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.

Identification of the Extracellular Nuclease Influencing Soaking RNA Interference Efficiency in Bursaphelenchus xylophilus.

RNA interference (RNAi) efficiency dramatically varies among different nematodes, which impacts research on their gene function and pest control. Bursaphelenchus xylophilus is a pine wood nematode in which RNAi-mediated gene silencing has unstable interference efficiency through soaking in dsRNA solutions, the factors of which remain unknown. Using agarose gel electrophoresis, we found that dsRNA can be degraded by nematode secretions in the soaking system which is responsible for the low RNAi efficiency. Based on the previously published genome and secretome data of B. xylophilus, 154 nucleases were screened including 11 extracellular nucleases which are potential factors reducing RNAi efficacy. To confirm the function of nucleases in RNAi efficiency, eight extracellular nuclease genes (BxyNuc1-8) were cloned in the genome. BxyNuc4, BxyNuc6 and BxyNuc7 can be upregulated in response to dsGFP, considered as the major nuclease performing dsRNA degradation. After soaking with the dsRNA of nucleases BxyNuc4/BxyNuc6/BxyNuc7 and Pat10 gene (ineffective in RNAi) simultaneously for 24 h, the expression of Pat10 gene decreased by 23.25%, 26.05% and 11.29%, respectively. With soaking for 36 h, the expression of Pat10 gene decreased by 43.25% and 33.25% in dsBxyNuc6+dsPat10 and dsBxyNuc7+dsPat10 groups, respectively. However, without dsPat10, dsBxyNuc7 alone could cause downregulation of Pat10 gene expression, while dsBxyNuc6 could not disturb this gene. In conclusion, the nuclease BxyNuc6 might be a major barrier to the RNAi efficiency in B. xylophilus.

Aphelenchoides besseyi Ab-FAR-1 Interacts with Arabidopsis thaliana AtADF3 to Interfere with Actin Cytoskeleton, and Promotes Nematode Parasitism and Pathogenicity.

Fatty acid and retinol binding proteins (FAR) are unique proteins found in nematodes and are considered potential targets for controlling these parasites. However, their functions in nematode parasitism and pathogenicity and interaction with hosts are still unclear. In this study, we investigated the specific roles of rice white tip nematodes (RWTNs), Aphelenchoides besseyi, and a protein, Ab-FAR-1, to elucidate the parasitic and pathogenic processes of nematodes. The results showed that the expression level of Ab-far-1 was significantly up-regulated after A. besseyi infection of the plant. The immunofluorescence and subcellular localisation showed that Ab-FAR-1 was secreted into plant tissues mainly through the body wall of nematodes and might act in the nucleus and cytoplasm of plant cells. The pathogenicity of RWTNs was enhanced in Arabidopsis thaliana overexpressing Ab-FAR-1 and inhibited in Ab-far-1 RNAi A. thaliana. Yeast two-hybrid, Co-IP, BiFC, and nematode inoculation experiments showed that Ab-FAR-1 could interact with the A. thaliana actin-depolymerizing factor protein AtADF3, and the A. thaliana adf3 mutant was more susceptible to nematodes. An in vitro actin filament depolymerisation assay demonstrated that Ab-FAR-1 could inhibit AtADF3-mediated depolymerisation of actin filaments, and the turnover process of cellular actin filaments was also affected in A. thaliana overexpressing Ab-FAR-1. In addition, flg22-mediated host defence responses were suppressed in A. thaliana overexpressing Ab-FAR-1 and adf3 mutants. Therefore, this study confirmed that RWTNs can affect the turnover of actin filament remodelling mediated by AtADF3 through Ab-FAR-1 secretion and thus inhibit plant PAMP-triggered immunity (PTI), promoting the parasitism and pathogenicity of nematodes.

Toxicity of Bacillus thuringiensis Strains Derived from the Novel Crystal Protein Cry31Aa with High Nematicidal Activity against Rice Parasitic Nematode Aphelenchoides besseyi.

