minutum cells.     

Process length variation in cysts of a dinoflagellate,Lingulodinium machaerophorum,in surface sediments: Investigating its potential as salinity proxy

A biometrical analysis of the dinoflagellate cyst Lingulodinium machaerophorum [Deflandre, G., Cookson, I.C., 1955. Fossil microplankton from Australia late Mesozoic and Tertiary sediments. Australian journal of Marine and Freshwater Research 6: 242–313.] Wall, 1967 in 144 globally distributed surface sediment samples revealed that the average process length is related to summer salinity and temperature at a water depth of 30 m by the equation (salinity/temperature) = (0.078?average process length + 0.534) with R² = 0.69. This relationship can be used to reconstruct palaeosalinities, albeit with caution. The particular ecological window can be associated with known distributions of the corresponding motile stage Lingulodinium polyedrum (Stein) Dodge, 1989. Confocal laser microscopy showed that the average process length is positively related to the average distance between process bases (R² = 0.78), and negatively related to the number of processes (R² = 0.65). These results document the existence of two end members in cyst formation: one with many short, densely distributed processes and one with a few, long, widely spaced processes, which can be respectively related to low and high salinity/temperature ratios. Obstruction during formation of the cysts causes anomalous distributions of the processes. From a biological perspective, processes function to facilitate sinking of the cysts through clustering.     

Alexandrium and Scrippsiella cyst viability and cytoplasmic fullness in a 60-cm sediment core from Sequim Bay,WA

Many marine protists produce a benthic resting stage during their life history. This non-motile cyst stage can either germinate near the sediment surface to provide the inoculum for subsequent blooms or, be buried by sediment deposits over time and entrained into the sedimentary record. Buried cysts can be resuspended into the water column by mixing events (e.g., storms) or other disturbances (e.g., dredging). It is not clear how long cysts can survive while buried in the sediments and still be capable of germinating given favorable conditions. Here, the germination success of cysts produced by the potentially toxic dinoflagellate genus Alexandrium and the non-toxic dinoflagellate genus Scrippsiella is reported from a 60-cm sediment core collected in Sequim Bay, WA, in December 2011. Cysts of Alexandrium spp. and Scrippsiella spp. were isolated from 2-cm sections of the core, placed in individual wells of a 96-well plate with growth medium, imaged, incubated at favorable conditions and monitored for germination. An image analysis program, DinoCyst, was used to quantitatively measure the amount of granular storage products, presumed energy stores, inside the cytoplasm to test the hypothesis that older cysts located deeper in the sediment core will have fewer energy stores available and will be less likely to germinate. An index of the area of the cytoplasm occupied with granular storage products relative to cyst size, termed ‘cytoplasmic fullness’, and age, based on ²¹⁰Pb dating of surrounding sediments, was compared with germination success or failure. This research indicates that cysts of Alexandrium spp. and Scrippsiella spp. can remain viable in sediments for 60 years or longer, show little visual evidence of cytoplasmic deterioration over this timescale (as measured by cytoplasmic fullness), and that germination success is statistically similar for cysts isolated from 0–60 cm deep in the sediment core. These results suggest that a cyst's cytoplasmic fullness is not indicative of viability and that cysts located as deep as 60 cm in the sediments are as likely to germinate as surface cysts given favorable conditions.     

Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment

Morphological observations have confirmed that cysts are produced by dinoflagellates. However, finding a seed bed or unknown cysts in field samples by microscopy is extremely time consuming. Real-time PCR has been used to facilitate the detection of dinoflagellate cysts in sediment. However, DNA from dead vegetative cells remaining on the surface sediment may persist for a long period of time, which can cause false positive DNA detection. In this study, a non-quantitative RNA targeted probe using real-time RT-PCR was developed for detection of viable cysts in sediment. Large-subunit rRNA was used to develop a species-specific RNA targeted probe for the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. The sediment samples were sieved and incubated at 30 °C for 3 h prior to RNA extraction to remove RNA from dead cells remaining in the sediment. Nested-PCR was conducted to maximize assay sensitivity. A field survey to determine the distribution of cysts at 155 sampling stations in the western and southern part of the Korean peninsula showed that C. polykrikoides cysts were detected at five sampling stations.     

