Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

The main advantage of this culturing technique is that you can obtain virgin copepods while controlling their pairing experience and tracking their development. The following behavior analysis method can help controlled and reproducible experiments on the mate-guarding behavior of the Tigriopus copepods which are common zooplankton in rocky tide pools. Though these methods can provide insight into the reproductive behavior of Tigriopus, they can also be used to look at how genetic and environmental factors impact the physiology and behavior of these copepods.

To begin, use a Pasteur pipette to collect gravid females carrying clear orange eggs from the stock culture. Rinse the females with clean culture medium via gentle pipetting. Next, place each female into an individual well of a six-well cell culture plate with clean culture medium.

Then place the plate in an incubator to culture the females until they release egg sacs. After the egg sacs are released, use a Pasteur pipette to remove the females from the wells of the plate. As soon as animals at the first copepodid stage or CI start to emerge, use a P-10 micropipette to collect them.

Every two to three days, monitor the wells for molted exuviae. After this, sex the animals by examining their antenna morphology. To correctly identify adult females, make sure that they have already molted five exuviae in culture wells because differences in shape of antennae between adult females and juveniles are not visibly significant in some populations.

After selecting animals of the appropriate stage and sex, feed the selected individuals with finely ground fish food. While the animals eat, prepare two 48-well flat bottom cell culture plates as testing chambers. After 30 minutes have passed, rinse each animal by pipetting it into a succession of four wells of a 24-well plate filled with clean artificial sea water.

Then place the rinsed individuals in the wells of plates A and B on the LED light pad. Allow the animals to acclimate for 30 minutes, then use a Pasteur pipette to transfer the targets from plate B to plate A.Allow a male and a target to interact in each well for the designated observation time before stopping the video recording. First, examine the video for the timing when each target was transferred into a well on plate A.Next, define the initiation of a guarding attempt by a male as a contact of the male's antenna with any body part of the target.

Define the termination of guarding attempt by a male as the point when both of the male's antenna detach from the target's body. Then, define initiation of copulation by a dorsal body bend of a male, followed by a repetitive press of the urosome against that of the female. First, generate a video clip featuring the episode of interest by trimming the movie with movie editing software.

Then, open ImageJ and import the video clip. Select the covert 8-bit grayscale option to reduce the memory required for image processing. Next, select the straight line selection tool for spacial calibration and draw a selection line along the diameter of the well.

Open the set scale menu and enter the known length into the known distance box and the unit in the unit of length box. After this, open the properties menu and enter the frame interval in the frame interval box. Open the MTrackJ plugin by selecting plugins and MtrackJ.

Then, click the tracking button in the dialog window to open the tracking configuration menu. Check apply local cursor snapping during track and select dark centroid as a snap feature. Next, click the add button in the MTrackJ dialog window to start tracking.

Position a cursor to cover the individuals in the video clip to be tracked. Then, click to detect the dark centroid of the object within the square. To generate data table, click the measure table in MTrackJ dialog window, then click the movie button to generate a movie with the tracked trajectory drawn as a colored line.

Finally, click the save button in the MTrackJ dialog window to save the results, including the data in the tracked two dimensional trajectory. In this experiment, mate-guarding behavior of Tigriopus copepods was recorded and observed. Analysis shows a difference in the average duration of guarding attempts between male-female pairs and male-male pairs of Tigriopus californicus where males in the male-male pairs exhibited relatively shorter duration of attempts than those in male-female pairs.

Two dimension spatial tracking of newly formed guarding pairs of Tigriopus californicus revealed that male-male pairs tended to show higher velocity than male-female pairs in the first three seconds of guarding attempts. After its development, this technique paved the way for researchers in the fields of ethology, biochemistry, and evolutionary genetics to explore the unique mating system of these type of copepods.