The plant parasitic nematode, Aphelenchoides besseyi, is a serious pest causing severe damage to various crop plants and vegetables. The Bacillus thuringiensis (Bt) strains, GBAC46 and NMTD81, and the biological strain, FZB42, showed higher nematicidal activity against A. besseyi, by up to 88.80, 82.65, and 75.87%, respectively, in a 96-well plate experiment. We screened the whole genomes of the selected strains by protein-nucleic acid alignment. It was found that the Bt strain GBAC46 showed three novel crystal proteins, namely, Cry31Aa, Cry73Aa, and Cry40ORF, which likely provide for the safe control of nematodes. The Cry31Aa protein was composed of 802 amino acids with a molecular weight of 90.257 kDa and contained a conserved delta-endotoxin insecticidal domain. The Cry31Aa exhibited significant nematicidal activity against A. besseyi with a lethal concentration (LC50) value of 131.80 µg/mL. Furthermore, the results of in vitro experiments (i.e., rhodamine and propidium iodide (PI) experiments) revealed that the Cry31Aa protein was taken up by A. besseyi, which caused damage to the nematode's intestinal cell membrane, indicating that the Cry31Aa produced a pore-formation toxin. In pot experiments, the selected strains GBAC46, NMTD81, and FZB42 significantly reduced the lesions on leaves by up to 33.56%, 45.66, and 30.34% and also enhanced physiological growth parameters such as root length (65.10, 50.65, and 55.60%), shoot length (68.10, 55.60, and 59.45%), and plant fresh weight (60.71, 56.45, and 55.65%), respectively. The number of nematodes obtained from the plants treated with the selected strains (i.e., GBAC46, NMTD81, and FZB42) and A. besseyi was significantly reduced, with 0.56, 0.83., 1.11, and 5.04 seedling mL-1 nematodes were achieved, respectively. Moreover, the qRT-PCR analysis showed that the defense-related genes were upregulated, and the activity of hydrogen peroxide (H2O2) increased while malondialdehyde (MDA) decreased in rice leaves compared to the control. Therefore, it was concluded that the Bt strains GBAC46 and NMTD81 can promote rice growth, induce high expression of rice defense-related genes, and activate systemic resistance in rice. More importantly, the application of the novel Cry31Aa protein has high potential for the efficient and safe prevention and green control of plant parasitic nematodes.

Metabolic Analysis of the Development of the Plant-Parasitic Cyst Nematodes Heterodera schachtii and Heterodera trifolii by Capillary Electrophoresis Time-of-Flight Mass Spectrometry.

The cyst nematodes Heterodera schachtii and Heterodera trifolii, whose major hosts are sugar beet and clover, respectively, damage a broad range of plants, resulting in significant economic losses. Nematodes synthesize metabolites for organismal development and social communication. We performed metabolic profiling of H. schachtii and H. trifolii in the egg, juvenile 2 (J2), and female stages. In all, 392 peaks were analyzed by capillary electrophoresis time-of-flight mass spectrometry, which revealed a lot of similarities among metabolomes. Aromatic amino acid metabolism, carbohydrate metabolism, choline metabolism, methionine salvage pathway, glutamate metabolism, urea cycle, glycolysis, gluconeogenesis, coenzyme metabolism, purine metabolism, pyrimidine metabolism, and tricarboxylic acid (TCA) cycle for energy conversion (ß-oxidation and branched-chain amino acid metabolism) energy storage were involved in all stages studied. The egg and female stages synthesized higher levels of metabolites compared to the J2 stage. The key metabolites detected were glycerol, guanosine, hydroxyproline, citric acid, phosphorylcholine, and the essential amino acids Phe, Leu, Ser, and Val. Metabolites, such as hydroxyproline, acetylcholine, serotonin, glutathione, and glutathione disulfide, which are associated with growth and reproduction, mobility, and neurotransmission, predominated in the J2 stage. Other metabolites, such as SAM, 3PSer, 3-ureidopropionic acid, CTP, UDP, UTP, 3-hydroxy-3-methylglutaric acid, 2-amino-2-(hydroxymethyl-1,3-propanediol, 2-hydroxy-4-methylvaleric acid, Gly Asp, glucuronic acid-3 + galacturonic acid-3 Ser-Glu, citrulline, and γ-Glu-Asn, were highly detected in the egg stage. Meanwhile, nicotinamide, 3-PG, F6P, Cys, ADP-Ribose, Ru5P, S7P, IMP, DAP, diethanolamine, p-Hydroxybenzoic acid, and γ-Glu-Arg_divalent were unique to the J2 stage. Formiminoglutamic acid, nicotinaminde riboside + XC0089, putrescine, thiamine 2,3-dihydroxybenzoic acid, 3-methyladenine, caffeic acid, ferulic acid, m-hydrobenzoic acid, o- and p-coumaric acid, and shikimic acid were specific to the female stage. Overall, highly similar identities and quantities of metabolites between the corresponding stages of the two species of nematode were observed. Our results will be a valuable resource for further studies of physiological changes related to the development of nematodes and nematode-plant interactions.

