臺灣博碩士論文加值系統

摘要

本研究的目的是在探討四種微細藻（周氏扁藻Tetraselmis chuii、等鞭金藻Isochrysis galbana、海鏈藻Thalassiosira weissflogii、牟氏角毛藻Chaetoceros muelleri）做為多齒米蝦(Caridina multidentata)幼蟲餌料並分別於飼育於29℃和25℃兩個溫度下對於發育及成長的影響。蒐集剛從抱卵母蝦釋出之第一期眼幼蟲（Zoea 1）置於100升的 FRP桶中，密度30隻/升，鹽度20 ‰。進行了一個三重複的複因子試驗(4×2) 。結果發現變態蝦苗活存率與培育溫度(25℃，29℃)無關。而變態蝦苗活存率以飼育扁藻(35％ 25℃，35％ 29℃)及海鏈藻(38％ 25℃，36％ 29℃)者間無顯差異，但顯著高於飼育牟氏角毛藻(13％ 25℃，12％ 29℃)者，而以等鞭金藻(9％ 25℃，9％ 29℃)組最差。水溫無論是25℃或29℃對於幼苗成長並無影響；而飼予周氏扁藻T. chui及海鏈藻T. weissflogii之多齒米蝦幼蟲在同一時間軸無論是體長或步足及泳足長度均高於餵予C. muelleri和I. galbana者，換言之有較好的成長速率。
因此周氏扁藻及海鏈藻是多齒米蝦幼苗最適餌料生物，餌料密度2x105cell/ml，飼育密度30隻/升，鹽度20 ‰，溫度介於25-29℃間。
關鍵詞：觀賞蝦、幼苗培育、微細藻、多齒米蝦、生長發育、存活率

ABSTRACT

The objectives of this study were to evaluate four different algae species, Tetraselmis chuii, Isochrysis galbana, Thalassiosira weissflogii, Chaetoceros muelleri as live feed fed to the Amano shrimp (Caridina multidentata) larvae at equal density of 2 x105cells/ml (three replicates) under two temperature conditions on the development, growth and survivals. A triplicated 4x2 factorial experiment was designed. The newly released shrimp larvae (Zoea1) obtained from pouched brooders were placed in 100 L Fiberglass reinforced plastics (FRP) at a density of 30 larvae/liter and a salinity of 20‰ for each experimental unit. Of the survival rate through covariance analysis, there existed no temperature effect between 29°C and 25°C, with the respective values of 9%, 12%, 35%, 36% for the larvae received C. muelleri, I. galbana, T. chuii, T. weissflogii at high temperature condition (29°C) compared to the corresponding values of 9%, 13%, 35%, 38% at low temperature (25°C). It was found that the metamorphosed juveniles with averaged body length of 5.33±0.04 mm on the larvae fed T. chui at day 19, which significantly higher than the corresponding 19-day-age length values of larvae fed T. weissflogii (4.76±0.09 mm), I. galbana (3.68±0.29 mm) and C. muelleri (3.28 ± 0.15 mm) at 29° C. At 25° C, the metamorphosed juveniles appeared on day 19 with averaged body length of 5.30±0.14 mm for those fed T. chui, which were significantly higher than those larvae on day 19 fed T. weissflogii (4.90 ± 0.02 mm), I. galbana (4.02 ± 0.01 mm) and Chaetoceros muelleri (3.28 ± 0.15 mm) respectively. At 29 °C and at day 19, the average length of pereiopod were 1.15±0.21 mm, 0.95±0.09 mm, 0.68±0.03 mm and 0.62±0.02 mm for T. chui, T. weissflogii, I. galbana and C. muelleri respectively. At 25°C and at day 19, in the treatment of Tetraselmis chui, the average length of pereiopods were 1.28 ± 0.12 mm while it was 0.45 ± 0.05 mm, 0.68 ± 0.08 mm and 0.95 ± 0.12 mm for C. muelleri, I. galbana and T. weissflogii respectively. At 29 °C and at day 19, in the treatment of Tetraselmis chui, the average length of pleopod were 0.84 ± 0.06 mm while it was 0.72 ± 0.04 mm, 0.22 ± 0.09 mm and 0.13 ± 0.02 mm for T. weissflogii, I. galbana and C. muelleri respectively. At 25 °C and at day 19, the results were 0.82 ± 0.07 mm, 0.72 ± 0.09 mm, 0.29 ± 0.01 mm and 0.31 ± 0.01 mm for T. chui, T. weissflogii, I. galbana and C. muelleri. In conclusion, both C. muelleri and I. galbana were recommended live diets for planktonic larvae of C. multidentata when culture condition was well control for: temperature 25-29℃, larvae density 30 individuals/L, algae density 2 x 105 cells/ ml, salinity 20‰.
