The hydrogen peroxide impact on larval settlement and metamorphosis of abalone Haliotis diversicolor supertexta

Abalone Haliotis diversicolor supertexta is an important economic mollusk.The settlement and metamorphosis are two critical stages during its development period,which has direct influence on abalone survival and production.The influence of reactive oxygen species(hydrogen peroxide) on abalone embryo and juvenile development were examined in this study.Larvae of Haliotis diversicolor supertexta were induced to settlement and metamorphose by exposure to seawater supplemented with hydrogen peroxide.They had the best performance at 800 μmol/L.The concentration of 1 000 μmol/L or higher was toxic to the larvae,as the larvae could settle down only at benthic diatom plates without complete metamorphosis.In addition,H2O2 adding time was critical to the larval performance.24h after two-day post-fertilization was proved to be the optimal adding time.In this paper,two action mechanisms of hydrogen peroxide are discussed:(1) hydrogen peroxide has direct toxicity to ciliated cells,thus cause apoptosis;(2) hydrogen peroxide,as a product from catecholamines' autoxidation process in vivo,can reverse this process to produce neuro-transmitters to induce abalone metamorphosis.     

Toxic effects of several phthalate esters on the embryos and larvae of abalone Haliotis diversicolor supertexta

As the most widely used plasticizers in the world, phthalate esters (PAEs) are potential endocrine disruption compounds (EDCs). In the present study, the toxicity of dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), di (2-ethylhexyl) phthalate (DEHP) on embryogenesis and larvae development of the marine univalve Haliotis diversicolor supertexta was examined in laboratory. The results show that the malformation of embryos appeared during the experiment, such as embryos died or lysed, small transparent flocculent rings studded on the periphery of the embryo, and the larvae could failed to hatch. In embryo toxic test, embryos incubated at the highest concentration of DMP, DEP and DBP solutions showed significantly high abnormal rate compared with the control, while DEHP solutions displayed no significant difference. In larval toxic test, in all concentrations of DMP, DEP and DBP solutions, larval settlement rates were low significantly than that of the control. Similarly, DEHP solutions show nearly no effect on the larval settlement. The order of toxicity on embryos and larvae is DBP>DEP>DMP>DEHP. Being a simple and easy stimulation to indoor spawn, sensitive to environmental factors, and short culture time, the embryos of H. diversicolor supertexta can be used to indicate toxicity of the PAEs. Supported by National Natural Science Foundation of China (No. 40476057)     

Induction of larval settlement and metamorphosis ofHaliotis discus hannai Ino (Gastropoda,Mollusca)

Conspecific foot mucus, excessive [K⁺] and gamma-aminobutyric acid (GABA) showed different metamorphosis-inductive effect on the veliger ofHaliotis discus hannai. The inductive effect of excessive [K⁺] and GABA was developmental stage-dependent and dose-dependent, while that of conspecific foot mucus was only developmental stage-dependent. At 20°C the veliger larvae became competent within 4 days after fertilization.H. discus hannai larvae showed gregarious settlement pattern on the conspecific foot mucus under the conditions of either presence or absence of KCl or GABA. The present studies showed that the effect of conspecific foot mucus on abalone larvae metamorphosis could be dose-independent.     

Characterization of defensin gene from abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis> and its deduced protein

Defensin is one of preserved ancient host defensive materials formed in biological evolution. As a regulator and effector molecule, it is very important in animals＇ acquired immune system. This paper reports the defensin gene from the mixed liver and kidney cDNA library of abalone Haliotis discus hannai Ino. Sequence analysis shows that the gene sequence of full-length cDNA encodes 42 mature peptides （including six Cys）, molecular weight of 4 323 Da, and pI of 8.02. Amino acid sequence homology analysis shows that the peptides are highly similar （70% in common） to other insects defensin. Because of a typical insect-defensin structural character of mature peptide in the secondary structure, the polypeptide named Haliotis discus defensin （hd-def）, a novel of antimicrobial peptides, belongs to insects defensin subfamily. The RT-PCR result of Haliotis discus defensin shows that the gene can be expressed only in the hepatopancreas by Gram-negative and positive bacteria stimulation, which is ascribed to inducible expression. Therefore, it is revealed that the Haliotis discus defensin gene expression was related to the antibacterial infection of Haliotis discus hannai Ino.     

