Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

1. Mouth form Phenotyping

1. Mouth Form Identification on Agarose Pads
   Note: In order to visualize the nematode mouth morphs, immobilize the worms with a mild anesthetic treatment on agarose pads and observe as follows.
   1. Grow and maintain nematode cultures such as P. pacificus on 6 cm standard nematode growth media (NGM) plates and feed on a bacterial lawn of E. coli OP50 ¹⁹.
   2. Make agarose pads by first adding 0.06 g agarose to 3 ml H₂O in a 15 ml tube to make 3 ml 2% agarose solution. This can be stored for up to a year at 4 ^°C.
   3. Mix and melt the agarose thoroughly in a microwave or alternatively use a heat block set to > 88^°C.
   4. Once fully melted, add 10 µl of a 10 % sodium azide solution to the agarose and mix thoroughly. CAUTION: Dry sodium azide is reactive and all forms are toxic.
   5. Using a 1 ml micropipette, place a drop of no less than 300 µl of the liquid agarose azide mix onto the middle of a standard glass microscope slide.
   6. Before the agar cools, quickly place a second microscope slide on top of the drop in order to flatten the agarose which forms a pad upon cooling. Repeat for as many pads as is required.
   7. Just prior to use, peel apart the glass microscope slides by sliding them off one another. Note: If agarose pads are prepared too far in advance they can be excessively dry and may damage the nematodes.
   8. To transfer worms to the anesthetic agarose pads, place a drop of M9 buffer (2 – 3 µl) onto the center of the pad. Pick 2 – 3 young P. pacificus adults into the drop of M9 before placing a cover slip carefully over the pad. The P. pacificus nematodes will be immobilized in the agarose and ready to visualize.
   9. Transfer the microscope slide containing the anesthetized worm to an appropriate microscope and observe under 63X Nomarski optics. Categorize morph identities based on the following features: the presence of an additional sub-ventral tooth, enlarged dorsal tooth and wide mouth opening is indicative of a eurystomatous mouth morph animal, while the presence of a single dorsal tooth and narrower mouth opening indicates a stenostomatous animal (Figure 1).
      Note: In order to maintain the health of the animal, worms should be maintained on the agar pad for no more than 5 min.
   10. After mouth morph identification, recover either eurystomatous or stenostomatous nematodes as required by removing the cover slip by gently sliding it off the agarose pad. Carefully pick the selected animals from the agarose pad (E. coli OP50 can be used on the pick to help make it stickier) onto fresh NGM plates. Allow recovery from the anesthetic until normal motile behavior has resumed upon which animals are ready for further predatory assays.
2. Rapid Mouth Phenotyping
   Note: Alternatively, with more experience, mouth form type can be assayed without the need for any anesthetic treatment via a stereomicroscope with high magnification (150X).
   1. Place nematodes on standard NGM plates with a bacterial lawn of E. coli OP50 onto the microscope viewing area.
   2. Detect differences in mouth size and width. Note: At this magnification no tooth like structures are observable therefore mouth morph identification is solely based on wide mouths versus narrow mouths.

2. Bite Assay

Note: Biting assays permit a detailed predatory behavioral analysis.

1. Grow and maintain nematode cultures on standard NGM plates (6 cm) and feed on a bacterial lawn of E. coli OP50 ¹⁹.
2. Make assay plates by growing a large quantity of selected prey nematode larvae such as C. elegans or alternatively an appropriate ecologically relevant prey. Note: Adult C. elegans are too large to be suitable prey so it is important to use the larval stage.
   1. Maintain C. elegans or other potential prey species on standard NGM plates and feed on a bacterial lawn of E. coli OP50 until the population is freshly starved, resulting in an abundance of young L1 larvae.
      Note: Time for starvation is dependent on numerous environmental and experimental factors, including number of nematodes used to start the culture, the amount of E. coliOP50 added, and the ambient temperature.
3. Wash four or more freshly starved prey plates with M9 and pass the worm solution through two 20 µm filters to remove all large animals and any remaining eggs before collection in a 15 ml tube. Only small larvae should remain in the solution.
4. To form a larval pellet centrifuge the filtered prey at 377 x g for 1 min.
5. Pipette 3 µl of pure worm pellet onto a 6 cm NGM plate with no E. coliOP50 present and wait at least 30 min for larvae to spread out sufficiently to generate an assay plate.
   Note: 3 µl of pure worm pellet on the standard assay plates contain > 3,000 prey larvae. This is sufficient to generate frequent contact between predators and prey.
6. Screen predatory nematodes for the required mouth morph (protocol 1).
7. Using standard worm picking techniques and a light stereomicroscope ¹⁹, transfer correctly classified predators on to the assay plate. Take care to transfer as little OP50 bacteria as possible to the assay plate when transferring predators in order to minimize bacterial contamination. Wait 15 min to allow the worm to recover from the stress of being transferred and check for wild-type motile behavior to ensure worms have not been damaged from the transfer.
   Note: There is no need to starve P. pacificus, as they are highly efficient predators of other nematode larvae even while well fed upon bacteria.
8. After recovery, observe the predator using a light stereomicroscope for 10 min. With this equipment, observe and characterize distinct feeding events such as biting, characterized by the predator restricting the movement of the prey; killing, where by an opening of the prey cuticle is detected; and feeding, categorized by an observable consumption of the prey innards (Figure 2A, B and Movie 1).
9. Repeat the assay by screening and observing a minimum of 10 individual predatory nematodes to ensure accuracy.

3. Corpse Assay

Note: Corpse assays facilitate a more rapid quantification of predatory behavior.

1. Grow and maintain nematode cultures on standard NGM plates and feed on a bacterial lawn of E. coli OP50 ¹⁹. Generate triplicates of the assay plates mentioned previously (protocol 2.1 – 2.5).
2. Screen predatory nematodes for the required mouth morph as described in protocol 1. Using standard worm picking techniques and a light stereomicroscope transfer 5 predatory nematodes with the required mouth morph to each assay plate. Leave the predators together with the prey for 2 hr.
3. After 2 hr screen the assay plate for the presence of emptied corpses (Figure 2B and C). Identify corpses by the absence of motility along with obvious morphological defects including leaking innards or missing worm fragments.

4. Analysis of Pharyngeal and Tooth Movement

1. Grow and maintain nematode cultures on standard NGM plates and feed on a bacterial lawn of E. coli OP50 ¹⁹. Generate assay plates as mentioned previously (protocol 2.1 – 2.5). If standard 6 cm NGM plates do not fit between the objective and the microscope stage, use the lid of small 35 mm petri dishes containing 2 ml NGM as a suitable alternative.
2. Screen predatory nematodes for the required mouth morph as described in protocol 1. Using standard worm picking techniques and a light stereomicroscope, transfer a single correctly classified predator on to the assay plate. Wait 15 min to allow the worm to recover from the stress of being transferred.
3. Observe predatory animals on a microscope at 40 – 63X Normaski, with a high-speed camera (Movies 2 and 3). Record pharyngeal pumping and tooth movement over 15 sec, at 50 Hz in at least 20 animals to ensure accurate quantification. Replay recorded movies at the desired speed in order to count individual pumps and tooth events.
   Note: Pumping is observed in the corpus, located at the middle of the pharynx, while tooth movement is detectable in the mouth opening and is only observed from the dorsal tooth.