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Volume 39 Issue 3
Apr.  2020
Article Contents

Citation:

Trophic upgrading of essential fatty acids by Oxyrrhis marina

  • Received Date: 2019-12-27
    Accepted Date: 2020-01-15
  • Heterotrophic dinoflagellates serve as an intermediate bridge connecting zooplankton and phytoplankton, and play a key role in material recycling and energy flowing in marine ecosystems.In this paper, fatty acid of heterotrophic dinoflagellate Oxyrrhis marina was analyzed after feeding on four phytoplankton, including Dunaliella tertiolecta, Heterosigma akashiwo, Phaeocystis globosa, and Navicula salinarum.Research shows that O.marina can feed on four kinds of phytoplankton, but ingestion rate on D.tertiolecta was higher than those on other three species.DHA (docosahexaenoic acid) was not observed in all prey, but found in the O.marina fed on them with the content up to (86.98±7.44) μg/mg C.These results indicate that O.marina can synthesize DHA independently, which means that O.marina had the ability to upgrade the nutritional value of phytoplankton, thereby efficiently delivery docosahexaenoic acid to the next trophic level.
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  • [1] LEE W J, PATTERSON D J.Diversity and geographic distribution of free-living heterotrophic flagellates-analysis by PRIMER[J].Protist, 1998, 149(3):229-244. doi: 10.1016/S1434-4610(98)70031-8
    [2] CORLISS J O, PATTERSON D J, LARSEN J.The biology of free-living heterotrophic flagellates[J].Transactions of the American Microscopical Society, 1992, 111(3):267-268. doi: 10.2307/3226616
    [3] WEISSE T, MÜLLER H.Planktonic protozoa and the microbial food web in Lake Constance[J].Lake Constance:Characterization of an Ecosystem in Transition, 1998, 53:223-254.
    [4] GIEBELHAUSEN B, LAMPERT W.Temperature reaction norms of Daphnia magna:the effect of food concentration[J].Freshwater Biology, 2001, 46(3):281-289. doi: 10.1046/j.1365-2427.2001.00630.x
    [5] WEISSE T, STADLER P, LINDSTRÖM E S, et al.Interactive effect of temperature and food concentration on growth rate:a test case using the small freshwater ciliate Urotricha farcta[J].Limnology and Oceanography, 2002, 47(5):1447-1455. doi: 10.4319/lo.2002.47.5.1447
    [6] BRETELER W C M K, SCHOGT N, BAAS M, et al.Trophic upgrading of food quality by protozoans enhancing copepod growth:role of essential lipids[J].Marine Biology, 1999, 135(1):191-198. doi: 10.1007/s002270050616
    [7] MÜLLER-NAVARRA D C, BRETT M T, PARK S, et al.Unsaturated fatty acid content in seston and tropho-dynamic coupling in lakes[J].Nature, 2004, 427(6969):69-72. doi: 10.1038/nature02210
    [8] KOSKI M, BRETELER W K, SCHOGT N.Effect of food quality on rate of growth and development of the pelagic copepodPseudocalanus elongatus (Copepoda, Calanoida)[J].Marine Ecology Progress Series, 1998, 170:169-187. doi: 10.3354/meps170169
