The accurate measurement of gene expression via quantitative real-time reverse transcription PCR（q RT-PCR）heavily relies on the choice of valid reference gene（s） for data normalization. Resting cyst is the dormant...The accurate measurement of gene expression via quantitative real-time reverse transcription PCR（q RT-PCR）heavily relies on the choice of valid reference gene（s） for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of q RT-PCR to the HAB-forming group. In this study, six candidate reference genes,18 S ribosomal RNA（18S）, glyceraldehyde-3-phosphate dehydrogenase（GAPDH）, α-tubulin（TUA）, β-tubulin（TUB）, actin（ACT） and cytochrome oxidase subunit 1（COX1）, were evaluated for their expression stability with q RT-PCR and three statistical algorithms（Ge Norm, Norm Finder, and Best Keeper） for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages（resting cysts）, different growth stages, in darkness, exposed to abscisic acid（ABA） and exposed to temperature stress. The results indicated that TUA, 18 S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18 S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle,particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates r展开更多
Polychaetes provide an excellent food resource for fi sh and represent the dominant zoobenthos in marine ecosystems. Diel variation in the rates of metabolism and ammonia-N excretion of Marphysa sanguinea were studied...Polychaetes provide an excellent food resource for fi sh and represent the dominant zoobenthos in marine ecosystems. Diel variation in the rates of metabolism and ammonia-N excretion of Marphysa sanguinea were studied. The worms were grouped according to their wet body weight into small（S; 1.24±0.06 g）, medium（M; 4.00±0.30 g）, and large（L; 8.54±1.08 g） categories. Their weight-specifi c metabolic rates, based on aerobic respiration（ R）, were measured at 16℃（±0.2℃） and classed as either routine（ RR） or standard（ RS） rates. Both respiration types decreased with increasing body weight. Respiration was described by R = a W^b, where b was-0.400 9 and-0.532 0 for RR and RS, respectively. Diurnal changes in RS for each group was relatively flat, with a slightly increasing trend with time, but was relatively stable as a whole. RR of the diurnal variation of worms was higher than RS, but both had similar overall trends. The peak values of specifi c dynamic action（SDA）（ RSDA） in the S, M, and L groups were 2.704, 1.149, and 0.682 mg/（g·h）, respectively. The durations of SDA were 13, 6, and 6 h, respectively and the energy expenditures of SDA were 377.98, 117.34, and 74.94 J/g, respectively. These data indicate that the metabolic rates were higher in smaller individuals, which is advantageous for their rapid growth.展开更多
Harmful algal blooms (HABs), caused by the overgrowth of certain phytoplankton species, have negative effects on marine environments and coastal fisheries. In addition to cell-counting methods using phytoplankton nets...Harmful algal blooms (HABs), caused by the overgrowth of certain phytoplankton species, have negative effects on marine environments and coastal fisheries. In addition to cell-counting methods using phytoplankton nets, a hydroacoustic technique based on acoustic backscattering has been proposed for the detection of phytoplankton blooms. However, little is known of the acoustic properties of HAB species. In this study, as essential data to support this technique, we measured the acoustic properties of two HAB species, Akashiwo sanguinea and Alexandrium affine, which occur in the South Sea off the coast of Korea. Due to the small size of the target, we used ultrasound for the measurements. Experiments were conducted under laboratory and field conditions. In the laboratory experiment, the acoustic signal received from each species was directly proportional to the cell abundance. We derived a relationship between the cell abundance and acoustic signal received for each species. The measured signals were compared to predictions of a fluid sphere scattering model. When A. sanguinea blooms appeared at an abundance greater than 3 500 cells/mL, the acoustic signals varied with cell abundance, showing a good correlation. These results confirm that acoustic measurements can be used to detect HAB species.展开更多