Diatom diet selectivity by early post-larval abalone Haliotis diversicolor supertexta under hatchery conditions

Benthic diatoms constitute the primary diet of abalone during their early stages of development. To evaluate the dietary preferences of early post-larval abalone, Haliotis diversicolor supertexta, we analyzed the gut contents of post-larvae that settled on diatom films. We compared the abundance and species diversity of diatom assemblages in the gut to those of the epiphytic diatom assemblages on the attachment films, and identified 40 benthic diatom species in the gut contents of post-larvae 12 to 24 d after settlement. The most abundant taxa in the gut contents were Navicula spp., Amphora copulate, and Amphora coffeaeformis. Navicula spp. accounted for 64.0% of the cell density. In the attachment films, we identified 110 diatom species belonging to 38 genera. Pennate diatoms were the dominant members including the species Amphiprora alata, Cocconeis placentula var. euglypta, Cylindrotheca closterium, Navicula sp. 2, and A. coffeaeformis. Nano-diatoms (<20 μm in length) accounted for a considerable proportion of the total species number and cell density of the diatom assemblages in the gut contents and on the films. This suggests that nano-diatoms are important to the efficient production of abalone seed. The difference of the composition and abundance of diatoms between in the guts and on the biofilms suggests that early post-larval grazing was selective. An early post-larval abalone preferred nano-diatoms and the genera Navicula and Amphora during the month after settlement.     

The effects of nitrogen,phosphorus, vitamins and trace metals on the growth of the red tide organismProrocentrum Micans

Laboratory culture experiments showed that <100μ mol/L nitrate, amonium or mixture of amino acids promote the growth of the red tide organismProrocentrum micans Ehrenb, but that >100μmol/L of ammonium, or mixture of glycine and glutamate was harmful to growth, and that orthophosphate wasP. micans’ main phosphorous source in the ocean. Presence of 80μ mol/L EDTA, 0.5 to 1 μmol/L Fe³⁺, 1.0 to 20.0 μ mol/L Mn²⁺ 0.1 to 0.4 μmol/L Co²⁺ in the culture medium could improve the growth ofP. micans. Vitamin B₁ promoted growth, but vitamin B₁₂ and biotin did not. The estimated minimum cell quotas (q _o) for nitrogen and phosphorus being 0.74 pmole/cell and 0.045 pmole/cell show that phosphorus (more than nitrogen) limits the growth ofP. micans in the study area. This project was supported by the Natural Science Foundation of Zhejiang Province.     

Effects of tributyltin （TBT） on enzyme activity and oxidative stress in hepatopancreas and hemolymph of small abalone, Haliotis diversicolor supertexta

We investigated the effect of tributyltin (TBT) exposure on the concentration of malondialdehyde (MDA) and the activity levels of the superoxide dismutase (SOD), catalase (CAT), and acid and alkaline phosphatase (ACP and AKP) enzymes in the small abalone, Haliotis diversicolor supertexta. We collected samples of the hepatopancreas and hemolymph 2, 6, 24, 48, 96, and 192 h after exposure to 0.35 μg (Sn)/L TBT. In the hepatopancreas, ACP activity was significantly higher in animals exposed to TBT 2, 24, and 96 h post-exposure compared with the control animals. AKP activity was also higher after 2 h, but SOD and CAT activity was unchanged. The concentration of MDA in the hemolymph was significantly higher than the control animals 2 and 6 h post-exposure. In the hemolymph of animals exposed to TBT, ACP activity was significantly lower than in the control animals 192 h post-exposure, whereas AKP activity was significantly lower 2 and 192 h post-exposure. Hemolymph SOD activity and levels of MDA were significantly lower than in the control animals 24 h after exposure but significantly higher after 96 h. Our results demonstrate that exposure to TBT cause rapid changes in ACP and AKP activity as well as altering the concentration of MDA in the hepatopancreas and hemolymph. SOD and CAT do not appear to be involved in the detoxification of TBT in the hepatopancreas of small abalone.     