    [9] TANG K W, JAKOBSEN H H, VISSER A W.Phaeocystis globosa (Prymnesiophyceae) and the planktonic food web:feeding, growth, and trophic interactions among grazers[J].Limnology and Oceanography, 2001, 46(8):1860-1870. doi: 10.4319/lo.2001.46.8.1860
    [10] LACOSTE A, POULET S A, CUEFF A, et al.New evidence of the copepod maternal food effects on reproduction[J].Journal of Experimental Marine Biology and Ecology, 2001, 259(1):85-107. doi: 10.1016/S0022-0981(01)00224-6
    [11] BROGLIO E, JÓNASDÓTTIR S H, CALBET A, et al.Effect of heterotrophic versus autotrophic food on feeding and reproduction of the calanoid copepodAcartia tonsa:relationship with prey fatty acid composition[J].Aquatic Microbial Ecology, 2003, 31(3):267-278.
    [12] GUILLARD R R L.Culture of phytoplankton for feeding marine invertebrates[M]//SMITH W L, CHANLEY M H.Culture of marine Invertebrate Animals.Boston, MA: Springer, 1975: 29-60.
    [13] O'HALLORAN C, SILVER M W, HOLMAN T R, et al.Heterosigma akashiwo in central California waters[J].Harmful Algae, 2006, 5(2):124-132. doi: 10.1016/j.hal.2005.06.009
    [14] ROUSSEAU V, MATHOT S, LANCELOT C.Calculating carbon biomass of Phaeocystis sp.from microscopic observations[J].Marine Biology, 1990, 107(2):305-314. doi: 10.1007/BF01319830
    [15] ADMIRAAL W, BOUWMAN L A, HOEKSTRA L, et al.Qualitative and quantitative interactions between microphytobenthos and herbivorous meiofauna on a brackish intertidal mudflat[J].Internationale Revue der gesamten Hydrobiologie und Hydrographie, 1983, 68(2):175-191. doi: 10.1002/iroh.19830680203
    [16] DODGE J D, CRAWFORD R M.Fine structure of the dinoflagellateOxyrrhis marina II.The flagellar system[J].Protistologica, 1971, 7:399-409.
    [17] FROST B W.Effects of size and concentration of food particles on the feeding behavior of the marine planktonic copepod calanus pacificus[J].Limnology and Oceanography, 1972, 17(6):805-815. doi: 10.4319/lo.1972.17.6.0805
    [18] FENCHEL T.Protozoan filter feeding[J].Progress in Protistology, 1986, 1:65-113.
    [19] HANSEN P J.Prey size selection, feeding rates and growth dynamics of heterotrophic dinoflagellates with special emphasis on Gyrodinium spirale[J].Marine Biology, 1992, 114(2):327-334. doi: 10.1007/BF00349535
    [20] TARRAN G A.Aspects of grazing behaviour of the marine dinoflagellateOxyrrhis marina, Dujardin[D].Soton: University of Southampton, 1991.
    [21] VELOZA A J, CHU F L E, TANG K W.Trophic modification of essential fatty acids by heterotrophic protists and its effects on the fatty acid composition of the copepodAcartia tonsa[J].Marine Biology, 2006, 148(4):779-788. doi: 10.1007/s00227-005-0123-1
    [22] SARGENT J R, WHITTLE K J.Lipids and hydrocarbons in the marine food web[M]//LONGHURST A R.Analysis of Marine Ecosystems.London: Academic Press, 1981: 491-533.
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Trophic upgrading of essential fatty acids by Oxyrrhis marina