Analysis of meiosis in Pristionchus pacificus reveals plasticity in homolog pairing and synapsis in the nematode lineage.

Meiosis is conserved across eukaryotes yet varies in the details of its execution. Here we describe a new comparative model system for molecular analysis of meiosis, the nematode Pristionchus pacificus, a distant relative of the widely studied model organism Caenorhabditis elegans. P. pacificus shares many anatomical and other features that facilitate analysis of meiosis in C. elegans. However, while C. elegans has lost the meiosis-specific recombinase Dmc1 and evolved a recombination-independent mechanism to synapse its chromosomes, P. pacificus expresses both DMC-1 and RAD-51. We find that SPO-11 and DMC-1 are required for stable homolog pairing, synapsis, and crossover formation, while RAD-51 is dispensable for these key meiotic processes. RAD-51 and DMC-1 localize sequentially to chromosomes during meiotic prophase and show nonoverlapping functions. We also present a new genetic map for P. pacificus that reveals a crossover landscape very similar to that of C. elegans, despite marked divergence in the regulation of synapsis and crossing-over between these lineages.

Tokorhabditis n. gen. (Rhabditida, Rhabditidae), a comparative nematode model for extremophilic living.

Life in extreme environments is typically studied as a physiological problem, although the existence of extremophilic animals suggests that developmental and behavioral traits might also be adaptive in such environments. Here, we describe a new species of nematode, Tokorhabditis tufae, n. gen., n. sp., which was discovered from the alkaline, hypersaline, and arsenic-rich locale of Mono Lake, California. The new species, which offers a tractable model for studying animal-specific adaptations to extremophilic life, shows a combination of unusual reproductive and developmental traits. Like the recently described sister group Auanema, the species has a trioecious mating system comprising males, females, and self-fertilizing hermaphrodites. Our description of the new genus thus reveals that the origin of this uncommon reproductive mode is even more ancient than previously assumed, and it presents a new comparator for the study of mating-system transitions. However, unlike Auanema and almost all other known rhabditid nematodes, the new species is obligately live-bearing, with embryos that grow in utero, suggesting maternal provisioning during development. Finally, our isolation of two additional, molecularly distinct strains of the new genus-specifically from non-extreme locales-establishes a comparative system for the study of extremophilic traits in this model.

Molecular characterization of Ditylenchus destructor voltage-gated calcium channel α1 subunits and analysis of the effect of their knockdown on nematode activity.