Keywords: Algae, Caridina multidentata, development, growth, larvae culture, Ornamental shrimp,

TABLE OF CONTENTS
DECLARATION i
DEDICATION ii
ACKNOWLEDGEMENTS iii
摘要 iv
ABSTRACT v
TABLE OF CONTENTS vii
LIST OF ABREVIATION INITIALS ix
LIST OF TABLES x
LIST OF FIGURES xii
1 INTRODUCTION 1
2 LITERATURE REVIEW 4
2.1 Microalgae and aquaculture 4
2.1.1 Introduction 4
2.1.2 Commonly used microalgae in Aquaculture 5
2.1.3 Characteristic of Tetraselmis chui 5
2.1.4 Ecology of Tetraselmis chui 5
2.1.5 Characteristic of Thalassiosira weissflogii 6
2.1.6 Ecology of Thalassiosira weissflogii 6
2.1.7 Characteristic of Isochrysis galbana 6
2.1.8 Description of Chaetoceros muelleri 6
2.1.9 Ecology of Chaetoceros muelleri 7
2.2 Caridina multidentata 7
2.2.1 Classification 7
2.2.2 Importance 7
2.2.3 Habitat and environment 8
2.2.4 Reproduction and larvae culture 8
3 MATERIALS AND METHODS 10
3.1 Source of shrimp and microalgae 10
3.2 Design of experiment 10
3.3 Brood stock management and environmental control 10
3.4 Handling the spawners 11
3.5 Larvae collection 11
3.6 Larvae culture 11
3.7 Control of culture tank 11
3.8 Microalgae manipulation 12
3.8.1 Pure culture maintenance 12
3.8.2 Scale up procedure 12
3.9 Sampling and data collection 12
3.10 Calculation 13
3.11 Statistical analysis 13
4 RESULTS 14
4.1 Development 14
4.2 Survival Rate 14
4.3 Growth 15
4.3.1 Body length 15
4.3.2 Pereiopods 16
4.3.3 Pleopods 17
5 DISCUSSION 18
6. CONCLUSION 20
7. REFERENCE 21

LIST OF TABLES
Table 1. Commercial algal culture and its applications 27
Table 2. Composition and preparation of Walne medium (1974). 28
Table 3 Characteristic morphologic of the developmental stages of Caridina multidentata analyzed through a factorial experiment (algae x temperature, 4 x 2) by feeding each of the respective algae density at 2x105cells/ml and salinity at 20‰ during 25 day-culture trials, the experiment terminated when all survivors entering into metamorphosed juvenile stage. 29
Table 4 The survival rate of Amano shrimp between the four algae species at two temperatures analyzed through a factorial experiment (algae x temperature, 4 x 2) after 25 days of culture with all survivors entering into metamorphosed juvenile stage at salinity 20‰, algae density 2x105cells/ml. 30
Table 5. The ANOVA main table of time dependent covariance analysis of a factorial experiment (Algae x Temperature, 4 x 2) on the body length (daily documented) for 25 days of feeding each of different algae species, all at a density of 2x105cells/ml and a salinity at 20‰. 31
Table 6. The body length of Amano shrimp between the four algae species at high temperature analyzed through a factorial experiment (algae x temperature, 4 x 1) after 25 days of culture with all survivors entering into metamorphosed juvenile stage at salinity 20‰, algae density 2x105cells/ml. 32
Table 7. The body length of Amano shrimp between the four algae species at low temperature analyzed through a factorial experiment (algae x temperature, 4 x 1) after 25 days of culture with all survivors entering into metamorphosed juvenile stage at salinity 20‰, algae density 2x105cells/ml. 33
Table 8 Orthogonal contrasts of the LS means of body length of Amano shrimp larvae on the four algae fed to them across the two temperature levels and temperature (25° c vs 29° c) comparison across all four species of algae fed to them. 34
Table 9 The ANOVA main table of time dependent covariance analysis of a factorial experiment (Algae x Temperature, 4 x 2) on the length of pereiopod (daily documented) for 25 days of feeding each of different algae species, all at a density of 2x105cells/ml and a salinity at 20‰. 35
Table 10 The length of pereiopod of Amano shrimp between the four algae species at high temperature analyzed through a factorial experiment (algae x temperature, 4 x 1) after 25 days of culture with all survivors entering into metamorphosed juvenile stage at salinity 20‰, algae density 2x105cells/ml. 36