  • Research Center for Harmful Algal and Marine Biology, Jinan University, Guangzhou 510632, China

Abstract: Heterotrophic dinoflagellates serve as an intermediate bridge connecting zooplankton and phytoplankton, and play a key role in material recycling and energy flowing in marine ecosystems.In this paper, fatty acid of heterotrophic dinoflagellate Oxyrrhis marina was analyzed after feeding on four phytoplankton, including Dunaliella tertiolecta, Heterosigma akashiwo, Phaeocystis globosa, and Navicula salinarum.Research shows that O.marina can feed on four kinds of phytoplankton, but ingestion rate on D.tertiolecta was higher than those on other three species.DHA (docosahexaenoic acid) was not observed in all prey, but found in the O.marina fed on them with the content up to (86.98±7.44) μg/mg C.These results indicate that O.marina can synthesize DHA independently, which means that O.marina had the ability to upgrade the nutritional value of phytoplankton, thereby efficiently delivery docosahexaenoic acid to the next trophic level.

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  • 异养甲藻(heterotrophic dinoflagellates)是一类以异养方式从外界获取物质和能量的原生生物,种类繁多,分布广泛[1]。细胞表面存在鞭毛,依靠鞭毛的运动进行运动和摄食[2]。海洋中异养甲藻摄食范围较为广泛[3],主要摄食海洋细菌和微型浮游植物,甚至能够摄食同自身粒径相近的食物。异养甲藻摄食对海洋细菌以及浮游植物的数量、结构和空间分布均有显著的影响。异养甲藻本身则被桡足类等中型浮游动物摄食,从而将物质和能量传递到更高营养级生物[4-5],成为连接微生物环和经典食物链的中枢环节。

    长链ω3多不饱和脂肪酸(long-chain n-3 polyunsaturated fatty acids, LCn-3PUFA),尤其是二十碳五烯酸(eicosapentaenoic acid, EPA; 20:5ω3)和二十二碳六烯酸(docosahexaenoic acid, DHA; 22:6ω3)是影响浮游动物生殖发育的重要因素[6-9]。然而浮游动物自身无法合成这类必需脂肪酸,必须从食物中获取。由于很多浮游植物往往缺乏该类必需脂肪酸,当桡足类摄食这些浮游植物时则会导致其生殖效率下降。桡足类在摄食缺乏EPA和DHA的杜氏藻(Dunaliella tertiolecta)和球形棕囊藻(Phaeocystis globosa)后显示出较低的产卵率、卵孵化率和较高的幼体死亡率[8-10]。但是在摄食异养甲藻后,桡足类的产卵率和卵的孵化率显著提高[9, 11],更多无节幼体发育为成体[6]。这表明异养甲藻细胞可能含有浮游植物体内缺乏的必需脂肪酸,从而更好的支持了浮游动物的生殖发育。但是异养甲藻同样摄食浮游植物,也会面临食物中脂肪酸缺乏等问题,那么异养甲藻的必需脂肪酸从何而来?异养甲藻是否具有独立合成必需脂肪酸的能力?

    本文选择海洋尖尾藻(Oxyrrhis marina)作为异养甲藻的代表,摄食不同种类浮游植物,研究海洋尖尾藻是否具有合成必需脂肪酸的功能。本研究有助于进一步了解异养甲藻对浮游动物种群结构和数量的影响。

1.   材料与方法

    1.1.   材料

  • 选取土生杜氏藻(Dunaliella tertiolecta)、赤潮异弯藻(Heterosigma akashiwo)、球形棕囊藻(Phaeocystis globosa)和盐生舟形藻(Navicula salinarum)作为海洋尖尾藻摄食的4种微型浮游植物(表 1),藻种均培养于暨南大学赤潮与海洋生物学研究中心。使用f/2培养基培养,培养温度20 ℃,盐度30,光照强度100 μmol photons/(m2·s),光循环12 h: 12 h。

    Table 1.  The equivalent spherical diameter, carbon content of each cell and collection site of phytoplankton

  • 1.2.   实验方法

  • 土生杜氏藻、赤潮异弯藻、盐生舟形藻和球形棕囊藻半连续培养在1 L的锥形瓶中,培养体积为500 mL,设置3个平行样,每天计数到达稳定期后取样测定脂肪酸种类及含量。

    海洋尖尾藻培养在1 L的锥形瓶中,培养体积为500 mL,设置3个平行样。在f/2培养基中半连续培养并分别投喂上述藻类,黑暗条件下培养24 h,计数24 h前后尖尾藻和藻类细胞浓度,海洋尖尾藻的摄食率的计算公式为[17]

    式中:I为海洋尖尾藻摄食率; [C]为实验过程中浮游植物的平均浓度; F为海洋尖尾藻的清滤率。

    清滤率的计算公式为:

