Plasmodium Sporozoite Motility on Flat Substrates

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PLOS Pathogens
Jul 2016



Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro.

Keywords: Plasmodium (疟原虫), Plasmodium berghei (伯氏疟原虫), Sporozoites (孢子体), Salivary gland isolation (唾液腺分离), Dissection (解剖), Gliding motility (滑翔运动), Malaria (疟疾), Mosquitoes (蚊), Anopheles (疟蚊)


Sporozoites of the phylum Plasmodium, the causative agent of malaria, are transmitted into the skin of their vertebrate host through the bite of an infectious mosquito. Sporozoite motility is a key prerequisite for parasite transmission and successful infection of the vertebrate host. Motility constitutes the first parasite mechanism that can be inhibited and is thus of interest for intervention strategies. Genetic modifications affecting gliding motility or motility modulating compounds can be readily investigated using 2D in vitro assays.

Part I: Isolation of salivary gland sporozoites

Materials and Reagents

  1. 15 ml conical centrifugation tube
  2. Two 10 ml Petri dishes
  3. Two 27 G needles
  4. Two 1 ml syringes
  5. Glass slide
  6. 1.5 ml plastic reaction tube
  7. Disposable polypropylene pestles (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F19923-0001 )
  8. Mosquitoes
  9. Ice
  10. RPMI (Thermo Fisher Scientific, GibcoTM, catalog number: 11835063 )
  11. 70% ethanol
  12. 1x PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 18912014 )
  13. Bovine serum albumin (BSA) (Carl Roth, catalog number: 8076 )
  14. BSA/RPMI 3% (see Recipes)


  1. Vacuum pump
  2. Styrofoam box
  3. Forceps
  4. Micropipette with disposable tips
  5. Binocular microscope (ZEISS, model: Stemi 305 or a comparable binocular microscope from any other manufacturer e.g., Nikon, Olympus, Leica)
  6. Light microscope with phase contrast 40x objective (ZEISS, model: Axio Lab.A1 or a similar device from any other manufacturer e.g., Nikon, Olympus, Leica)
  7. Hemocytometer


  1. Aspirate mosquitoes with the aid of a vacuum pump into a 15 ml conical centrifugation tube and place it on ice for five minutes in order to tranquilise the mosquitoes.
  2. Prepare one reaction tube with 50-100 µl of RPMI for collection of the salivary glands. Place it on ice.
  3. Fill one 10 ml Petri dish with 70% ethanol and a second Petri dish with 1x PBS.
  4. Attach the 27 G needles to the syringes.
  5. Transfer the cooled and therefore immobilised mosquitoes into the Petri dish filled with 70% ethanol for up to 1 min. This will on the one hand reduce bacteria contamination and on the other hand reduce their hydrophobicity. Afterwards, use forceps to transfer the mosquitoes into the Petri dish with 1x PBS.
  6. For isolation of the salivary glands, place a female mosquito in a drop of 1x PBS on a glass slide under the binocular microscope. Gently immobilise the thorax of the mosquito with the help of one needle (needle 1), while the other needle (needle 2) is placed at the intersection of head and thorax (Figure 1).

    Figure 1. Scheme of a female mosquito with placement of needles and their movement indicated

    Slowly, but firmly, pull the head apart from the body of the mosquito. Ideally, the salivary glands should stay connected to the head and are easily distinguished from the other tissue by their shining appearance (Figure 2).

    Figure 2. Salivary glands of a female Anopheles mosquito. The glands on the left are still attached to the head (large dark object at lower left). Scale bar = 50 µm.

    While fixating the head, cut off the salivary glands and place them into the prepared 1.5 ml reaction tube. Proceed with the remaining mosquitoes and collect all isolated salivary glands in the same reaction tube.
  7. Use the disposable polypropylene pestle to disrupt the tissue in the reaction tube and hence free the sporozoites. Grind gently for approximately 2 min until the solution is homogenous.
  8. To determine the total number of sporozoites, make 20 µl of a 1:10 dilution in RPMI. Alternatively, set up the 1:10 dilution with activated sporozoites by using RPMI supplemented with 3% BSA. (see Note 1)
  9. Transfer 10 µl of the dilution into a hemocytometer and allow the sporozoites to settle for 10 min at room temperature. Store the remaining sporozoites on ice.
  10. Count the number of sporozoites in four big counting squares using a light microscope with phase contrast 40x objective (Figure 3).