Voltage-gated calcium channels (VGCCs) mediate the entry of Ca2+ ions into cells in response to membrane depolarization and play fundamental roles in the nervous system, and the α1 subunits are the main subunits of Ca2+ channels. Caenorhabditis elegans possesses genes encoding α1 subunits; however, very few of these genes have been cloned in plant-parasitic nematodes (PPNs). Ditylenchus destructor is a PPN that has been proposed as a new model for studying the biology and control of PPNs. To understand the structure and function of the VGCCs of this PPN, we first cloned and identified three full-length cDNAs of VGCC α1 subunit genes in D. destructor with the defining structural and conserved features of Cav1 (L-type), Cav2 (non-L-type) and Cav3 (T-type). In situ hybridization assays demonstrated that the Cav1 VGCC α1 subunit gene (DdCα1D) was expressed within body wall muscles. The Cav2 VGCC α1 subunit (DdCα1A) was expressed in the oesophageal gland, vulva and vas deferens of the worm, and the Cav3 VGCC α1 subunit (DdCα1G) was localized to the oesophagus and median bulb. In addition, on the basis of the in vitro knockdown of L-, non-L- and T-type genes via RNAi, these genes were predicted to play a key role in the modulation of locomotion, feeding and reproduction. After the silencing of DdCα1G, the median bulb muscle of D. destructor was obviously contracted, and its feeding and reproduction abilities were significantly inhibited. This study provides insight into the structure and function of VGCC α1 subunits in D. destructor.

PIPE4: Fast PPI Predictor for Comprehensive Inter- and Cross-Species Interactomes.

The need for larger-scale and increasingly complex protein-protein interaction (PPI) prediction tasks demands that state-of-the-art predictors be highly efficient and adapted to inter- and cross-species predictions. Furthermore, the ability to generate comprehensive interactomes has enabled the appraisal of each PPI in the context of all predictions leading to further improvements in classification performance in the face of extreme class imbalance using the Reciprocal Perspective (RP) framework. We here describe the PIPE4 algorithm. Adaptation of the PIPE3/MP-PIPE sequence preprocessing step led to upwards of 50x speedup and the new Similarity Weighted Score appropriately normalizes for window frequency when applied to any inter- and cross-species prediction schemas. Comprehensive interactomes for three prediction schemas are generated: (1) cross-species predictions, where Arabidopsis thaliana is used as a proxy to predict the comprehensive Glycine max interactome, (2) inter-species predictions between Homo sapiens-HIV1, and (3) a combined schema involving both cross- and inter-species predictions, where both Arabidopsis thaliana and Caenorhabditis elegans are used as proxy species to predict the interactome between Glycine max (the soybean legume) and Heterodera glycines (the soybean cyst nematode). Comparing PIPE4 with the state-of-the-art resulted in improved performance, indicative that it should be the method of choice for complex PPI prediction schemas.

Identification and Characterization of a Fatty Acid- and Retinoid-Binding Protein Gene (Ar-far-1) from the Chrysanthemum Foliar Nematode, Aphelenchoides ritzemabosi.

The chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi, is a migratory, plant-parasitic nematode that is widely distributed and infects the aboveground parts of many plants. The fatty acid- and retinoid-binding proteins (FAR) are nematode-specific proteins that are involved in the development, reproduction, and infection of nematodes and are secreted into the tissues to disrupt the plant defense reaction. In this study, we obtained the full-length sequence of the FAR gene (Ar-far-1) from CFN, which is 727 bp and includes a 546 bp ORF that encodes 181 amino acids. Ar-FAR-1 from CFN has the highest sequence similarity to Ab-FAR-1 from A. besseyi, and they are located within the same branch of the phylogenetic tree. Fluorescence-based ligand-binding analysis confirmed that recombinant Ar-FAR-1 was bound to fatty acids and retinol. Ar-far-1 mRNA was expressed in the muscle layer, intestine, female genital system, and egg of CFN, and more highly expressed in females than in males among the four developmental stages of CFN. We demonstrated that the reproduction number and infection capacity of CFN decreased significantly when Ar-far-1 was effectively silenced by in vitro RNAi. Ar-far-1 plays an important role in the development, reproduction, infectivity, and pathogenesis of CFN and may be used as an effective target gene for the control of CFN. The results provide meaningful data about the parasitic and pathogenic genes of CFN to study the interaction mechanism between plant-parasitic nematodes and hosts.

Infective juveniles of entomopathogenic nematodes (Steinernema and Heterorhabditis) secrete ascarosides and respond to interspecific dispersal signals.