Iris sangumea Dorm ex Horn. is a perennial cold-tolerant herbs of the Iris genus of Iridaceae, which has a well-formed, bright color and adaptability. Through understanding the fl ower bud differentiation, Sporogenesi...Iris sangumea Dorm ex Horn. is a perennial cold-tolerant herbs of the Iris genus of Iridaceae, which has a well-formed, bright color and adaptability. Through understanding the fl ower bud differentiation, Sporogenesis, gametogenesis, fertilization and embryogenesis of I. sangumea, we developed fl owering regulation and control breeding. I. sangumea fl ower bud differentiation was observed in April for 21 days; in this stage, the early differentiation process was slow, temperature was more than 5 °C, if the temperature is shorter than this, most fl ower bud will have no differentiation, and turn to vegetative growth; later differentiation is faster and required temperature is from 5 to 8 °C and gradually rose to 10-12 °C. The temperature in April plays the defi nitely role in the number of bud differentiation. Three stamina contain tetrasporangiate anthers and a glandular tapetum. The anther wall is composed of four cell layers inclduding the epidermis, the endothecium, one middle layer and the secretory tapetum. The type of anther wall development the dicotyledonous type. After pollen maturation, the anther wall forms a double-layer containing only epidermis and endothecium. Simultaneous cytokinesis is of the continuous type during meiosis of microspore mother cell results in a zygomorphous or tetragonal tetrad. Mature pollen grains are two-celled. I. sangumea ’s stylar canal is hollow, open type style, and the gynoecium has three carpels. The ovaries have axial placentas. The ovule is anatropous, bitegmic and crassinucellate. The primary sporogenous cell differentiates directly as the megaspore. The megaspore tetrads are linear, T-shaped or juxtaposed. Generally, the fi rst of the tetrad megaspores at the chalaza becomes functional while the other three degenerate. A mature embryo sac with seven cells and eight nuclei embryo sac is of the polygonum type. For dichogamy, the male gametophyte matures about 2 days earlier than the female gametophyte. The pollen tube entered into the embryo sac for 22–24 h af展开更多
Rapid amplification of cDNA ends (RACE) and real-time polymerase chain reaction (RT-PCR) were carried out to analyze the CYP4 gene expression in polychaete Marphysa sanguinea exposed to benzo[a]pyrene (BaP) in this st...Rapid amplification of cDNA ends (RACE) and real-time polymerase chain reaction (RT-PCR) were carried out to analyze the CYP4 gene expression in polychaete Marphysa sanguinea exposed to benzo[a]pyrene (BaP) in this study. The full length of MsCYP4 cDNA was 2 470 bp, and it encoded 512 amino acids. The deduced amino acid sequence showed 47% identity with CYP4F from frog Xenopus tropicalis and shared high homology with other known CYP4 sequences. To analyse the role of CYP4 in protecting M. sanguinea from BaP exposure, three BaP groups were established: 0.5, 5 and 50 μg/L. Polychaetes were sampled after 3, 7 and 12 d. At 0.5 μg/L, the effect of BaP on MsCYP4 gene expression increased with time prolonged. MsCYP4 gene expression curve showed Ushaped trend with time in 5 and 50 μg/L BaP groups. Therefore, MsCYP4 gene may play an important role in maintaining the balance of cellular metabolism and protecting M. sanguinea from BaP toxicity.展开更多
Blooms of Akashiwo sanguinea frequently break out around the world,causing huge economic losses to the aquaculture industry and seriously damaging coastal ecosystems.However,the formation mechanisms of A.sanguinea blo...Blooms of Akashiwo sanguinea frequently break out around the world,causing huge economic losses to the aquaculture industry and seriously damaging coastal ecosystems.However,the formation mechanisms of A.sanguinea blooms remain unclear.We investigated the allelopathic effects of A.sanguinea on multiple phytoplankton species,explored the mode of allelochemicals action and the way of nutrient factors regulation of the allelopathic activity.Results show that strains of A.sanguinea could inhibit the growth of co-occurring phytoplankton including Scrippsiella trochoidea,Phaeocystis globosa,and Rhodomonas salina,but inhibition of Prorocentrum micans was not obvious.The inhibition rates on phytoplankton were positively correlated with the cell densities of A.sanguinea.The highest inhibition rate of 94%on R.salina was for A.sanguinea CCMA256 culture of 2000 cells/mL at 72 h.We observed that cells of S.trochoidea,Ph.g lobosa,and R.sali na were lysed when co-cultured with A.sanguinea,with the shortest time for S.trochoidea.Additionally,the growth rates of A.sanguinea were promoted by coculturing with S.trochoidea,Ph.globosa,and R.salina.Four components of A.sanguinea culture were all able to inhibit growth of R.salina:the strongest inhibitory effect was found in the sonicated culture,followed by whole-cell culture,filtrates of sonicated culture,and filtrate culture.The crude extract of A.sanguinea culture also lysed cells of R.salina,and the inhibition rates on R.salina increased with the increasing dose of crude extract.It was shown that both nutrient enrichment and nitrogen:phosphorus ratio imbalance enhanced remarkably the allelopathic activity of A.sanguinea.The highest inhibition rate on R.salina of 70%occurred in A.sanguinea JX13 treatment at 2000 cells/mL under high nutrient condition in 48 h.In JX14 treatment at 2000 cells/mL for N:P of 10:1,the inhibition rate increased by 1.7 times of that for N:P of 20:1.In addition,the allelopathy of A.sanguinea could not only be a competitive strategy but also a nutrition stra展开更多