    Figure 3. Neubauer hemocytometer with schematic sporozoites in the four big squares marked in red, which are counted

  11. Calculate the total number of sporozoites using following equation:

    Ntotal= ([Ncounted/4] x 10 [dilution factor] x 10 [chamber factor]) x sporozoite solution (µl)

    In addition, the number of sporozoites per salivary gland can be determined by dividing Ntotal by the amount of dissected female mosquitoes.

Part II: Motility assay

Materials and Reagents

  1. 15 ml conical centrifugation tube
  2. 1.5 ml reaction tubes
  3. 96-well or 384-well optical flat bottom plate (NuncTM MicroWellTM 96 well plates [Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 267342 ]; NuncTM 384 well plates [Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 240074 ])
  4. Latex bulb
  5. Isolated Plasmodium sporozoites in a reaction tube (see Part I) on ice
  6. Incomplete RPMI (Thermo Fisher Scientific, GibcoTM, catalog number: 11835063 )
  7. 17% w/v solution of Accudenz (Accurate Chemical & Scientific, catalog number: AN7050/BLK ) in distilled deionised water
  8. Bovine serum albumin (Carl Roth, catalog number: 8076 )
  9. BSA/RPMI 6% (see Recipes)
  10. 17% Accudenz in distilled deionised water (see Recipes)


  1. Micropipette and disposable tips
  2. Glass Pasteur pipette
  3. Table top microcentrifuge (Eppendorf)
  4. Heraeus Multifuge 1 SR (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM MultifugeTM 1 SR ) or comparable centrifuge
  5. Zeiss Axiovert 200M (ZEISS, model: Axiovert 200M ) inverted microscope or equivalent device from any other manufacturer e.g., Nikon, Leica, Olympus; 25x (NA 0.8) or 10x (NA 0.25) objective


1. Fiji (download at


Note: If using sporozoites from 5 or less well-infected female mosquitoes gliding assays can be performed directly. Accudenz purification of sporozoites (Kennedy et al., 2012) is needed when sporozoites derived from more than 5 salivary glands are used due to impurity with mosquito debris.

  1. Accudenz purification
    1. Adjust the volume of the undiluted sporozoite solution to 1 ml with RPMI.
    2. Load the sporozoite solution onto a 3 ml Accudenz cushion in a 15 ml conical centrifugation tube.
    3. Spin the tube at 2,500 x g without brake for 20 min at room temperature.
    4. Collect the interface comprising of purified sporozoites with a glass Pasteur pipette and transfer the solution into a clean 1.5 ml reaction tube (see Figure 1A in Kennedy et al., 2012).
    5. Spin for 3 min at 17,000 x g (maximum speed) at room temperature in a table top microcentrifuge.
    6. Discard supernatant and resuspend the pelleted sporozoites in 50 µl incomplete RPMI.
    7. Count sporozoites as described in Part I steps 8-11.

  2. Motility assay
    1. Prepare a 6% solution of bovine serum albumin in RPMI (see Note 1).
    2. Determine an appropriate volume of the sporozoite solution and transfer it into a clean 1.5 ml reaction tube. A total number of about 2,500 sporozoites per 50 µl using a 96-well and 500 sporozoites in 25 µl in a 384-well plate is convenient for a motility assay. Adjust the volume using incomplete RPMI but consider the volume of the drugs, activators or inhibitors, which are to be tested.
    3. If appropriate, add drug, activator, or inhibitor in the concentration to be tested (see Note 2)
    4. Activate the sporozoites by adding 50 µl or 25 µl 6% BSA in RPMI, for the 96- or 384-well plate, respectively (final concentration of 3% BSA), into the reaction tube and transfer the solution into a well of the preferred plate.
    5. Spin the 96- or 384-well plate for 3 min at 200 x g in a centrifuge at room temperature.
    6. Transfer the plate to the Axiovert 200M microscope and start recording 1 image every 3 sec for 3 min using the 25x objective for a 96-well plate (Video 1) and a 10x objective for a 384-well plate. Record at least 3 movies each from 3 independent experiments to get an appropriate number of sporozoites to analyse. Be aware that it is absolutely necessary to keep the same settings between different experiments as otherwise results cannot be compared.

