Available via license: CC BY
Content may be subject to copyright.
RESEARCH ARTICLE
Kin-recognition and predation shape
collective behaviors in the cannibalistic
nematode Pristionchus pacificus
Fumie HiramatsuID
1,2
, James W. LightfootID
1
*
1Max Planck Research Group Genetics of Behavior, Max Planck Institute for Neurobiology of Behavior–
caesar, Bonn, Germany, 2International Max Planck Research School for Brain and Behavior, Bonn,
Germany
*james.lightfoot@mpinb.mpg.de
Abstract
Kin-recognition is observed across diverse species forming an important behavioral adapta-
tion influencing organismal interactions. In many species, the molecular mechanisms
involved are difficult to characterize, but in the nematode Pristionchus pacificus molecular
components regulating its kin-recognition system have been identified. These determine its
predatory behaviors towards other con-specifics which prevents the killing and cannibaliza-
tion of kin. Importantly, their impact on other interactions including collective behaviors is
unknown. Here, we explored a high altitude adapted clade of this species which aggregates
abundantly under laboratory conditions, to investigate the influence of the kin-recognition
system on their group behaviours. By utilizing pairwise aggregation assays between distinct
strains of P.pacificus with differing degrees of genetic relatedness, we observe aggregation
between kin but not distantly related strains. In assays between distantly related strains, the
aggregation ratio is frequently reduced. Furthermore, abolishing predation behaviors
through CRISPR/Cas9 induced mutations in Ppa-nhr-40 result in rival strains successfully
aggregating together. Finally, as Caenorhabditis elegans are found naturally occurring with
P.pacificus, we also explored aggregation events between these species. Here, aggregates
were dominated by P.pacificus with the presence of only a small number of predators prov-
ing sufficient to disrupt C.elegans aggregation dynamics. Thus, aggregating strains of P.
pacificus preferentially group with kin, revealing competition and nepotism as previously
unknown components influencing collective behaviors in nematodes.
Author summary
Many species are able to recognize their relatives from other organisms and this influences
how they behave with one another. In some species, this kin-recognition ability results in
cooperation while in others it can induce more competitive and aggressive behaviors.
Importantly, in many species we do not know the mechanisms which regulate these inter-
actions. In the predatory nematode (round worm) Pristionchus pacificus, we have previ-
ously observed that they attack and kill other nematode larvae but their kin-recognition
PLOS GENETICS
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 1 / 20
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Hiramatsu F, Lightfoot JW (2023) Kin-
recognition and predation shape collective
behaviors in the cannibalistic nematode
Pristionchus pacificus. PLoS Genet 19(12):
e1011056. https://doi.org/10.1371/journal.
pgen.1011056
Editor: Kaveh Ashrafi, University of California San
Francisco, UNITED STATES
Received: July 24, 2023
Accepted: November 8, 2023
Published: December 14, 2023
Copyright: ©2023 Hiramatsu, Lightfoot. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All data is available in
the main text or the supplementary materials.
Funding: This work was funded by the Max Planck
Society and funded by the Deutsche
Forschungsgemeinschaft (DFG, German Research
Foundation) - project number 495445600 (JWL).
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: All authors declare they have
no competing interests.
abilities prevent them from harming their close relatives. Importantly, in P.pacificus we
have an abundance of genetic and molecular tools and can begin to understand how they
are able to carry out this recognition behavior. Here, we have shown that their kin-recog-
nition and predatory abilities also influence other interactions including how these nema-
todes group together. Many nematode species form aggregates to help them find food or
to avoid harmful environmental conditions. In P.pacificus, we have observed that they
preferentially aggregate with their close relatives and avoid grouping with more distantly
related strains. This may ensure that they give their relatives an advantage within their
ecological niche. Therefore, our work will help us understand more about the significance
of kin-recognition behaviors and their evolution.
Introduction
Kin-recognition is observed across many species and is associated with a diversity of different
behaviors. This includes behaviors in single celled organisms such as swarming behaviors in
bacteria [1], flocculation in yeast [2], and altruism in the amoeba Dictyostelium [3–5]. Addi-
tionally, behaviors in more complex organisms are also thought to be reliant on kin-recogni-
tion including cooperation in insects [6,7] and lizards [8,9], colony fusions events in tunicates
[10,11], cannibalism in several amphibian species [12,13], as well as nest mate preference in
rodents [14,15]. In nematodes a kin-recognition system was also recently identified in Pris-
tionchus pacificus. This is an omnivorous and cannibalistic nematode species and is capable of
attacking and killing the larvae of other nematodes as well as other P.pacificus con-specifics
[16,17]. These predatory behaviors likely provide a supplementary nutrient source as well as a
mechanism to remove potential competitors from their environment as both territorial behav-
iors and surplus killing have been described [18–20]. Furthermore, while P.pacificus can be a
voracious predator of other nematode larvae, it avoids killing its close relatives and progeny
through the existence of a hypervariable small peptide mediated kin-recognition system
[21,22]. However, previous P.pacificus kin-recognition studies have focused on the interac-
tions between predators and larvae, and little is known of its impact on larger scale group
behaviors. Importantly, these larger scale dynamics are likely to occur frequently due to the
boom-and-bust life history strategy employed by P.pacificus in its natural ecological setting
which temporarily results in a large concentration of nematodes around a confined niche [23].
While few studies on larger scale collective dynamics have taken place in P.pacificus, aggre-
gation behaviors have previously been reported in a high-altitude adapted clade as a mecha-
nism to avoid hyperoxia although it is likely other as yet unstudied factors will also induce
aggregation in this species [24–27]. Instead, nematode aggregation behaviors have been
intensely studied at the molecular and neuronal level in the model nematode Caenorhabditis
elegans. These behaviors in C.elegans are induced in response to various factors including
hyperoxia, aversive stimuli, food quantity and population density [28–31]. Accordingly, many
wild isolates of C.elegans show aggregation behaviors in which they group together on a bacte-
rial lawn and even exhibit dynamic swarming over longer time periods [32,33]. In contrast, the
C.elegans laboratory reference strain N2 does not aggregate due to the presence of a gain-of-
function mutation in the npr-1 gene and is considered to be a solitary strain [28,34]. This
behavioral difference is caused by a single amino acid change in this neuropeptide receptor
with gregarious wild isolates carrying a 215F allele version of npr-1 while in solitary strains a
215V version is found instead which suppresses this behavior. However, both variants have
been shown to confer fitness advantages depending on food availability and dispersal strategy
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 2 / 20
[35,36]. In addition to npr-1, aggregation is mediated through various receptors found in sen-
sory neurons which depend on the intraflagellar transport (IFT) machinery, several soluble
guanylate cyclases and components of the transforming growth factor beta (TGF-β) family
[29,30,37,38]. This includes the TGF-βdaf-7 pathway which acts in parallel to npr-1. Further-
more, pheromone signaling through small molecule ascarosides also promotes attraction and
aggregation which converge on the same neuronal circuits [39–41]. Intriguingly, while the IFT
system is essential for aggregation in P.pacificus, neither Ppa-npr-1 nor Ppa-daf-7 are required
for these behaviors [26,27,42]. Therefore, aggregation in P.pacificus is dependent on a distinct
mechanistic process that has evolved independently from those described in C.elegans.
Here, by exploring the specialized high-altitude adapted and aggregating clade of P.pacifi-
cus we identify kin-recognition as an essential component shaping aggregate formation. We
find aggregating strains of P.pacificus preferentially group with their own kin or close relatives
and avoid more divergent strains. Moreover, pairwise interactions between distantly related
strains reveal that the aggregation ratio is frequently reduced across both strains. This territori-
ality also extends to interactions of P.pacificus with other aggregating species including C.ele-
gans and reveals kin-recognition and competition as a previously unknown component
influencing collective behaviors in nematodes.
Results
Collective behaviors are found in many nematode species indicating its high degree of evolu-
tionary conservation [43]. Conversely, in P.pacificus most strains behave in a solitary manner
with aggregation only predominantly observed in one of the main P.pacificus evolutionary lin-
eages referred to as clade B [25]. However, it is likely that other unexplored factor may also
induce aggregation in this species (Fig 1A–1C). Strains belonging to this clade are frequently
found in a high-altitude environment and have adapted to lower oxygen concentrations [25].
