Culturing and Genetically Manipulating Entomopathogenic Nematodes.

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Nuclear hormone receptor NHR-49 acts in parallel with HIF-1 to promote hypoxia adaptation in Caenorhabditis elegans.

The response to insufficient oxygen (hypoxia) is orchestrated by the conserved hypoxia-inducible factor (HIF). However, HIF-independent hypoxia response pathways exist that act in parallel with HIF to mediate the physiological hypoxia response. Here, we describe a hypoxia response pathway controlled by Caenorhabditis elegans nuclear hormone receptor NHR-49, an orthologue of mammalian peroxisome proliferator-activated receptor alpha (PPARα). We show that nhr-49 is required for animal survival in hypoxia and is synthetic lethal with hif-1 in this context, demonstrating that these factors act in parallel. RNA-seq analysis shows that in hypoxia nhr-49 regulates a set of genes that are hif-1-independent, including autophagy genes that promote hypoxia survival. We further show that nuclear hormone receptor nhr-67 is a negative regulator and homeodomain-interacting protein kinase hpk-1 is a positive regulator of the NHR-49 pathway. Together, our experiments define a new, essential hypoxia response pathway that acts in parallel with the well-known HIF-mediated hypoxia response.

Cell nonautonomous roles of NHR-49 in promoting longevity and innate immunity.

Aging and immunity are inextricably linked and many genes that extend life span also enhance immunoresistance. However, it remains unclear whether longevity-enhancing factors modulate immunity and longevity by discrete or shared mechanisms. Here, we demonstrate that the Caenorhabditis elegans pro-longevity factor, NHR-49, also promotes resistance against Pseudomonas aeruginosa but modulates immunity and longevity distinctly. NHR-49 expression increases upon germline ablation, an intervention that extends life span, but was lowered by Pseudomonas infection. The immunosusceptibility induced by nhr-49 loss of function was rescued by neuronal NHR-49 alone, whereas the longevity diminution was rescued by expression in multiple somatic tissues. The well-established NHR-49 target genes, acs-2 and fmo-2, were also differentially regulated following germline elimination or Pseudomonas exposure. Interestingly, neither gene conferred immunity toward Gram-negative Pseudomonas, unlike their known functions against gram-positive pathogens. Instead, genes encoding antimicrobial factors and xenobiotic-response proteins upregulated by NHR-49 contributed to resistance against Pseudomonas. Thus, NHR-49 is differentially regulated by interventions that bring about long-term changes (life span extension) versus short-term stress (pathogen exposure) and in response it orchestrates discrete outputs, including pathogen-specific transcriptional programs.

NHR-49/PPAR-α and HLH-30/TFEB cooperate for C. elegans host defense via a flavin-containing monooxygenase.

The model organism Caenorhabditis elegans mounts transcriptional defense responses against intestinal bacterial infections that elicit overlapping starvation and infection responses, the regulation of which is not well understood. Direct comparison of C. elegans that were starved or infected with Staphylococcus aureus revealed a large infection-specific transcriptional signature, which was almost completely abrogated by deletion of transcription factor hlh-30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene, fmo-2/FMO5. Deletion of fmo-2/FMO5 severely compromised infection survival, thus identifying the first FMO with innate immunity functions in animals. Moreover, fmo-2/FMO5 induction required the nuclear hormone receptor, NHR-49/PPAR-α, which controlled host defense cell non-autonomously. These findings reveal an infection-specific host response to S. aureus, identify HLH-30/TFEB as its main regulator, reveal FMOs as important innate immunity effectors in animals, and identify the mechanism of FMO regulation through NHR-49/PPAR-α during S. aureus infection, with implications for host defense and inflammation in higher organisms.

Transcriptomic analysis of hookworm Ancylostoma ceylanicum life cycle stages reveals changes in G-protein coupled receptor diversity associated with the onset of parasitism.

