2.1 Movement on agar
Movement on agar was observed in 9-cm Petri dishes containing a 2-3 mm thick layer of 1% water agar. A single male was placed on the agar and observed. The various characteristics of the tracks inscribed on the agar were photographed. Five individuals were studied separately.
2.2 Locomotory behaviour characteristics
Aspects of locomotory behaviour were observed in plain water agar in 5-cm Petri dishes. A single male worm was placed in the centre of the agar plate and was allowed to acclimatize for 5 min before its behavior was recorded. All behavioural analysis was based on two minute video recordings made on a Zeiss Discovery V20 Stereo microscope with an AxioCam MRc5 camera was used.
2.3 Movement characteristics
i) Forward movement: When nematode crawl on agar it is due to alternate contraction and relaxation of the somatic muscles. Such contraction waves are initiated anteriorly and pass down the body resulting in forward movement.
ii) Backward movement: This movement is brought about by the forward waves initiated in the tail region. Forward waves down the whole body result in a “reversal bout” and it causes the nematode to move tail first. Forward movement is periodically and spontaneously interrupted by backward movements.
iii) Pause: After a brief forward or backward movement, nematodes come to rest for few seconds. Normally it occurs at regular period.
iv) Omega: Occasionally a backward wave is generated which causes the nematode to move extremely to one side, until the head touched or almost touches the tail end. This is followed by a further normal backward wave, and characteristically inscribes something like an “omega”. The nematode body bends in an obtuse turning angle resulting in a sharp change in the direction of motion.
2.4 Wave characteristics
i) Wavelength: The distance between two successive amplitudes in the same phase is defined as the wavelength. The wavelength is the distance the wave travels before repeating itself, typically referred to as one oscillation.
ii) Amplitude: The amplitude represents average central displacement over the entire track. It is the distance from each point in the skeleton to the line connecting the lip region and the tail.
iii) Average velocity: It is calculated as the distance of each point along a worm's spine as it moves between successive frames, divided by the time between successive frames.
iv) Bending angle: It is a measure of the curvature along the worm. Angles are measured from the head towards the tail, and are in the range of ± 180°, indicating an inclination to the left (positive) or to the right (negative).
2.5 Software
Detection, tracking, imaging and analysis were done with the WormLab (MBF Bioscience) software. This novel application of tracking procedure enables the extraction and analysis of parallel multiple parameters characterizing the behavior of nematodes such as size, speed, wavelength, amplitude, etc. The video generated from the ZEN software was transferred to WormLab tracking software. For tracking data are analysis Microsoft Visual C++ (6.0), using custom algorithm written in MATLAB (Math Works, Inc., Natick, MA, USA) was used.