Divergent combinations of cis-regulatory elements control the evolution of phenotypic plasticity.

The widespread occurrence of phenotypic plasticity across all domains of life demonstrates its evolutionary significance. However, how plasticity itself evolves and how it contributes to evolution is poorly understood. Here, we investigate the predatory nematode Pristionchus pacificus with its feeding structure plasticity using recombinant-inbred-line and quantitative-trait-locus (QTL) analyses between natural isolates. We show that a single QTL at a core developmental gene controls the expression of the cannibalistic morph. This QTL is composed of several cis-regulatory elements. Through CRISPR/Cas-9 engineering, we identify copy number variation of potential transcription factor binding sites that interacts with a single intronic nucleotide polymorphism. Another intronic element eliminates gene expression altogether, mimicking knockouts of the locus. Comparisons of additional isolates further support the rapid evolution of these cis-regulatory elements. Finally, an independent QTL study reveals evidence for parallel evolution at the same locus. Thus, combinations of cis-regulatory elements shape plastic trait expression and control nematode cannibalism.

Kin-recognition and predation shape collective behaviors in the cannibalistic nematode Pristionchus pacificus.

Kin-recognition is observed across diverse species forming an important behavioral adaptation 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 cannibalization 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 proving 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.

N-terminal acetylation promotes synaptonemal complex assembly in C. elegans.

N-terminal acetylation of the first two amino acids on proteins is a prevalent cotranslational modification. Despite its abundance, the biological processes associated with this modification are not well understood. Here, we mapped the pattern of protein N-terminal acetylation in Caenorhabditis elegans, uncovering a conserved set of rules for this protein modification and identifying substrates for the N-terminal acetyltransferase B (NatB) complex. We observed an enrichment for global protein N-terminal acetylation and also specifically for NatB substrates in the nucleus, supporting the importance of this modification for regulating biological functions within this cellular compartment. Peptide profiling analysis provides evidence of cross-talk between N-terminal acetylation and internal modifications in a NAT substrate-specific manner. In vivo studies indicate that N-terminal acetylation is critical for meiosis, as it regulates the assembly of the synaptonemal complex (SC), a proteinaceous structure ubiquitously present during meiosis from yeast to humans. Specifically, N-terminal acetylation of NatB substrate SYP-1, an SC structural component, is critical for SC assembly. These findings provide novel insights into the biological functions of N-terminal acetylation and its essential role during meiosis.

Evolution and Diversity of TGF-ß Pathways are Linked with Novel Developmental and Behavioral Traits.

Transforming growth factor-ß (TGF-ß) signaling is essential for numerous biologic functions. It is a highly conserved pathway found in all metazoans including the nematode Caenorhabditis elegans, which has also been pivotal in identifying many components. Utilizing a comparative evolutionary approach, we explored TGF-ß signaling in nine nematode species and revealed striking variability in TGF-ß gene frequency across the lineage. Of the species analyzed, gene duplications in the DAF-7 pathway appear common with the greatest disparity observed in Pristionchus pacificus. Specifically, multiple paralogues of daf-3, daf-4 and daf-7 were detected. To investigate this additional diversity, we induced mutations in 22 TGF-ß components and generated corresponding double, triple, and quadruple mutants revealing both conservation and diversification in function. Although the DBL-1 pathway regulating body morphology appears highly conserved, the DAF-7 pathway exhibits functional divergence, notably in some aspects of dauer formation. Furthermore, the formation of the phenotypically plastic mouth in P. pacificus is partially influenced through TGF-ß with the strongest effect in Ppa-tag-68. This appears important for numerous processes in P. pacificus but has no known function in C. elegans. Finally, we observe behavioral differences in TGF-ß mutants including in chemosensation and the establishment of the P. pacificus kin-recognition signal. Thus, TGF-ß signaling in nematodes represents a stochastic genetic network capable of generating novel functions through the duplication and deletion of associated genes.

Conserved nuclear hormone receptors controlling a novel plastic trait target fast-evolving genes expressed in a single cell.