As such, under normal laboratory conditions, aggregate formation is induced and animals
localize at the border of the bacterial lawn to avoid hyperoxia [24–27]. Furthermore, unlike in
C.elegans this aggregation behavior is observed even in the absence of a bacterial food source
with the exception of the C.elegans dauer state (S1 Fig) [31]. Importantly, like all other strains
of P.pacificus sampled so far, isolates from these locations are also predatory and kill the larvae
of other nematodes including other P.pacificus con-specifics (Fig 1D). They also possess kin-
recognition behaviors which prevent the killing of their own progeny and close relatives while
enabling the killing of more distantly related strains [21,22]. Therefore, we exploited the P.
pacificus clade B propensity to aggregate to investigate the influence of kin-recognition and its
associated predation on group behaviors in these nematodes. Accordingly, we selected four
strains from one high altitude location to investigate their interactions and ability to form
aggregates together. These strains were all originally isolated from the same area on the Nez de
Bœuf volcano peak (>2089 m above sea level) on La Reunion Island (Fig 1E) [44] and as such
also represent potentially ecologically relevant interactions. We selected RSB001 as it has previ-
ously formed the basis of numerous molecular studies [25,27,45,46], as well as a close relative
RSB005. We also selected another pair of strains (RSA075 and RSB033) which are distantly
related to RSB001 and RSB005 but are closely related to one another (Fig 1C).
Kin-recognition and predation influence aggregation behaviors
Having identified aggregating strains with differing degrees of genetic relatedness, we next
explored if aggregation behaviors occurred between these mixed P.pacificus populations. To
assess this, we established aggregation assays in which 120 worms (60 worms from each strain)
were placed onto a defined OP50 Escherichia coli bacterial lawn of 70 μl for 3 hrs. In order to
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 3 / 20
RSB001
PS312
La Reunion
Killing Picture
RSC002
RSB008
RSC001
RSC008
RSC003
RSB037
RSC045
RSB040
RSC100
RSC173
RSC006
RSB001
RSC005
RSB035
RSC010
RSC004
RSB005
RSC013
RS5361
RSA075
RSC172
RSC012
RSB038
RSB034
RSC007
RSC011
RSC036
RSB033
RSC035
RSB013
RSC009
B
A
1
A
3
A
2
C1
C2
0.02
PS312
AB
C
DE
Fig 1. P.pacificus aggregation behaviors are prevalent in a high altitude adapted clade. (A) The main laboratory P.
pacificus wild type strain, PS312 does not aggregate under standard laboratory conditions. (B) RSB001 is a high altitude
adapted strain which aggregates under standard laboratory conditions. Scale bar = 2000 μm. (C) P.pacificus
phylogenetic tree representing the genetic relationship between 323 wild isolates. Image adapted from Ro¨delsperger
et al 2017 [46]. The four strains selected for further analysis are highlighted in bold as well as the main laboratory strain
PS312. (D) SEM image of a P.pacificus predator (large right nematode) killing a C.elegans larvae (smaller left
nematode). (E) Strains selected for analysis were all isolated from the region circled which is a high-altitude
environment on La Reunion Island. Figure made with GeoMapApp [44].
https://doi.org/10.1371/journal.pgen.1011056.g001
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 4 / 20
differentiate and define strain specific behavioral interactions in mixed populations, we uti-
lized a previously established staining method using fluorescent vital dyes that has been dem-
onstrated to have no effect on the animal health [47] or its aggregation behavior (S2A–S2C
Fig). Under these conditions, all four strains aggregated strongly in control assays consisting of
only their own strain compared to a solitary strain. (Figs 2A, 2B and S2B–S2E). However, in
mixed assays consisting of two strains, we observed striking differences in aggregation depend-
ing on the combination of strains assessed. Specifically, aggregates between populations of the
relatively closely related pair RSB001 and RSB005 and the other related pair RSA075 and
RSB033 were consistent with controls and mostly aggregated together. However, assays
between RSB001 or RSB005 together with the more distantly related RSA075 or RSB033,
resulted in a significant decrease in aggregate formation (Fig 2A and 2B and S1 Movie). Fur-
thermore, the composition of aggregates also changed in mixed populations with aggregation
in one strain frequently reduced more than in the opposing strain (Fig 2B and 2C). Therefore,
between strain competition is a key element defining aggregate composition with P.pacificus
preferentially grouping with their own kin and close relatives and not more distantly related
strains.
As aggregation behaviors were disrupted between more distantly related strains but not
close relatives, we next investigated if this may be due to aggressive predatory behaviors
between strains. P.pacificus attacks other nematodes including other P.pacificus strains which
can result in the killing of these larvae; however, the thicker cuticle of the adults usually pre-
vents fatal interactions and instead induces an escape response (S2 Movie) [16,19]. Addition-
ally, the kin-recognition system prevents any aggressive interactions between a strain’s own
progeny as well as their close relatives [21,22]. Therefore, we utilized previously established
assays [16] to determine the predation outcome between our selected strains. We observed no
killing of self-progeny in all strains as expected and evidence of only very limited predatory
interactions between the closely related RSB001 and RSB005 as well as RSA075 together with
RSB033. However, predation between any of the more distantly related strains resulted in an
aggressive response with large numbers of larvae killed (Fig 2D). Therefore, between strain
aggregation is consistent with their kin-recognition and associated predatory interactions.
Thus, closely related strains such as RSB001 and RSB005 as well as RSA075 and RSB033 per-
ceive one another as kin, do not attack and are capable of aggregating together. Conversely,
more distantly related strains are regarded as non-kin, aggressively predate one another and
aggregate formation is disrupted.
Phenotypic plasticity and predatory interactions enforce kin aggregation
We next investigated the significance of the non-kin associated aggressive interactions on
aggregation behaviors in more detail by removing the ability of these strains to predate one
another. One mechanism to suppress the predatory behaviors in P.pacificus is through manip-
ulating their phenotypically plastic mouth as during development P.pacificus form one of two
distinct mouth morphs which are also associated with specific feeding behaviors [16]. This
irreversible developmental process results in the formation of either the stenostomatous (St)
mouth type which is strictly microbivorous and is characterized by a narrower mouth opening
and a single small dorsal tooth. Alternatively, animals can develop the eurystomatous (Eu)
morph which promotes an omnivorous diet including predatory behaviors and is defined by a
wider, shallower mouth structure, an enlarged dorsal tooth and an additional sub-ventral
tooth [48] (Fig 3A). The majority of P.pacificus strains exhibit an Eu bias under most condi-
tions assessed which suggests a propensity for these strains to aggressively compete with other
nematodes in their environment via predatory interactions [22,49]. Importantly, the P.
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 5 / 20
Fig 2. P.pacificus aggregates with kin and displaces non-kin. (A) Representative images of the aggregation
phenotypes of the P.pacificus strain RSB001 control as well as mixed cultures of RSB001 with RSB005 and RSB001
with RSB075. Scale bar = 2000 μm. (B) Quantification of aggregation ratios of P.pacificus cultures including RSB001,
RSB005, RSA075 and RSB033 controls as well as all pairwise permutations. Aggregation is disrupted between more
distantly related strains. Significant decreases in aggregation ratios were observed in assays between distantly-related
strains. Statistical tests were performed against control, unless there are bars above the boxplots for any between-pair
comparisons (Mann-Whitney-U-test with Bonferroni corrections). n = 10 per condition. (C) Heatmaps of pairwise
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 6 / 20
pacificus mouth morph fate is influenced by a panoply of well characterized genetic and envi-
ronmental factors providing a mechanism to manipulate the mouth form and subsequently
the P.pacificus predatory behavior [49–53]. The nuclear hormone receptor Ppa-nhr-40 is
required for the Eu mouth morph fate [51], and as such mutations in this gene result in 100%
of animals forming the St mouth form. As all our selected strains are highly Eu, we mutated
Ppa-nhr-40 in both RSB001 and RSA075 which succeeded in abolishing their predatory behav-
ior (Figs 3B,3C,S3A and S3B).
With the Ppa-nhr-40
RSB001
and Ppa-nhr-40
RSA075
non-predatory variants established, we
next explored the influence of their predatory behaviors on group selectivity and aggregate for-
mation. Mixed Ppa-nhr-40
RSB001
and Ppa-nhr-40
RSA075
mutant cultures show higher frequen-
cies of between strain aggregates than RSB001 and RSA075 wild type strains (Fig 3D–3F).