Bioaccumulation of Pb<Superscript>2+</Superscript> and its effects on growth,morphology and pigment contents of <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

A laboratory experiment was conducted to assess the bioaccumulation of Pb²⁺ and its effects on growth, morphology and pigment contents of Spirulina (Arthrospira) platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations (0, 5, 10, 30, 50 and 100 μg mL⁻¹). The growth of S. platensis was adversely affected by Pb²⁺ at high concentrations (30, 50 and 100 μg mL⁻¹). However, at low concentrations (5 μg mL⁻¹), Pb²⁺ could stimulate its growth slightly. The pigment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner. The highest reductions (67% and 53% respectively in chlorophyll α and β carotene) were observed in 100 μg mL⁻¹ treatment group. The LC₅₀ (96 h) of Pb²⁺ was measured as 75.34 μg mL⁻¹. Apart from a few cases of filament breakages at elevated concentrations (50 and 100 μg mL⁻¹), morphological abnormalities are not specific. Metal bioaccumulation increased with Pb²⁺ concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mg g⁻¹ dry weight. The bioconcentration factor (BCF) reached to a peak at day 2, followed by a gradual reduction for all the exposure concentrations. S. platensis is able to tolerate considerably high Pb²⁺ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.     

DIETARY PHOSPHORUS REQUIREMENT OF YOUNG ABALONE, HALIOTIS DISCUS HANNAI INO

An experiment was performed to determine the dietary phosphorus requirement of the young abalone,Haliotis discus hannai. Five semi-purified diets were formulated to provide a series of graded levels of dietary total phosphorus (0.23%–1.98) from monobasic potassium phosphate (KH2P04). The brown alga,Laminaria japonica, was used as a control diet. Similar size abalone were distributed in a single-pass, flow-through system using a completely randomized design with six treatments and three replicates each treatment. The abalone were hand-fed to satiation with appropriate diets in excess, once daily at 17:00. The feeding trial was run for 120-d. Survival rate and soft-body to shell ratio (SB/S) were constantly maintained regardless of dietary treatment. However, the weight gain rate (WGR), daily increment in shell length (DISL), muscle RNA to DNA ratio (RNA/DNA), carcass levels of lipid and protein, soft-body alkaline phosphatase (SBAKP), and phosphorus concentrations of whole body (WB) and soft body (SB) were significantly (ANOVA, P<0.05) affected by the dietary phosphorus level. The dietary phosphorus requirements of the abalone were evaluated from the WGR, DISL, and RNA/DNA ratio respectively, by using second-order polynomial regression analysis. Based on these criteria, about 1.0%–1.2% total dietary phosphorus, i.e. 0.9%–1.1% dietary available phosphorus is recommended for the maximum growth of the abalone. Project 39670572 supported by the NSFC.     

AVAILABILITY OF PHOSPHORUS FROM SELECTED INORGANIC PHOSPHATES TO JUVENILE ABALONE, HALIOTIS DISCUS HANNAI INO

The availability of phosphorus to juvenile abalone,Haliotis discus hannai from primary, secondary and tertiary calcium phosphate, primary sodium or potassium phosphate separately or in combination was determined in a 120-day feeding trial. Seven semi-purified diets were formulated to contain relatively constant dietary phosphorus, ranging from 0.78%–0.82%. The concentrations of dietary phosphorus were presumed to be slightly below the requirement for abalone. A brown alga,Laminaria japonica, was used as a control diet. Abalone juveniles of silimar size (average weight 1.18 g; average shell length 18.74 mm) were distributed in a single-pass, flow-through system using a completely randomized design with eight treatments and three replicates each treatment. The survival ranged from 71.1% to 81.1%, and was not significantly (P>0.05) related to dietary treatment. However, the weight gain rate (41.72%–65.02%), daily increment in shell length (36.87–55.07 μm) and muscle RNA-DNA ratio (3.44–4.69) were significantly (P<0.05) affected by dietary treatment. Soft body alkaline phosphatase activity (10.9–19.8 U/g wet tissue) and carcass levels of lipid (7.71%–9.33%) and protein (46.68%–49.35%) were significantly (P<0.05) responsive to available phosphorus of the diets. Dietary treatment had significant effect (P<0.05) on concentrations of phosphorus in the whole body (WB) and soft body (SB). Apparent digestibility coefficients (45%–97%) of phosphorus were also significantly (P<0.05) different between dietary treatments. The results indicated that among these inorganic compounds, primary calcium, potassium and sodium phosphates separately or in combination could be utilized effectively by juvenile abalone as dietary phosphorus sources (availability ranging from 87%–97%). However, secondary and tertiary calcium phosphates were found to be low in availability, the values being 45% and 77%, repectively. Project 39670572 supported by the Natural Science Foundation of China (NSFC).     