      Video 1. Representative video of gliding sporozoites on glass recorded with a frame rate of 1 image every 1.5 sec for a time frame of 90 sec. Scale bar = 10 µm.

Data analysis

  1. Analysis of individual sporozoite speeds can be performed by manual tracking using the manual tracking plugin in Fiji (Carey et al., 2013). Alternatively the number of circles a gliding sporozoite accomplishes in 100 sec can be determined and used as a proxy for speed (Hegge et al., 2010).
  2. For sporozoite movement pattern classification, z-projection of individual movies can be performed using Fiji (Image->Stacks->z-Project->Max intensity->OK), followed by manually assigning the pattern into attached, waving or gliding (Figure 4).

    Figure 4. Motility assay. A. First slide of movie and B. z-projection; Examples of C. continuous gliding (red squares), D. Waving (blue squares) and E. patch gliding (orange squares). Max projection = maximum projection.

  3. For in-depth analysis, movies have to be investigated manually in full-length to additionally determine other forms of motion or whether sporozoites are gliding clockwise or counterclockwise.
  4. Movies of fluorescent sporozoites recorded with a 10x objective can also be analysed with the automated tracking plugin ToAST (Hegge et al., 2009) also present in Fiji. The output data record speed of individual sporozoites, average speed as well as detailed movement patterns for all tracked sporozoites.


  1. Always prepare fresh 3% or 6% BSA/RPMI solution before starting the experiment.
  2. When testing different compounds or concentrations, plan at least 10-15 min at the microscope to assess the gliding for each reaction mix. To ensure identical incubation times, the addition of compounds and/or BSA can be delayed until the measurement of the previous sample is completed.
  3. Generally, sporozoites from rodent infecting malaria model species such as P. berghei or P. yoelii show robust motility and are mostly investigated in order to understand the underlying molecular mechanisms. Sporozoites expressing fluorescent proteins in their cytoplasm are the most easy to handle. However, also non-fluorescent sporozoites from human infecting species can be analysed.
  4. It is important to dissect salivary glands as pure as possible given that impurities like mosquito debris can influence gliding and complicate image analysis.


  1. BSA/RPMI 3%
    Weigh out 0.03 mg BSA, ideally directly in the reaction tube
    Add 1 ml RPMI
    Vortex until the albumin crystals are completely dissolved
    Store on ice during the experiment
  2. BSA/RPMI 6%
    Weigh out 0.06 mg BSA, ideally directly in the reaction tube
    Add 1 ml RPMI
    Vortex until the albumin crystals are completely dissolved
    Store on ice during the experiment
  3. 17% Accudenz in distilled deionised water
    Weigh out 1.7 mg Accudenz in a 15 ml conical centrifugation tube
    Add 10 ml distilled deionised water
    Vortex until complete dissolving of the powder
    The solution can be stored at 4 °C for at least a week


Funding: collaborative research center SFB 1129 of the Deutsche Forschungsgemeinschaft.