Table 11 The length of pereiopod of Amano shrimp between the four algae species at low temperature analyzed through a factorial experiment (algae x temperature, 4 x 1) after 25 days of culture with all survivors entering into metamorphosed juvenile stage at salinity 20‰, algae density 2x105cells/ml. 37
Table 12 Orthogonal contrasts of the LS means of pereiopod of Amano shrimp larvae on the four algae fed to them across the two temperature levels and temperature (25° c vs 29° c) comparison across all four species of algae fed to them. There existed no temperature effect between 25° C and 29° C. 38
Table 13 The ANOVA main table of time dependent covariance analysis of a factorial experiment (Algae x Temperature, 4 x 2) on the length of pleopod (daily documented) for 25 days of feeding each of different algae species, all at a density of 2x105cells/ml and a salinity at 20‰. 39
Table 14. The length of pleopod of Amano shrimp between the four algae species at high temperature analyzed through a factorial experiment (algae x temperature, 4 x 1) after 25 days of culture with all survivors entering into metamorphosed juvenile stage at salinity 20‰, algae density 2x105cells/ml. 40
Table 15. The length of pleopod of Amano shrimp between the four algae species at low temperature analyzed through a factorial experiment (algae x temperature, 4 x 1) after 25 days of culture with all survivors entering into metamorphosed juvenile stage at salinity 20‰, algae density 2x105cells/ml. 41
Table 16 Orthogonal contrasts of the LS means of pleopod of Amano shrimp larvae on the four algae fed to them across the two temperature levels and temperature (25° c vs 29° c) comparison across all four species of algae fed to them. There existed no temperature effect between 25° C and 29° C. 42


LIST OF FIGURES
Figure 1. Scale up facility for microalgae 43
Figure 2. New released pouched larvae at zoea stage I (Day 0) 44
Figure 3. Larvae at zoea stage (day 1) by feeding Isochrysis galbana at 2x105cells/ml algae density and salinity at 20‰ at high temperature
45
Figure 4. Larvae at mysis stage (day 7) by feeding Tetraselmis chui at 2x105cells/ml algae density and salinity at 20‰ at high temperature
46
Figure 5. Larvae at post-larvae stage (day 12) by feeding Thalassiorales weissflogii at 2x105cells/ml algae density and salinity at 20‰ at high temperature
47
Figure 6. Larvae at juvenile stage (day 19) by feeding Tetraselmis chui at 2x105cells/ml algae density and salinity at 20‰ at low temperature
48
Figure 7. Time related growth line on the body length of Amano shrimp larvae analyzed through a factorial experiment (algae x temperature, 4 x 2) by feeding each of the respective algae density at 2x105cells/ml and salinity at 20‰ during 25 day-culture trials, the experiment terminated when all survivors entering into metamorphosed juvenile stage. 49
Figure 8. Time related line on the length of pereiopod of Amano shrimp larvae analyzed through a factorial experiment (algae x temperature, 4 x 2) by feeding each of the respective algae density at 2x105cells/ml and salinity at 20‰ during 25 day-culture trials, the experiment terminated when all survivors entering into metamorphosed juvenile stage.. 50
Figure 9. Time related line on the length of pleopod of Amano shrimp larvae analyzed through a factorial experiment (algae x temperature, 4 x 2) by feeding each of the respective algae density at 2x105cells/ml and salinity at 20‰ during 25 day-culture trials, the experiment terminated when all survivors entering into metamorphosed juvenile stage. 51

7. REFERENCE
Andersen R.A. 2005. Algal Culturing Techniques. First edition, Academic Press. p596.
Anger, K. 1998. “Patterns of growth and chemical composition of decapod crustacean larvae”. Invertebrate Reproduction and Development, 33: 159.-76.
