Edible Peanut Worm(Sipunculus nudus) in the Beibu Gulf: Resource,Aquaculture, Ecological Impact and Counterplan

Sipunculus nudus is an important economic species because of its high nutritional and medicinal values. The exploitation and utilization of S. nudus primarily occur in the coastal regions of the Beibu Gulf. However, wild resource of S. nudus is rapidly decreasing because of the overexploitation, which has led to considerable developments of artificial breeding techniques. The cultivation scale of S. nudus has increased in response to successful artificial breeding; however, methods for culturing S. nudus in tidal flats or ponds require further study. Most studies have focused on the breeding, nutrition, medical value and ecological impact of these worms. Studies on the distribution, sediment requirements, nutrition characteristics, breeding techniques and aquaculture ecology of this species are summarized in this paper to promote the development of the aquaculture industry for S. nudus. The high biomass of S. nudus in the Beibu Gulf is positively correlated with the sediment characteristics and water quality of the region. The production of peanut worm has improved to some extent through culturing; however, the nutrient value and ecological environment problems have been observed, which reflect the over exploitation of trace elements and the sediment. These problems will worsen unless they are resolved, and the release of organic materials, nitrogen and phosphorus during harvesting impacts the coastal environment. Moreover, genetic erosion is a potential risk for larvae in artificial breeding programs in tidal flats. Therefore, culturing and collecting methods should be improved and the wild resource conservation should be implemented to promote the sustainable development of the peanut worm.     

Distribution of sediment chloroplastic pigments in the Southern Yellow Sea,China

The distribution of sediment chloroplastic pigments (Chl-a, i.e. chlorophyll a and Pha-a, i.e. phaeophorbide a) in the Southern Yellow Sea of China was studied. Samples were collected from four cruises in January and June 2003, and January and June 2004. The results show that the vertical distribution of Chl-a and Pha-a in the sediment layers 0-2cm, 2-5cm and 5-8cm, follows a stable ratio, 5:3:2. The average ratio of Pha-a to Chl-a in sediment is 2.83. Spearman 2-tailed rank correlation analysis shows that Chl-a and Pha-a contents in each sediment layer have a highly significant correlation. The average contents of Chl-a and Pha-a in the sediment of the 0-8cm layer in the investigated area are 0.31 -0.47μgg-1 and 1.28-1.40 μgg-1 sediment (dry weight), respectively. The average Chl-a and Pha-a contents in sediment are higher in summer than in winter. ANOVA analysis shows that there is a highly significant variation among the Chl-a contents (P = 0.002 <0.01) of the four cruies, but this is not true for the case of Pha-a content (P = 0.766>0.05). The average Chl-a and Pha-a contents in the 2 sediment layers (0-2cm and 2-5cm) have significant or highly significant correlations with organic matter (OM), median diameter (Mdφ), silt plus clay percentage in the January 2003 cruise. In the June 2003 cruise, the average Chl-a content in the 3 sediment layers (0-2cm, 2-5cm, and 5-8cm) has a significant correlation with meiofauna biomass, and Pha-a content has highly significant correlations with water depth, bottom water temperature, OM and Mdφ The contents of Chl-a and Pha-a are lower than those in estuaries and intertidal areas, but close to those in the same area studied previously.     

Characterization of bacterial communities associating with larval development of Yesso Scallop (<Emphasis Type="Italic">Patinopecten yessoensisis</Emphasis> Jay, 1857) by high-throughput sequencing

Bacterial community presumably plays an essential role in inhibiting pathogen colonization and maintaining the health of scallop larvae, but limiting data are available for Yesso scallop (Patinopecten yessoensisis Jay, 1857) larval development stages. The aim of this study was to characterize and compare the bacterial communities associating with Yesso scallop larval development at fertilized egg S1, trochophora S2, D-shaped larvae S3, umbo larvae S4, and juvenile scallop S5 stages by Illumina high-throughput sequencing. Genomic DNA was extracted from the larvae and their associating bactera, and a gene segment covering V3-V4 region of 16S rRNA gene was amplified and sequenced using an Illumina Miseq sequencer. Overall, 106760 qualified sequences with an average length of 449 bp were obtained. Sequences were compared with those retrieved from 16S rRNA gene databases, and 4 phyla, 7 classes, 15 orders, 21 families, 31 genera were identified. Proteobacteria was predominant phylum, accounting for more than 99%, at all 5 larval development stages. At genus level, Pseudomonas was dominant at stages S1 (80.60%), S2 (87.77%) and S5 (68.71%), followed by Photobacterium (17.06%) and Aeromonas (1.64%) at stage S1, Serratia (6.94%), Stenotrophomonas (3.08%) and Acinetobacter (1.2%) at stage S2, Shewanella (25.95%) and Pseudoalteromonas (4.57%) at stage S5. Moreover, genus Pseudoalteromonas became dominant at stages S3 (44.85%) and S4 (56.02%), followed by Photobacterium (29.82%), Pseudomonas (11.86%), Aliivibrio (8.60%) and Shewanella (3.39%) at stage S3, Pseudomonas (18.16%), Aliivibrio (14.29%), Shewanella (4.11%), Psychromonas (4.04%) and Psychrobacter (1.81%) at stage S4. From the results, we concluded that the bacterial community changed significantly at different development stages of Yesso Scallop larvae.     