Effect of lead on cytoskeletal protein stability in crucian carp <Emphasis Type="Italic">Carassius auratus</Emphasis>

Inorganic lead （Pb） is one of the most common environmental pollutants. Much evidence indicates that Pb exposure could directly affect fish growth and development. In this study, we investigated the cytotoxic effects of Pb on cytoskeletal protein stability at both protein and mRNA level in crucian carp Carassius auratus. Pb（NO3）2 treatment in concentration of 100 μmol/L resulted in decreased expression of both α- and β-tubulin but γ-tubulin as assayed with SDS-PAGE, Western Blot, and ELISA. In vivo and in vitro analyses on protein expression of tubulins are consistent. The effect of Pb on mRNA expression varied among different tissues. Our results suggest that cytotoxicity of Pb at protein translation level is stronger than at mRNA expression level.     

Effects of salinity, light and temperature on growth rates of two species of Gracilaria （Rhodophyta）

Effects of temperature, salinity and light intensity on growth rates of Gracilaria lichenoides and G. tenuistipitata var. liui Zhang et Xia were tested. Eight to ten levels of each factor were first tested separately. The best growth rate was obtained under the conditions of 32°C, 30 and 240 μmol/(m²·s) for G. lichenoides, and 24°C, 20 and 200 μmol/(m²·s) for G. tenuistipitata, respectively. Then a uniform design was used to evaluate the optimal combinations of the three factors. The best conditions for the highest daily specific growth rates (% increase in wet weight) are determined to be 31.30°C, 32.10, and 287.23 μmol/(m²·s) for G. lichenoides (16.26%/d), and 25.38°C, 21.10, and 229.07 μmol/(m²·s) for G. tenuistipitata (14.83%/d), respectively. Supported by the 908 Special Program (908-02-04-07), the National Basic Research Program of China (973 Program, No. 2006CB400608), and K. C. Wong Magna Fund in Ningbo University     

Effects of dietary carbohydrates sources on lipids compositions in abalone, Haliotis discus hannai Ino

A study was conducted to evaluate the effects of dietary carbohydrates on triglyceride, cholesterol and fatty acid concentrations in abalone, Haliotis discus hannai Ino. Six semi-purified diets with different carbohydrates (dextrin, heat-treated wheat starch, wheat starch, corn starch, tapioca starch and potato starch, respectively), all containing a carbohydrate level of 33.5%, were fed to abalone (initial shell length: 29.98 mm ± 0.09 mm; initial weight: 3.42 g ± 0.02 g) for 24 weeks in a recirculation system. The results indicate that serum triglyceride concentrations were significantly (P < 0.05) higher in the abalone fed with dextrin, heat-treated wheat starch and wheat starch than those fed with corn starch, and serum cholesterol concentrations were significantly (P < 0.05) higher in the abalone fed with dextrin, heat-treated wheat starch than those fed with corn starch. Fatty acid C20:4n-6 in the foot muscles were significantly (P < 0.05) lower in the abalone fed with dextrin than those fed with wheat starch, corn starch, tapioca starch and potato starch. Fatty acid C20:4n-6 in hepatopancreas was significantly (P < 0.05) lower in abalone fed with heat-treated wheat starch than those fed with corn starch, tapioca starch and potato starch. Fatty acid C22:6n-3 in the foot muscles were significantly (P < 0.05) lower in the abalone fed with dextrin and heat-treated wheat starch than those fed with wheat starch and potato starch.     

