The overall goal of these procedures is to reliably analyze complex feeding behaviors in predatory nematodes by using several novel assays. These methods can help answer key questions in the field of behavioral neurobiology, such as the regulation of novel complex behaviors and indeed, also the evolution. The main advantages of these techniques is that they provide a rapid, easily repeated methods, in order to observe and quantify predatory feeding behaviors in nematodes, including the satellite model organism, Pristionchus pacificus.
In order to visualize the nematode mouth morphs, first immobilize the worms with a mild anesthetic treatment on a freshly made 2%agarose pad. To make a pad, carefully add 10 microliters of a 10%sodium azide solution to three milliliters of melted agarose and mix thoroughly. Sodium azide is reactive and toxic so take proper precautions.
Then, drop at least 300 microliters of the mixture onto the middle of a standard glass microscope slide and use another slide to flatten the agarose. Leave the top slide in position until the pad is about to be used. To prepare the pad for use, remove the top slide and add a drop of M9.Then pick two or three worms, place them into the drop and carefully apply a cover slip.
Observe the worms with 63x Nomarski optics to categorize their morph identities. An enlarged dorsal tooth, an additional subventral tooth or an enlarged wide mouth opening are all features of a eurystomatous animal. Whereas a single dorsal tooth and narrower mouth opening indicates a stenostomatous animal.
Within five minutes, remove the nematodes from the anesthetic pad. Gently slide off the cover slip and transfer them onto a fresh MGM plate. Wait until they show motile behavior before any further analysis.
With more experience the morph can be assayed without anesthetic treatment. Transfer 10 to 20 nematodes from a standard culture plate onto a fresh plate seeded with a bacterial lawn of OP50 for rapid mouth form phenotyping using a stereo microscope with high magnification. Then detect differences in mouth size and width.
At this magnification, no tooth like structures are observable so identify mouth morphs based on their widths. Thus distinguish wide mouths from narrow mouths. Make the assay plates by growing a large quantity of selected prey nematode larvae such as larval stage C.elegans or an appropriate ecologically relevant prey.
Starve four or more plates of the prey species to collect the L1 larvae. Later, wash the plates with M9 and pass the collection through two 20 micron filters. Only the small larvae should remain in the solution.
Pellet the larvae at 377 g for a minute. Remove M9 supernatant into a waste beaker and take a three microliter aliquot of pure worm pellet. Deposit the worms at the center of a six centimeter MGM plate without E.coli and let them spread out for about half an hour.
Later, use the larvae for the bite assay. Transfer the predators onto the assay plate with as little of the OP50 bacteria as possible. For P.pacificus, add five predators to the plate.
Wait 15 minutes to allow the transferred animals to recover from the stress. After 15 minutes, the worms should move about normally or they may have been damaged. Now observe and record distinct events by one of the predators for 10 minutes.
Biting is characterized by the predator restricting the movement of the prey. Killing occurs when the prey cuticle is openend. Feeding is characterized by an observable consumption of the prey innards.
Observe at least 10 individual predatory nematodes to ensure accuracy. Pharyngeal pumping and tooth movement can be observed under Nomarski optics at 40 to 63x magnification and recorded with a high speed camera at 50 hertz. After recording 15 second clips of at least 20 animals, the videos can be analyzed reliably.
Pumping is observed in the corpus, located at the middle of the pharynx. Tooth movement is detectable in the mouth opening and is only observed from the dorsal tooth. After two hours of predation, screen the plate for empty corpses.
Identify the corpses by their absence of motility and obvious morphological defects including leaking innards or missing worm fragments. Following successful identification of the appropriate mouth morph in P.pacificus, clear differences between eurystomatous and stenostomatous animals were observed. Only eurystomatous animals engaged in killing behavior.
Furthermore, the differences in the tooth activity and pharyngeal pumping of eurystamatous animals on bacteria and prey are also evident. During predatory feeding, the pumping rate is reduced below that observed during bacterial feeding and tooth movement is even with pharyngeal pumping. After watching this video, you should have a good understanding of how to combine these different assays in order to characterize complex predatory feeding behaviors in nematodes including in the satellite model organism Pristionchus pacificus.