Effects of different phosphorus concentrations and N/P ratios on the growth and photosynthetic characteristics of <Emphasis Type="Italic">Skeletonema costatum</Emphasis> and <Emphasis Type="Italic">Prorocentrum donghaiense</Emphasis>

The effects of different phosphorus (P) concentrations (0.36, 3.6, and 36 μmol/L corresponding to low-, middle-, and high-P concentration groups, respectively) and nitrogen (N)/P ratios on the growth and photosynthetic characteristics of Skeletonema costatum and Prorocentrum donghaiense were studied. For both species, the high-P (HP) concentration group showed the greatest algal density and highest specific growth rate. Changes in the maximum efficiency of photosystem II (F _v /F _m ) were monitored under the various P and N/P conditions. The largest decrease in F _v /F _m was in the low-P (LP) group in S. costatum and in the HP group in P. donghaiense. There were high rapid light curves and photochemical quantum yields (Φ _PSII) for S. costatum in the HP group, while the actual photosynthetic capacity was higher in P. donghaiense than in S. costatum in the MP group. Under eutrophic but relatively P-restricted conditions, P. donghaiense had higher photosynthetic activity and potential, which could cause this dinoflagellate to increasingly dominate the phytoplankton community in these conditions. Under the same P concentration and N/P ratio, P. donghaiense had a larger relative maximum rate of electron transport and higher Φ _{PS II} values than those of S. costatum. These differences between P. donghaiense and S. costatum may explain the interaction and succession patterns of these two species in the Changjiang (Yangtze) River estuary from a photosynthesis perspective.     

Biodeposition,respiration, and excretion rates of an introduced clam <Emphasis Type="Italic">Mercenaria mercenaria</Emphasis> in ponds with implications for potential competition with the native clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis> in Shuangtaizi estuary,China

This study aimed to evaluate the potential impacts of an introduced clam Mercenaria mercenaria on estuarine ecosystem, and implications for the niche competition with a native clam Meretrix meretrix. The biodeposition, respiration, and excretion rates of M. mercenaria were determined seasonally using a sediment trap and a closed respirator in field. The biodeposition rates of M. mercenaria were 0.06–0.37 g/ (ind.·d), and the respiration rates were 0.31–14.66 mg/(ind.·d). The ammonia and phosphate excretion rates were 0.18–36.70 and 1.44–14.87 μg/(ind.·d), respectively. The hard clam M. mercenaria may discharge dry deposits up to 2.1×10⁵ t, contribute 18.3 t ammonia and 9.0 t phosphate to culture ponds, and consume 7.9×10³ t O₂ from ponds annually. It suggested that the hard clam M. mercenaria might play an important role in pelagic-benthic coupling in pond ecosystem through biodeposition and excretion. A comparison of the key physiological parameters of the introduced clam M. mercenaria and the native clam Meretrix meretrix suggested that M. mercenaria had a niche similar to that of Meretrix meretrix in Shuangtaizi estuary and might have a potential competition with Meretrix meretrix for habitat and food if M. mercenaria species escaped from the culture pond or artificially released in estuarine ecosystem.     