Bardach, J.E., J.H. Ryther, and W.O. McLarney. 1972. “The farming and husbandry of freshwater and marine organisms”. Wiley-Interscience, 686 pp.
Bauer, R. T. 2011a. “Amphidromy and migrations of freshwater shrimps. I. Costs, benefits, evolutionary origins, and an unusual case of amphidromy,” New Frontiers in Crustacean Biology, pp. 145-156.
Becker, W. 2004. “The nutritional value of microalgae for aquaculture.” Handbook of Micro algal Culture, pp. 380–391.
Becker, B., Marin, B. and M. Melkonian, 1994: Structure, composition, and biogenesis of prasinophyte cell coverings. Protoplasma, 181: 233-244.
Belay A., T. Kato, and Y. Ota. 1996. “Spirulina (Arthrospira): potential application as an animal feed supplement”. Journal of Applied Phycology; 8:303–311.
Benemann, J.R. B. Koopman, J. Weissman, D. Eisenberg, and R. Goebel. 1980. “Development of microalgae harvesting and high-rate pond technologies in California”. Elsevier/North-Holland Biomedical Press, 457-495.
Borowitzka, M. A. 2005. “Culturing microalgae in outdoor ponds”. Algal Culturing Techniques, 205‐218.
Byrne, M., R. Przeslawski, 2013. “Multistressor studies of the impacts of warming and acidification of the ocean on marine invertebrates, life histories.” Integrative and Comparative Biology, 53, 582-596.
Cai, Y., S. Shokita, and K. Satake. 1860. “On the species of Japanese atyid shrimps (Decapoda: Caridea),” Journal of Crustacean Biology, 26(3); 392-419.
Calado, R., L. Narciso, S. Morais, A.L. Rhyne, J. Lin. 2003. “A rearing system for the culture of ornamental decapod crustacean larvae,” Aquaculture (18) 329-339.
Chase, F. A. 1997. “The caridean shrimps (Crustacea: Decapoda) of the Albatross,” Smithsonian Contributions to Zoology, 587: 1-96.
Cohen, J., T. M. Samocha, J. M. Fox, and A. L. Lawrence. 2005. “Characterization of water quality factors during intensive raceway production of juvenile Litopenaeus vannamei using limited discharge and biosecure management tools.” Aquacultural engineering, 32:425–442.
De Rivera, C.E., G.M. Ruiz, N.G. Hitchcock, S.A. Teck, B. Steves, and A.H. Hines. 2007. “Larval development rate predicts range expansion of an introduced crab.” Marine Biology, 150:1275-1288.
FAO, 2004 “The hatchery culture of bivalves: a practical manual.” Fisheries and Aquaculture Department. p. 201.
FAO, 2005. “The numbers represent the average unit value of imports for 1994–2003,” Fishery Statistics, 83–97.
FAO, 2010. “The ornamental fish trade. Production and commerce of ornamental fish: technical-managerial and legislative aspects.” Globefish Research Programme, volumes 102, p. 134
Hayashi, K. I. and T. Hamano. 1984. “The complete larval development of Caridina japonica De Man (Decapoda, Caridea, Atyidae) reared in the laboratory”. Zoological Science, 1:571–589.
Heerbrandt, T. C. and J. Lin. 2006. “Larviculture, of Red Front Shrimp, Caridina gracilirostris (Atyidae, Decapoda),” Journal of the World Aquaculture Society, 37(2), 186-190.
Hu, Q., M. Sommerfeld, E.Jarvis, M Ghirardi, M. Posewitz, M. Seibert, and A. Darzins, 2008. “Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances”, The Plant Journal, 54, 621–639.
Jamali, H., N. Ahmadifard, and D. Abdollahi. 2015. “Evaluation of growth, survival and body composition of larval white shrimp (Litopenaeus vannamei) fed the combination of three types of algae.” International Aquaculture, 7：115-122.
Johansen, J.R and S.R. Rushforth. 1985. “A contribution to the taxonomy of Chaetoceros muelleri Lemmermann (Bacillariophyceae) and related taxanomy,” Phycologia, Vol. 24, No. 4, pp. 437-447.
Kiatmetha, P., W. Siangdang, B. Bunnag, S. Senapin, and B. Withyachumnarnkul. 2011. “Enhancement of survival and metamorphosis rates of Penaeus monodon larvae by feeding with the diatom Thalassiosira weissflogii.” Aquaculture International. 19(4): 599-609.