Environment shapes development through a phenomenon called developmental plasticity. Deciphering its genetic basis has potential to shed light on the origin of novel traits and adaptation to environmental change. However, molecular studies are scarce, and little is known about molecular mechanisms associated with plasticity. We investigated the gene regulatory network controlling predatory vs. non-predatory dimorphism in the nematode Pristionchus pacificus and found that it consists of genes of extremely different age classes. We isolated mutants in the conserved nuclear hormone receptor nhr-1 with previously unseen phenotypic effects. They disrupt mouth-form determination and result in animals combining features of both wild-type morphs. In contrast, mutants in another conserved nuclear hormone receptor nhr-40 display altered morph ratios, but no intermediate morphology. Despite divergent modes of control, NHR-1 and NHR-40 share transcriptional targets, which encode extracellular proteins that have no orthologs in Caenorhabditis elegans and result from lineage-specific expansions. An array of transcriptional reporters revealed co-expression of all tested targets in the same pharyngeal gland cell. Major morphological changes in this gland cell accompanied the evolution of teeth and predation, linking rapid gene turnover with morphological innovations. Thus, the origin of feeding plasticity involved novelty at the level of genes, cells and behavior.

Cilia drive developmental plasticity and are essential for efficient prey detection in predatory nematodes.

Cilia are complex organelles involved in a broad array of functions in eukaryotic organisms. Nematodes employ cilia for environmental sensing, which shapes developmental decisions and influences morphologically plastic traits and adaptive behaviours. Here, we assess the role of cilia in the nematode Pristionchus pacificus, and determine their importance in regulating the developmentally plastic mouth-form decision in addition to predatory feeding and self-recognition behaviours, all of which are not present in Caenorhabditis elegans. An analysis of a multitude of cilia-related mutants including representatives of the six protein subcomplexes required in intraflagellar transport (IFT) plus the regulatory factor X transcription factor daf-19 revealed that cilia are essential for processing the external cues influencing the mouth-form decision and for the efficient detection of prey. Surprisingly, we observed that loss-of-function mutations in the different IFT components resulted in contrasting mouth-form phenotypes and different degrees of predation deficiencies. This observation supports the idea that perturbing different IFT subcomplexes has different effects on signalling downstream of the cilium. Finally, self-recognition was maintained in the cilia deficient mutants tested, indicating that the mechanisms triggering self-recognition in P. pacificus may not require the presence of fully functional cilia.

Germline organization in Strongyloides nematodes reveals alternative differentiation and regulation mechanisms.

Nematodes of the genus Strongyloides are important parasites of vertebrates including man. Currently, little is known about their germline organization or reproductive biology and how this influences their parasitic life strategies. Here, we analyze the structure of the germline in several Strongyloides and closely related species and uncover striking differences in the development, germline organization, and fluid dynamics compared to the model organism Caenorhabditis elegans. With a focus on Strongyloides ratti, we reveal that the proliferation of germ cells is restricted to early and mid-larval development, thus limiting the number of progeny. In order to understand key germline events (specifically germ cell progression and the transcriptional status of the germline), we monitored conserved histone modifications, in particular H3Pser10 and H3K4me3. The evolutionary significance of these events is subsequently highlighted through comparisons with six other nematode species, revealing underlying complexities and variations in the development of the germline among nematodes.

An intronic microRNA links Rb/E2F and EGFR signaling.

The importance of microRNAs in the regulation of various aspects of biology and disease is well recognized. However, what remains largely unappreciated is that a significant number of miRNAs are embedded within and are often co-expressed with protein-coding host genes. Such a configuration raises the possibility of a functional interaction between a miRNA and the gene it resides in. This is exemplified by the Drosophila melanogaster dE2f1 gene that harbors two miRNAs, mir-11 and mir-998, within its last intron. miR-11 was demonstrated to limit the proapoptotic function of dE2F1 by repressing cell death genes that are directly regulated by dE2F1, however the biological role of miR-998 was unknown. Here we show that one of the functions of miR-998 is to suppress dE2F1-dependent cell death specifically in rbf mutants by elevating EGFR signaling. Mechanistically, miR-998 operates by repressing dCbl, a negative regulator of EGFR signaling. Significantly, dCbl is a critical target of miR-998 since dCbl phenocopies the effects of miR-998 on dE2f1-dependent apoptosis in rbf mutants. Importantly, this regulation is conserved, as the miR-998 seed family member miR-29 repressed c-Cbl, and enhanced MAPK activity and wound healing in mammalian cells. Therefore, the two intronic miRNAs embedded in the dE2f1 gene limit the apoptotic function of dE2f1, but operate in different contexts and act through distinct mechanisms. These results also illustrate that examining an intronic miRNA in the context of its host's function can be valuable in elucidating the biological function of the miRNA, and provide new information about the regulation of the host gene itself.