Free-living nematodes respond to variable and unpredictable environmental stimuli whereas parasitic nematodes exist in a more stable host environment. A positive correlation between the presence of environmental stages in the nematode life cycle and an increasing number of G-protein coupled receptors (GPCRs) reflects this difference in free-living and parasitic lifestyles. As hookworm larvae move from the external environment into a host, they detect uncharacterized host components, initiating a signalling cascade that results in the resumption of development and eventual maturation. Previous studies suggest this process is mediated by GPCRs in amphidial neurons. Here we set out to uncover candidate GPCRs required by a hookworm to recognise its host. First, we identified all potential Ancylostoma ceylanicum GPCRs encoded in the genome. We then used life cycle stage-specific RNA-seq data to identify differentially expressed GPCRs between the free-living infective L3 (iL3) and subsequent parasitic stages to identify receptors involved in the transition to parasitism. We reasoned that GPCRs involved in host recognition and developmental activation would be expressed at higher levels in the environmental iL3 stage than in subsequent stages. Our results support the model that a decrease in GPCR diversity occurs as the larvae develop from the free-living iL3 stage to the parasitic L3 (pL3) in the host over 24-72 h. We find that overall GPCR expression and diversity is highest in the iL3 compared with subsequent parasitic stages. By 72 h, there was an approximately 50% decrease in GPCR richness associated with the moult from the pL3 to the L4. Taken together, our data uncover a negative correlation between GPCR diversity and parasitic development in hookworm. Finally, we demonstrate proof of principal that Caenorhabditis elegans can be used as a heterologous system to examine the expression pattern of candidate host signal chemoreceptors (CRs) from hookworm. We observe expression of candidate host signal CRs in C. elegans, demonstrating that C. elegans can be effectively used as a surrogate to identify expressed hookworm genes. We present several preliminary examples of this strategy and confirm a candidate CR as neuronally expressed.

The longevity-promoting factor, TCER-1, widely represses stress resistance and innate immunity.

Stress resistance and longevity are positively correlated but emerging evidence indicates that they are physiologically distinct. Identifying factors with distinctive roles in these processes is challenging because pro-longevity genes often enhance stress resistance. We demonstrate that TCER-1, the Caenorhabditis elegans homolog of human transcription elongation and splicing factor, TCERG1, has opposite effects on lifespan and stress resistance. We previously showed that tcer-1 promotes longevity in germline-less C. elegans and reproductive fitness in wild-type animals. Surprisingly, tcer-1 mutants exhibit exceptional resistance against multiple stressors, including infection by human opportunistic pathogens, whereas, TCER-1 overexpression confers immuno-susceptibility. TCER-1 inhibits immunity only during fertile stages of life. Elevating its levels ameliorates the fertility loss caused by infection, suggesting that TCER-1 represses immunity to augment fecundity. TCER-1 acts through repression of PMK-1 as well as PMK-1-independent factors critical for innate immunity. Our data establish key roles for TCER-1 in coordinating immunity, longevity and fertility, and reveal mechanisms that distinguish length of life from functional aspects of aging.

Isolation and characterization of a naturally occurring multidrug-resistant strain of the canine hookworm, Ancylostoma caninum.

Soil-transmitted nematodes infect over a billion people and place several billion more at risk of infection. Hookworm disease is the most significant of these soil-transmitted nematodes, with over 500 million people infected. Hookworm infection can result in debilitating and sometimes fatal iron-deficiency anemia, which is particularly devastating in children and pregnant women. Currently, hookworms and other soil-transmitted nematodes are controlled by administration of a single dose of a benzimidazole to targeted populations in endemic areas. While effective, people are quickly re-infected, necessitating frequent treatment. Widespread exposure to anthelmintic drugs can place significant selective pressure on parasitic nematodes to generate resistance, which has severely compromised benzimidazole anthelmintics for control of livestock nematodes in many areas of the world. Here we report, to our knowledge, the first naturally occurring multidrug-resistant strain of the canine hookworm Ancylostoma caninum. We reveal that this isolate is resistant to fenbendazole at the clinical dosage of 50 mg/kg for 3 days. Our data shows that this strain harbors a fixed, single base pair mutation at amino acid 167 of the ß-tubulin isotype 1 gene, and by using CRISPR/Cas9 we demonstrate that introduction of this mutation into the corresponding amino acid in the orthologous ß-tubulin gene of Caenorhabditis elegans confers a similar level of resistance to thiabendazole. We also show that the isolate is resistant to the macrocyclic lactone anthelmintic ivermectin. Understanding the mechanism of anthelmintic resistance is important for rational design of control strategies to maintain the usefulness of current drugs, and to monitor the emergence of resistance. The isolate we describe represents the first multidrug-resistant strain of A. caninum reported, and our data reveal a resistance marker that can emerge naturally in response to heavy anthelminthic treatment.