However, to investigate this further, we also conducted assays between nhr-40 mutants
together with their rival strain Eu morph. With only one strain capable of predatory behaviors
in these assays we predicted the Eu strain would dominate and form aggregates at the expense
of its Ppa-nhr-40 mutant opponent. Indeed, in both pairwise assays involving the St variant
with its Eu rival, aggregation in the St strain was significantly impaired (Fig 3E–3F). Thus, phe-
notypic plasticity and its associated predatory interactions are key components influencing
aggregation behaviors.
Mutations in self-1 are insufficient to disrupt aggregation
With aggregate formation observed between animals of the same genotype as well as between
kin but not more distantly related strains, we next explored the importance of the P.pacificus
molecular kin-recognition components for these interactions. The only molecular component
involved in this process that has been described so far is self-1 which encodes for a small pep-
tide containing a hypervariable C-terminus that is thought to be important for generating
between strain specificity. Mutations in self-1 cause a mild kin-recognition defect resulting in
predators erroneously killing their own offspring [21]. self-1 has been previously identified in
all of our four selected assay strains [22] (S4 Fig). As RSB001 has previously formed the basis
of numerous molecular studies we focused on this strain [25,27,45]. We generated a putative
self-1 null mutant (self-1.1) in RSB001, however surprisingly no kin-recognition defect was
observed (Fig 4A and 4B). Therefore, we analyzed the RSB001 genome further and identified a
further potential self-1 paralogue which shares 72.9% sequence identity at the amino acid level
outside of the hypervariable domain (Fig 4A). This additional copy was designated self-1.2and
a subsequent CRISPR/Cas9 induced putative null mutation successfully phenocopied the mod-
est kin-defective phenotype previously described in self-1 mutants in other strains (Fig 4A and
4B) [21]. Additionally, a self-1.1; self-1.2double mutant revealed a stronger kin-killing defect
than the self-1.2single mutant alone (Fig 4B). Therefore, we next assessed if the kin-recogni-
tion defect associated with these self-1 mutations was sufficient to also disrupt aggregation
behaviors. In aggregation assays consisting of either mutants alone, or assays containing an
equal amount of RSB001 together with self-1 mutants, aggregation was maintained at wild type
control levels despite the kin-recognition defect (Fig 4B–4E). Therefore, the mild kin-recogni-
tion defect caused by these mutations is insufficient to disrupt aggregation. With recent studies
interactions revealing the proportion of each strain in an aggregate. The two axes each represents the number of
animals from the two interacting strains present in an aggregate, with the color indicating the percentage of the
population found there. (D) Quantification of predation assays revealing killing between all possible pairwise
permutations. Increased killing is observed between more distantly related strains. n = 21 for RSB001 predators on
RSB001 prey, n = 15 for RSB001 predators on RSB005 prey, n = 13 for RSB001 predators on RSA075 prey, RSB005
predators on RSB005 prey, and n = 10 for each of the other conditions.
https://doi.org/10.1371/journal.pgen.1011056.g002
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 7 / 20
eurystomatous
eurystomatous
stenostomatous
stenostomatous
dorsal
subventral
AB
E
D
RSB001
RSA075
% eurystomatous
100
80
60
40
20
0
RSA075
RSB001
RSB001 RSA075
Predators:
Prey:
50
40
30
20
10
0
number of corpses
nhr-40 RSB001 nhr-40 RSA075 nhr-40 RSB001
nhr-40 RSA075
C
nhr-40
(RSA075)
nhr-40
(RSB001)
nhr-40
(RSA075)
nhr-40
(RSB001)
F
nhr-40 (RSA075) (animals/aggregate)
RSB001 (animals/aggregate)
RSA075 (animals/aggregate)
nhr-40 (RSB001) (animals/aggregate)
nhr-40 (RSA075) (animals/aggregate)
nhr-40 (RSB001) (animals/aggregate)
total number of animals (%)
RSA075
RSB001
RSA075
RSB001
RSB001
RSA075
nhr-40
(RSB001)
nhr-40
(RSA075)
nhr-40
(RSA075)
nhr-40
(RSA075)
nhr-40
(RSB001)
nhr-40
(RSB001)
control pairwise pairwise pairwise pairwise
ns ns ns
ns ns ns
** **
**
***
***
** ***
*
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 8 / 20
demonstrating that additional as yet unidentified components must also contribute to the kin-
recognition signal [22], we predict that mutations in these other elements may result in stron-
ger kin-recognition defects which could be sufficient to also influence and disrupt aggregation
behavior.
P.pacificus territorial biting prevents C.elegans aggregation
Interactions between con-specific P.pacificus likely occur frequently in their natural habitat as
strains compete to occupy their preferred ecological niche. Additionally, P.pacificus also
comes into contact with other nematode species which frequently includes rhabditid species
and rarely C.elegans although, as yet, C.elegans has not been found in the high-altitude envi-
ronments where the clade B strains are located [54,55]. The P.pacificus predatory ability how-
ever has previously been implicated in territorial behaviors against C.elegans [19,56].
Therefore, we next assessed conspecific aggregation interactions between diverse C.elegans
strains as well as the impact of P.pacificus on the aggregation ability of these wild C.elegans
isolates. We selected two strains of C.elegans which both aggregate strongly, CB4856 isolated
from the Hawaiian Islands, and JU2001 from La Reunion Island (Fig 5A). Utilizing the same
fluorescent dye method employed to stain P.pacificus [47], we firstly explored pairwise aggre-
gation assays between these con-specific C.elegans strains. Here, both strains successfully
aggregated with one another at similar levels to with their own strain indicating the presence
of another distantly related rival did not disrupt aggregation in C.elegans (Fig 5A–6C). Next,
we challenged the C.elegans aggregating strains by pairing them with the aggregating P.pacifi-
cus strain RSB001, a non-aggregating P.pacificus strain PS312, or a P.pacificus St mutant
strain Ppa-nhr-40
PS312
which could therefore not predate. In these mixed species assays, we
observed C.elegans failed to aggregate and instead behaved in a more solitary manner only
when it was challenged with P.pacificus strains capable of predation (Fig 6A–6C). Finally, as P.
pacificus strongly displaces C.elegans from aggregates, we analyzed the potency of P.pacificus
for influencing these C.elegans behaviors by reducing the representation of P.pacificus present
in mixed species pairwise assays. Here, we observed C.elegans aggregation behaviors were dis-
rupted when only 17% of the assay population was P.pacificus (Fig 6D–6E). Additionally,
under these conditions C.elegans aggregates were mostly only maintained when they consisted
of large numbers of animals. Therefore, C.elegans aggregates above a density threshold may be
sufficient to resist predation induced displacement and may represent an ecological relevant
strategy to counter the effects of P.pacificus predation (S5A and S5B Fig). Thus, a minimal P.
pacificus presence is capable of shaping the aggregation behaviors of other species such as C.
elegans. These interactions may therefore represent a territorial behavior conferring an ecolog-
ically relevant and advantageous state for P.pacificus within their shared environmental niche.