Ascarosides are a modular series of signalling molecules that are widely conserved in nematodes where they function as pheromones with both behavioural and developmental effects. Here we show that the developmentally arrested infective juvenile (IJ) stage of entomopathogenic nematodes (EPN) secrete ascarosides into the surrounding medium. The exometabolome of Steinernema carpocapsae and Heterorhabditis megidis was examined at 0, 1, 7 and 21 days of storage. The concentration of several ascarosides (ascr#11, ascr#9, ascr#12, ascr#1 and ascr#14 for both species, plus ascr#10 for H. megidis) showed a progressive increase over this period, while the concentration of longer chain ascarosides increased up to day 7, with an apparent decline thereafter. Ascr #9 was the main ascaroside produced by both species. Similar ascarosides were found over a 7-day period for Steinernema longicaudum and S. feltiae. Ascaroside blends have previously been shown to promote nematode dispersal. S. carpocapsae and H. megidis IJs were stored for up to 12 weeks and assayed at intervals. IJs where exometabolome was allowed to accumulate showed higher dispersal rates than those where water was changed frequently, indicating that IJ exometabolome maintained high dispersal. Infectivity was not affected. IJ exometabolome accumulated over 7 days promoted dispersal of freshly harvested IJs, both of their own and other EPN species. Similarly, extracts of nematode-infected cadavers promoted dispersal of con- and heterospecific IJs. Thus, IJs are encouraged to disperse from a source cadaver or from other crowded conditions by public information cues, a finding that may have application in enhancing biocontrol. However, the complexity of the ascaroside blend produced by IJs suggests that it may have ecological functions other than dispersal.

Influence of symbiotic and non-symbiotic bacteria on pheromone production in Steinernema nematodes (Nematoda, Steinernematidae).

In this study, we assessed the effect of symbiotic (cognate and non-cognate) and non-symbiotic bacteria on ascaroside production of first-generation adults in two Steinernema spp.: S. carpocapsae All strain and S. feltiae SN strain. Each nematode species was reared under three bacterial scenarios: (1) cognate symbiotic, (2) non-cognate symbiotic strain and (3) non-cognate symbiotic species. Our results showed S. carpocapsae produced four quantifiable ascaroside molecules: asc-C5, asc-C6, asc-C7 and asc-C11, whereas in S. feltiae only three molecules were detected: asc-C5, asc-C7 and asc-C11. Bacterial conditions did not significantly affect the quantity of the secreted ascarosides in first-generation adults of S. carpocapsae However, in S. feltiae, Xenorhabdus nematophila All strain influenced the production of two ascaroside molecules: asc-C5 and asc-C11.

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.

Young genes have distinct gene structure, epigenetic profiles, and transcriptional regulation.

Species-specific, new, or "orphan" genes account for 10%-30% of eukaryotic genomes. Although initially considered to have limited function, an increasing number of orphan genes have been shown to provide important phenotypic innovation. How new genes acquire regulatory sequences for proper temporal and spatial expression is unknown. Orphan gene regulation may rely in part on origination in open chromatin adjacent to preexisting promoters, although this has not yet been assessed by genome-wide analysis of chromatin states. Here, we combine taxon-rich nematode phylogenies with Iso-Seq, RNA-seq, ChIP-seq, and ATAC-seq to identify the gene structure and epigenetic signature of orphan genes in the satellite model nematode Pristionchus pacificus Consistent with previous findings, we find young genes are shorter, contain fewer exons, and are on average less strongly expressed than older genes. However, the subset of orphan genes that are expressed exhibit distinct chromatin states from similarly expressed conserved genes. Orphan gene transcription is determined by a lack of repressive histone modifications, confirming long-held hypotheses that open chromatin is important for new gene formation. Yet orphan gene start sites more closely resemble enhancers defined by H3K4me1, H3K27ac, and ATAC-seq peaks, in contrast to conserved genes that exhibit traditional promoters defined by H3K4me3 and H3K27ac. Although the majority of orphan genes are located on chromosome arms that contain high recombination rates and repressive histone marks, strongly expressed orphan genes are more randomly distributed. Our results support a model of new gene origination by rare integration into open chromatin near enhancers.