Inbreeding depression on growth and survival of full-sib family of Manila clam (<Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>)

In present study, the inbreeding depression (ID) of growth and survival of Manila clam (Ruditapes philippinarum) was investigated at larval and juvenile stages. Nine inbred families (A ₂, B ₂, C ₂, D ₂, E ₂, F ₂, G ₂, H ₂ and I ₂) were established by mating within nine full-sib families with expected inbreeding coefficient of 0.25. Inbred families showed significant differences in shell length and hatching rate of D-larvae (straight-hinged larvae). The larvae of the nine inbred families grew slower than those of control group (CG), and their ID value ranged from 0.81% ± 6.09% to 16.10% ± 1.49%. The ID value of larval survival rate varied between 27.47% ± 9.36% and 70.50% ± 13.66%. The ID was also detected for juvenile growth in A ₂, B ₂, C ₂, and D ₂, which ranged from 4.60 ± 2.21 to 17.71 ± 7.73. The A ₂ family maintained the highest juvenile survival rate, whereas the other inbred families exhibited ID values varying between 62.79% ± 4.54% and 96.14% ± 0.87%. The linear relationship of estimated ID between growth and survival was negatively correlated (R = ?0.434, P < 0.05). The results of this study suggested that the ID of growth was common at the larval stage but was less prevalent at juvenile stage. In contrast, the ID of survival increased from larval to juvenile stage. A better understanding of the effect of inbreeding may aid to selective breeding of Manila clam.     

Succession and seasonal variation in epilithic biofilms on artificial reefs in culture waters of the sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

Periphytic biofilms in aquaculture waters are thought to improve water quality, provide an additional food source, and improve the survival and growth of some reared animals. In the Asia- Pacific region, particularly in China, artificial reefs are commonly used in the commercial farming of sea cucumbers. However, few studies have examined the epilithic biofilms on the artificial reefs. To gain a better understanding of the succession of epilithic biofilms and their ecological processes in sea cucumber culture waters, two experiments were conducted in culture waters of the sea cucumber Apostichopus japonicus in Rongcheng, China, using artificial test panels. On the test panels of succession experiment, more than 67 species were identified in the biofilms. On the test panels of seasonal variation experiment, more than 46 species were recorded in the biofilms. In both experiments, communities of epilithic biofilms were dominated by diatoms, green algae and the annelid Spirorbis sp. In the initial colonization, the dominant diatoms were Cocconeis sp., Amphora spp. and Nitzschia closterium in June, which were succeeded by species of Navicula, Cocconeis and Nitzschia (July to September), and then by Licmophora abbreviata, Nitzschia closterium and Synedra spp. in the following months. A diatom bloom in the autumn and filamentous green algae burst in the summer were also observed. Ecological indices well annotated the succession and seasonal changes in epilithic communities. Multidimensional scaling (MDS) analysis found significant differences in diatom community composition among months and seasons. Fast growth of biofilms was observed in the summer and autumn, whereas the biomass of summer biofilms was largely made up of filamentous green algae. Present results show that the components of epilithic biofilms are mostly optimal foods of A. japonicus, suggesting that biofilms on artificial reefs may contribute important nutritional sources for sea cucumbers during their growth seasons. Future works should include quantitative determination of the contribution of epilithic biofilms to the diet of A. japonicus, potential roles of epilithic biofilms in regulating the water quality of sea cucumber ponds, and the regulation of epilithic biofilms in sea cucumber culture ponds.     

Early embryo and larval development of inviable intergeneric hybrids derived from <Emphasis Type="Italic">Crassostrea angulata</Emphasis> and <Emphasis Type="Italic">Saccostrea cucullata</Emphasis>

To detect the intergeneric hybridization between the oyster Crassostrea angulata and Saccostrea cucullata coexisting along the southern coast of China, reciprocal crosses were conducted between the two species. Barriers for sperm recognizing, binding, penetrating the egg, and forming the pronucleus were detected by fluorescence staining. From the results, although fertilization success was observed in hybrid crosses, the overall fertilization rate was lower than that of intraspecific crosses. A large number of hybrid larvae died at 6–8 d after hatching, and those survived could not complete metamorphosis. C. angulata ♀× S. cucullata ♂ larvae had a growth rate similar to that of the maternal species, whereas S. cucullata ♀ × C. angulata ♂ larvae grew the slowest among all crosses. Molecular genetics analysis revealed that hybrid progeny were amphimixis hybrids. This study demonstrated that hybrid embryos generated by crossing C. angulata and S. cucullata could develop normally to the larval state, but could not complete metamorphosis and then develop to the spat stage. Thus, there is a post-reproductive isolation between C. angulata and S. cucullata.     

