Bacterially produced metabolites protect C. elegans neurons from degeneration.

Caenorhabditis elegans and its cognate bacterial diet comprise a reliable, widespread model to study diet and microbiota effects on host physiology. Nonetheless, how diet influences the rate at which neurons die remains largely unknown. A number of models have been used in C. elegans as surrogates for neurodegeneration. One of these is a C. elegans strain expressing a neurotoxic allele of the mechanosensory abnormality protein 4 (MEC-4d) degenerin/epithelial Na+ (DEG/ENaC) channel, which causes the progressive degeneration of the touch receptor neurons (TRNs). Using this model, our study evaluated the effect of various dietary bacteria on neurodegeneration dynamics. Although degeneration of TRNs was steady and completed at adulthood in the strain routinely used for C. elegans maintenance (Escherichia coli OP50), it was significantly reduced in environmental and other laboratory bacterial strains. Strikingly, neuroprotection reached more than 40% in the E. coli HT115 strain. HT115 protection was long lasting well into old age of animals and was not restricted to the TRNs. Small amounts of HT115 on OP50 bacteria as well as UV-killed HT115 were still sufficient to produce neuroprotection. Early growth of worms in HT115 protected neurons from degeneration during later growth in OP50. HT115 diet promoted the nuclear translocation of DAF-16 (ortholog of the FOXO family of transcription factors), a phenomenon previously reported to underlie neuroprotection caused by down-regulation of the insulin receptor in this system. Moreover, a daf-16 loss-of-function mutation abolishes HT115-driven neuroprotection. Comparative genomics, transcriptomics, and metabolomics approaches pinpointed the neurotransmitter γ-aminobutyric acid (GABA) and lactate as metabolites differentially produced between E. coli HT115 and OP50. HT115 mutant lacking glutamate decarboxylase enzyme genes (gad), which catalyze the conversion of GABA from glutamate, lost the ability to produce GABA and also to stop neurodegeneration. Moreover, in situ GABA supplementation or heterologous expression of glutamate decarboxylase in E. coli OP50 conferred neuroprotective activity to this strain. Specific C. elegans GABA transporters and receptors were required for full HT115-mediated neuroprotection. Additionally, lactate supplementation also increased anterior ventral microtubule (AVM) neuron survival in OP50. Together, these results demonstrate that bacterially produced GABA and other metabolites exert an effect of neuroprotection in the host, highlighting the role of neuroactive compounds of the diet in nervous system homeostasis.

Diapause induces functional axonal regeneration after necrotic insult in C. elegans.

Many neurons are unable to regenerate after damage. The ability to regenerate after an insult depends on life stage, neuronal subtype, intrinsic and extrinsic factors. C. elegans is a powerful model to test the genetic and environmental factors that affect axonal regeneration after damage, since its axons can regenerate after neuronal insult. Here we demonstrate that diapause promotes the complete morphological regeneration of truncated touch receptor neuron (TRN) axons expressing a neurotoxic MEC-4(d) DEG/ENaC channel. Truncated axons of different lengths were repaired during diapause and we observed potent axonal regrowth from somas alone. Complete morphological regeneration depends on DLK-1 but neuronal sprouting and outgrowth is DLK-1 independent. We show that TRN regeneration is fully functional since animals regain their ability to respond to mechanical stimulation. Thus, diapause induced regeneration provides a simple model of complete axonal regeneration which will greatly facilitate the study of environmental and genetic factors affecting the rate at which neurons die.

Modeling amyloid beta and tau pathology in human cerebral organoids.

The typical abnormalities observed in the brain of Alzheimer's disease (AD) patients include synaptic alterations, neuronal death, brain inflammation, and the accumulation of protein aggregates in the form of amyloid plaques and neurofibrillary tangles. Despite the development of many animal and in vitro models for AD, there is a lack of an experimental approach that fully recapitulates essential aspects of the disease in human cells. Here, we report the generation of a new model to study AD, consisting of cerebral organoids (COs) produced from human-induced pluripotent stem cells (iPSCs). Under our experimental conditions, COs grow to form three-dimensional (3D) structures containing neural areas with cortical-like organization. Analysis of COs by histological and biochemical methods revealed that organoids produced from iPSCs derived from patients affected by familial AD or Down syndrome (DS) spontaneously develop over time pathological features of AD, including accumulation of structures highly reminiscent to amyloid plaques and neurofibrillary tangles. These pathological abnormalities were not observed in COs generated from various controls, including human iPSCs from healthy individuals, human iPSCs from patients affected by Creutzfeldt-Jakob disease, mouse embryonic stem cells (ESCs), or mouse iPSCs. These findings enable modeling genetic AD in a human cellular context in a 3D cortical-like tissue developed in vitro from patient-specific stem cells. This system provides a more relevant disease model compared to pre-existing methods and offers a new platform for discovery of novel targets and screening of drugs for therapeutic intervention.