Fig 3. Mouth form plasticity and associated predation behaviors influence aggregation. (A) P.pacificus mouth dimorphism. The
eurystomatous mouth has a wide buccal cavity with two teeth and is omnivorous feeding on both bacteria and other nematodes while the
stenostomatous mouth is narrow with a single tooth and microbivorous. Scale bar = 5 μm. (B) Mutations in Ppa-nhr-40 in both RSB001 and
RSA075 change the mouth form frequency from highly Eu to 100% St. (C) Killing assays comparing the ability of RSA075 and Ppa-nhr-40
RSA075
to predate upon RSB001 prey and the reciprocal assays of RSB001 and Ppa-nhr-40
RSB001
to predate upon RSA075 prey. Mutations in
Ppa-nhr-40
RSB001
and Ppa-nhr-40
RSA075
remove the ability of these strains to predate one another. n = 10 per condition. (D) Representative
images of the aggregation phenotypes of the P.pacificus Ppa-nhr-40
RSB001
and Ppa-nhr-40
RSA075
controls as well as mixed cultures of Ppa-
nhr-40
RSB001
and Ppa-nhr-40
RSA075
which aggregate together. Scale bar = 2000 μm. (E) Quantification of aggregation ratios in P.pacificus
cultures including of RSB001, RSA075, Ppa-nhr-40
RSB001
, and Ppa-nhr-40
RSA075
controls as well as all pairwise permutations. Significant
decreases in aggregation ratios of Ppa-nhr-40 mutants were observed when assays were performed between the mutants and the wildtype of a
distantly-related strain. Statistical tests were performed against control, unless there are bars above the boxplots for any between-pair
comparisons (Mann-Whitney-U-test with Bonferroni corrections). n = 10 replicates. (F) Heatmaps of pairwise interactions between different
combinations of Ppa-nhr-40 mutants and the corresponding wildtype strains, revealing the proportion of each strain in an aggregate.
https://doi.org/10.1371/journal.pgen.1011056.g003
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 9 / 20
self-1.1/1.2 RSB001 RSB001
self-1.1/1.2 RSB001
BC
E
4 bp deletion
49bp insertion and 3bp deletion
MWKIFVALLALIGLAASAQFEQSSGVQAIGSDATSPLIMRLKRKPAGWETQGHRSKRKVRVG
MWKIFVALLALIGLAASAQFEQSSG AQSAL*
MWKILVALLALFALAASAQFEQTSGVQAVASEAAFAAPLSLRVRRKPAGWETQGHRIKRVGQNGKK
MWKILVALLALFAL H*
self-1.1
RSB001
self-1.2
RSB001
WT
bnn5
WT
bnn10
A
D
self-1.1
(RSB001)
self-1.2
(RSB001)
self-1.1/1.2
(RSB001)
RSB001
self-1.1
(RSB001)
RSB001
RSB001
self-1.2
(RSB001)
RSB001
self-1.1/1.2
(RSB001)
control pairwise pairwise pairwise
ns ns ns ns
*
*
RSB001
self-1.1
(RSB001)
self-1.2
(RSB001)
self-1.1/1.2
(RSB001)
RSB005
RSA075
RSB033
RSB001
Prey:
Predator:
self-1.1 (RSB001) (animals/aggregate)
RSB001 (animals/aggregate)
self-1.2 (RSB001) (animals/aggregate)
RSB001 (animals/aggregate)
self-1.1/1.2 (RSB001) (animals/aggregate)
RSB001 (animals/aggregate)
total number of animals (%)
Fig 4. The kin-recognition component SELF-1 is dispensable for aggregate formation. (A) Predicted gene structure of self-1.1and self-1.2 in strain
RSB001. CRISPR/Cas9 target sites are highlighted across both genes. Mutations generated resulted in severely truncated proteins and putative null
mutants. Predicted wildtype protein sequences as well as associated mutations are shown. Scale bar = 100 bp, (Scissors image from openclipart.org).
(B) Quantification of predation assays revealing the modest kin-killing phenotype caused by mutations in self-1. n = 21 for RSB001 prey, n = 13 for
RSB005 prey, n = 12 for RSA075 prey and n = 10 for RSB033 prey, self-1.1
RSB001
prey, self-1.2
RSB001
prey and self-1.1; self-1.2
RSB001
prey. (C)
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 10 / 20
Discussion
Group behavior in nematodes including aggregate formation emerge from local interactions
between individuals [33]. In P.pacificus however, an additional layer of complexity is evident
through its predatory capacity which functions to broaden potential food opportunities and as
a mechanism to remove competitors from their local environment [18–22] as well as its kin-
recognition ability which prevents attacks on close relatives [21,22]. In our work, we show
these elements combine to also impact their collective behaviors as aggregation is favored
between kin while predatory attacks between distantly related con-specifics result in the dis-
placement of their rivals and the induction of less preferable behavioral strategies. Moreover,
in pairwise assays between distantly related strains, we also observed the frequency of mono-
typic aggregation increases. Importantly, the exclusion of potential competitors from optimal
Representative images of the aggregation phenotypes of the self-1.1; self-1.2
RSB001
mutant as well as mixed cultures of self-1.1; self-1.2
RSB001
with
RSB001. Scale bar = 2000 μm. (D) Quantification of aggregation ratios of P.pacificus cultures including of RSB001 and self-1 mutants alone as well as
mixed cultures of RSB001 together with self-1 mutants. Mutations in self-1.1; self-1.2
RSB001
are not sufficient to disrupt aggregation. Statistical tests
were performed against control (Mann-Whitney-U-test with Bonferroni corrections). n = 10 per condition. (E) Heatmaps of pairwise interactions
between RSB001 and self-1.1
RSB001
, RSB001 and self-1.2
RSB001
, and RSB001 and self-1.1; self-1.2
RSB001
revealing the proportion of each strain in an
aggregate. Mixed RSB001 and the self-1 mutants cultures aggregate together.
https://doi.org/10.1371/journal.pgen.1011056.g004
JU2001
CB4856 JU2001
CB4856
A
CB ns
ns
control pairwise
Fig 5. C.elegans pairwise interactions between aggregating strains. (A) Representative images of the aggregation phenotypes of the C.
elegans strains CB4856 (Hawaiian) and JU2001 (La Reunion) controls as well as mixed cultures of CB4856 and JU2001 together. Scale
bar = 2000 μm. (B) Quantification of aggregation ratios of C.elegans cultures including of CB4856 and JU2001alone as well as mixed
cultures of CB4856 and JU2001 together. Both C. elegans strains aggregate together abundantly. Statistical tests were performed against
control (Mann-Whitney-U-test with Bonferroni corrections). n = 10 per condition.(C) Heatmap of pairwise interactions between of
CB4856 and JU2001 revealing the proportion of each strain in an aggregate. Mixed CB4856 and JU2001 cultures form frequent aggregates
together.
https://doi.org/10.1371/journal.pgen.1011056.g005
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 11 / 20
***
*** ***
*** ns
***
RSB001
RSB001
RSB001
JU2001
JU2001
JU2001
JU2001
CB4856
CB4856
CB4856
CB4856
PS312
PS312
PS312
nhr-40
(PS312)
nhr-40
(PS312)
nhr-40
(PS312)
control pairwise pairwise pairwise pairwise pairwise pairwise
ns
ns ns
*
**
**
***
***
***
***
***
***
ns
***
** ***
***
CB4856 (animals/aggregate)
RSB001 (animals/aggregate)
CB4856 (animals/aggregate)
PS312 (animals/aggregate)
CB4856 (animals/aggregate)
nhr-4
0
(PS312) (animals/aggregate)
JU2001 (animals/aggregate)
RSB001 (animals/aggregate)
JU2001 (animals/aggregate)
PS312 (animals/aggregate)
JU2001 (animals/aggregate)
nhr-4
0
(PS312) (animals/aggregate)
total number of animals (%)
Fig 6. pacificus interferes with C.elegans aggregation behaviors. P.(A) Representative images of the aggregation
phenotypes with P.pacificus in mixed cultures together with C.elegans strains CB4856 (Hawaiian) or JU2001 (La
Reunion). Scale bar = 2000 μm. (B) Quantification of aggregation ratios of P.pacificus aggregating strain RSB001,
solitary strain PS312, the St mutant strain Ppa-nhr-40
PS312
and C.elegans CB4856 and JU2001 controls as well as mixed
cultures of each P.pacificus strain together with each of the C.elegans strain. Both RSB001 and PS312 disrupts the
aggregation ability of both C.elegans strains while Ppa-nhr-40
PS312
does not. Statistical tests were performed against
control, unless there are bars above the boxplots for any between-pair comparisons (Mann-Whitney-U-test with
Bonferroni corrections). n = 10 per condition. (C) Heatmap of pairwise interactions between P.pacificus RSB001,
PS312, and Ppa-nhr-40
PS312
together with C.elegans CB4856 or JU2001 revealing the proportion of each strain in an
aggregate. Increased aggregations were observed between the St mutant strain and C.elegans strains. (D)
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 12 / 20
kin groupings is not unique to P.pacificus and has been observed in many organisms. This
includes in slime molds which aggregate together in fruiting bodies under stressful conditions.
This behavior depends on the between strains compatibility of the cell surface csA gene which
ultimately facilitates admission to the fruiting body [5,57]. Additionally, in social yeast species,
robust cell adhesion molecules promote efficient homogenous aggregate and biofilm forma-
tion while weak adhesive forces between non-kin result in their exclusion [2,58]. However,
these mechanisms of selectivity and omission depend on preventing aggregations between
non-kin which differs from our observations in P.pacificus whereby predation provides an
active mechanism to displace competitors.