A Colpoda aspera isolate from animal faeces: In vitro cultivation and identification

A colpodean ciliate was found in the faeces of experimental rabbits. It was initially cultivated in medium mixed with 2% (w/v) rabbit faeces. Subsequently, two chemically defined media, designated CA-1 and CA-2, were found to be suitable for axenical cultivation of the ciliate. The maximum abundance of the ciliate isolate in the CA media was 1–2 × 10⁵ cells/ml. The ciliate isolate was further identified with silver impregnation and molecular analysis. Features of the left oral polykinetid, somatic dikinetids, and sliverline pattern were similar to those of Colpoda aspera as described by Foissner (1993). The 18S small subunit ribosomal RNA gene of the ciliate isolate shared 99% sequence identity with that of C. aspera, with 100% coverage, and formed a sister clade in the phylogenetic tree with the reference C. aspera isolate. In addition, the trophozoite of C. aspera could proliferate over a temperature range from 25–37 °C. When resting cysts were cultivated in CA-1 medium at 30–35 °C, 98.2% of the trophozoites were detached from the cyst wall after 7 h.     

Contribution of several nitrogen sources to growth of Alexandrium catenella during blooms in Thau lagoon,southern France

A monitoring program with a weekly sampling frequency over a 15-month period indicates that urea concentrations above a certain threshold level may trigger the blooms of Alexandrium catenella in Thau lagoon. However, urea concentrations were also sometimes related to ammonium and dissolved organic nitrogen concentrations, indicating that the role of urea may not be a direct one. An original approach is used to assess the relative contribution of several nitrogen sources (nitrate, nitrite, ammonium, urea) to growth of A. catenella by comparing nitrogen uptake rates to nitrogen-based growth rates estimated from dilution experiments during four blooms over a 4-year period (2001–2004) in Thau lagoon. Nitrate and nitrite contributed 0.1–14% and 0.1–5% respectively of growth requirements. Ammonium and urea were the main N sources fueling growth of A. catenella (30–100% and 2–59%, respectively). Indirect estimates indicated that an unidentified N source could also contribute significantly to growth at specific times. Concerning ammonium and urea uptake kinetics, half-saturation constants varied between 0.2 and 20 μgat N L⁻¹ for ammonium and between 0.1 and 44 μgat N L⁻¹ over the 4-year period, indicating that A. catenella can have a competitive advantage over other members of the phytoplankton even under low concentrations of ammonium and urea. However, the observed large changes in ammonium and urea uptake kinetics on a short time scale (days) during blooms preclude more precise estimates of those contributions to growth and require further investigation.     

Towards an optimal sampling strategy for Alexandrium catenella (Dinophyceae) benthic resting cysts

The study proposes methodological developments to optimize sampling strategy of resting cysts of Alexandrium catenella to estimate their abundance with a predefined error. This work also aims to provide information on spatial distribution of resting cysts in sediments. The distribution mode of A. catenella resting cysts related to the abundance variability was studied through sediment cores sampling on four different spatial scales and using Ludox CLX gradient density method. The quantification method underestimates by a factor of 2 the resting cysts abundance in one gram of sediment. Application of Taylor's power law allowed us to define a compromise between sampling effort and abundance estimation error. In the case of A. catenella resting cysts from Thau lagoon, the optimal sampling strategy consists of sampling 10 stations on a surface of 2 km² for a given coefficient of variability (C) of 15%, sampling 3 sediment cores at each station (C = 30%) and counting only one replicate by core (C = 18%). Results related to the application of Taylor's power law are closely dependent on resting cyst density and aggregation in a given sediment. In our area, A. catenella resting cysts are mainly observed in the upper 3 cm of sediment. Horizontally, their heterogeneity is lower on 10 cm² surface and tends to stabilize itself beyond a surface of 10 m². Each author has to carry out this pre-sampling effort for his own resting cysts-forming species, in his own area, in order to increase accuracy of resting cyst mapping.     