Diapause formation and downregulation of insulin-like signaling via DAF-16/FOXO delays axonal degeneration and neuronal loss.

Axonal degeneration is a key event in the pathogenesis of neurodegenerative conditions. We show here that mec-4d triggered axonal degeneration of Caenorhabditis elegans neurons and mammalian axons share mechanistical similarities, as both are rescued by inhibition of calcium increase, mitochondrial dysfunction, and NMNAT overexpression. We then explore whether reactive oxygen species (ROS) participate in axonal degeneration and neuronal demise. C. elegans dauers have enhanced anti-ROS systems, and dauer mec-4d worms are completely protected from axonal degeneration and neuronal loss. Mechanistically, downregulation of the Insulin/IGF-1-like signaling (IIS) pathway protects neurons from degenerating in a DAF-16/FOXO-dependent manner and is related to superoxide dismutase and catalase-increased expression. Caloric restriction and systemic antioxidant treatment, which decrease oxidative damage, protect C. elegans axons from mec-4d-mediated degeneration and delay Wallerian degeneration in mice. In summary, we show that the IIS pathway is essential in maintaining neuronal homeostasis under pro-degenerative stimuli and identify ROS as a key intermediate of neuronal degeneration in vivo. Since axonal degeneration represents an early pathological event in neurodegeneration, our work identifies potential targets for therapeutic intervention in several conditions characterized by axonal loss and functional impairment.

Conditional gene expression and RNAi using MEC-8-dependent splicing in C. elegans.

We describe a method for conditional regulation of gene expression based on the processing of an intron cassette. The RNA processing factor MEC-8 is necessary for the function of the Caenorhabditis elegans touch receptor neurons; mec-8 mutants are touch insensitive. We show here that this insensitivity involves the loss of MEC-8-dependent splicing of mec-2, which encodes a component of the mechanosensory transduction complex. MEC-8 is needed to remove the ninth intron in mec-2 pre-mRNA to form the longest of three mRNAs, mec-2a. Without MEC-8, splicing causes the termination of the transcript. Inclusion of mec-2 intron 9 is sufficient to convey mec-8-dependent regulation on other genes and, in mec-8(u218ts) mutants, resulted in their temperature-dependent expression. Because mec-8 is expressed ubiquitously in embryos and extensively in larvae, this system should produce temperature-sensitive expression for most genes. As an example, we report a strain that exhibits temperature-dependent RNA interference.

Enhanced neuronal RNAi in C. elegans using SID-1.

We expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNA interference (RNAi), in C. elegans neurons. This expression increased the response of neurons to double-stranded (ds)RNA delivered by feeding. Mutations in the lin-15b and lin-35 genes enhanced this effect. Worms expressing neuronal SID-1 showed RNAi phenotypes when fed with bacteria expressing dsRNA for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes. Neuronal expression of sid-1 decreased nonneuronal RNAi, suggesting that neurons expressing transgenic sid-1(+) served as a sink for dsRNA. This effect, or a sid-1(-) background, can be used to uncover neuronal defects for lethal genes. Expression of sid-1(+) from cell-specific promoters in sid-1 mutants results in cell-specific feeding RNAi. We used these strains to identify a role for integrin signaling genes in mechanosensation.

The Conscious Nematode: Exploring Hallmarks of Minimal Phenomenal Consciousness in Caenorhabditis Elegans.