Subsequently, we also demonstrated that the aggressive displacement of potential competi-
tors from aggregates is at least partially dependent on the predatory Eu mouth form. This is
the most prevalent morph found in wild P.pacificus isolates including those from the high alti-
tude adapted clade utilized in these studies [22,49]. As all strains of P.pacificus are capable of
also developing the non-predatory St morph, aggregation behaviors are therefore theoretically
possible even between highly divergent strains which may prove to be beneficial under certain
environmental conditions. In addition to the role of P.pacificus predation, previous studies
have identified several genetic components that are necessary for P.pacificus aggregation. In
particular, mutations in the IFT system cause a solitary strain of P.pacificus to aggregate [25–
27,37]. Therefore, in the future, it will be important to assess the impact of these mutations on
con-specific aggregation behaviors which may aid in establishing more of the sensory mecha-
nisms involved however, due to their substantial influence on mouth morph fate, these experi-
ments will not be straightforward.
While predation and kin-recognition appear to play an integral role in aggregation behav-
iors in P.pacificus, neither behavior has been observed in C.elegans. Accordingly, we observed
aggregation between distantly related social strains of C.elegans with no apparent effect on
their behaviors. This is consistent with previous studies in which a mixed population of C.ele-
gans including an aggregating strain and an evolutionary distinct solitary strain, each main-
tained their specific behavioral phenotypes when grown together [35]. However, in mixed
assays between aggregating C.elegans and P.pacificus strains, aggregation was hindered in C.
elegans while maintained in P.pacificus. Furthermore, the presence of relatively few P.pacificus
predators proved sufficient to modulate the aggregation behavior in C.elegans suggesting a
minimal amount of P.pacificus animals are capable of altering the group dynamics of a much
larger competing nematode population. Additionally, as P.pacificus develops slower and have
a smaller brood size than C.elegans [59,60], the ability to alter the behavior and outcompete
other nematodes such as C.elegans may be highly beneficial. Moreover, this may represent an
additional facete to the previously characterized P.pacificus territorial behaviors which have
been shown to disrupt C.elegans ability to approach and enter shared bacterial lawns [19]. In
the future, it will be essential to explore other effects which may influence these aggregation
dynamics and which are likely to be influenced by a suite of sensory inputs. These may include
signals as diverse as oxygen [25], kin-relevant signals [21], chemical messages such as ascaro-
sides [61,62], and alarm pheromones which are released from injured nematodes and which
may be triggered by the predatory events [63].
Representative images of the aggregation phenotypes with a reduced ratio of P.pacificus to C.elegans CB4856 in mixed
cultures assays. Scale bar = 2000 μm. (E) Quantification of aggregation assays with a reduced ratio of P.pacificus
RSB001 to C.elegans CB4856. Even a minimal P.pacificus presence is capable of shaping the aggregation behaviors of
C.elegans. Statistical tests were performed against control, unless there are bars above the boxplots for between-pair
comparisons (Mann-Whitney-U-test with Bonferroni corrections). n = 13 for 100% CB4856 and n = 10 for each of the
other conditions.
https://doi.org/10.1371/journal.pgen.1011056.g006
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 13 / 20
With our work revealing a previously unknown influence for kin-recognition and its associ-
ated predation on collective behaviors in P.pacificus, questions remain regarding what other
behaviors may also be affected by these abilities. For example, recent work in C.elegans has
demonstrated that their swimming gait is influenced by the presence of other nearby animals
[64]. Additionally, more diverse collective behaviors including swarming have been described
in C.elegans [32,33] and groups of both C.elegans and P.pacificus are capable of forming large
3D tower like structures thought to aid in their dispersal to new environments [54,65]. There-
fore, how predation and kin-recognition may influence these other collective behaviors
remains unknown and will form the basis of future studies. Thus, kin-recognition abilities in
P.pacificus facilitate a diverse behavioral repertoire of interactions. This includes the territorial
removal of competitors via predatory events, as well as between kin-aggregation in which indi-
viduals nepotistically favor grouping with relatives as rivals take on less preferable behavioral
strategies. Furthermore, with the wealth of molecular tools available, P.pacificus offers a pow-
erful means for understanding the genetic and neural mechanisms behind these kin-recogni-
tion mediated interactions and their evolution.
Materials and methods
Nematode Husbandry
All nematodes used were maintained on standard NGM plates on a diet of Escherichia coli
OP50. All strains used in this study can be found in the S1 Table.
Aggregation assays
Aggregation assays were utilized to quantify aggregation behaviors between different strains.
Assay plates were prepared two days prior to the experiment. 6 cm NGM plates were seeded
with 70 μl of OP50 bacteria and incubated at room temperature. All animals were maintained
on NGM plates seeded with OP50 bacteria until freshly starved, resulting in an abundance of
young larvae. These plates were washed with M9 and passed through two 20 μm filters to iso-
late pure cultures of larvae. They were centrifuged and transferred onto new NGM plates
seeded with OP50 bacteria and incubated at 20˚C for three days until they become young
adults. They were then washed with M9 and transferred into 1.5 ml tubes, containing 50 μM of
dye (CellTracker Green BODIPY Dye or CellTracker Orange CMRA Dye), and incubated on a
rotator in dark at 20˚C for 3 h. The worms were then washed with M9 four times and trans-
ferred into an unseeded NGM plate. For controls, 120 worms from one strain were picked and
transferred to an assay plate, outside of the OP50 bacterial lawn. For pairwise aggregation
assays, 60 worms per strain from two strains were transferred to an assay plate. The plates
were incubated at 20˚C in dark for 3 h, then imaged using a fluorescent microscope ZEISS
Axio Zoom.V16 with ZEN (blue edition) software. Images were analyzed manually with Fiji. It
was considered an aggregate when two or more animals were in contact along 50% of their
body length. The staining with CellTracker dyes method was adapted from Werner et al [47]
and pairwise aggregation assay was adapted from Moreno et al [25]. The number of animals in
solitary state as well as in each aggregate are manually scored from a static image of the assay
plate. The aggregation ratio is calculated by: (total number of animals of strain A in aggregates)
/ (total number of animals of strain A found on the assay plate).
Off food aggregation assay
Off food aggregation assays were utilized to investigate aggregation behaviors of nematodes in
an environment without bacterial food (OP50). Animals are well-fed and are washed prior to
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 14 / 20
the experiment. 360 worms of the same strain were transferred on a 3.5 cm NGM plate. The
plates were incubated at 20˚C for 1 h. Images were taken using brightfield on microscope
ZEISS Axio Zoom.V16 with ZEN (blue edition) software.
CRISPR/Cas9 induced mutations
Mutations were induced in candidate genes via CRISPR/Cas9. Gene specific crRNA and uni-
versal trans-activating CRISPR RNA (tracrRNA) was purchased from Integrated DNA Tech-
nologies and 5 μL of each 100 μM stock mixed and denatured at 95˚C for 5 min before cooling
at room temperature to anneal. Cas9 (Integrated DNA Technologies) was added to the hybrid-
ized product and incubated at room temperature for 5 mins. This was subsequently diluted
with TE buffer to a final concentration of 18.1 μM for the sgRNA and 2.5 μM Cas9. This was
injected into the germline of the required P.pacificus strain. Eggs from injected P0s were
recovered up to 16 h post injection. After hatching and 2 days’ growth these F1 were segregated
onto individual plates until they had also developed sufficiently and egg laying had been initi-
ated. The genotype of the F1 animals were subsequently analyzed via Sanger sequencing and
mutations identified before re-isolation in homozygosis. sgRNAs and associated primers uti-
lized in this study can be found in S2 Table.
Mouth-form
Mouth form phenotyping was achieved by observation of the nematode buccal cavity using
ZEISS Axio Zoom.V16 microscope with morph identities based on previous described species
characteristics [49]. Final mouth-form frequencies are the mean of 3 independent replicates,
each assaying 100 animals.