    式中:cp为尖尾藻最终浓度;c0ct分别为藻类初始和最终浓度;t为培养时间。

    当尖尾藻培养到稳定期后,停止投喂食物继续黑暗培养,保证食物被尖尾藻完全摄食后收取样品,储存在-80 ℃直到脂质分析。

    采用乙酰氯-甲醇法提取样品中的脂肪酸,将样品加入1 mg/mL C17脂肪酸20 μL作为内标后加入2 mL w(NaOH/MeOH)=2.5%溶液,80 ℃水浴15 min,加入2 mL φ(AcCL/MeOH)=10%溶液,继续水浴15 min,室温冷却后加入1 mL w(K2CO3)=6%溶液,最后加入1 mL n-Hexane(正己烷),混匀后静置分层,取上清液N2吹干,利用GCMS(Agilent 7890B/5977 GCMSD)检测。

    以52种脂肪酸甲脂作为外标,C17脂肪酸作为内标,定性定量最终样品中的脂肪酸种类和含量。

  • 1.3.   统计分析方法

  • 应用Origin 2017软件对数据进行统计分析,利用ONE WAY-ANOVA检验摄食率和脂肪酸浓度的差异,显著性水平设置为P < 0.05。

2.   结果与讨论

    2.1.   结果

    2.1.1.   海洋尖尾藻的摄食率
  • 海洋尖尾藻对四种浮游植物均可摄食(图 1),其中海洋尖尾藻对杜氏藻的摄食率达到(24.64±3.44)pg C/(cell·d),明显高于其他3种浮游植物。当以球形棕囊藻为食物时,异养甲藻出现最低的摄食率,仅为(3.08±0.57)pg C/(cell·d)。

    Figure 1.  The ingestion rate of Oxyrrhis marina feeding on different phytoplankton

  • 2.1.2.   浮游植物的脂肪酸
  • 4种浮游植物细胞内均含有较高浓度的C16:0饱和脂肪酸(表 2),其中舟形藻的C16:0浓度最高,达到(83.98±12.95)μg/mg C。除杜氏藻之外,其他3种浮游植物中均检测出C14:0。球形棕囊藻的C14:0最为丰富,高达(87.57±3.37)μg/mg C。杜氏藻和赤潮异弯藻的单不饱和脂肪酸含量较少,而球形棕囊藻中含有较高浓度的单不饱和脂肪酸C18:1,浓度达到(35.91±3.21)μg/mg C。舟形藻中则含有最高浓度的C16:1,浓度为(112.34±2.73)μg/mg C。球形棕囊藻中未检测出任何多不饱和脂肪酸,杜氏藻中检测出C18:2ω6、C18:3ω6和C18:3ω3,C18:3ω3浓度最高,为(35.89±3.48) μg/mg C。赤潮异弯藻和舟形藻中均检测出了二十碳五烯酸(EPA),浓度分别为(20.67±2.11) μg/mg C和(31.77±5.45) μg/mg C。但是4种浮游植物中均未检出二十二碳六烯酸(DHA)。

    Table 2.  The fatty acid composition and concentration of different phytoplankton

  • 2.1.3.   海洋尖尾藻的脂肪酸
  • 海洋尖尾藻检出了C14:0和C16:0两种饱和脂肪酸,C16:0浓度较高。摄食杜氏藻和舟形藻后,尖尾藻细胞内的C16:0浓度分别达到了(87.37±7.10) μg/mg C和(161.51±22.45) μg/mg C。摄食舟形藻和球形棕囊藻后海洋尖尾藻中检测出单不饱和脂肪酸C16:1和C18:1以及C24:1。EPA仅在摄食球形棕囊藻的尖尾藻细胞内检出,摄食其余藻类并未检出。尖尾藻在分别摄食4种浮游植物后均产生了DHA,浓度范围为(18.47±2.50)~(86.98±7.44)μg/mg C。摄食杜氏藻的海洋尖尾藻C18:3ω3和DHA含量均为最高,分别达到(51.46±4.20) μg/mg C和(86.98±7.44)μg/mg C。

    Table 3.  The fatty acid composition and concentration of Oxyrrhis marina diet of different kinds of food

