Published: September 4th, 2016
Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.
This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.
Nematodes with their small but complex nervous systems have proved powerful tools for understanding many aspects of neurobiology including behavior. Much of this research has focused on the model organism Caenorhabditis elegans in which a wealth of different behaviors have been successfully dissected and analyzed. These include mechanosensory 3, chemotactic 4, thermotactic 5,6 and magnetotactic 7 influencing mating 8,9, learning 10 and feeding behaviors 11. However, other more distantly related nematode species display behaviors which are not observed in the rhabditid C. elegans or alternatively show additional levels of complexity, which raises pertinent questions regarding their evolution and regulation. One such instance of this can be observed in the distantly related diplogastrid nematode Pristionchus pacificus, which displays much more complex feeding behaviors and rhythms than are observed in C. elegans 1. This is despite the two species sharing homologous pharyngeal neurons 12. Coinciding with these additional feeding behaviors, P. pacificus also displays an expanded dietary range, as they are avid predators, capable of supplementing their bacterial diet by also feeding on the larvae of other nematodes. Fortunately, P. pacificus has been developed as a model for comparative and integrative evolutionary biology and therefore many molecular and genetic tools are now available. These include a fully sequenced and annotated genome 13, molecular and genetic tools including transgenes 14 and CRISPR/Cas9 15,16 as well as a detailed and well annotated phylogeny 17 with over 25 closely related species including its newly discovered sister species. In addition, the ecology of numerous Pristionchus species including P. pacificus is well defined with many species having now been described sharing a necromenic association with scarab beetles, a host they frequently share with other nematode species 18. P. pacificus therefore provides an excellent model system with which to dissect the evolution of novel behaviors and their ecological significance.
In order to analyze predatory feeding behaviors in nematode species such as P. pacificus we developed several novel behavioral assays for easy observation and quantification of predatory actions. As P. pacificus displays a dimorphic mouth structure, which strongly influences predatory behavior, identification of the correct morphotype is essential 1,2. The narrow mouthed stenostomatous morph contains a single blunt dorsal tooth and does not engage in any predatory feeding. Alternatively, the wide mouthed eurystomatous morph includes a much larger claw shaped dorsal tooth and an additional opposing sub-ventral tooth, which together operate to efficiently open the cuticle of their prey. The ratio of the predatory eurystomatous to the non-predatory stenostomatous form varies among Pristionchus species and also within P. pacificus, however, the percentage of eurystomatous mouth morph in the P. pacificus wild type strain (PS312) is usually 70 - 90% 2. Additionally, the mouth form ratios can fluctuate depending on differing environmental influences (both known, including starvation and some small molecule signaling as well as unknown factors), thus correct identification and isolation of the predatory eurystomatous mouth form is essential for successful predatory assays.
Alongside the description of the predatory mouth form we have developed a "bite assay" for direct observation and quantification of predatory behaviors including biting, killing and feeding events. Here prey nematodes are isolated through filtering of newly starved cultures and exposed to predatory adult P. pacificus, which are observed together over a short time span. In addition, we have also developed a high throughput "corpse assay" to facilitate rapid screening of predatory behavior through indirect observation of predatory events. This takes advantage of the presence of larval corpses as a tool to screen for predation. Both assays provide easy and highly repeatable methods for observing and measuring predatory behavior in nematode species such as P. pacificus.
1. Mouth form Phenotyping
2. Bite Assay
Note: Biting assays permit a detailed predatory behavioral analysis.
3. Corpse Assay
Note: Corpse assays facilitate a more rapid quantification of predatory behavior.
4. Analysis of Pharyngeal and Tooth Movement
Following successful identification of the appropriate mouth morph in P. pacificus, clear differences between eurystomatous and stenostomatous animals can be detected (Figure 3) with only the eurystomatous animals engaging in killing behavior. In stenostomatous animals this behavior seems to be suppressed entirely. Furthermore, differences in the tooth activity and pharyngeal pumping of eurystomatous animals on bacteria and prey (Figure 4 and Movies 2 and 3) are also evident. While predatory feeding, the pumping rate is reduced below that observed during bacterial feeding and tooth movement is detected in a one to one ratio with the pharyngeal pumping. This is potentially indicative of key regulatory mechanisms modulating the behavioral response to differing diet.
Figure 1. P. pacificus has a Mouth Dimorphism which Influences Feeding Behavior. (A) The eurystomatous mouth form is capable of predation and has a wide mouth opening with a large claw shaped dorsal tooth (false-colored red) and (B) a large opposing hook shaped sub-ventral tooth (false-colored blue). (C) The stenostomatous mouth form is only able to feed on bacteria and has a narrower mouth opening with a flint shaped dorsal tooth (false-colored red) and (D) no sub-ventral tooth (*). Normaski images are 63X and scale bar represents 10 µm. Please click here to view a larger version of this figure.
Figure 2. Predation Assays. (A) P. pacificus bites and kills the larvae of other nematodes such as C. elegans. (B) For biting assays, the number of bitesby predators (*) can be observed using a light stereomicroscope and successful killing and feeding events also recorded. Corpses are also clearly visible (circles). (C) For corpse assays, larval carcasses (arrows) can be easily identified compared to living larvae. Scale bar represents 1 mm in B and 150 µm in C. Please click here to view a larger version of this figure.
Figure 3. Results of Bite and Corpse Assays on C. elegans Prey. (A) Biting behavior is only evident in the eurystomatous mouth form with this behavior not displayed in stenostomatous animals. Error bar represents standard deviation of 10 replicates. (B) Coinciding with no biting behavior evident from stenostomatous animals, corpse assays also reveal carcasses only on assay plates of eurystomatous animals. Error bar represents standard deviation of 5 replicates. Please click here to view a larger version of this figure.