  1. Carey, A. F., Menard, R. and Bargieri, D. Y. (2013). Scoring sporozoite motility. Methods Mol Biol 923: 371-383.
  2. Hegge, S., Kudryashev, M., Smith, A. and Frischknecht, F. (2009). Automated classification of Plasmodium sporozoite movement patterns reveals a shift towards productive motility during salivary gland infection. Biotechnol J 4(6): 903-913.
  3. Hegge, S., Munter, S., Steinbuchel, M., Heiss, K., Engel, U., Matuschewski, K. and Frischknecht, F. (2010). Multistep adhesion of Plasmodium sporozoites. FASEB J 24(7): 2222-2234.
  4. Kennedy, M., Fishbaugher, M. E., Vaughan, A. M., Patrapuvich, R., Boonhok, R., Yimamnuaychok, N., Rezakhani, N., Metzger, P., Ponpuak, M., Sattabongkot, J., Kappe, S. H., Hume, J. C. and Lindner, S. E. (2012). A rapid and scalable density gradient purification method for Plasmodium sporozoites. Malar J 11: 421.


疟原虫子孢子是由按蚊按蚊传播的疟疾寄生虫的传染性,高度活跃的形式。 可以在唾液腺分泌唾液腺和体外分离子孢子之后评估子孢子运动性。
【背景】疟原虫的病原体疟原虫的子孢子通过感染性蚊子的叮咬传播到其脊椎动物宿主的皮肤中。 子孢子运动是寄生虫传播和成功感染脊椎动物宿主的关键前提。 动力构成第一个可以被抑制的寄生虫机制,因此对于干预策略是有意义的。 影响滑动运动性或运动性调节化合物的遗传修饰可以使用体外实验来容易地进行研究。

关键字:疟原虫, 伯氏疟原虫, 孢子体, 唾液腺分离, 解剖, 滑翔运动, 疟疾, 蚊, 疟蚊



  1. 15 ml锥形离心管
  2. 两个10 ml培养皿
  3. 两个27 G针
  4. 两个1毫升注射器
  5. 玻璃幻灯片
  6. 1.5 ml塑料反应管
  7. 一次性聚丙烯杵(SP Scienceware - Bel-Art产品 - H-B仪器,目录号:F19923-0001)
  8. 蚊子

  9. RPMI(Thermo Fisher Scientific,Gibco TM ,目录号:11835063)
  10. 70%乙醇
  11. 1x PBS(Thermo Fisher Scientific,Gibco TM ,目录号:18912014)
  12. 牛血清白蛋白(BSA)(Carl Roth,目录号:8076)
  13. BSA / RPMI 3%(参见食谱)


  1. 真空泵
  2. 泡沫塑料盒
  3. 镊子
  4. 具有一次性提示的微型移液器
  5. 双目显微镜(ZEISS,型号:Stemi 305或任何其他制造商例如,Nikon,Olympus,Leica的可比双目显微镜)
  6. 具有相差40倍物镜的光学显微镜(ZEISS,型号:Axio Lab.A1或任何其他制造商的类似装置,例如,尼康,奥林巴斯,徕卡)
  7. 血细胞计数器


  1. 用真空泵将吸入的蚊子吸入15 ml锥形离心管中,并将其放在冰上5分钟,以使蚊子平静。
  2. 准备一个具有50-100μlRPMI的反应管,用于收集唾液腺。放在冰上。
  3. 填充一个10 ml培养皿与70%乙醇和第二培养皿与1x PBS。
  4. 将27 G针连接到注射器。
  5. 将冷却的固定的蚊子转移到装有70%乙醇的培养皿中长达1分钟。这将一方面减少细菌污染,另一方面降低它们的疏水性。之后,用镊子将蚊子用1x PBS转移到培养皿中
  6. 为了分离唾液腺,在双眼显微镜下的玻璃载玻片上放置一滴1x PBS的雌性蚊子。用一针(针1)的帮助轻轻地固定蚊子的胸部,而另一根针(针2)放置在头部和胸部的交点处(图1)。




  7. 使用一次性聚丙烯杵来破坏反应管中的组织,从而释放子孢子。轻轻研磨约2分钟,直到溶液均匀。
  8. 为了确定子孢子的总数,在RPMI中制成20μl的1:10稀释液。或者,使用补充有3%BSA的RPMI建立活化子孢子的1:10稀释。 (见注1)
  9. 将10μl稀释液转移到血细胞计数器中,并允许子孢子在室温下沉降10分钟。将剩余的子孢子储存在冰上。
  10. 使用具有相差40倍物镜的光学显微镜计数四个大计数平方的子孢子数(图3)。