Refined ab initio gene predictions of Heterorhabditis bacteriophora using RNA-seq.

Interest has recently grown in developing the entomopathogenic nematode Heterorhabditis bacteriophora as a model to genetically dissect the process of parasitic infection. Despite the availability of a full genome assembly, there is substantial variation in gene model accuracy. Here, a methodology is presented for leveraging RNA-seq evidence to generate improved annotations using ab initio gene prediction software. After alignment of reads and subsequent generation of a RNA-seq supported annotation, the new gene prediction models were verified on a selection of genes by comparison with sequenced 5' and 3' rapid amplification of cDNA ends products. By utilising a whole transcriptome for genome annotation, the current reference annotation was enriched, demonstrating the importance of coupling transcriptional data with genome assemblies.

Identification of candidate infection genes from the model entomopathogenic nematode Heterorhabditis bacteriophora.

BACKGROUND: Despite important progress in the field of innate immunity, our understanding of host immune responses to parasitic nematode infections lags behind that of responses to microbes. A limiting factor has been the obligate requirement for a vertebrate host which has hindered investigation of the parasitic nematode infective process. The nematode parasite Heterorhabditis bacteriophora offers great potential as a model to genetically dissect the process of infection. With its mutualistic Photorhabdus luminescens bacteria, H. bacteriophora invades multiple species of insects, which it kills and exploits as a food source for the development of several nematode generations. The ability to culture the life cycle of H. bacteriophora on plates growing the bacterial symbiont makes it a very exciting model of parasitic infection that can be used to unlock the molecular events occurring during infection of a host that are inaccessible using vertebrate hosts. RESULTS: To profile the transcriptional response of an infective nematode during the early stage of infection, we performed next generation RNA sequencing on H. bacteriophora IJs incubated in Manduca sexta hemolymph plasma for 9 h. A subset of up-regulated and down-regulated genes were validated using qRT-PCR. Comparative analysis of the transcriptome with untreated controls found a number of differentially expressed genes (DEGs) which cover a number of different functional categories. A subset of DEGs is conserved across Clade V parasitic nematodes revealing an array of candidate parasitic genes. CONCLUSIONS: Our analysis reveals transcriptional changes in the regulation of a large number of genes, most of which have not been shown previously to play a role in the process of infection. A significant proportion of these genes are unique to parasitic nematodes, suggesting the identification of a group of parasitism factors within nematodes. Future studies using these candidates may provide functional insight into the process of nematode parasitism and also the molecular evolution of parasitism within nematodes.

Correction: DAF-16 and TCER-1 Facilitate Adaptation to Germline Loss by Restoring Lipid Homeostasis and Repressing Reproductive Physiology in C. elegans.

[This corrects the article DOI: 10.1371/journal.pgen.1005788.].

Nuclear hormone receptors as mediators of metabolic adaptability following reproductive perturbations.

Previously, we identified a group of nuclear hormone receptors (NHRs) that promote longevity in the nematode Caenorhabditis elegans following germline-stem cell (GSC) loss. This group included NHR-49, the worm protein that performs functions similar to vertebrate PPARα, a key regulator of lipid metabolism. We showed that NHR-49/PPARα enhances mitochondrial ß-oxidation and fatty acid desaturation upon germline removal, and through the coordinated enhancement of these processes allows the animal to retain lipid homeostasis and undergo lifespan extension. NHR-49/PPARα expression is elevated in GSC-ablated animals, in part, by DAF-16/FOXO3A and TCER-1/TCERG1, two other conserved, pro-longevity transcriptional regulators that are essential for germline-less longevity. In exploring the roles of the other pro-longevity NHRs, we discovered that one of them, NHR-71/HNF4, physically interacted with NHR-49/PPARα. NHR-71/HNF4 did not have a broad impact on the expression of ß-oxidation and desaturation targets of NHR-49/PPARα. But, both NHR-49/PPARα and NHR-71/HNF4 were essential for the increased expression of DAF-16/FOXO3A- and TCER-1/TCERG1-downstream target genes. In addition, nhr-49 inactivation caused a striking membrane localization of KRI-1, the only known common upstream regulator of DAF-16/FOXO3A and TCER-1/TCERG1, suggesting that it may operate in a positive feedback loop to potentiate the activity of this pathway. These data underscore how selective interactions between NHRs that function as nodes in metabolic networks, confer functional specificity in response to different physiological stimuli.