Predation and Kin-recognition assays
Corpse assays facilitated rapid quantification of predatory behaviors between different
strains. Prey were maintained on NGM plates seeded with OP50 bacteria until freshly
starved, resulting in an abundance of young larvae. These plates were washed with M9 and
passed through two 20 μm filters to isolate pure cultures of larvae. They were subsequently
centrifuged before being deposited on to an unseeded assay plate by pipetting 1.5 μl of P.
pacificus or 1.0 μl of C.elegans larval pellet on to a 6 cm NGM unseeded plate. Five preda-
tory nematodes were screened for the appropriate mouth morph and added to assay plates
for prey assays. They were then permitted to feed on the prey for 2 h and the plate was sub-
sequently screened for the presence of corpses. As self-1 mutants are only mildly kin-recog-
nition defective it is necessary to conduct predation assays over 20 h before screening the
plate for the presence of corpses.
Statistical analysis
Statistical tests were performed using Python with Scipy and statsmodels libraries. Box plot
represents the first quartile (Q1) to the third quartile (Q3) of the data with a line at the median
(Q2). Q1, Q2, and Q3 are the values which lie at 25%, 50% and 75% of the data points. Whis-
kers represent the range in which most values are found, whereas values outside this range are
presented as outliers. The statistical tests were always performed against the control condition
of the same strain. Non-significant (ns), p-value �0.05 (*), p-value �0.01 (**), p-
value �0.001 (***), p-value �0.0001 (****).
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 15 / 20
Supporting information
S1 Fig. The solitary C.elegans strain N2 and social CB4856 both do no aggregate in the
absence of bacteria. The P.pacificus PS312 solitary strain also does not aggregate in the
absence of food however the social RSB001 continues to aggregate. Scale bar = 2000 μm.
(PDF)
S2 Fig. (A) Vital dyes allow fluorescent labelling of distinct populations for identification in
mixed cultures. Scale bar = 200 μm. (B) Aggregation is not affected by either CellTracker
Green BODIPY Dye or CellTracker Orange CMRA Dye staining. Aggregation for RSB001 is
shown. (C) All strains form frequent aggregates under standard laboratory conditions and are
not affected by CellTracker Orange CMRA Dye (left image) or CellTracker Green BODIPY
Dye (right image). Scale bar = 2000 μm. (D) Heatmaps of aggregation size distribution in con-
trol aggregation assays of each strain. (E) Quantification of aggregation ratios in the non-
aggregating strain PS312 and the aggregating strain RSB001. Minimal transient aggregates
occur in PS312. n = 10 per strain.
(PDF)
S3 Fig. (A) Gene structure and CRISPR target site for Ppa-nhr-40 mutations in both RSB001
and RSA075. Mutations were successfully generated in both strains. Scale bar = 1kb. (Scissors
image from openclipart.org) (B) Mutations result in a frame shift in both RSB001 and RSA075
strains which leads to a putative premature stop codon and a truncated protein.
(PDF)
S4 Fig. self-1 is present in all four strains used in the study. This includes variable copy
numbers and distinct hypervariable regions in RSB001, RSB005, RSA075 and RSB033.
(PDF)
S5 Fig. (A) Representative aggregation assay with a reduced ratio of P.pacificus RSB001 to C.
elegans CB4856. Occasionally C.elegans aggregates were established consisting of large num-
bers of C.elegans (marked *) which may be sufficient to resist predation induced displacement.
Scale bar = 2000 μm. (B) Heatmaps of pairwise interactions between reduced ratio of P.pacifi-
cus RSB001 and increased ratio of C.elegans CB4856 revealing the proportion of each strain in
an aggregate.
(PDF)
S1 Table. List of all strains used and alleles associated with the mutations.
(XLSX)
S2 Table. List of primers and CRISPR/Cas9 associated sequences for generating mutants.
(XLSX)
S1 Movie. Movie showing pairwise assay between two competing strains. RSA075 is stained
yellow and RSB001 is stained in red.
(AVI)
S2 Movie. Movie showing biting interaction between adults of the distantly related strains
RSB001 and RSA075. A RSB001 predator (top most animal) approaches and makes contact
with the RSA075 aggregate. Nose contact and a probable bite causes one RSA075 to temporar-
ily leave the aggregate before returning.
(MOV)
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 16 / 20
Acknowledgments
We would like to thank Marianne Roca and Monika Scholz for discussion and critical reading
of the manuscript as well as Nurit Zorn for strain maintenance (MPI Neurobiology of Behav-
ior–caesar, Bonn). Additionally, we wish to thank the Sommer lab for P.pacificus strains,
Christian Ro¨delsperger for phylogenetic tree data and bioinformatic discussions (MPI for Biol-
ogy, Tu¨bingen), Marie-Anne Felix and Aure
´lien Richaud for C.elegans strain JU2001 (IBENS,
Paris) and Bogdan Sieriebriennikov (New York University) for mouth form images. Finally,
some strains were provided by the CGC, which is funded by NIH Office of Research Infra-
structure Programs (P40 OD010440).
Author Contributions
Conceptualization: Fumie Hiramatsu, James W. Lightfoot.
Formal analysis: Fumie Hiramatsu.
Investigation: Fumie Hiramatsu.
Methodology: Fumie Hiramatsu, James W. Lightfoot.
Supervision: James W. Lightfoot.
Visualization: Fumie Hiramatsu.
Writing – original draft: James W. Lightfoot.
Writing – review & editing: Fumie Hiramatsu, James W. Lightfoot.
References
1. Gibbs KA, Urbanowski ML, Greenberg EP. Genetic Determinants of Self Identity and Social Recogni-
tion in Bacteria. Science. 2008; 321(5886):256–9. https://doi.org/10.1126/science.1160033 PMID:
18621670
2. Smukalla S, Caldara M, Pochet N, Beauvais A, Guadagnini S, Yan C, et al. FLO1 Is a Variable Green
Beard Gene that Drives Biofilm-like Cooperation in Budding Yeast. Cell. 2008; 135(4):726–37. https://
doi.org/10.1016/j.cell.2008.09.037 PMID: 19013280
3. Strassmann JE, Zhu Y, Queller DC. Altruism and social cheating in the social amoeba Dictyostelium
discoideum. Nature. 2000; 408(6815):965–7. https://doi.org/10.1038/35050087 PMID: 11140681
4. Hirose S, Benabentos R, Ho HI, Kuspa A, Shaulsky G. Self-Recognition in Social Amoebae Is Mediated
by Allelic Pairs of Tiger Genes. Science. 2011; 333(6041):467–70. https://doi.org/10.1126/science.
1203903 PMID: 21700835
5. Queller DC, Ponte E, Bozzaro S, Strassmann JE. Single-Gene Greenbeard Effects in the Social
Amoeba Dictyostelium discoideum. Science. 2003; 299(5603):105–6. https://doi.org/10.1126/science.
1077742 PMID: 12511650
6. Keller L, Ross KG. Selfish genes: a green beard in the red fire ant. Nature. 1998; 394(6693):573–5.
7. Arnold G, Quenet B, Cornuet JM, Masson C, Schepper BD, Estoup A, et al. Kin recognition in honey-
bees. Nature. 1996; 379(6565):498–498.
8. Sinervo B, Chaine A, Clobert J, Calsbeek R, Hazard L, Lancaster L, et al. Self-recognition, color signals,
and cycles of greenbeard mutualism and altruism. Proc National Acad Sci. 2006; 103(19):7372–7.
https://doi.org/10.1073/pnas.0510260103 PMID: 16651531
9. Sinervo B, Clobert J. Morphs, Dispersal Behavior, Genetic Similarity, and the Evolution of Cooperation.
Science. 2003; 300(5627):1949–51. https://doi.org/10.1126/science.1083109 PMID: 12817150
10. Voskoboynik A, Newman AM, Corey DM, Sahoo D, Pushkarev D, Neff NF, et al. Identification of a Colo-
nial Chordate Histocompatibility Gene. Science. 2013; 341(6144):384–7. https://doi.org/10.1126/
science.1238036 PMID: 23888037
11. Tomaso AWD, Nyholm SV, Palmeri KJ, Ishizuka KJ, Ludington WB, Mitchel K, et al. Isolation and char-
acterization of a protochordate histocompatibility locus. Nature. 2005; 438(7067):454–9. https://doi.org/
10.1038/nature04150 PMID: 16306984
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 17 / 20
12. Pfennig DW, Reeve HK, Sherman PW. Kin recognition and cannibalism in spadefoot toad tadpoles.
Anim Behav. 1993; 46(1):87–94.