Resting cysts,and effects of temperature and salinity on the growth of vegetative cells of the potentially harmful species Alexandrium insuetum Balech (Dinophyceae)

The potentially harmful species Alexandrium insuetum established by the incubation of resting cysts isolated from sediment trap samples collected at Jinhae-Masan Bay, Korea was characterized by morphological and phylogenetic analysis. The effects of temperature and salinity on the growth of A. insuetum were also investigated. The resting cysts are characterized by a spherical shape, a small size (20–25 μm) and the presence of either three or four red accumulation bodies. The similarity of morphological features of the resting cysts to those of other species of the minutum group (consisting of Alexandrium minutum and A. tamutum) indicates that the morphological features of resting cysts might improve the accuracy of the grouping of Alexandrium species. A. insuetum germinated from the resting cysts is morphologically consistent with vegetative cells reported from Korean and Japanese coastal areas, and has an partial large subunit (LSU) rDNA sequence identical to that from Japanese strains. The growth of A. insuetum was observed between salinity 20 and 35, with increasing temperature; however at 25 °C, A. insuetum could grow even at the salinity of 15. The highest growth rate (0.60 d⁻¹) was observed at 25 °C and the salinity of 25, which is higher than the previously reported growth rate of A. tamarense, which is responsible for outbreaks of paralytic shellfish poisoining and blooms in Jinhae-Masan Bay. These results suggest that the proliferation of A. insuetum in Jinhae-Masan Bay is likely to be highest during the summer.     

The germination characteristics of Alexandrium minutum (Dinophyceae), a toxic dinoflagellate from the Fal estuary (UK)

The germination characteristics of Alexandrium minutum cysts from the Fal estuary were studied at different conditions of temperature (4–24 °C) and salinity (15–35‰) and in the dark and low light intensity (2 μmol^?2 s^?1). Sediment sub-samples were directly cultured and processed at the end of the experiment for counts of non-germinated cysts. A decrease in the number of cysts was interpreted as germination that was calculated by comparison of the number of cysts over time with that of initial counts. The 50% germination time (time at which 50% of the total initial number of cysts had germinated) was calculated for each condition. A. minutum did not germinate in the dark but it germinated under all other conditions studied. Highest germination occurred at salinities of 30 psu and 35 psu and temperatures from 8 °C to 24 °C (germination rate—expressed as the inverse of the 50% germination time: 1.1–1.2). Lowest germination occurred at 15 psu and 4 °C and 24 °C (germination rate: 3.9–3.8). However, little variation in germination rates occurred across the conditions studied. As these conditions represent those likely in the estuary it is probable that A. minutum cysts on the surface of the sediments represent a constant source of cells to the water column and sediment disturbance (revealing buried cysts) could rapidly inoculate the water column with vegetative cells. This data was used to develop a model for Alexandrium germination from coastal sediments.     

Which species,Alexandrium catenella (Group I) or A. pacificum (Group IV), is really responsible for past paralytic shellfish poisoning outbreaks in Jinhae-Masan Bay,Korea?