Chromosome movements promoted by the mitochondrial protein SPD-3 are required for homology search during Caenorhabditis elegans meiosis.

Pairing of homologous chromosomes during early meiosis is essential to prevent the formation of aneuploid gametes. Chromosome pairing includes a step of homology search followed by the stabilization of homolog interactions by the synaptonemal complex (SC). These events coincide with dramatic changes in nuclear organization and rapid chromosome movements that depend on cytoskeletal motors and are mediated by SUN-domain proteins on the nuclear envelope, but how chromosome mobility contributes to the pairing process remains poorly understood. We show that defects in the mitochondria-localizing protein SPD-3 cause a defect in homolog pairing without impairing nuclear reorganization or SC assembly, which results in promiscuous installation of the SC between non-homologous chromosomes. Preventing SC assembly in spd-3 mutants does not improve homolog pairing, demonstrating that SPD-3 is required for homology search at the start of meiosis. Pairing center regions localize to SUN-1 aggregates at meiosis onset in spd-3 mutants; and pairing-promoting proteins, including cytoskeletal motors and polo-like kinase 2, are normally recruited to the nuclear envelope. However, quantitative analysis of SUN-1 aggregate movement in spd-3 mutants demonstrates a clear reduction in mobility, although this defect is not as severe as that seen in sun-1(jf18) mutants, which also show a stronger pairing defect, suggesting a correlation between chromosome-end mobility and the efficiency of pairing. SUN-1 aggregate movement is also impaired following inhibition of mitochondrial respiration or dynein knockdown, suggesting that mitochondrial function is required for motor-driven SUN-1 movement. The reduced chromosome-end mobility of spd-3 mutants impairs coupling of SC assembly to homology recognition and causes a delay in meiotic progression mediated by HORMA-domain protein HTP-1. Our work reveals how chromosome mobility impacts the different early meiotic events that promote homolog pairing and suggests that efficient homology search at the onset of meiosis is largely dependent on motor-driven chromosome movement.

Predatory feeding behaviour in Pristionchus nematodes is dependent on phenotypic plasticity and induced by serotonin.

Behavioural innovation and morphological adaptation are intrinsically linked but their relationship is often poorly understood. In nematodes, a huge diversity of feeding morphologies and behaviours can be observed to meet their distinctive dietary and environmental demands. Pristionchus and their relatives show varied feeding activities, both consuming bacteria and also predating other nematodes. In addition, Pristionchus nematodes display dimorphic mouth structures triggered by an irreversible developmental switch, which generates a narrower mouthed form with a single tooth and a wider mouthed form with an additional tooth. However, little is known about the specific predatory adaptations of these mouth forms or the associated mechanisms and behaviours. Through a mechanistic analysis of predation behaviours, in particular in the model organism Pristionchus pacificus, we reveal multifaceted feeding modes characterised by dynamic rhythmic switching and tooth stimulation. This complex feeding mode switch is regulated by the neurotransmitter serotonin in a previously uncharacterised role, a process that appears conserved across several predatory nematode species. Furthermore, we investigated the effects of starvation, prey size and prey preference on P. pacificus predatory feeding kinetics, revealing predation to be a fundamental component of the P. pacificus feeding repertoire, thus providing an additional rich source of nutrition in addition to bacteria. Finally, we found that mouth form morphology also has a striking impact on predation, suppressing predatory behaviour in the narrow mouthed form. Our results therefore hint at the regulatory networks involved in controlling predatory feeding and underscore P. pacificus as a model for understanding the evolution of complex behaviours.

Novel use of a disposable digital pressure transducer to increase the safety of pericardiocentesis.

Cardiac tamponade represents a medical emergency necessitating emergent pericardiocentesis. Use of two-dimensional echocardiography (ECHO) has improved the safety of pericardiocentesis, but procedural challenges may occur when performed in an emergent manner outside of the catheterization laboratory without availability of fluoroscopy and readily available pressure transducers. The most problematic situation is the initial finding of bloody fluid on aspiration where intrapericardial versus intravascular location of the needle must be determined. We report two cases of cardiac tamponade managed with the use of a novel, disposable lightweight digital pressure transducer to directly measure intrapericardial pressures during an ECHO guided pericardiocentesis. In both cases the fluid initially encountered was grossly bloody and rapid definition of whether this was pericardial fluid versus an inappropriately located needle in the vascular space was critical. This type of novel, disposable self contained manometer has the potential to further minimize complications associated with pericardiocentesis. It offers a cost effective alternative and answers questions about the shifting point of service for pericardiocentesis from the invasive cath lab to less costly locations (Drummond, et al., J Am Soc Echocardiogr 1998;11:433-435).