Kim, S., M. Pan, and H. Cheol. 2012. "Fucoxanthin as a major carotenoid in Isochrysis aff. galbana: Characterization of extraction for commercial application". Journal of the Korean Society for Applied Biological Chemistry, 55:477-483.
Kumlu, M., O.T. Eroldogan, M. Aktas. “Effects of temperature and salinity on larval growth, survival and development of Penaeus semisulcatus,” Aquaculture, 188 2000 167–173.
Laramore, S.; C. R. Laramore, and J. Scarpa. 2001. “Effect of low salinity on growth and survival of postlarvae and juvenile Litopenaeus vannamei”. Journal of the World Aquaculture Society, 32:385-392.
Li, E.C., L.Q. Chen, C. Zeng, X.M. Chen, N. Yu, Q.M. Lai, and J.G. Qin. 2007. “Growth, body composition, respiration and ambient ammonia nitrogen tolerance of the juvenile white shrimp, Litopenaeus vannamei, at different salinities.” Aquaculture, 265 (2007), pp. 385-390.
Liao, I.C., H.M. Su, J.H. Lin. 1993. “Larval foods for Penaeid prawns”. In: McVey, J. P. CRC Handbook of Mariculture: Crustacean Aquaculture 2nd ed. Vol.1, Crustacean Aquaculture. 29-59.
Lin, J., D. Zhang, and A. L. Rhyne. 2002. “Broodstock and larval nutrition of marine ornamental shrimp”. Avances en Nutrición Acuícola VI. Memorias del VI Simposium Internacional de Nutrición Acuícola, pp. 277-280.
Lober, M. and C. Zeng. 2009. “Effect of microalgae concentration on larval survival, development and growth of an Australian strain of giant freshwater prawn Macrobrachium rosenbergii”. Aquaculture, 289: 95-100.
Mustafa, M.G. and Y.H. Nakagawa. 1995. “A review: dietary benefits of algae as an additive in fish feed”. Israeli Journal of Aquaculture, 47, 155–162.
Nakahara, Y., A. Hagiwara, Y. Miya, and K. Hirayama. 2005. “Larval rearing of three amphidromous shrimp species (Atyidae) under different feeding and salinity,” Aquaculture Science, 53: 305-310.
Napolitano, G.E., R. G. Ackman, W.M.N. Ratnayake. 1990. “Fatty acid composition of three cultured algal species (Isochvysis galbana, Chaetoceros gracilis and Chaetoceros calcitrans) used as food for bivalve larvae,” Journal of the World Aquaculture Society, 21: 122–130.
Nhan, D. T., M. Wille, L. T. Hung, and P. Sorgeloos. 2010. Effects of larval stocking density and feeding regime on larval rearing of giant freshwater prawn (Macrobrachium rosenbergii). Aquaculture. 300(1): 80-86.
Nelson, S. G., D. A. Armstrong, A. W. Knight, and H. W. Li. 1977. “The effect of temperature and salinity on the metabolic rate of juvenile Macrobrachium rosenbergii (Crustacea: Palaemonidae)”, Composition of Biochemistry Physiology, 56: 533-537.
New, M.B., 2000. “History and global status of freshwater prawn farming. In: New, M.B., Valenti, W.C. (Eds.), Freshwater Prawn Culture, The Farming of Macrobrachium rosenbergii.”, pp. 1 –11.
Norris, R.E., T. Hori, and M. Chihara. 1980. “Revision of the Genus Tetraselmis (Class Prasinophyceae),” Botanical Magasine. 93: 317-339.
O'Brien, C.J. 1994. The effects of temperature and salinity on growth and survival of juvenile tiger prawn Penaeus esculentus,” Journal of Experimental Marine Biology and Ecology, 183, pp. 133-145.
Okauchi M, K. Kawamura, and Y. Mizukami. 1997. “Nutritive value of ‘Tahiti Isochrysis’ Isochrysis sp. for larval greasy back shrimp, Metapenaeus ensis. Bulletin of National Research Institute of Aquaculture, 30: 1–11.