  11. 使用以下等式计算子孢子总数:

    总数 =([N / 4]×10 [稀释因子]×10 [室因子])x子孢子溶液(μl)
    此外,每个唾液腺的子孢子数可以通过将N <总计除以女性蚊子的数量来确定。



  1. 15 ml锥形离心管
  2. 1.5ml反应管
  3. 96孔或384孔光学平底板(NuncMicroWell96孔板[Thermo Fisher Scientific,Thermo ScientificSupest TM,目录号编号:267342]; Nunc TM 384孔板[Thermo Fisher Scientific,Thermo Scientific TM,目录号:240074])
  4. 乳胶灯泡
  5. 在冰上在反应管(见第一部分)中分离疟原虫子孢子
  6. 不完整的RPMI(Thermo Fisher Scientific,Gibco TM ,目录号:11835063)
  7. Accudenz(Accurate Chemical&amp; Scientific,目录号:AN7050 / BLK)在蒸馏去离子水中的17%w / v溶液
  8. 牛血清白蛋白(Carl Roth,目录号:8076)
  9. BSA / RPMI 6%(见配方)
  10. 蒸馏去离子水中的17%Accudenz(见食谱)


  1. 微型移液器和一次性提示
  2. 玻璃巴斯德吸管
  3. 台式微量离心机(Eppendorf)
  4. Heraeus Multifuge 1 SR(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Multifuge 1 SR)或类似的离心机
  5. 蔡司Axiovert 200M(ZEISS,型号:Axiovert 200M)倒置显微镜或任何其他制造商例如Nikon,Leica,Olympus的等效装置; 25x(NA 0.8)或10x(NA 0.25)目标


1.斐济(从 下载)



  1. Accudenz净化
    1. 用RPMI将未稀释的子孢子溶液的体积调节至1ml。
    2. 将子孢子溶液装入3ml Accudenz垫中的15ml锥形离心管中
    3. 在2,500 x g的情况下,在室温下不用刹车旋转管20分钟
    4. 用玻璃巴斯德吸管收集包含纯化的子孢子的界面,并将溶液转移到干净的1.5ml反应管中(参见Kennedy等人,2012年的图1A)。
    5. 在桌面微量离心机中,室温下以17,000 x g(最高速度)旋转3分钟。
    6. 弃去上清液,将沉淀的子孢子重悬于50μl不完全RPMI中
    7. 按照第I部分步骤8-11中所述计算子孢子。

  2. 运动测定
    1. 在RPMI中制备6%牛血清白蛋白溶液(见注1)
    2. 确定适当体积的子孢子溶液并将其转移到干净的1.5ml反应管中。在384孔板中使用96孔和500个子孢子在25μl中每50μl总共约2,500个子孢子便于运动测定。使用不完全RPMI调整体积,但考虑要测试的药物,激活剂或抑制剂的体积。
    3. 如果适当,加入待测浓度的药物,活化剂或抑制剂(见注2)
    4. 通过在RPMI中分别加入50μl或25μl6%BSA(96孔或384孔板)(最终浓度为3%BSA)来激活子孢子,并将溶液转移到优选的孔中板。
    5. 在室温下在离心机中以200×g旋转96孔或384孔板3分钟。
    6. 将板转移到Axiovert 200M显微镜,并使用25孔目标的96孔板(视频1)和3×3目的用于384孔板,每3秒开始记录1次图像。 从3个独立实验中记录至少3部电影,以获得适当数量的子孢子进行分析。 请注意,绝对有必要在不同实验之间保持相同的设置,否则不能比较结果。

      Video 1. Representative video of gliding sporozoites on glass recorded with a frame rate of 1 image every 1.5 sec for a time frame of 90 sec. Scale bar = 10 µm.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