While subcellular components of cognition and affectivity that involve the interaction between experience, environment, and physiology -such as learning, trauma, or emotion- are being identified, the physical mechanisms of phenomenal consciousness remain more elusive. We are interested in exploring whether ancient, simpler organisms such as nematodes have minimal consciousness. Is there something that feels like to be a worm? Or are worms blind machines? 'Simpler' models allow us to simultaneously extract data from multiple levels such as slow and fast neural dynamics, structural connectivity, molecular dynamics, behavior, decision making, etc., and thus, to test predictions of the current frameworks in dispute. In the present critical review, we summarize the current models of consciousness in order to reassess in light of the new evidence whether Caenorhabditis elegans, a nematode with a nervous system composed of 302 neurons, has minimal consciousness. We also suggest empirical paths to further advance consciousness research using C. elegans.

Mientras que los componentes subcelulares de fenómenos cognitivos y afectivos que involucran la interacción entre experiencia, ambiente y fisiología -tales como aprendizaje, trauma, o emociones- son identificados con cada vez mayor detalle, los mecanismos biofísicos de la consciencia fenoménica permanecen elusivos. Nos interesa explorar si organismos sencillos como los nemátodos presentan consciencia mínima. ¿Hay algo que se sienta como ser un gusano? ¿O acaso los gusanos son máquinas carentes de toda experiencia? Los modelos "sencillos" nos permiten extraer datos de múltiples niveles en simultáneo: dinámica neuronal rápida y lenta, conectividad estructural, dinámica molecular, conducta, toma de decisiones, etc., y así testear predicciones de las propuestas teóricas actuales en disputa. En esta revisión compendiamos los modelos actuales de consciencia para evaluar, considerando la evidencia reciente, si Caenorhabditis elegans, un nemátodo con un sistema nervioso de 302 neuronas, tiene consciencia mínima. Sugerimos además vías empíricas para desarrollar investigaciones en consciencia utilizando C. elegans.

Microbiota and Diapause-Induced Neuroprotection Share a Dependency on Calcium But Differ in Their Effects on Mitochondrial Morphology.

The balance between the degeneration and regeneration of damaged neurons depends on intrinsic and environmental variables. In nematodes, neuronal degeneration can be reversed by intestinal GABA and lactate-producing bacteria, or by hibernation driven by food deprivation. However, it is not known whether these neuroprotective interventions share common pathways to drive regenerative outcomes. Using a well established neuronal degeneration model in the touch circuit of the bacterivore nematode Caenorhabditis elegans, we investigate the mechanistic commonalities between neuroprotection offered by the gut microbiota and hunger-induced diapause. Using transcriptomics approaches coupled to reverse genetics, we identify genes that are necessary for neuroprotection conferred by the microbiota. Some of these genes establish links between the microbiota and calcium homeostasis, diapause entry, and neuronal function and development. We find that extracellular calcium as well as mitochondrial MCU-1 and reticular SCA-1 calcium transporters are needed for neuroprotection by bacteria and by diapause entry. While the benefits exerted by neuroprotective bacteria require mitochondrial function, the diet itself does not affect mitochondrial size. In contrast, diapause increases both the number and length of mitochondria. These results suggest that metabolically induced neuronal protection may occur via multiple mechanisms.

The Assembly of Bacteria Living in Natural Environments Shapes Neuronal Integrity and Behavioral Outputs in Caenorhabditis elegans.

Bacterivore nematodes are the most abundant animals in the biosphere, largely contributing to global biogeochemistry. Thus, the effects of environmental microbes on the nematodes' life-history traits are likely to contribute to the general health of the biosphere. Caenorhabditis elegans is an excellent model to study the behavioral and physiological outputs of microbial diets. However, the effects of complex natural bacterial assemblies have only recently been reported, as most studies have been carried out with monoxenic cultures of laboratory-reared bacteria. Here, we quantified the physiological, phenotypic, and behavioral traits of C. elegans feeding on two bacteria that were coisolated with wild nematodes from a soil sample. These bacteria were identified as a putative novel species of Stenotrophomonas named Stenotrophomonas sp. strain Iso1 and a strain of Bacillus pumilus designated Iso2. The distinctive behaviors and developmental patterns observed in animals fed with individual isolates changed when bacteria were mixed. We studied in more depth the degeneration rate of the touch circuit of C. elegans and show that B. pumilus alone is protective, while the mix with Stenotrophomonas sp. is degenerative. The analysis of the metabolite contents of each isolate and their combination identified NAD+ as being potentially neuroprotective. In vivo supplementation shows that NAD+ restores neuroprotection to the mixes and also to individual nonprotective bacteria. Our results highlight the distinctive physiological effects of bacteria resembling native diets in a multicomponent scenario rather than using single isolates on nematodes. IMPORTANCE Do behavioral choices depend on animals' microbiota? To answer this question, we studied how different bacterial assemblies impact the life-history traits of the bacterivore nematode C. elegans using isolated bacteria found in association with wild nematodes in Chilean soil. We identified the first isolate, Iso1, as a novel species of Stenotrophomonas and isolate Iso2 as Bacillus pumilus. We find that worm traits such as food choice, pharyngeal pumping, and neuroprotection, among others, are dependent on the biota composition. For example, the neurodegeneration of the touch circuit needed to sense and escape from predators in the wild decreases when nematodes are fed on B. pumilus, while its coculture with Stenotrophomonas sp. eliminates neuroprotection. Using metabolomics analysis, we identify metabolites such as NAD+, present in B. pumilus yet lost in the mix, as being neuroprotective and validated their protective effects using in vivo experiments.