Changes of Biogenic Elements in <Emphasis Type="Italic">Phragmites australis</Emphasis> and <Emphasis Type="Italic">Suaeda salsa</Emphasis> from Salt Marshes in Yellow River Delta,China

Little information is available on biogenic elements (carbon, nitrogen, phosphorus and sulfur) and the ecological stoichiometric characteristics of plants in coastal wetlands. To investigate the contents of carbon, nitrogen, phosphorus and sulfur of plants, and their ecological stoichiometric characteristics in the Yellow (Huanghe) River Delta, plant samples were collected from two typical salt marshes (Suaeda salsa and Phragmites australis wetlands) during the period of from August to October in 2007, and the ratios of C/N, C/P, N/P, C/N/P and C/N/P/S were calculated. Results showed that during the studying period, plant C, N and P were lower than the global average values, and plant N and P were lower than the China’s average values. Leaf C and S in Suaeda salsa were significantly lower than those in Phragmites australis (P < 0.05), and leaf N and P in Suaeda salsa and Phragmites australis showed no significant differences (P > 0.05). Average C/N ratios were 23.75 in leaf, 73.36 in stem, 65.67 in root of Suaeda salsa, and 33.77 in leaf, 121.68 in stem, 97.13 in root of Phragmites australis. Average C/N ratios of Suaeda salsa and Phragmites australis were all great than 25, indicating the salt marsh in the Yellow River Delta is an N limitation system. Average C/P ratios were 276.78 in leaf, 709.28 in stem and 1031.32 in root of Suaeda salsa, and 536.94 in leaf, 768.13 in stem and 875.22 in root of Phragmites australis. The average N/P ratios of Suaeda salsa were 12.92 in leaf, 10.77 in stem and 10.91 in root, and the average N/P ratios of Phragmites australis were 16.40 in leaf, 7.40 in stem and 6.92 in root, indicating the Suaeda salsa wetlands were N limited and Phragmites australis wetlands were N limited in August and P limited in October in 2007. The average C/N, C/P and C/N/P ratios in Suaeda salsa and Pragmites australis were higher than the global average values, indicating the lower quality of organic matter provided by wetland plants in the Yellow River delta.     

Effect of an escape vent in accordion-shaped traps on the catch and size of Asian paddle crabs <Emphasis Type="Italic">Charybdis japonica</Emphasis> in an artificial reef area

Accordion-shaped traps are widely used in China to catch the Asian paddle crab Charybdis japonica but traps of conventional design often catch juvenile crabs. A new type of accordion-shaped trap with an escape vent (L×W=4.3 cm×3.0 cm) was designed and a comparative study between the newly designed and conventional traps was performed in the artificial reef area of Zhuwang, Laizhou Bay, China from June to August 2012. The mean catch per unit effort (CPUE) of undersized crabs was significantly lower in the vented traps than in the conventional traps (paired t -test, n=30, P<0.001), while the CPUE of marketable crabs was significantly higher in the vented traps (paired t-test, n=30, P<0.001). The mean size of crabs (carapace length) caught in the vented traps was significantly larger than in conventional traps (paired t-test, n=29, P<0.001). The ratio of undersized crabs was 35.05±2.57% in conventional traps and 12.53±0.69% in vented traps (significantly lower, paired t-test, n=29, P<0.001). Therefore, a 4.3 cm×3.0 cm escape vent was considered appropriate for C. japonica fishing in the artificial reef area. This finding will assist the development of more sustainable and efficient crab fishing methods using accordion-shaped traps.     

Biodegradation of <Emphasis Type="Italic">Enteromorpha</Emphasis> polysaccharides by intestinal micro-community from <Emphasis Type="Italic">Siganus oramin</Emphasis>

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal micro-communities involved in the biodegradation of Enteromorpha polysaccharides (EP) were seldom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U mL^?1 in 32 hours. With denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading micro- community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.     

Effects of a Potential Autochthonous Probiotic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> 2-1 on the Growth and Intestinal Microbiota of Juvenile Sea Cucumber, <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> Selenka

The effects of Bacillus subtilis 2-1 from the intestine of healthy sea cucumber on the growth, digestive enzyme activities and intestinal microbiota of juvenile sea cucumber (Apostichopus japonicus) were determined in the present study. Sea cucumber was fed with Sargassum thunbergii powder supplemented with B. subtilis 2-1 at different concentrations varying among 0 (control), 10⁵, 10⁷, and 10⁹ CFU g^?1 for 8 weeks. Results showed that the growth performance and intestinal amylase and trypsin activities were significantly increased by dietary B. subtilis 2-1 at 10⁹ CFU g^?1 (P < 0.05). However, dietary B. subtilis 2-1 had no significant influence on the lipase activity in sea cucumber (P > 0.05). The polymerase chain reaction denaturing gradient gel electrophoresis and 16S rRNA gene sequencing analysis indicated that dietary B. subtilis 2-1 at 10⁵ and 10⁷ CFU g^?1 inhibited most of the Proteobacteria including those in genus Vibrio. Dietary B. subtilis 2-1 at 10⁹ CFU g^?1 not only decreased the abundance and species of genus Vibrio, but also increased the intensity of genera Psychrobacter and Bacillus. A specific dosage of dietary B. subtilis 2-1 could increase the growth and modulate the intestinal microbiota of sea cucumber; thus it might be a novel probiotic for keeping the health of sea cucumber.     