Figure 4. Eurystomatous Pumping Rate and Tooth Movement during Predatory Feeding. Tooth movement is only observed while eurystomatous animals are engaged in predatory feeding. This also coincides with a reduction in pharyngeal pumping. Error bar represents standard deviation of 10 replicates. Please click here to view a larger version of this figure.
Nematodes provide a powerful system for understanding neurobiology and behavior with C. elegans thus far being the primary tool. However, numerous nematode species including P. pacificus display behaviors, which are absent or vary in complexities from the model organism C. elegans and therefore raise fascinating questions regarding the evolution and regulation of these behaviors. One such additional behavior found in many other nematode species including P. pacificus is the capacity to supplement their bacterial diet by engaging in predatory feeding 1, 20. We have therefore developed and described a detailed protocol for easy and rapid characterization of these previously unanalyzed predatory behaviors in nematodes.
Firstly, we have provided methods to screen for variations in feeding apparatus within the nematode mouth. The identification of the correct mouth type is an essential first step for successful predation assays as, at least within the Pristionchus genus only eurystomatous animals are capable of predatory feeding. It is best to identify mouth morphs with the "rapid mouth phenotyping" protocol described in protocol 1.2 as this method is much less invasive and therefore it is less likely that predatory behaviors may be perturbed. However, it is recommended to first become familiar with the different mouth structures by identification with anesthetized animals on agar pads (protocol 1.1).
Following identification of the desired mouth morph, we have described two assays for quantifying predatory feeding. These are a rapid, high throughput "corpse assay" (protocol 3) and a more time consuming but more in-depth behavioral analysis through the "bite assay" (protocol 2). Both of these protocols are highly flexible allowing for several modifications in order to optimize the assays depending on the experimental requirements. For bite assays using P. pacificus predators on C. elegans prey, observations of predatory behavioral interactions for a time window of 10 min was sufficient to quantify a significant amount of bites along with other feeding events. For "corpse assays" again utilizing P. pacificus predators on C. elegans prey, 5 predators for 2 hr produced easily quantifiable and consistent corpse numbers allowing for rapid behavioral analysis. However, it should be noted different species of predatory nematode move at different speeds, eat at different rates and generally demonstrate a large diversity in other behaviors 1. Additionally, different prey species may also be eaten at different rates for similar reasons. It is therefore recommended to optimize the assays based upon the nematode species tested both as predators and prey, and also for any differences in environmental conditions. During both "bite" and "corpse" assays it is critical that both prey and predators are healthy, as stressed or injured predators will not kill efficiently. In addition, fresh assay plates are essential as older plates can become dried out which adversely affects the health of the nematodes leading to erroneous assays. It is also hoped that future iterations of these predatory assays will be able to take advantage of recent advances in technology in order to automate much of the analysis as has been accomplished for investigating many behaviors observed in C. elegans21, 22. Currently problems are likely to arise in nematodes such as P. pacificus as they appear much more sensitive to contact, making isolation and immobilization in microfluidic chambers likely to abrogate predatory feeding. Overcoming this may prove challenging but would facilitate individual nematodes to be screened for subtler predatory behaviors.
Finally, we have also provided methods for examining the nematode feeding apparatus itself facilitating comparisons between predatory and bacterial feeding modes by quantifying the tooth and pharyngeal pumping kinetics using a high-speed camera (protocol 4). The quantification of pharyngeal pumping rates in C. elegans has been utilized to monitor feeding for many years 23, however, C. elegans lacks any form of mouth denticle and also lacks predatory behaviors. Through combining the quantification of pharyngeal pumping with that of tooth activity, any innervation of the teeth specific to predation can be also observed. Due to the magnification required to observe the tooth movement the animals often move out of the focal plane, thus it is usually only possible to observe the tooth for short time windows. Additionally, unlike C. elegans, the pharynx of P. pacificus does not continuously pump, rather it engages in spells of pumping and feeding. Therefore, for accurate pharyngeal pumping rates while feeding to be determined, it is important to record 15 sec of continuous feeding.
These methods presented here therefore provide the first framework for investigating predatory behaviors in nematode systems. Moreover, they may also be adaptable for use in investigating other interactions within the nematode ecosystem including the influence of additionally ecologically relevant organisms on predation including microorganisms, fungi and mites. Thus they provide a means to dissect how these predatory behaviors are regulated, how they may have evolved and also their ecological significance.
The authors declare no financial or competing interests.
We thank Dr. Daniel Bumbarger for the predation behavior picture. This work was funded by the Max-Planck Society.
|Nylon net filters (20 um)
|Merck Millipore Ltd
|Used to filter worms just leaving larvae for use as prey.
|PP Funnel for filter (54mm)
|Used to filter worms just leaving larvae for use as prey.
|Small petri dish (35/10 mm)
|For imaging on High speed camera
|Zeiss SteREO Discovery V12
|For mouth form identificaton
|For mouth form identificaton
|Motion Scope M3 Highspeed camera
|High speed camera
|Video zoom 44 ENG 1/2" 0.5x to 2.4x
|High speed camera zoom
|Nematode Growth Medium (NGM) ingredients:
|Sodium chloride (NaCl)
|Calcium chloride dihydrate (CaCl2)
|Cholesterol from lanolin
|F 26732 00050
|Magnesium sulfate heptahydrate (MgSO4)
|Potassium dihydrogen phosphate (KH2PO4)
|6 cm petri dish
|3.5 cm petri dish
|Potassium dihydrogen phosphate (KH2PO4)
|Sodium hydrogen phosphate heptahydrate (NaHPO4)
|Sodium chloride (NaCl)
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