Effects of Mg2+, NaCl, citric acid, and other factors on synthesis and accumulation of β-carotene inDunaliella salina

The effect of Mg²⁺, NaCl and citric acid on the accumulation of β-carotene inDunaliella Salina was studied. The experimental results showed that 10.5 mmol/L Mg²⁺, 5 mol/L NaCl, 3 μmol/L citric acid, and CO₂ are favorable forDunaliella Salina cell growth and β-carotene accumulation. After 144 h culture under the above conditions, theDunaliella Salina biomass increased by 7.18 times; β-carotene reached 9.61%. Project 89023990 supported by the Shandong Natural Science Fund.     

The antitumor effect of bromophenol derivatives <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">Leathesia nana</Emphasis> extract <Emphasis Type="Italic">in vivo</Emphasis>

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-(2,3-dibromo-4,5-dihydroxybenzyl)-2,3-dibromo-4,5-dihydroxy benzyl methyl ether (1), (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroisobenzofuran (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethyl-pyrocatechol (3), 2,2′,3,3′-tetrabromo-4,4′,5,5′-tetrahydroxy-diphenylmethane (4), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (5), 2,2′,3-tribromo-3′,4,4′,5-tetrahydroxy-6′-ethyloxymethyldiphenylmethane (6) were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTT assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402 and HCT-8. Their inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana (EELN) was evaluated on S₁₈₀-bearing mice. All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC₅₀ below 10 μg/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4%, respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenol derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer. Supported by the National High Technology Research and Development Program of China (863 Program, No. 2007AA09Z410) and Knowledge Innovation Program of Chinese Academy of Sciences (No. KZCX2-YW-209)     

Study on gametophyte vegetative growth ofUndaria pinnatifida and its applications

When cultured under certain environmental conditions (25°C, light intensity 80 μmol/m²·s, LD 12/12, in enriched seawater medium with 7×10⁻⁴ mol/L NO₃−N, 1.56×10⁻⁴ mol/L, PO₄−P and supplements of other elements like Mn, Fe, I, etc.), male and female gametophytes ofU. pinnatifida kept growing vegetatively and propagated fast at average daily fresh weight increase rate of about 20%. The empirical formulaG _m=G _o·3^m was established to estimate the output of vegetative gametophytes. Vigorous vegetative gametophyte cells began to form reproductive structures (oogonium and spermatangium, when the temperature was lower than 25°C and other environmental factors were kept optimal. The sufficient supply of gametophyte cells provided enough seeds for raisingUndaria sporelings on production scale. Controlled cross-breeding experiments using selected male and female gametophyte clones which increase their cell number by mitosis instead of meiosis were also carried out in vitro. Juvenile sporophytes from the cross-breeding had almost the same length and width increase rates as those of the control. The fact that vegetative gametophytes can be purposely selected, propagated quickly, cross-bred, with the resulting sporophytes having almost the same characteristics leads to a new way to select desiredUndaria strains for long time use without losing the desired economic characteristics. Contribution No. 2696 from the Institute of Oceanology, Chinese Academy of Sciences. Supported by National Natural Science Foundation of China.     

Impacts of CO<Subscript>2</Subscript> enrichment on growth and photosynthesis in freshwater and marine diatoms