RNAi-mediated gene knockdown by microinjection in the model entomopathogenic nematode Heterorhabditis bacteriophora.

BACKGROUND: Parasitic nematodes threaten the health of humans and livestock and cause a major financial and socioeconomic burden to modern society. Given the widespread distribution of diseases caused by parasitic nematodes there is an urgent need to develop tools that will elucidate the genetic complexity of host-parasite interactions. Heterorhabditis bacteriophora is a parasitic nematode that allows simultaneous monitoring of nematode infection processes and host immune function, and offers potential as a tractable model for parasitic nematode infections. However, molecular tools to investigate these processes are required prior to its widespread acceptance as a robust model organism. In this paper we describe microinjection in adult H. bacteriophora as a suitable means of dsRNA delivery to knockdown gene transcripts. METHODS: RNA interference was used to knockdown four genes by injecting dsRNA directly into the gonad of adult hermaphrodite nematodes. RNAi phenotypes were scored in the F1 progeny on the fifth day post-injection, and knockdown of gene-specific transcripts was quantified with real-time quantitative RT-PCR (qRT-PCR). RESULTS: RNAi injection in adult hermaphrodites significantly decreased the level of target transcripts to varying degrees when compared with controls. The genes targeted by RNAi via injection included cct-2, nol-5, dpy-7, and dpy-13. In each case, RNAi knockdown was confirmed phenotypically by examining the progeny of injected animals, and also confirmed at the transcriptional level by real-time qRT-PCR. CONCLUSIONS: Here we describe for the first time the successful use of microinjection to knockdown gene transcripts in H. bacteriophora. This technique can be used widely to study the molecular basis of parasitism.

DAF-16 and TCER-1 Facilitate Adaptation to Germline Loss by Restoring Lipid Homeostasis and Repressing Reproductive Physiology in C. elegans.

Elimination of the proliferating germline extends lifespan in C. elegans. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with major physiological perturbations. Here, we demonstrate that two conserved transcription regulators essential for the longevity of germline-less adults, DAF-16/FOXO3A and TCER-1/TCERG1, concurrently enhance the expression of multiple genes involved in lipid synthesis and breakdown, and that both gene classes promote longevity. Lipidomic analyses revealed that key lipogenic processes, including de novo fatty acid synthesis, triglyceride production, desaturation and elongation, are augmented upon germline removal. Our data suggest that lipid anabolic and catabolic pathways are coordinately augmented in response to germline loss, and this metabolic shift helps preserve lipid homeostasis. DAF-16 and TCER-1 also perform essential inhibitory functions in germline-ablated animals. TCER-1 inhibits the somatic gene-expression program that facilitates reproduction and represses anti-longevity genes, whereas DAF-16 impedes ribosome biogenesis. Additionally, we discovered that TCER-1 is critical for optimal fertility in normal adults, suggesting that the protein acts as a switch supporting reproductive fitness or longevity depending on the presence or absence of the germline. Collectively, our data offer insights into how organisms adapt to changes in reproductive status, by utilizing the activating and repressive functions of transcription factors and coordinating fat production and degradation.

Germline signals deploy NHR-49 to modulate fatty-acid ß-oxidation and desaturation in somatic tissues of C. elegans.

