2 Key Laboratory of Marine Biotechnology of Zhejiang Province, Ningbo University, Ningbo 315211, China;
3 Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, Ningbo University, Ningbo 315211, China;
4 Senior High School of Yudu, Ganzhou 343200, China
With the problems such as high eutrophication, poor water exchange, low biodiversity, marine aquaculture ponds become areas where some specific notorious harmful microalgal species blooms were most likely triggered (Stanley et al., 2005; Martínez-Porchas et al., 2010; Granéli et al., 2012; Place et al., 2012). In return, the blooms caused tremendous damages to the cultured organisms and economical losses (Deeds et al., 2002; Houdan et al., 2004; Roelke et al., 2011; Martinez-Porchas and Martinez-Cordova, 2012).
Harmful microalgae in aquaculture ponds face complicated interactions with different cultured organisms. During the interactions, toxic materials may be released from the algal cell or just keep being content inside the cell. On account of the toxins, harmful effects may be caused by definite algal toxins or normal metabolites that are not generally thought as toxins, such as predation detergents DMS (P) (Kiene et al., 2000). It is difficult to answer the question that how toxic microalgae produce toxin or anti-predation detergent when predator present. Simulation of these processes is necessary to analyze the instant moment when the toxic species are physically disturbed or damaged because of the activities of cultured animals.
Harmful microalgae, such as Prymnesium parvum, Pleurochrysis elongata, Karlodinium veneficum, were usually found in some aquaculture ponds in Sanmen Bay, Zhejiang province, blooming or coblooming. Cell densities ranged 102–107 cells/mL occasionally. Prymnesium parvum is a wide-spread species and can cause mortality to many cultured animals such as fish, shellfish, shrimp and crab (Edvardsen and Paasche, 1998; Igarashi et al., 1998; Sasaki et al., 2001; Edvardsen and Imai, 2006; Baker et al., 2007). Many substances were thought to be responsible to its toxic potency (Henrikson et al., 2010). However, toxic effect of P. parvum was highly labile from case to case (Granéli and Salomon, 2010). Modes of action, potency of toxins or toxic effects may change in response to the ambient physical, chemical and biological factors such as salinity, light and temperature (Larsen et al., 1998; Brooks et al., 2010), pH (Valenti et al., 2010), metal ion availability and photolysis (James et al., 2011). Quantification of prymnesins is not analytically feasible because of different geographical strain of different toxic potency existed (Schug et al., 2010; Manning and La Claire, 2010).
As P. parvum, calcified coccolithophorid specie P. elongata belong to Prymnesiophyceae. Genus Pleurochrysis is drawing much attention because that some species of this Genus typically bloom in aquaculture pools in a way of long lasting notorious foam bloom. Pleurochrysis elongata is lethal to aquatic organisms such as Artemia salina (Houdan et al., 2004). However, few studies are done to clarify its toxic mechanism. Unlike the former one with some identified toxins, Pleurochrysis has no definite chemical compounds that could be classified as toxin(s). Mechanism of the lethal effect of Pleurochrysis is still unknown (Not et al., 2012).
Small dinoflagellate K. veneficum contains toxins (karlotoxins) of multiple toxic characteristic, such as hemolytic, ichthyotoxic, and cytotoxic activities (Deeds et al., 2002; Kempton et al., 2002; Zhou et al., 2011). Feature and activity model of the toxins differ from different strains and have not been completely characterized (Bachvaroff et al., 2009; Calbet et al., 2011). The content and structure of karlotoxins vary significantly with different strains, locations, environmental conditions (Place et al., 2012).
It is difficult to detect instant interaction between prey and predator in situ. In order to simulate the possible origination of the harmful effects, in this study, different algal samples were prepared to analysis the toxic potency based on brine shrimp toxicological bioassay. Toxic assessment based on crustaceans Artemia nauplii has been widely used for long time (Michael et al., 1956; Vanhaecke et al., 1981; Nunes et al., 2005; Hameed et al., 2009). When simultaneous assessment of multiple ecotoxicological significance of harmful species with multiple toxic characteristics is needed, Artemia nauplii are perfect model candidates (Nunes et al., 2006; Waggett et al., 2008; Faimali et al., 2012).2 MATERIAL AND METHOD 2.1 Microalgal cultures and culture conditions
Three harmful microalgal species, namely P. parvum, P. elongata, K. veneficum, were isolated from aquaculture pond in Sanmen Bay, typical aquaculture area in Zhejiang Province, in the coastal area of the East China Sea. Identification was done through analyses of morphology and Nucleic acid sequencing and SEM. Pure isolates were kept in Microalgae Collection Center in Ningbo University, China. Accession code in NCBI for P. parvum is MF039887. Strain number of P. elongate and K. veneficum are NMBjih026-1 and NMBjah047-1 respectively in the Collection Center. Isochrysis galbana (strain NMBjih021-1) was used as non-toxic species control.
