The overall goal for this procedure is to observe the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus. This method can help advance psychological studies of the nematode. It specifically deals with observance of mating behavior of the pinewood nematode.
The main advantage of this technique is that virgin adults can be obtained, which makes it much easier to observe adult mating behavior. The implications of this technique purely extend towards the broader study of nematodes, because they have similar life cycles behaviors. For the demonstration of this method is critical, as acquisition of virgin adult nematodes is difficult because constrains of developmental nematodes takes practice and it's not easy to precisely distinguish males from females in early first stage juveniles.
To culture pinewood nematodes, first prepare potato dextrose agar plates. To inoculate a plate, punch a piece of fungal mat with a diameter of 10 millimeters from a betritus senaria mat, and put it in the middle of a new PDA plate. Culture the fungal mat for five days at 25 degrees Celsius in the dark.
After five days, add the pinewood nematodes to the prepared Petri dish and culture them for three days at 25 degrees Celsius in the dark. To isolate the nematodes from the fungus, first prepare a collection tool. Place a clamped rubber tube below a funnel, and hold the prepared funnel in a rack to assemble the baermann funnel apparatus.
Then, place two layers of filter paper instead of gauze in the mouth of the funnel. Now, transfer the fungal cultures to the funnel setup and add water until the fungal mat is immersed. After two hours, the nematodes will come out of the fungal culture, cross the filter paper, and settle down in the lowest part of the baermann funnel.
Collect the nematodes by placing a 15 milliliter tube below the funnel, then slowly releasing the clamp to collect a few drops of water, which will contain the nematodes. Centrifuge the nematode collections at 3, 000 G for two minutes at room temperature. Then asparate the upper level of water, and re-suspend the worms in one milliliter of water.
Transfer the nematodes'suspension to a six centimeter glass dish, and add three milliliters of water to help the nematodes swim freely. Now, collect eggs to make virgin stocks. Transfer the dish to a dark incubator, and let the pregnant females lay eggs for 10 minutes.
Because of their glycoprotein surface, the eggs will stick to the glass. Next remove the water with the worms carefully to avoid disturbing the eggs stuck to the dish. Then, quickly add back 3 milliliters of water to the dish to prevent the eggs from drying out.
Repeat this process of cleaning eggs at least three times, to remove all the larvae and adults and obtain the pure eggs. To the washed pure egg collection, add five milliliters of water, and proceed with developing the eggs in the dark at 25 degrees Celsius. 24 hours after starting the egg culture, collect the hatched J2 larvae.
Carefully transfer the J2 larvae into a 15 milliliter tube. Then centrifuge the collection at 3, 000 G for two minutes. Asparate the upper layer of water, and add back 200 microliters of water.
Then, culture the J2 larvae on a PDA plate prepared with a mat of fungus. After 52 hours, most of the J2 larvae will have developed into early stage J4 larvae. Assemble a baermann funnel, and extract the nematodes from the fungal cultures for two hours, as previously described.
While waiting, prepare a needle to pick the worms. Draw a tip from a glass capillary heated over an alcohol burner. The tip needs to be about 50 microns in diameter, roughly the body width of an adult nematode.
After two hours, transfer the J4 larvae into a clean Petri dish as previously described. Now, sex the nematodes under a stereo microscope. About two thirds of the larvae are usually female.
The female's vulva is obviously different from the male's anatomy, but because the worms are very active, sexing will take practice. The most critical step to this protocol, is to precisely distinguish and separate males from females in early first stage. Otherwise, there'll be mixed and bad, once they become adult.
And that makes it difficult to observe the mating. Gradually, pick and transfer the worms to a drop of water on a slide for males or females. To ensure success, two different people should double check the sex allocation.
Now, transfer the water drop containing the sexed worms to appropriate fungal culture plate. Then, culture the virgins for another 24 hours before studying their mating behavior. To observe pinewood nematode mating behavior, first prepare several concave slides by adding 200 microliters of water to each of their wells.
Next, pick a single prepared virgin adult and transfer it to a prepared slide. Keep the virgin adults inside the well for an hour to let them adapt to the new aqueous environment. After an hour, add a single virgin adult of the opposite sex to the well, using a pipette.
Then, observe the mating behavior under a stereo microscope, and film the behavior for quantitative analysis. The whole mating process is usually over in two hours. Every 30 minutes, add 50 microliters of water to compensate for evaporation.
To investigate mating choice and intersexual competition, group and observe virgin nematodes of two different combinations. The mating success rate of virgin adults was about 87%which was much higher than that of the randomly selected adults. Pairs took an average of 83 minutes to complete the entire mating process, which contains four different phases.
Searching, contacting, copulating, and lingering. Within these phases, many specific patterns of behavior can be observed. When one female met several males, there was an obvious mating choice.
The female usually uses her head to contact one male, usually at the head, body, tail, and genital region. The female then copulated with the male, or left him quickly, and swam to another male. Mating success rates were measured after the first contact for the different nematode combinations.
Intersexual competition also occurred. When one virgin female was mixed with three virgin males, the males tried to approach and contact the female as much as possible, to get the chance of mating with her. When the vulva was occupied, the other males tried to disturb the mating process using violent body swings, or dragging to separate them.
Female intersexual competition was seen to be more or less similar to male intersexual competition. Because of the intersexual competition, made copulation frequencies with the different combinations were significantly different. After its development, this technique paved the way for exploration of the behavioral ecology in nematodes.
Once mastered, this procedure can be completed in 12 days, if it's performed properly. While attempting this procedure, it's important to remember to precisely separate males from females, the early four stage juveniles to obtain virgin adults.