The physiological responses of Nitzschia palea Kvtzing, a freshwater diatom, to elevated CO2 were investigated and compared with those of a marine diatom, Chaetoceros muelleri Lemmermann previously reported. Elevated CO2 concentration to 700 μl/L increased the dissolved inorganic carbon （D!C） and lowered the pH in the cultures of N. palea, thus enhancing the growth by 4%-20% during the whole growth period. High CO2-grown N. palea cells showed lower levels of dark respiration rates and higher Ik values. Light-saturated photosynthetic rates and photosynthetic efficiencies decreased in N. palea with the doubling CO2 concentration in airflow to the bottom of cultures, although the doubling CO2 concentration in airflow to the surface cultures had few effects on these two photosynthetic parameters. N. palea cells were found to be capable of using HCO5 in addition to gaseous CO2, and the CO2 enrichment decreased their affinity for HCO5 and CO2. Although doubled CO2 level would enhance the biomass of N. patea and C. muelleri to different extents, compared with the marine diatom, it had a significant effect on the specific growth rates of N. palea. In addition, the responses of photosynthetic parameters of IV. palea to doubled CO2 concentration were almost opposite to those of C. muelleri.     

DYNAMIC CHANGES OF INORGANIC NITROGEN AND ASTAXANTI—IIN ACCUMULATION IN HAEMATOCOCCUS PLUVIALIS

This study on dynamic changes of culture color,astaxanthin and chlorophylls,inorganic N including N-NO3^-,N-NO2^- and N-NH4^ in batch culture of Haematococcus pluvialis exposed to different additive nitrate concentration showed(1)ast/chl ratio was over 0.8 for brown and red algae,but was usually less than 0.5 for green and yellow algae;(2)N-NO3^-,in general,was unstable and decreased,except for a small unexpected increase in nitrate enriched treatment groups;(3)measurable amounts of N-NO2^- and N-NH4^ were observed respectively with three change modes although no extemal nitrite and ammonia were added into the culture;(4)a non-linear correlation between ast/chl ratio(or color)changes and the levels of N-NO3^-,N-NO2^-,N-NH4^ in H.pluvialis culture;(5)up and down variation of the ast/chl ratio occurred simultaneously with a perceptible color change from yellow to brown(or red)when N-NO3^-,N-NO2^- and N-NH4^ fluctuated around 30,5,5μmol/L respectively;(6)existence of three dynamic modes of N-NO3^-,N-NO2^- and N-NH4^ changes,obviously associated with initial extemal nitrate;(7)the key level of total inorganic N concentration regulating the above physiological changes during indoor cultivation was about 50 μmol/L;and(8)0.5-10mmol/L of nitrate was theoretically conducive to cell growth in batch culture.     

Effects of iron electrovalence and species on growth and astaxanthin production of Haematococcus pluvialis

To increase the cell concentration and the accumulation of astaxanthin in the cultivation of Haematococcus pluvialis, effects of different iron electrovalencies (Fe²⁺-EDTA and Fe³⁺-EDTA) and species (Fe-EDTA, Fe(OH)_x^32x and FeC₆H₅O₇) addition on cell growth and accumulation of astaxanthin were studied. Results show that different iron electrovalencies have various effects on cell growth and astaxanthin accumulation of H. pluvialis. Compared with Fe³⁺-EDTA, Fe²⁺-EDTA stimulate more effectively the formation of astaxanthin. The maximum astaxanthin content (30.70 mg/g biomass cell) was obtained under conditions of 18 μmol/L Fe²⁺-EDTA, despite the lower cell density (2.3×10⁵ cell/ml) in such condition. Fe³⁺-EDTA is more effective than Fe²⁺-EDTA in improving the cell growth. Especially, the maximal steady-state cell density, 2.9×10⁵ cell/ml was obtained at 18 μmol/L Fe³⁺-EDTA addition. On the other hand, all the various species of iron (EDTA-Fe, Fe(OH)_x^32x, FeC₆H₅O₇) are capable to improve the growth of the algae and astaxanthin production. Among the three iron species, FeC₆H₅O₇ performed the best. Under the condition of a higher concentration (36 μmol/L) of FeC₆H₅O₇, the cell density and astaxanthin production is 2 and 7 times higher than those of iron-limited group, respectively. The present study demonstrates that the effects of the stimulation with different iron species increased in the order of FeC₆H₅O₇, Fe(OH)_x^32x and EDTA-Fe. Supported by Xiamen Scientific and Technologic Projects (No. 3052Z20031086), Xiamen University Alumni Association Foundation in Singapore, and the First National College Students Innovative Experimental Project