DAF-19/RFX controls ciliogenesis and influences oxygen-induced social behaviors in Pristionchus pacificus.

Cilia are complex organelles involved in sensory perception and motility with intraflagellar transport (IFT) proteins being essential for cilia assembly and function, but little is known about cilia in an evo-devo context. For example, recent comparisons revealed conservation and divergence of IFT components in the regulation of social feeding behaviors between the nematodes Caenorhabditis elegans and Pristionchus pacificus. Here, we focus on the P. pacificus RFX transcription factor daf-19, the master regulator of ciliogenesis in C. elegans. Two CRISPR/Cas9-induced Ppa-daf-19 mutants lack ciliary structures in amphid neurons and display chemosensory defects. In contrast to IFT mutants, Ppa-daf-19 mutants do not exhibit social behavior. However, they show weak locomotive responses to shifts in oxygen concentration, suggesting partial impairment in sensing or responding to oxygen. To identify targets of Ppa-daf-19 regulation we compared the transcriptomes of Ppa-daf-19 and wild-type animals and performed a bioinformatic search for the X-box RFX binding-site across the genome. The regulatory network of Ppa-DAF-19 involves IFT genes but also many taxonomically restricted genes. We identified a conserved X-box motif as the putative binding site, which was validated for the Ppa-dyf-1 gene. Thus, Ppa-DAF-19 controls ciliogenesis, influences oxygen-induced behaviors and displays a high turnover of its regulatory network.

Duplication of hsp-110 Is Implicated in Differential Success of Globodera Species under Climate Change.

Managing the emergence and spread of crop pests and pathogens is essential for global food security. Understanding how organisms have adapted to their native climate is key to predicting the impact of climate change. The potato cyst nematodes Globodera pallida and G. rostochiensis are economically important plant pathogens that cause yield losses of up to 50% in potato. The two species have different thermal optima that may relate to differences in the altitude of their regions of origin in the Andes. Here, we demonstrate that juveniles of G. pallida are less able to recover from heat stress than those of G. rostochiensis. Genome-wide analysis revealed that while both Globodera species respond to heat stress by induction of various protective heat-inducible genes, G. pallida experiences heat stress at lower temperatures. We use C. elegans as a model to demonstrate the dependence of the heat stress response on expression of Heat Shock Factor-1 (HSF-1). Moreover, we show that hsp-110 is induced by heat stress in G. rostochiensis, but not in the less thermotolerant G. pallida. Sequence analysis revealed that this gene and its promoter was duplicated in G. rostochiensis and acquired thermoregulatory properties. We show that hsp-110 is required for recovery from acute thermal stress in both C. elegans and in G. rostochiensis. Our findings point towards an underlying molecular mechanism that allows the differential expansion of one species relative to another closely related species under current climate change scenarios. Similar mechanisms may be true of other invertebrate species with pest status.

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.

Serotonin Drives Predatory Feeding Behavior via Synchronous Feeding Rhythms in the Nematode Pristionchus pacificus.

Feeding behaviors in a wide range of animals are regulated by the neurotransmitter serotonin, although the exact neural circuits and associated mechanism are often unknown. The nematode Pristionchus pacificus can kill other nematodes by opening prey cuticles with movable teeth. Previous studies showed that exogenous serotonin treatment induces a predatory-like tooth movement and slower pharyngeal pumping in the absence of prey; however, physiological functions of serotonin during predation and other behaviors in P. pacificus remained completely unknown. Here, we investigate the roles of serotonin by generating mutations in Ppa-tph-1 and Ppa-bas-1, two key serotonin biosynthesis enzymes, and by genetic ablation of pharynx-associated serotonergic neurons. Mutations in Ppa-tph-1 reduced the pharyngeal pumping rate during bacterial feeding compared with wild-type. Moreover, the loss of serotonin or a subset of serotonergic neurons decreased the success of predation, but did not abolish the predatory feeding behavior completely. Detailed analysis using a high-speed camera revealed that the elimination of serotonin or the serotonergic neurons disrupted the timing and coordination of predatory tooth movement and pharyngeal pumping. This loss of synchrony significantly reduced the efficiency of successful predation events. These results suggest that serotonin has a conserved role in bacterial feeding and in addition drives the feeding rhythm of predatory behavior in Pristionchus.