Neuroscience: Hedonic worms gorge on high-energy foods.

Cannabinoids can enhance the preference for calorific foods through hedonic feeding behaviors. A new study identifies and characterizes these indulgent behaviors in the nematode Caenorhabditis elegans, providing insights into the mechanisms of their regulation.

Genomic integration of transgenes using UV irradiation in Pristionchus pacificus.

Transgenes are widely used throughout molecular biology for numerous applications. In Caenorhabditis elegans, stable transgenes are usually generated by microinjection into the germline establishing extrachromosomal arrays. Furthermore, numerous technologies exist to integrate transgenes into the C. elegans genome. In the nematode Pristionchus pacificus, transgenes are possible, however, their establishment is less efficient and dependent on the formation of complex arrays containing the transgene of interest and host carrier DNA. Additionally, genomic integration has only been reported via biolistic methods. Here we describe a simple technique using UV irradiation to facilitate the integration of transgenes into the P. pacificus genome.

Sex or cannibalism: Polyphenism and kin recognition control social action strategies in nematodes.

Resource polyphenisms, where single genotypes produce alternative feeding strategies in response to changing environments, are thought to be facilitators of evolutionary novelty. However, understanding the interplay between environment, morphology, and behavior and its significance is complex. We explore a radiation of Pristionchus nematodes with discrete polyphenic mouth forms and associated microbivorous versus cannibalistic traits. Notably, comparing 29 Pristionchus species reveals that reproductive mode strongly correlates with mouth-form plasticity. Male-female species exhibit the microbivorous morph and avoid parent-offspring conflict as indicated by genetic hybrids. In contrast, hermaphroditic species display cannibalistic morphs encouraging competition. Testing predation between 36 co-occurring strains of the hermaphrodite P. pacificus showed that killing inversely correlates with genomic relatedness. These empirical data together with theory reveal that polyphenism (plasticity), kin recognition, and relatedness are three major factors that shape cannibalistic behaviors. Thus, developmental plasticity influences cooperative versus competitive social action strategies in diverse animals.

Improving Transgenesis Efficiency and CRISPR-Associated Tools Through Codon Optimization and Native Intron Addition in Pristionchus Nematodes.

A lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, clustered regularly interspaced short palindromic repeats (CRISPR)-associated engineering, and related tools are fundamental in the modern life sciences, but their applications are still limited to a few model organisms. In the phylum Nematoda, transgenesis can only be performed in a handful of species other than Caenorhabditis elegans, and additionally, other species suffer from significantly lower transgenesis efficiencies. We hypothesized that this may in part be due to incompatibilities of transgenes in the recipient organisms. Therefore, we investigated the genomic features of 10 nematode species from three of the major clades representing all different lifestyles. We found that these species show drastically different codon usage bias and intron composition. With these findings, we used the species Pristionchus pacificus as a proof of concept for codon optimization and native intron addition. Indeed, we were able to significantly improve transgenesis efficiency, a principle that may be usable in other nematode species. In addition, with the improved transgenes, we developed a fluorescent co-injection marker in P. pacificus for the detection of CRISPR-edited individuals, which helps considerably to reduce associated time and costs.

Bacterial vitamin B12 production enhances nematode predatory behavior.

Although the microbiota is known to affect host development, metabolism, and immunity, its impact on host behavior is only beginning to be understood. In order to better characterize behavior modulation by host-associated microorganisms, we investigated how bacteria modulate complex behaviors in the nematode model organism Pristionchus pacificus. This nematode is a predator that feeds on the larvae of other nematodes, including Caenorhabditis elegans. By growing P. pacificus on different bacteria and testing their ability to kill C. elegans, we reveal large differences in killing efficiencies, with a Novosphingobium species showing the strongest enhancement. This enhanced killing was not accompanied by an increase in feeding, which is a phenomenon known as surplus killing, whereby predators kill more prey than necessary for sustenance. Our RNA-seq data demonstrate widespread metabolic rewiring upon exposure to Novosphingobium, which facilitated screening of bacterial mutants with altered transcriptional responses. We identified bacterial production of vitamin B12 as an important cause of such enhanced predatory behavior. Although vitamin B12 is an essential cofactor for detoxification and metabolite biosynthesis, shown previously to accelerate development in C. elegans, supplementation with this enzyme cofactor amplified surplus killing in P. pacificus, whereas mutants in vitamin B12-dependent pathways reduced surplus killing. By demonstrating that production of vitamin B12 by host-associated microbiota can affect complex host behaviors, we reveal new connections between animal diet, microbiota, and nervous system.