In C. elegans, removal of the germline extends lifespan significantly. We demonstrate that the nuclear hormone receptor, NHR-49, enables the response to this physiological change by increasing the expression of genes involved in mitochondrial ß-oxidation and fatty-acid desaturation. The coordinated augmentation of these processes is critical for germline-less animals to maintain their lipid stores and to sustain de novo fat synthesis during adulthood. Following germline ablation, NHR-49 is up-regulated in somatic cells by the conserved longevity determinants DAF-16/FOXO and TCER-1/TCERG1. Accordingly, NHR-49 overexpression in fertile animals extends their lifespan modestly. In fertile adults, nhr-49 expression is DAF-16/FOXO and TCER-1/TCERG1 independent although its depletion causes age-related lipid abnormalities. Our data provide molecular insights into how reproductive stimuli are integrated into global metabolic changes to alter the lifespan of the animal. They suggest that NHR-49 may facilitate the adaptation to loss of reproductive potential through synchronized enhancement of fatty-acid oxidation and desaturation, thus breaking down some fats ordained for reproduction and orchestrating a lipid profile conducive for somatic maintenance and longevity.

The C. elegans lifespan assay toolkit.

Since the discovery of single gene mutations that double its lifespan, the nematode Caenorhabditis elegans has provided remarkable insights into the biology of aging. The precisely measurable lifespan of worms has proven to be an efficient tool to assess the impact of various genetic, physiological and environmental factors on organismal aging. In this article, we describe methods to set up and monitor experiments to determine worm lifespan. We include procedures used for classical, small-scale lifespan assays that are generally performed on solid media, and review recent advances in high-throughput, automated longevity experiments conducted in liquid culture and microfluidic devices. In addition, tools that help analyze this data to obtain survival statistics are summarized, and C. elegans strains that offer particular advantages for lifespan studies are listed.

The C. elegans healthspan and stress-resistance assay toolkit.

A wealth of knowledge on the genetic mechanisms that govern aging has emerged from the study of mutants that exhibit enhanced longevity and exceptional resilience to adverse environmental conditions. In these studies, lifespan has been an excellent proxy for establishing the rate of aging, but it is not always correlated with qualitative measures of healthy aging or 'healthspan'. Although the attributes of healthspan have been challenging to define, they share some universal features that are increasingly being incorporated into aging studies. Here we describe methods used to determine Caenorhabditis elegans healthspan. These include assessments of tissue integrity and functionality and resistance to a variety of biotic and abiotic stressors. We have chosen to include simple, rapid assays in this collection that can be easily undertaken in any C. elegans laboratory, and can be relied on to provide a preliminary but thorough insight into the healthspan of a population.

The many landscapes of recombination in Drosophila melanogaster.

Recombination is a fundamental biological process with profound evolutionary implications. Theory predicts that recombination increases the effectiveness of selection in natural populations. Yet, direct tests of this prediction have been restricted to qualitative trends due to the lack of detailed characterization of recombination rate variation across genomes and within species. The use of imprecise recombination rates can also skew population genetic analyses designed to assess the presence and mode of selection across genomes. Here we report the first integrated high-resolution description of genomic and population variation in recombination, which also distinguishes between the two outcomes of meiotic recombination: crossing over (CO) and gene conversion (GC). We characterized the products of 5,860 female meioses in Drosophila melanogaster by genotyping a total of 139 million informative SNPs and mapped 106,964 recombination events at a resolution down to 2 kilobases. This approach allowed us to generate whole-genome CO and GC maps as well as a detailed description of variation in recombination among individuals of this species. We describe many levels of variation in recombination rates. At a large-scale (100 kb), CO rates exhibit extreme and highly punctuated variation along chromosomes, with hot and coldspots. We also show extensive intra-specific variation in CO landscapes that is associated with hotspots at low frequency in our sample. GC rates are more uniformly distributed across the genome than CO rates and detectable in regions with reduced or absent CO. At a local scale, recombination events are associated with numerous sequence motifs and tend to occur within transcript regions, thus suggesting that chromatin accessibility favors double-strand breaks. All these non-independent layers of variation in recombination across genomes and among individuals need to be taken into account in order to obtain relevant estimates of recombination rates, and should be included in a new generation of population genetic models of the interaction between selection and linkage.