  1. 通过使用斐济的手动跟踪插件(Carey等人,2013)可以通过人工跟踪来进行个体子孢子速度的分析。或者,可以确定在100秒内实现滑翔子孢子的圆数,并将其用作速度的代理(Hegge等人,2010)。
  2. 对于子孢子运动模式分类,可以使用斐济(Image-&gt; Stacks-&gt; z-Project-&gt; Max intensity-&gt; OK)来执行各个动画的z投影,然后手动地将模式分配给附着的挥动或滑翔(图4)。

    图4.运动测定。 :一种。电影和B. z投影的第一张幻灯片; C.连续滑行(红色方块),D.挥动(蓝色方块)和E.补片滑翔(橙色方块)的例子。最大投影=最大投影。

  3. 为了进行深入分析,必须手动对电影进行全面调查,以确定其他形式的运动,或子孢子是顺时针还是逆时针滑行。
  4. 使用10x目标记录的荧光子孢子的电影也可以使用也存在于斐济的自动跟踪插件ToAST(Hegge等人,2009)进行分析。每个子孢子的输出数据记录速度,平均速度以及所有追踪子孢子的详细运动模式。


  1. 在开始实验之前,始终准备新鲜的3%或6%BSA / RPMI溶液。
  2. 当测试不同的化合物或浓度时,在显微镜下计划至少10-15分钟以评估每个反应混合物的滑翔。为了确保相同的孵育时间,可以延迟添加化合物和/或BSA,直到完成先前样品的测量。
  3. 一般来说,来自啮齿动物的子孢子感染疟疾模型物种如P. berghei 或 P。 yoelii 显示出强大的运动性,并且大多被调查以了解潜在的分子机制。在细胞质中表达荧光蛋白的子孢子是最容易处理的。然而,也可以分析来自人感染物种的非荧光子孢子。
  4. 清除唾液腺是很重要的,因为像蚊子杂质这样的杂质会影响滑翔和复杂的图像分析。


  1. BSA / RPMI 3%
    称量0.03毫克BSA,理想情况下直接在反应管中 加入1ml RPMI
    涡旋直至白蛋白晶体完全溶解 实验期间存放在冰上
  2. BSA / RPMI 6%
    称量0.06毫克BSA,理想情况下直接在反应管中 加入1ml RPMI
    涡旋直至白蛋白晶体完全溶解 实验期间存放在冰上
  3. 蒸馏去离子水中的17%Accudenz
    加入10ml蒸馏去离子水 旋转直至粉末完全溶解 该溶液可以在4°C下储存至少一周


资助:德意志联邦研究中心的SFB 1129合作研究中心。


  1. Carey,AF,Menard,R.and Bargieri,DY(2013)。&nbsp; 计数子孢子运动。方法Mol Biol 923:371-383。
  2. Hegge,S.,Kudryashev,M.,Smith,A.和Frischknecht,F。(2009)。疟原虫子孢子运动模式的自动分类显示在唾液腺感染期间向生产性运动转变。生物技术J 4 (6):903-913。
  3. Hegge,S.,Munter,S.,Steinbuchel,M.,Heiss,K.,Engel,U.,Matuschewski,K.and Frischknecht,F。(2010)。&lt; a class =“ke-insertfile”href =“”target =“_ blank”>疟原虫子孢子之间的多步粘附。FASEB J 24(7):2222-2234。
  4. Kennedy,M.,Fishbaugher,ME,Vaughan,AM,Patrapuvich,R.,Boonhok,R.,Yimamnuaychok,N.,Rezakhani,N.,Metzger,P.,Ponpuak,M.,Sattabongkot,J.,Kappe, SH,Hume,JC和Lindner,SE(2012)。&lt; a class =“ke-insertfile”href =“”target =“_ blank” >用于疟原虫子孢子的快速且可扩展的密度梯度纯化方法。疟疾J 11:421.
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引用:Prinz, H. L., Sattler, J. M. and Frischknecht, F. (2017). Plasmodium Sporozoite Motility on Flat Substrates. Bio-protocol 7(14): e2395. DOI: 10.21769/BioProtoc.2395.