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Fig. S1. Bar graphs showing comparison between normal and drug-treated parameters of ookinete motility in the presence of microtubule inhibitors Oryzalin (1 mM) and Colchicine (1 mM). Boxplot error bars indicate 5th to 95th percentiles with outliers shown individually.

Fig. S2. A schematic representation for computing ookinete motility parameters.

A. A right-handed helical primitive (in a right-handed Cartesian co-ordinate system) comprise three consecutive vectors (A, B, C) oriented as shown in the figure. Here vectors A and B lie in the figure plane, and vector C is directed either away from the reader (left panel) or towards the reader (right panel). X is a cross-product of vectors A and B. In the right-handed helical primitive the angle between X and C is smaller than π/2.

B. Estimation of the radius and pitch of a single helical loop from three trajectory points Pi, Pi+T/2, and Pi+T, where T is the estimated track period.

Fig. S3. The role of force in determining ookinete helical motion. A model for how organized force contributes to ookinete motion. Here V is the translational velocity and ω is the rotational velocity. Flat and Frear denote lateral and rearwards forces respectively.

Supplementary Discussion

Fig. S4. Parameter estimates are not sensitive to mild deviations in chosen threshold values. Each group represents wild-type ookinete parameters computed using a different combination of thresholds. Boxplot error bars indicate 5th to 95th percentiles with outliers shown individually.

Fig. S5. The estimates of chirality for the same group of ookinetes varies with threshold, but stabilizes after the value of 10 frames. Each group represents wild-type ookinete parameters computed using a different threshold for chirality computation. Boxplot error bars indicate 5th to 95th percentiles with outliers shown individually.

Fig. S6. An example that illustrates various properties of a moving ookinete (blue ellipse). The ookinete moves along some real smooth trajectory (blue curve), whereas a movie of five frames contains only five points from the trajectory (points connected by the red segments). Higher frame rates enable a better approximation of the real trajectory (bottom panel, yellow segments). In contrast, total displacement (green line) is not very sensitive to frame rate, and is characteristic of directionality of motion only.

Fig. S7. 3D reference object. In a right-handed system such that the y-axis coincides with the vertical stroke, and z-axis is perpendicular to the plane of the symbol (and to the plane of the tissue), in the reference spatial configuration, going from point A to point B would increase both x and y co-ordinates, while going from tissue to symbol plane would increase z co-ordinate. After acquiring the object using a confocal microscope and inspecting the resulting z-stack during image processing (see Experimental procedures), while y and z co-ordinates increased as expected, x co-ordinates decreased when going from A to B. This implies that in the test system the image is digitized using a left-handed co-ordinate system.

Supplementary Code and Data. Code and data is available on request including the source output from the Imaris tracking for different experiments, and the code that reads these data, which computes locomotion parameters and compares different conditions.

cmi12283-sup-0002-movie_s1.mov131K

Movie S1. 40 × 3D motion path of a single P. berghei ookinete in Matrigel tracked using the Imaris v.7.5.1.

cmi12283-sup-0003-movie_s2.mov378K

Movie S2. 20 × 3D motion of P. berghei ookinetes in a single well from a 96-well plate pre-loaded with Matrigel.

cmi12283-sup-0004-movie_s3.mov2091K

Movie S3. Tracking of Movie S3 using Imaris v.7.5.1.

cmi12283-sup-0005-movie_s4.mov11769K

Movie S4. Tracking of three dimensional motion of P. berghei ookinetes from an explanted Anopheles stephensi-infected midgut using Imaris v.7.5.1.

cmi12283-sup-0006-movie_s5.mov1028K

Movie S5. Magnification of Movie S5.

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Movie S6. Rotation of single wild-type P. berghei ookinete rendered from focused ion beam (FIB) electron microscopy (EM) sections.

cmi12283-sup-0008-movie_s7.mov949K

Movie S7. Rotation of single IMC1h-KO P. berghei ookinete rendered from FIB-EM sections.

cmi12283-sup-0009-movie_s8.mov1252K

Movie S8. Rotation of an additional IMC1h-KO P. berghei ookinete rendered from FIB-EM sections.

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