Lewy Body-like Pathology and Loss of Dopaminergic Neurons in Midbrain Organoids Derived from Familial Parkinson's Disease Patient.

Progressive accumulation of α-Synuclein (αSyn) in Lewy bodies (LBs) and loss of dopaminergic (DA) neurons are the hallmark pathological features of Parkinson's disease (PD). Although currently available in vitro and in vivo models have provided crucial information about PD pathogenesis, the mechanistic link between the progressive accumulation of αSyn into LBs and the loss of DA neurons is still unclear. To address this, it is critical to model LB formation and DA neuron loss, the two key neuropathological aspects of PD, in a relevant in vitro system. In this study, we developed a human midbrain-like organoid (hMBO) model of PD. We demonstrated that hMBOs generated from induced pluripotent stem cells (hiPSCs), derived from a familial PD (fPD) patient carrying αSyn gene (SNCA) triplication accumulate pathological αSyn over time. These cytoplasmic inclusions spatially and morphologically resembled diverse stages of LB formation and were composed of key markers of LBs. Importantly, the progressive accumulation of pathological αSyn was paralleled by the loss of DA neurons and elevated apoptosis. The model developed in this study will complement the existing in vitro models of PD and will provide a unique platform to study the spatiotemporal events governing LB formation and their relation with neurodegeneration. Furthermore, this model will also be beneficial for in vitro screening and the development of therapeutic compounds.

Interspecies effectors of a transgenerational memory of bacterial infection in Caenorhabditis elegans.

The inheritance of memory is an adaptive trait. Microbes challenge the immunity of organisms and trigger behavioral adaptations that can be inherited, but how bacteria produce inheritance of a trait is unknown. We use Caenorhabditis elegans and its bacteria to study the transgenerational RNA dynamics of interspecies crosstalk leading to a heritable behavior. A heritable response of C. elegans to microbes is the pathogen-induced diapause (PIDF), a state of suspended animation to evade infection. We identify RsmY, a small RNA involved in quorum sensing in Pseudomonas aeruginosa as a trigger of PIDF. The histone methyltransferase (HMT) SET-18/SMYD3 and the argonaute HRDE-1, which promotes multi-generational silencing in the germline, are also needed for PIDF initiation. The HMT SET-25/EHMT2 is necessary for memory maintenance in the transgenerational lineage. Our work is a starting point to understanding microbiome-induced inheritance of acquired traits, and the transgenerational influence of microbes in health and disease.

The Heritability of Behaviors Associated With the Host Gut Microbiota.

What defines whether the interaction between environment and organism creates a genetic memory able to be transferred to subsequent generations? Bacteria and the products of their metabolism are the most ubiquitous biotic environments to which every living organism is exposed. Both microbiota and host establish a framework where environmental and genetic factors are integrated to produce adaptive life traits, some of which can be inherited. Thus, the interplay between host and microbe is a powerful model to study how phenotypic plasticity is inherited. Communication between host and microbe can occur through diverse molecules such as small RNAs (sRNAs) and the RNA interference machinery, which have emerged as mediators and carriers of heritable environmentally induced responses. Notwithstanding, it is still unclear how the organism integrates sRNA signaling between different tissues to orchestrate a systemic bacterially induced response that can be inherited. Here we discuss current evidence of heritability produced by the intestinal microbiota from several species. Neurons and gut are the sensing systems involved in transmitting changes through transcriptional and post-transcriptional modifications to the gonads. Germ cells express inflammatory receptors, and their development and function are regulated by host and bacterial metabolites and sRNAs thus suggesting that the dynamic interplay between host and microbe underlies the host's capacity to transmit heritable behaviors. We discuss how the host detects changes in the microbiota that can modulate germ cells genomic functions. We also explore the nature of the interactions that leave permanent or long-term memory in the host and propose mechanisms by which the microbiota can regulate the development and epigenetic reprogramming of germ cells, thus influencing the inheritance of the host. We highlight the vast contribution of the bacterivore nematode C. elegans and its commensal and pathogenic bacteria to the understanding on how behavioral adaptations can be inter and transgenerational inherited.