13. Pfennig DW, Collins JP. Kinship affects morphogenesis in cannibalistic salamanders. Nature. 1993;
362(6423):836–8. https://doi.org/10.1038/362836a0 PMID: 8479520
14. Green JP, Holmes AM, Davidson AJ, Paterson S, Stockley P, Beynon RJ, et al. The Genetic Basis of
Kin Recognition in a Cooperatively Breeding Mammal. Curr Biol. 2015; 25(20):2631–41. https://doi.org/
10.1016/j.cub.2015.08.045 PMID: 26412134
15. Clemens AM, Wang H, Brecht M. The lateral septum mediates kinship behavior in the rat. Nat Commun.
2020; 11(1):3161. https://doi.org/10.1038/s41467-020-16489-x PMID: 32572024
16. Wilecki M, Lightfoot JW, Susoy V, Sommer RJ. Predatory feeding behaviour in Pristionchus nematodes
is dependent on phenotypic plasticity and induced by serotonin. J Exp Biol. 2015; 218(9):1306–13.
https://doi.org/10.1242/jeb.118620 PMID: 25767144
17. Ishita Y, Chihara T, Okumura M. Different combinations of serotonin receptors regulate predatory and
bacterial feeding behaviors in the nematode Pristionchus pacificus. G3 Genes Genomes Genetics.
2021;11(2):jkab011-.
18. Akduman N, Lightfoot JW, Ro
¨seler W, Witte H, Lo WS, Ro
¨delsperger C, et al. Bacterial vitamin B12 pro-
duction enhances nematode predatory behavior. Isme J. 2020; 14(6):1494–507. https://doi.org/10.
1038/s41396-020-0626-2 PMID: 32152389
19. Quach KT, Chalasani SH. Flexible reprogramming of Pristionchus pacificus motivation for attacking
Caenorhabditis elegans in predator-prey competition. Curr Biol. 2022; https://doi.org/10.1016/j.cub.
2022.02.033 PMID: 35259340
20. Quach KT, Chalasani SH. Intraguild predation between Pristionchus pacificus and Caenorhabditis ele-
gans: a complex interaction with the potential for aggressive behaviour. J Neurogenet. 2020;1–16.
https://doi.org/10.1080/01677063.2020.1833004 PMID: 33054476
21. Lightfoot JW, Wilecki M, Ro
¨delsperger C, Moreno E, Susoy V, Witte H, et al. Small peptide–mediated
self-recognition prevents cannibalism in predatory nematodes. Science. 2019; 364(6435):86–9. https://
doi.org/10.1126/science.aav9856 PMID: 30948551
22. Lightfoot JW, Dardiry M, Kalirad A, Giaimo S, Eberhardt G, Witte H, et al. Sex or cannibalism: Polyphen-
ism and kin recognition control social action strategies in nematodes. Sci Adv. 2021; 7(35):eabg8042.
https://doi.org/10.1126/sciadv.abg8042 PMID: 34433565
23. Renahan T, Lo W, Werner MS, Rochat J, Herrmann M, Sommer RJ. Nematode biphasic ‘boom and
bust’ dynamics are dependent on host bacterial load while linking dauer and mouth-form polyphenisms.
Environ Microbiol. 2021; https://doi.org/10.1111/1462-2920.15438 PMID: 33587771
24. McGaughran A, Ro
¨delsperger C, Grimm DG, Meyer JM, Moreno E, Morgan K, et al. Genomic Profiles
of Diversification and Genotype–Phenotype Association in Island Nematode Lineages. Mol Biol Evol.
2016; 33(9):2257–72. https://doi.org/10.1093/molbev/msw093 PMID: 27189551
25. Moreno E, McGaughran A, Ro
¨delsperger C, Zimmer M, Sommer RJ. Oxygen-induced social behav-
iours in Pristionchus pacificus have a distinct evolutionary history and genetic regulation from Caenor-
habditis elegans. Proc Royal Soc B Biological Sci. 2016; 283(1825):20152263. https://doi.org/10.1098/
rspb.2015.2263 PMID: 26888028
26. Moreno E, Lenuzzi M, Ro
¨delsperger C, Prabh N, Witte H, Roeseler W, et al. DAF-19/RFX controlscilio-
genesis and influences oxygen-induced social behaviors in Pristionchus pacificus. Evol Dev. 2018; 20
(6):233–43. https://doi.org/10.1111/ede.12271 PMID: 30259625
27. Moreno E, Sieriebriennikov B, Witte H, Ro
¨delsperger C, Lightfoot JW, Sommer RJ. Regulation of hyper-
oxia-induced social behaviour in Pristionchus pacificus nematodes requires a novel cilia-mediated envi-
ronmental input. Sci Rep-uk. 2017; 7(1):17550. https://doi.org/10.1038/s41598-017-18019-0 PMID:
29242625
28. Bono M de, Bargmann CI. Natural Variation in a Neuropeptide Y Receptor Homolog Modifies Social
Behavior and Food Response in C. elegans. Cell. 1998; 94(5):679–89. https://doi.org/10.1016/s0092-
8674(00)81609-8 PMID: 9741632
29. Bono M de, Tobin DM, Davis MW, Avery L, Bargmann CI. Social feeding in Caenorhabditis elegans is
induced by neurons that detect aversive stimuli. Nature. 2002; 419(6910):899–903. https://doi.org/10.
1038/nature01169 PMID: 12410303
30. Gray JM, Karow DS, Lu H, Chang AJ, Chang JS, Ellis RE, et al. Oxygen sensation and social feeding
mediated by a C. elegans guanylate cyclase homologue. Nature. 2004; 430(6997):317–22. https://doi.
org/10.1038/nature02714 PMID: 15220933
31. Sugi T, Ito H, Nishimura M, Nagai KH. C. elegans collectively forms dynamical networks. Nat Commun.
2019; 10(1):683. https://doi.org/10.1038/s41467-019-08537-y PMID: 30778072
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 18 / 20
32. Demir E, Yaman YI, Basaran M, Kocabas A. Dynamics of pattern formation and emergence of swarm-
ing in Caenorhabditis elegans. Elife. 2020; 9:e52781. https://doi.org/10.7554/eLife.52781 PMID:
32250243
33. Ding SS, Schumacher LJ, Javer AE, Endres RG, Brown AE. Shared behavioral mechanisms underlie
C. elegans aggregation and swarming. Elife. 2019; 8:e43318. https://doi.org/10.7554/eLife.43318
PMID: 31021320
34. Rogers C, Reale V, Kim K, Chatwin H, Li C, Evans P, et al. Inhibition of Caenorhabditis elegans social
feeding by FMRFamide-related peptide activation of NPR-1. Nat Neurosci. 2003; 6(11):1178–85.
https://doi.org/10.1038/nn1140 PMID: 14555955
35. Gloria-Soria A, Azevedo RBR. npr-1 Regulates Foraging and Dispersal Strategies in Caenorhabditis
elegans. Curr Biol. 2008; 18(21):1694–9. https://doi.org/10.1016/j.cub.2008.09.043 PMID: 18993077
36. Zhao Y, Long L, Xu W, Campbell RF, Large EE, Greene JS, et al. Changes to social feeding behaviors
are not sufficient for fitness gains of the Caenorhabditis elegans N2 reference strain. Elife. 2018; 7:
e38675. https://doi.org/10.7554/eLife.38675 PMID: 30328811
37. Moreno E, Sommer RJ. A cilia-mediated environmental input induces solitary behaviour in Caenorhab-
ditis elegans and Pristionchus pacificus nematodes. Nematology. 2018; 20(3):201–9.