基金The National Natural Science Foundation of China-Shandong Joint Fund for Marine Science Research Centers under contract No.U1406403the State Key Program of National Natural Science of China under contract No.61533011China Postdoctoral Science Foundation under contract Nos 2014M551969 and 2015T80754
文摘The accurate measurement of gene expression via quantitative real-time reverse transcription PCR（q RT-PCR）heavily relies on the choice of valid reference gene（s） for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of q RT-PCR to the HAB-forming group. In this study, six candidate reference genes,18 S ribosomal RNA（18S）, glyceraldehyde-3-phosphate dehydrogenase（GAPDH）, α-tubulin（TUA）, β-tubulin（TUB）, actin（ACT） and cytochrome oxidase subunit 1（COX1）, were evaluated for their expression stability with q RT-PCR and three statistical algorithms（Ge Norm, Norm Finder, and Best Keeper） for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages（resting cysts）, different growth stages, in darkness, exposed to abscisic acid（ABA） and exposed to temperature stress. The results indicated that TUA, 18 S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18 S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle,particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates r
基金Supported by the National Natural Science Foundation of China(No.30901107)the National Marine Public Welfare Research Project(Nos.201305002,201305043)the Project of Marine Ecological Restoration Technology Research to the Penglai 19-3 Oil Spill Accident(No.19-3YJ09)
文摘Polychaetes provide an excellent food resource for fi sh and represent the dominant zoobenthos in marine ecosystems. Diel variation in the rates of metabolism and ammonia-N excretion of Marphysa sanguinea were studied. The worms were grouped according to their wet body weight into small（S; 1.24±0.06 g）, medium（M; 4.00±0.30 g）, and large（L; 8.54±1.08 g） categories. Their weight-specifi c metabolic rates, based on aerobic respiration（ R）, were measured at 16℃（±0.2℃） and classed as either routine（ RR） or standard（ RS） rates. Both respiration types decreased with increasing body weight. Respiration was described by R = a W^b, where b was-0.400 9 and-0.532 0 for RR and RS, respectively. Diurnal changes in RS for each group was relatively flat, with a slightly increasing trend with time, but was relatively stable as a whole. RR of the diurnal variation of worms was higher than RS, but both had similar overall trends. The peak values of specifi c dynamic action（SDA）（ RSDA） in the S, M, and L groups were 2.704, 1.149, and 0.682 mg/（g·h）, respectively. The durations of SDA were 13, 6, and 6 h, respectively and the energy expenditures of SDA were 377.98, 117.34, and 74.94 J/g, respectively. These data indicate that the metabolic rates were higher in smaller individuals, which is advantageous for their rapid growth.
基金project titled "Establishment and demonstration of red tide detection and prediction system for minimizing red tide damage" funded by the Ministry of Oceans and Fisheries, Korea (PM61410)
文摘Harmful algal blooms (HABs), caused by the overgrowth of certain phytoplankton species, have negative effects on marine environments and coastal fisheries. In addition to cell-counting methods using phytoplankton nets, a hydroacoustic technique based on acoustic backscattering has been proposed for the detection of phytoplankton blooms. However, little is known of the acoustic properties of HAB species. In this study, as essential data to support this technique, we measured the acoustic properties of two HAB species, Akashiwo sanguinea and Alexandrium affine, which occur in the South Sea off the coast of Korea. Due to the small size of the target, we used ultrasound for the measurements. Experiments were conducted under laboratory and field conditions. In the laboratory experiment, the acoustic signal received from each species was directly proportional to the cell abundance. We derived a relationship between the cell abundance and acoustic signal received for each species. The measured signals were compared to predictions of a fluid sphere scattering model. When A. sanguinea blooms appeared at an abundance greater than 3 500 cells/mL, the acoustic signals varied with cell abundance, showing a good correlation. These results confirm that acoustic measurements can be used to detect HAB species.