Olvera-Novoa, M.A., L.J. Dominguez-Cen, L. Olivera-Castillo, and C.A. Martínez-Palacios. 1998. “Effect of the use of the microalgae Spirulina maxima as fishmeal replacement in diets for tilapia, Oreochromis mossambicus,” Aquaculture research, 29: 709-715.
Patnaik S., T.M. Samocha, D.A. Davis, R.A. Bullis, and C.L. Browdy. “The use of HUFA rich algal meals in diets for Litopenaeus vannamei,” Aquaculture Nutrition, 12 (2006), pp. 395-401.
Ponce-Palafox, J., C.A. Martinez-Palacios, and L.G. Ross. 1997. “The effects of salinity and temperature on the growth and survival rates of juvenile white shrimp, Penaeus vannamei”, Boone, 1931. Aquaculture, 157: 107–115.
Quillfeldt, C.H.V. 2001. “Identification of Some Easily Confused Common Diatom Species in Arctic Spring Blooms.” Botanica Marina, Vol. 44: 375–389.
Regunathan, C., and S.G. Wesley. 2006. “Pigment deficiency correction in shrimp broodstock using Spirulina as a carotenoid source”. Aquaculture Nutrition 12(6): 425–432.
Rines, J.E.B., P. Boonruang, and E.C. Theriot. 2000. “Chaetoceros phuketensis sp. nov. (Bacillariophyceae): a new species from the Andaman Sea.” Phycological Research, 48 (3): 161–168.
Samocha, T.M., A.L. Lawrence and D. Pooser. 1998. “Growth and survival of juvenile Penaeus vannamei in low salinity water in a semi-closed recirculating system”. Israeli Journal of Aquaculture- Bamidgeh 50: 55–59.
Sanford, E., S.B. Holzman, R.A. Haney, D.M. Rand, and M.D. Bertness. 2006. “Larval tolerance, gene flow, and the northern geographic range limit of fiddler crabs.” Ecology, 87(11):2882–2894.
Shih, H.T., Y. Cai. 2007. “Two new species of the land-locked freshwater shrimps Genus, Neocaridina Kubo, 1938 (Decapoda: Caridea: Atyidae), from Taiwan, with notes on speciation on the island”. Zoological Studies, 46(6) ：680-694.
Takeuchi, T., J. Lu, G. Yoshizaki, and S. Satoh. 2002. “Effect on the growth and body composition of juvenile tilapia Oreochromis niloticus fed raw Spirulina”. Fisheries Science, 68, 34–40.
Thornton, T. and S.S. Lessen. 1978. “A temperature algorithm for modifying biological rate”. Transatlantic American Fishery Society, 107: 284-294.
Tobias-Quinitio, E., and C. T. Villegas. 1982. “Growth, survival and macronutrient composition of Penaeus monodon Fabricius larvae fed with Chaetoceros calcitrans and Tetraselmis chui”. Aquaculture, 29(3-4): 253-260.
Tomas, C. R., G. R Hasle., E. E. Syvertsen, K. A. Steidinger, K. Tangen, J. Throndsen, and B. R. Heimdal. 1997. Identifying Marine Phytoplankton, pp. 858.
Vrieling, E., L. Poort, T. Beelen, and W. Gieskes. “Growth and silica content of the diatoms Thalassiosira weissflogii and Navicula salinarum at different salinities and enrichments with aluminium,” European Journal of Phycology, vol. 34, no 3, ‎ 1999, p. 307-316.
Walne, P. R. 1974. “Culture of bivalve mollusks: 50 years' experience” at Conway Fishing News Books Ltd. Surrey, pp. 173.
Webb, K. L. and F.L. Chu. 1982. “Phytoplankton as a food source for bivalve larvae”, Biochemical and Physiological Approaches to Shellfish Nutrition. Wild Mariculture Society, 272-291.
Wilson, J.H. 1979. “Observations on the grazing rates and growth of Ostrea edulis L. larvae when fed algal cultures of different ages. Journal of Experimental Mariculture Biological Ecology, 38: 187-199.
Wujek, D.E. and M. Graebner. 1980. “A new freshwater species of Chaetoceros from the Great Lakes region.” Journal of Great Lakes Research, 6(3): 260-262.
Yen, P.T. and A.N. Bart. 2008. “Salinity effects on reproduction of giant freshwater prawn Macrobrachium rosenbergii (de Man)”. Aquaculture, 280：124-128.