Trophic Interaction in a <Emphasis Type="Italic">Portunus rituberculatus</Emphasis> Polyculture Ecosystem Based on Carbon and Nitrogen Stable Isotope Analysis

Trophic interaction among various biomass groups in a swimming crab Portunus trituberculatus polyculture pond was investigated using carbon and nitrogen stable isotope analysis. The polycultured animal species also included white shrimp Litopenaeus vannamei, short-necked clam Ruditapes philippinarum, and redlip mullet Liza haematochila. The mean δ¹³C value for all the biomass groups in polyculture ecosystem ranged from ?25.61‰ to ?16.60‰, and the mean δ¹⁵N value ranged from 6.80‰ to 13.09‰. Significant difference in the δ¹³C value was found between particulate organic matter (POM) and sediment organic matter (SOM) (P < 0.05), indicating that these two organic matter pools have different material sources. Assuming that a ¹³C-enrichment factor of 1.00‰ and a ¹⁵N-enrichment factor of 2.70‰ existed between consumer and prey, diets of the four cultured animals were estimated using a stable isotope mixing model. The estimated model results indicated that P. trituberculatus mainly feed on Aloidis laevis; L. vannamei mainly feed on shrimp feed; while A. laevis, R. philippinarum and L. haematochelia mainly feed on POM. Shrimp feed was also an important food source of R. philippinarum and L. haematochelia. The diets of P. trituberculatus, L. vannamei, R. philippinarum, and L. haematochila showed complementary effects in this polyculture ecosystem. Our finding indicated that the polyculture of these four organisms with suitable farming density could make an effective use of most of the food sources, which can make a highly efficient polyculture ecosystem.     

Diel variation in metabolism and ammonia excretion of <Emphasis Type="Italic">Marphysa sanguinea</Emphasis> (Polychaeta: Eunicidae)

Polychaetes provide an excellent food resource for fish 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-specific metabolic rates, based on aerobic respiration (R), were measured at 16°C (±0.2°C) and classed as either routine (R _R) or standard (R _S) 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 R _R and R _S, respectively. Diurnal changes in R _S for each group was relatively flat, with a slightly increasing trend with time, but was relatively stable as a whole. R _R of the diurnal variation of worms was higher than R S, but both had similar overall trends. The peak values of specific dynamic action (SDA) (R _SDA) 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.     

Change of digestive physiology in sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Selenka) induced by corn kernels meal and soybean meal in diets

The present study was conducted to determine the change of digestive physiology in sea cucumber Apostichopus japonicus(Selenka) induced by corn kernels meal and soybean meal in diets. Four experimental diets were tested, in which Sargassum thunbergii was proportionally replaced by the mixture of corn kernels meal and soybean meal. The growth performance, body composition and intestinal digestive enzyme activities in A. japonicus fed these 4 diets were examined. Results showed that the sea cucumber exhibited the maximum growth rate when 20% of S. thunbergii in the diet was replaced by corn kernels meal and soybean meal, while 40% of S. thunbergii in the diet can be replaced by the mixture of corn kernels meal and soybean meal without adversely affecting growth performance of A. japonicus. The activities of intestinal trypsin and amylase in A. japonicus can be significantly altered by corn kernels meal and soybean meal in diets. Trypsin activity in the intestine of A. japonicus significantly increased in the treatment groups compared to the control, suggesting that the supplement of corn kernels meal and soybean meal in the diets might increase the intestinal trypsin activity of A. japonicus. However, amylase activity in the intestine of A. japonicus remarkably decreased with the increasing replacement level of S. thunbergii by the mixture of corn kernels meal and soybean meal, suggesting that supplement of corn kernels meal and soybean meal in the diets might decrease the intestinal amylase activity of A. japonicus.     

Genetic transformation of <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> with a bacterial phleomycin resistance gene as dominant selective marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×10⁷ cells mL^?1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL^?1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.