  • 2.2.   讨论

  • 影响摄食者摄食率的因素有很多,食物粒径是关键因素之一[18-20]。海洋尖尾藻对于4种浮游植物均可摄食(图 1),但海洋尖尾藻明显偏好摄食舟形藻,杜氏藻和赤潮异弯藻,它们的粒径分别为7.25,7.40和16.00 μm,摄食率分别达到了(11.63±0.16)pg C/(cell·d),(24.64±3.44)pg C/(cell·d)和(10.67±0.57) pg C/(cell·d),显著高于粒径为5 μm的球形棕囊藻。食物粒径的大小与其碳含量是相关的,粒径小的细胞碳含量相对较小,为了最大化获得自身所需的能量,摄食者会选择在可摄食的范围内优先选择粒径相对较大的食物,本研究中尖尾藻偏好摄食的藻类粒径在7.25~16 μm,处于Hansen所建议的异养甲藻食物的粒径谱范围之间[19],而球形棕囊藻粒径仅有5 μm,接近异养甲藻的摄食粒径范围最低值,导致了尖尾藻对其的低摄食率。

    值得注意的是,本研究中4种浮游植物都缺乏DHA,但摄食了4种浮游植物的海洋尖尾藻均含有DHA,其中摄食了杜氏藻的尖尾藻细胞内DHA含量更是高达(86.98±7.44)μg/mg C,说明海洋尖尾藻能够自主合成DHA,这些结果与Veloza等人报道的异养甲藻多米尼环沟藻(Gyrodinium dominans)可以自主产生DHA的结果一致[21]。单细胞合成DHA的途径可能为:(1)细胞从头合成;(2)修饰细胞获得的长链ω3多不饱和脂肪酸前体。细胞利用三羧酸循环产生的乙酰辅酶A延伸形成硬脂酸(C18:0),再利用不同的脂肪酸延长酶和脱氢酶,交替进行碳链的延伸和去饱和反应,最终合成长链多不饱和脂肪酸,例如DHA。异养甲藻也可能利用已合成或者从食物中获取的C18:0,进行脱氢和延长合成长链多不饱和脂肪酸,该途径效率甚至高于第一种[22]。本研究无法完全证实第一种途径,但是能够证实异养甲藻可利用脱氢酶和延长酶将短链脂肪酸转化成长链脂肪酸。第一,尖尾藻摄食球形棕囊藻后,检测出C24:1,而球形棕囊藻中并未含有C24:1,说明尖尾藻必然能利用延长酶将短链脂肪酸合成长链脂肪酸。第二,尖尾藻中EPA含量显著低于所摄食的食物中EPA,这表明尖尾藻可能会利用EPA通过延伸和去饱和交替反应合成DHA。第三,浮游植物和异养甲藻中均含有C18脂肪酸,尖尾藻可通过延伸和去饱和反应合成C18:3ω3,进而合成DHA[22]

    很多研究表明当分别以缺乏必需脂肪酸的浮游植物和异养甲藻为食物时,摄食异养甲藻后的桡足类会表现出更高的产卵率、卵孵化率以及幼体成活率,甚至加快了幼体至成体的生长[6, 19]。这些研究均证实了多不饱和脂肪酸在桡足类生殖发育过程的功能,异养甲藻独立合成DHA的能力避免了浮游动物必需脂肪酸的营养限制问题,有助于提高浮游动物的生殖效率。异养甲藻提高了浮游动物获取的食物质量,更进一步显示出异养甲藻在食物链的有机碳传递中具有的重要作用。

3.   结论
  • 海洋尖尾藻对杜氏藻、赤潮异弯藻、舟形藻和球形棕囊藻4种浮游植物均可摄食,粒径不同会导致摄食率不同。4种浮游植物均不含有DHA,海洋尖尾藻在摄食4种浮游植物后均检测出DHA,表明海洋尖尾藻具有独立合成DHA的能力。海洋尖尾藻这种独立合成脂肪酸的能力实现了对低质量食物的营养升级。

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