Paralytic shellfish poisoning (PSP) caused the deaths of four people in coastal area of Korea, mainly Jinhae-Masan Bay and adjacent areas, in April 1986 and in 1996. The PSP outbreaks were caused by the consumption of mussels, Mytilus edulis. The organism that caused PSP was identified, from morphological data only, as Alexandrium tamarense which is recently renamed as A. catenella, however recent studies have shown that the morphological diagnostic characteristics used to identify Alexandrium species have uncertainties and molecular tools and other criteria should be considered as well. The organism that caused past PSP outbreaks and incidents in Korea therefore need to be carefully reconsidered. The aim of this study was to re-evaluate the species really responsible for past outbreaks of PSP in Jinhae-Masan Bay, Korea. The temporal production and fluxes of the resting cysts of Alexandrium species were investigated for one year (from March 2011 to February 2012) using a sediment trap, and the morphology and phylogeny of vegetative cells germinated from the resting cysts were analysed. The production of Alexandrium species peaked in August and November, when temporal discrepancies were found in the water temperature (22.4 and 22.7 °C in August, 19.1 and 19.6 °C in November) and salinity (29.5 and 26.1 psu in August, 30.5 and 31.8 psu in November). The morphological data revealed that Alexandrium species germinated from resting cysts collected in August have a ventral pore on the 1′ plate, whereas the 1′ plate in Alexandrium species germinated from resting cysts collected in November lacks a ventral pore. Molecular phylogenetic data for the vegetative cells from the germination experiments allowed the August and November peaks to be assigned to Alexandrium catenella (Group I) and A. pacificum (Group IV), respectively. This indicates that the production of resting cysts of A. catenella can be enhanced by relatively high water temperature. This result is not consistent with those of previous studies that A. catenella responsible for PSP outbreaks was found at relatively low water temperature. In addition, large subunit ribosomal sequences data revealed that A. pacificum isolates from Korea were closely related to those from Australia, Japan and New Zealand where the PSP toxicity of shellfish and blooms occurred in the 1990s, indicating that the introduction of toxic dinoflagellates were related to ballast water from bulk-cargo shipping. Based on these results, we concluded that past PSP outbreaks in Jinhae-Masan Bay of Korea could have been caused by A. pacificum rather than by A. catenella.     

Assessment of spatial distribution and cysts resources of Artemia in late autumn in Dangxiong Co salt lake

Based on the vertically interval sampling in 25 sampling sites in Dangxiong Co salt lake in 2011, a preliminary investigation on population spatial distribution and cysts resources of Artemia in the lake has been conducted. The study achieves four new progresses. First, the average density of Artemia and Artemia cysts in the lake is 4.157 × 10³ ind. m⁻² and 8.069 × 10⁴ ind. m⁻², respectively. Among Artemia, the adults account for 60.31%; Second, different from other salt lakes in horizontal distribution, the Artemia population mainly distributes in the open water, only a little in the shallow water, and there is no distribution in the longshore area in the north part and the estuary region; Third, in vertical distribution, 44.24% of individuals intensively distribute in the upper water layer within 2.0 m, especially 0–0.2 m, where the average density of Artemia and cysts are maximum (129.488 ind. L⁻¹ and 5.728 ind. L⁻¹, respectively). A decrease of distribution density is accompanied by an increase of water depth basically, the percentage of Artemia and cysts decrease to 0.68% and 4.60%, respectively; Fourth, the cysts resources of 14.96 t in the lake are assessed using contour map. 66.35% of them distributed in 0.0–2.0 m water layer and 49.06% concentrate in the 18.21% areas of the central water; Fifth, quantity of cysts suitable for development in the lake is 2.399 ± 0.320 t, with an upper limit of 0.879 t. The study can provide a reference for the sustainable development and exploitation of Artemia in Dangxiong Co salt lake.     

Comparison of macrofauna communities in sediments of the beds of vertical flow constructed wetlands planted with Panicum maximum (Jacq.) treating domestic wastewater

To improve our understanding of the ecological functioning of constructed wetlands, the macrofauna structure in the sediments of a constructed wetland planted with Panicum maximum treating domestic wastewater was studied. Two beds were planted with young P. maximum and two unplanted beds were used as controls. After 150 days of wastewater treatment on the beds, macrofauna was collected by taking five cores of sediment samples at the corners and the centre of each bed following three layers in the vertical profile. Globally, the planted beds removed COD, NH₄⁺, and PO₄^3? more (p < 0.05) than the control. Eleven taxa belonging to 6 classes and 11 orders were recorded. Macrofauna was significantly more diversified (Mann–Whitney test: p < 0.05) in terms of Shannon index of diversity in the planted beds (0.25–0.44 bits/ind.) than in the control (0.08–0.23 bits/ind.). But macrofauna settlement presents a relative homogeneity between the beds (index Jaccard = 0.63). Its abundance was three times higher in the planted bed than in the control. From the surface to the bottom of the beds, macrofauna diversity and abundance decreased and were heavily dominated by Annelida. The significant relationships were only observed between Insecta and Myriapoda in the control.