The cultures were grown in sterile natural seawater (salinity 25) enriched with NMB3 medium (Xu et al., 2012). Conditions of culture room were 20℃, 40 μmol photons/(m2·s) illumination with 12 h:12 h L:D cycle.2.2 Artemia nauplii preparation
Artemia cysts preserved in 4℃ were warmed up to room temperature before use. The cysts were incubated at 30℃ in an incubator with continuous light of 50 μmol photons/(m2·s). The cysts hatched gradually after 24 h. Active nauplii were picked and placed into fresh seawater to keep for an additional 24 h to make sure all the nauplii moulted into instar Ⅱ–Ⅲ stages. These nauplii were prepared in the study.2.3 Mortalities of Artemia as a function of microalgal cell densities
Experiment was performed using 24-well plate. Cell densities of the pre-cultures were 1.1×106, 2.8×106 and 9.8×105 cells/mL for P. parvum, P. elongata and K. veneficum respectively at their early stationary growth phase. The cultures were 10 fold serially diluted with fresh culture medium. Five groups of different densities were prepared for each species. Ten nauplii together with 10 μL seawater were added to each well with micropipette (Eppendorf, Germany). Two milliliter algal cultures were put into each well. Nauplii in fresh culture medium were used as starving control. All treatments were replicated four times. The plates were placed in the culture room of 20℃ with continuous illumination of 15 μmol photons/(m2·s). Conditions of nauplii were documented and the dead ones were counted under inverted microscope (Nikon Ti-U Microscope Type 108) at 24 and 48 h. Still nauplii on the bottom without any kind of moving were considered dead.2.4 Lethal effects of different algal samples on Artemia
Algal samples preparation. Various samples were prepared after the cultures grew to late exponential phase. For each microalgae strain, totally 5 sorts of samples from 4 aliquots of each culture were prepared for the bioassay:
Cell free mediums (CFM). There were two ways to get CFM: cell free supernatants of the cultures centrifuged at 4℃ by 4 700×g for 10 min (CR11GIII, Hitachi, Japan), CFMcf; cell free filtrates by natural gravity filtration through 0.3 μm GF/C film (Whatman, USA), CFMgf.
Cell extracts (CEcf). Cell sediments by centrifugation were re-suspended in fresh culture medium to original volume. The cells were ultrasonic ruptured for 10–30 min (20 kHz, 400 W, with 5 s interval at 4℃). Cells totally ruptured were confirmed by observing under microscope. The suspensions were centrifuged at 42 300×g (4℃) for 10 min. The supernatants were used as cell extracts, CEcf.
Cell debris (CD). Ultrasonic ruptured cell debris was got from sediments while CEcf was prepared. The debris was re-suspended to original volume with fresh culture medium before use.
Original cultures of live cells (LC) were used to compare the effects of the algal samples. Original cell densities of the cultures were: 3.3×106, 5.3×105, 7.5×105, 6.4×106 cells/mL for P. parvum, P. caterae, K. veneficum and I. galbana respectively.
Toxicity detection process was the same as described in Section 2.3. Effects on Artemia were observed at time point of 0, 5, 12, 24, 36, 48 and 72 h. Dead nauplii were counted at each time.
Artemia in fresh culture medium (DSW) was set as starving control in all the tests.2.5 Data handling, statistical analyses, LD50 and LT50
Data are present as means ±SD.
Mortality of Artemia nauplii was calculated as:
where Nt represents dead nauplii at time t.
Median lethal density (LD50) or median lethal time (LT50) was obtained by applying a linear regression between cell concentration (or time) and mortality of Artemia nauplii according the method of Hewlett and Plackett (1979). LD50 in 24 h and 48 h were analyzed in Section 2.3. LT50, the time when 50% mortality of Artemia nauplii occurred, was analyzed in 2.4.
Statistical analyses were conducted with Statistical Package for the Social Sciences (SPSS). Significant differences were determined by one-way ANOVA test.3 RESULT 3.1 Mortality of Artemia nauplii as a function of microalgal cell density
All the three cultures had density dependent lethal effects on Artemia nauplii. Linear regression relationship existed in 24 h and 48 h (Fig. 1). For P. parvm, linear relationship between cell density and mortality of Artemia nauplii was the most significant (R2=0.954 79 and 0.970 71 in 24 h and 48 h respectively). LD50 were 1.1×104 cells/mL in 24 h and 8 cells/mL in 48 h. For P. elongate, linear relationship was more significant in 48 h (R2=0.979 4) than in 24 h (R2=0.870 55). LD50 was 7.2×105 and 2.9×103 cells/mL in 24 h and 48 h respectively. At the early time of exposure, there was no significant difference among the mortalities in groups of cell density of 102–104 cells/mL (P > 0.05, ANOVA). Not until the cell density rise to magnitude of 105 did the mortality increase significantly. In 48 h, however, mortality increased with rising culture density in a way of significant linear relationship. For K. veneficum, LD50 in 24 h was 1.5×105 cells/mL. It was 1.3×104 cells/mL in 48 h. Linear regression relationship in both 24 h and 48 h were significant.
Table 1 compared toxic potency of the three harmful species based on simultaneously assessment of mortalities of Artemia in 24 h and 48 h. P. parvm had the highest lethal activities than the other two. In 24 h, K. veneficum showed higher toxic activity than P. elongata. In 48 h, P. elongata showed higher toxic activity than K. veneficum. No dead nauplii were observed in feed or starving controls.3.2 Lethal effects of different algal samples on Artermia nauplii 3.2.1 Effects of the cellular extracts and cell debris
By comparing with the effects of live cells (LC) of each culture, mortality of Artermia as function of cell extracts (CEcf) and cell debris (CD) of the three species were studied (Fig. 2).
For P. parvm, acute effects were observed in 5 h from live cells, cell extracts and cell debris groups, mortality rates were 20%–22% without significant difference. In 36 h, the mortality reached 100%.
For P. elongata, no lethal effect was observed in 5 h or 12 h. Lethal effects showed no significant difference in 24 h. From 36 to 72 h, cell debris and live cells groups had similar lethal activities without significant differences. Artemia mortality was 2 times higher than that of the cells extracts in 48 h. It reached 90% in 72 h.
For K. veneficum, no acute effect occurred in 5 h. Mortalities, as low as 2% to 10%, were no significant differences in 24 h. Similar mortality in live cell and cell debris groups ranged 15%–18%. That of cell extracts was the lowest (5%). However, from 48 to 72 h, mortalities in cell extracts and cell debris groups increased significantly, averaged 60%, which was 2 times higher than that in live cells.3.2.2 Toxic activities of cell free medium of the harmful microalgae