Differential Change Patterns of Main Antimicrobial Peptide Genes During Infection of Entomopathogenic Nematodes and Their Symbiotic Bacteria.

The expression of antimicrobial peptides (AMPs) as the main humoral defense reactions of insects during infection by entomopathogenic nematodes (EPNs) and their symbiont is addressed herein. Three AMPs, attacin, cecropin, and spodoptericin, were evaluated in the fifth instar larvae of Spodoptera exigua Hübner (beet armyworm) when challenged with Steinernema carpocapsae or Heterorhabditis bacteriophora. The results indicated that attacin was expressed to a greater extent than either cecropin or spodoptericin. While spodoptericin was expressed to a much lesser extent, this AMP was induced against Gram-positive bacteria, and thus not expressed after penetration of Xenorhabdus nematophila and Photorhabdus luminescens. Attacin and cecropin in the larvae treated with S. carpocapsae at 8 hr post-injection (PI) attained the maximum expression levels and were 138.42-fold and 65.84-fold greater than those of larvae infected with H. bacteriophora, respectively. Generally, the ability of H. bacteriophora to suppress attacin, cecropin, and spodoptericin was greater than that of S. carpocapsae. According to the results, the expression of AMPs by Sp. exigua larvae against S. carpocapsae was determined in the 4 statuses of monoxenic nematode, axenic nematode, live symbiotic bacterium, and dead symbiotic bacterium. The expression of attacin in larvae treated with a monoxenic nematode and live bacterium at 8 and 2 hr PI, respectively, were increased to the maximum amount. Live X. nematophila was the strongest agent for the suppression of attacin. The expression of cecropin against monoxenic nematodes and live symbiotic bacteria at 8 and 4 hr PI, respectively, reached the maximum amount while the expression levels of attacin and cecropin for axenic nematodes were lesser and stable. The results highlighted that the ability of P. luminescens in AMPs suppression was much more than X. nematophila. The results also showed that the effect of symbiotic bacterium in suppressing attacin and cecropin expression was greater than that of a monoxenic nematode; this result provided deep insight into the expression pattern parallels and fluctuations of the main AMPs during nematode infection.

Effect of inoculum age and physical parameters on in vitro culture of the entomopathogenic nematode Steinernema feltiae.

Entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae have a symbiotic association with bacteria which makes them virulent against insects. EPNs have been mass produced using in vivo and in vitro methods, including both solid and liquid fermentation. This study assessed the effect of nematode inoculum age on the production of Steinernema feltiae in liquid, solid and biphasic processes. Several physical parameters were also assessed: the effect of medium viscosity, flask size and aeration speed on the recovery and yield of infective juveniles (IJs). Inoculum age treatments included inoculum liquid cultures that were 7, 14, 21 and 28 days old. Nematodes from the same inoculum were added to one liquid medium (liquid culture), one solid medium with bacteria previously grown in sponge (solid culture) and a variation of the solid medium (a biphasic culture), in which the bacteria were first grown in liquid and, then, soaked into the sponges, with the purpose of providing a more homogeneous bacterial culture before nematode inoculation. Experiments were conducted in Erlenmeyer flasks. Eight treatments were established involving combinations of three variables: two media (with and without 0.2% agar), two flask sizes (250 and 150 ml) and two agitation speeds (180 and 280 rpm). The study showed increases in nematode yield for liquid cultures, but not for solid or biphasic cultures, with the advance of the inoculum age up to 28 days of growth. Furthermore, the addition of 0.2% agar to the liquid medium and increasing the aeration rate by using larger flasks with higher agitation speed may increase nematode recovery and final yield. The experiments were conducted using shake flasks but the results may also be applicable for bioreactors.