基金supported by the National Natural Science Foundation of China(No.31670344)the Fundamental Research Funds for the Central Universities(2572019DF08)
文摘Iris sangumea Dorm ex Horn. is a perennial cold-tolerant herbs of the Iris genus of Iridaceae, which has a well-formed, bright color and adaptability. Through understanding the fl ower bud differentiation, Sporogenesis, gametogenesis, fertilization and embryogenesis of I. sangumea, we developed fl owering regulation and control breeding. I. sangumea fl ower bud differentiation was observed in April for 21 days; in this stage, the early differentiation process was slow, temperature was more than 5 °C, if the temperature is shorter than this, most fl ower bud will have no differentiation, and turn to vegetative growth; later differentiation is faster and required temperature is from 5 to 8 °C and gradually rose to 10-12 °C. The temperature in April plays the defi nitely role in the number of bud differentiation. Three stamina contain tetrasporangiate anthers and a glandular tapetum. The anther wall is composed of four cell layers inclduding the epidermis, the endothecium, one middle layer and the secretory tapetum. The type of anther wall development the dicotyledonous type. After pollen maturation, the anther wall forms a double-layer containing only epidermis and endothecium. Simultaneous cytokinesis is of the continuous type during meiosis of microspore mother cell results in a zygomorphous or tetragonal tetrad. Mature pollen grains are two-celled. I. sangumea ’s stylar canal is hollow, open type style, and the gynoecium has three carpels. The ovaries have axial placentas. The ovule is anatropous, bitegmic and crassinucellate. The primary sporogenous cell differentiates directly as the megaspore. The megaspore tetrads are linear, T-shaped or juxtaposed. Generally, the fi rst of the tetrad megaspores at the chalaza becomes functional while the other three degenerate. A mature embryo sac with seven cells and eight nuclei embryo sac is of the polygonum type. For dichogamy, the male gametophyte matures about 2 days earlier than the female gametophyte. The pollen tube entered into the embryo sac for 22–24 h af
基金The Dalian Science Fund for Distinguished Young Scholars under contract No.2016RJ06the Natural Science Foundation of Liaoning Province under contract No.201602107+1 种基金the Superior College Science Technology Research Project of Liaoning Province under contract No.ZL201701the National Natural Science Foundation of China under contract No.41306138
文摘Rapid amplification of cDNA ends (RACE) and real-time polymerase chain reaction (RT-PCR) were carried out to analyze the CYP4 gene expression in polychaete Marphysa sanguinea exposed to benzo[a]pyrene (BaP) in this study. The full length of MsCYP4 cDNA was 2 470 bp, and it encoded 512 amino acids. The deduced amino acid sequence showed 47% identity with CYP4F from frog Xenopus tropicalis and shared high homology with other known CYP4 sequences. To analyse the role of CYP4 in protecting M. sanguinea from BaP exposure, three BaP groups were established: 0.5, 5 and 50 μg/L. Polychaetes were sampled after 3, 7 and 12 d. At 0.5 μg/L, the effect of BaP on MsCYP4 gene expression increased with time prolonged. MsCYP4 gene expression curve showed Ushaped trend with time in 5 and 50 μg/L BaP groups. Therefore, MsCYP4 gene may play an important role in maintaining the balance of cellular metabolism and protecting M. sanguinea from BaP toxicity.
基金Supported by the National Natural Science Foundation of China(NSFC)(Nos.41576159,41676099)。
文摘Blooms of Akashiwo sanguinea frequently break out around the world,causing huge economic losses to the aquaculture industry and seriously damaging coastal ecosystems.However,the formation mechanisms of A.sanguinea blooms remain unclear.We investigated the allelopathic effects of A.sanguinea on multiple phytoplankton species,explored the mode of allelochemicals action and the way of nutrient factors regulation of the allelopathic activity.Results show that strains of A.sanguinea could inhibit the growth of co-occurring phytoplankton including Scrippsiella trochoidea,Phaeocystis globosa,and Rhodomonas salina,but inhibition of Prorocentrum micans was not obvious.The inhibition rates on phytoplankton were positively correlated with the cell densities of A.sanguinea.The highest inhibition rate of 94%on R.salina was for A.sanguinea CCMA256 culture of 2000 cells/mL at 72 h.We observed that cells of S.trochoidea,Ph.g lobosa,and R.sali na were lysed when co-cultured with A.sanguinea,with the shortest time for S.trochoidea.Additionally,the growth rates of A.sanguinea were promoted by coculturing with S.trochoidea,Ph.globosa,and R.salina.Four components of A.sanguinea culture were all able to inhibit growth of R.salina:the strongest inhibitory effect was found in the sonicated culture,followed by whole-cell culture,filtrates of sonicated culture,and filtrate culture.The crude extract of A.sanguinea culture also lysed cells of R.salina,and the inhibition rates on R.salina increased with the increasing dose of crude extract.It was shown that both nutrient enrichment and nitrogen:phosphorus ratio imbalance enhanced remarkably the allelopathic activity of A.sanguinea.The highest inhibition rate on R.salina of 70%occurred in A.sanguinea JX13 treatment at 2000 cells/mL under high nutrient condition in 48 h.In JX14 treatment at 2000 cells/mL for N:P of 10:1,the inhibition rate increased by 1.7 times of that for N:P of 20:1.In addition,the allelopathy of A.sanguinea could not only be a competitive strategy but also a nutrition stra