38. Zimmer M, Gray JM, Pokala N, Chang AJ, Karow DS, Marletta MichaelA, et al. Neurons Detect
Increases and Decreases in Oxygen Levels Using Distinct Guanylate Cyclases. Neuron. 2009; 61
(6):865–79. https://doi.org/10.1016/j.neuron.2009.02.013 PMID: 19323996
39. Macosko EZ, Pokala N, Feinberg EH, Chalasani SH, Butcher RA, Clardy J, et al. Ahub-and-spoke cir-
cuit drives pheromone attraction and social behaviour in C. elegans. Nature. 2009; 458(7242):1171–5.
https://doi.org/10.1038/nature07886 PMID: 19349961
40. Jang H, Levy S, Flavell SW, Mende F, Latham R, Zimmer M, et al. Dissection of neuronal gap junction
circuits that regulate social behavior in Caenorhabditis elegans. Proc National Acad Sci. 2017; 114(7):
E1263–72. https://doi.org/10.1073/pnas.1621274114 PMID: 28143932
41. Srinivasan J, Reuss SH von, Bose N, Zaslaver A, Mahanti P, Ho MC, et al. A Modular Library of Small
Molecule Signals Regulates Social Behaviors in Caenorhabditis elegans. Plos Biol. 2012; 10(1):
e1001237. https://doi.org/10.1371/journal.pbio.1001237 PMID: 22253572
42. Lo WS, Roca M, Dardiry M, Mackie M, Eberhardt G, Witte H, et al. Evolution and Diversity of TGF-β
Pathways are Linked with Novel Developmental and Behavioral Traits. Mol Biol Evol. 2022; 39(12):
msac252.
43. McBride JM, Hollis JP. Phenomenon of Swarming in Nematodes. Nature. 1966; 211(5048):545–6.
https://doi.org/10.1038/211545b0 PMID: 5967505
44. Ryan WBF, Carbotte SM, Coplan JO, O’Hara S, Melkonian A, Arko R, et al. Global Multi-Resolution
Topography synthesis: GLOBAL MULTI-RESOLUTION TOPOGRAPHY SYNTHESIS. Geochem,
Geophys, Geosystems. 2009; 10(3):n/a-n/a.
45. Witte H, Moreno E, Ro
¨delsperger C, Kim J, Kim JS, Streit A, et al. Gene inactivation using the CRISPR/
Cas9 system in the nematode Pristionchus pacificus. Dev Genes Evol. 2015; 225(1):55–62. https://doi.
org/10.1007/s00427-014-0486-8 PMID: 25548084
46. Ro¨delsperger C, Meyer JM, Prabh N, Lanz C, Bemm F, Sommer RJ. Single-Molecule Sequencing
Reveals the Chromosome-Scale Genomic Architecture of the Nematode Model Organism Pristionchus
pacificus. Cell Reports. 2017; 21(3):834–44. https://doi.org/10.1016/j.celrep.2017.09.077 PMID:
29045848
47. Werner MS, Claaßen MH, Renahan T, Dardiry M, Sommer RJ. Adult Influence on Juvenile Phenotypes
by Stage-Specific Pheromone Production. Iscience. 2018; 10:123–34. https://doi.org/10.1016/j.isci.
2018.11.027 PMID: 30513394
48. Bento G, Ogawa A, Sommer RJ. Co-option of the hormone-signalling module dafachronic acid–DAF-12
in nematode evolution. Nature. 2010; 466(7305):494–7. https://doi.org/10.1038/nature09164 PMID:
20592728
49. Ragsdale EJ, Mu¨ller MR, Ro
¨delsperger C, Sommer RJ. A Developmental Switch Coupled to the Evolu-
tion of Plasticity Acts through a Sulfatase. Cell. 2013; 155(4):922–33. https://doi.org/10.1016/j.cell.
2013.09.054 PMID: 24209628
50. Bui LT, Ivers NA, Ragsdale EJ. A sulfotransferase dosage-dependently regulates mouthpart polyphen-
ism in the nematode Pristionchus pacificus. Nat Commun. 2018; 9(1):4119. https://doi.org/10.1038/
s41467-018-05612-8 PMID: 30297689
51. Sieriebriennikov B, Sun S, Lightfoot JW, Witte H, Moreno E, Ro
¨delsperger C, et al. Conserved nuclear
hormone receptors controlling a novel plastic trait target fast-evolving genes expressed in a single cell.
Plos Genet. 2020; 16(4):e1008687. https://doi.org/10.1371/journal.pgen.1008687 PMID: 32282814
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 19 / 20
52. Werner MS, Sieriebriennikov B, Loschko T, Namdeo S, Lenuzzi M, Dardiry M, et al. Environmental influ-
ence on Pristionchus pacificus mouth form through different culture methods. Sci Rep-uk. 2017; 7
(1):7207. https://doi.org/10.1038/s41598-017-07455-7 PMID: 28775277
53. Werner MS, Loschko T, King T, Reich S, Theska T, Franz-Wachtel M, et al. Histone 4 lysine 5/12 acety-
lation enables developmental plasticity of Pristionchus mouth form. Nat Commun. 2023; 14(1):2095.
https://doi.org/10.1038/s41467-023-37734-z PMID: 37055396
54. Fe
´lix MA, Duveau F. Population dynamics and habitat sharing of natural populations of Caenorhabditis
elegans and C. briggsae. Bmc Biol. 2012; 10(1):59. https://doi.org/10.1186/1741-7007-10-59 PMID:
22731941
55. Meyer JM, Baskaran P, Quast C, Susoy V, Ro
¨delsperger C, Glo
¨ckner FO, et al. Succession and
dynamics of Pristionchus nematodes and their microbiome during decomposition of Oryctes borbonicus
on La Re
´union Island. Environ Microbiol. 1476; 19(4):1476–89.
56. Fe
´lix MA, Ailion M, Hsu JC, Richaud A, Wang J. Pristionchus nematodes occur frequently in diverse rot-
ting vegetal substrates and are not exclusively necromenic, while Panagrellus redivivoides is found spe-
cifically in rotting fruits. Plos One. 2018; 13(8):e0200851. https://doi.org/10.1371/journal.pone.0200851
PMID: 30074986
57. Foster KR, Shaulsky G, Strassmann JE, Queller DC, Thompson CRL. Pleiotropy as a mechanism to
stabilize cooperation. Nature. 2004; 431(7009):693–6. https://doi.org/10.1038/nature02894 PMID:
15470429
58. Bru¨ckner S, Schubert R, Kraushaar T, Hartmann R, Hoffmann D, Jelli E, et al. Kin discrimination in
social yeast is mediated by cell surface receptors of the Flo11 adhesin family. Elife. 2020; 9:e55587.
https://doi.org/10.7554/eLife.55587 PMID: 32286952
59. Serobyan V, Ragsdale EJ, Sommer RJ. Adaptive value of a predatory mouth-form in a dimorphic nema-
tode. Proc Royal Soc B Biological Sci. 2014; 281(1791):20141334.
60. Dardiry M, Piskobulu V, Kalirad A, Sommer RJ. Experimental and theoretical support for costs of plas-
ticity and phenotype in a nematode cannibalistic trait. Evol Lett. 2023; 7(1):48–57. https://doi.org/10.
1093/evlett/qrac001 PMID: 37065436
61. Markov GV, Meyer JM, Panda O, Artyukhin AB, Claaßen M, Witte H, et al. Functional Conservation and
Divergence of daf-22 Paralogs in Pristionchus pacificus Dauer Development. Mol Biol Evol. 2016; 33
(10):2506–14. https://doi.org/10.1093/molbev/msw090 PMID: 27189572
62. Falcke JM, Bose N, Artyukhin AB, Ro
¨delsperger C, Markov GV, Yim JJ, et al. Linking Genomic and
Metabolomic Natural Variation Uncovers Nematode Pheromone Biosynthesis. Cell Chem Biol. 2018;
25(6):787–796.e12. https://doi.org/10.1016/j.chembiol.2018.04.004 PMID: 29779955
63. Zhou Y, Loeza-Cabrera M, Liu Z, Aleman-Meza B, Nguyen JK, Jung SK, et al. Potential Nematode
Alarm Pheromone Induces Acute Avoidance in Caenorhabditis elegans. Genetics. 2016; 206(3):1469–
78.
64. Yuan J, Raizen DM, Bau HH. Gait synchronization in Caenorhabditis elegans. Proc National Acad Sci.
2014; 111(19):6865–70. https://doi.org/10.1073/pnas.1401828111 PMID: 24778261
65. Penkov S, Ogawa A, Schmidt U, Tate D, Zagoriy V, Boland S, et al. A wax ester promotes collective
host finding in the nematode Pristionchus pacificus. Nat Chem Biol. 2014; 10(4):281–5. https://doi.org/
10.1038/nchembio.1460 PMID: 24584102
PLOS GENETICS
Kin-recognition and collective behaviors in nematodes
PLOS Genetics | https://doi.org/10.1371/journal.pgen.1011056 December 14, 2023 20 / 20