Correction: Bacterial vitamin B12 production enhances nematode predatory behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Small peptide-mediated self-recognition prevents cannibalism in predatory nematodes.

Self-recognition is observed abundantly throughout the natural world, regulating diverse biological processes. Although ubiquitous, often little is known of the associated molecular machinery, and so far, organismal self-recognition has never been described in nematodes. We investigated the predatory nematode Pristionchus pacificus and, through interactions with its prey, revealed a self-recognition mechanism acting on the nematode surface, capable of distinguishing self-progeny from closely related strains. We identified the small peptide SELF-1, which is composed of an invariant domain and a hypervariable C terminus, as a key component of self-recognition. Modifications to the hypervariable region, including single-amino acid substitutions, are sufficient to eliminate self-recognition. Thus, the P. pacificus self-recognition system enables this nematode to avoid cannibalism while promoting the killing of competing nematodes.

Regulation of hyperoxia-induced social behaviour in Pristionchus pacificus nematodes requires a novel cilia-mediated environmental input.

Social behaviours are frequently utilised for defence and stress avoidance in nature. Both Caenorhabditis elegans and Pristionchus pacificus nematodes display social behaviours including clumping and bordering, to avoid hyperoxic stress conditions. Additionally, both species show natural variation in social behaviours with "social" and "solitary" strains. While the single solitary C. elegans N2 strain has evolved under laboratory domestication due to a gain-of-function mutation in the neuropeptide receptor gene npr-1, P. pacificus solitary strains are commonplace and likely ancestral. P. pacificus therefore provides an opportunity to further our understanding of the mechanisms regulating these complex behaviours and how they evolved within an ecologically relevant system. Using CRISPR/Cas9 engineering, we show that Ppa-npr-1 has minimal influence on social behaviours, indicating independent evolutionary pathways compared to C. elegans. Furthermore, solitary P. pacificus strains show an unexpected locomotive response to hyperoxic conditions, suggesting a novel regulatory mechanism counteracting social behaviours. By utilising both forward and reverse genetic approaches we identified 10 genes of the intraflagellar transport machinery in ciliated neurons that are essential for this inhibition. Therefore, a novel cilia-mediated environmental input adds an additional level of complexity to the regulation of hyperoxia-induced social behaviours in P. pacificus, a mechanism unknown in C. elegans.

Comparative transcriptomics of the nematode gut identifies global shifts in feeding mode and pathogen susceptibility.

BACKGROUND: The nematode Pristionchus pacificus has been established as a model for comparative studies using the well known Caenorhabditis elegans as a reference. Despite their relatedness, previous studies have revealed highly divergent development and a number of morphological differences including the lack of a pharyngal structure, the grinder, used to physically lyse the ingested bacteria in C. elegans. RESULTS: To complement current knowledge about developmental and ecological differences with a better understanding of their feeding modes, we have sequenced the intestinal transcriptomes of both nematodes. In total, we found 464 intestine-enriched genes in P. pacificus and 724 in C. elegans, of which the majority (66%) has been identified by previous studies. Interestingly, only 15 genes could be identified with shared intestinal enrichment in both species, of which three genes are Hedgehog signaling molecules supporting a highly conserved role of this pathway for intestinal development across all metazoa. At the level of gene families, we find similar divergent trends with only five families displaying significant intestinal enrichment in both species. We compared our data with transcriptomic responses to various pathogens. Strikingly, C. elegans intestine-enriched genes showed highly significant overlaps with pathogen response genes whereas this was not the case for P. pacificus, indicating shifts in pathogen susceptibility that might be explained by altered feeding modes. CONCLUSIONS: Our study reveals first insights into the evolution of feeding systems and the associated changes in intestinal gene expression that might have facilitated nematodes of the P. pacificus lineage to colonize new environments. These findings deepen our understanding about how morphological and genomic diversity is created during the course of evolution.