Interspecies RNA Interactome of Pathogen and Host in a Heritable Defensive Strategy.

Communication with bacteria deeply impacts the life history traits of their hosts. Through specific molecules and metabolites, bacteria can promote short- and long-term phenotypic and behavioral changes in the nematode Caenorhabditis elegans. The chronic exposure of C. elegans to pathogens promotes the adaptive behavior in the host's progeny called pathogen-induced diapause formation (PIDF). PIDF is a pathogen avoidance strategy induced in the second generation of animals infected and can be recalled transgenerationally. This behavior requires the RNA interference machinery and specific nematode and bacteria small RNAs (sRNAs). In this work, we assume that RNAs from both species co-exist and can interact with each other. Under this principle, we explore the potential interspecies RNA interactions during PIDF-triggering conditions, using transcriptomic data from the holobiont. We study two transcriptomics datasets: first, the dual sRNA expression of Pseudomonas aeruginosa PAO1 and C. elegans in a transgenerational paradigm for six generations and second, the simultaneous expression of sRNAs and mRNA in intergenerational PIDF. We focus on those bacterial sRNAs that are systematically overexpressed in the intestines of animals compared with sRNAs expressed in host-naïve bacteria. We selected diverse in silico methods that represent putative mechanisms of RNA-mediated interspecies interaction. These interactions are as follows: heterologous perfect and incomplete pairing between bacterial RNA and host mRNA; sRNAs of similar sequence expressed in both species that could mimic each other; and known or predicted eukaryotic motifs present in bacterial transcripts. We conclude that a broad spectrum of tools can be applied for the identification of potential sRNA and mRNA targets of the interspecies RNA interaction that can be subsequently tested experimentally.

Mitochondrial transplant to replenish damaged mitochondria: A novel therapeutic strategy for neurodegenerative diseases?

Neurodegenerative diseases are currently some of the most debilitating and incurable illness, including highly prevalent disorders, such as Alzheimer's and Parkinson's disease. Despite impressive advances in understanding the molecular basis of neurodegenerative diseases, several clinical trials have failed in identifying drugs that successfully delay or stop disease progression. New targets are likely necessary to successfully combat these devastating diseases. In this chapter, we review the evidence indicating that impairment in the cellular energy machinery in the form of mitochondrial damage and dysfunction may be at the root of neurodegeneration. We also propose that transplant of functional isolated mitochondria may overcome the energetic damage and delay the progression of neurodegenerative diseases.

Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons from Familial Parkinson's Disease Patients Display α-Synuclein Pathology and Abnormal Mitochondrial Morphology.

Accumulation of α-synuclein (α-syn) into Lewy bodies (LBs) and mitochondrial abnormalities are the two cardinal pathobiological features of Parkinson's disease (PD), which are associated with the loss of dopaminergic neurons. Although α-syn accumulates in many different cellular and mouse models, these models generally lack LB features. Here, we generated midbrain dopaminergic (mDA) neuronal cultures from induced pluripotent stem cells (iPSCs) derived from familial PD (fPD) patients and healthy controls. We show that mDA neuronal cultures from fPD patients with A53T mutation and α-syn gene (SNCA) triplication display pathological α-syn deposits, which spatially and morphologically resemble LBs. Importantly, we did not find any apparent accumulation of pathological α-syn in mDA neuronal culture derived from a healthy donor. Furthermore, we show that there are morphological abnormalities in the mitochondrial network in mDA neuronal cultures from fPD patients. Consequently, these cells were more susceptible to mitochondrial damage compared with healthy donor-derived mDA neuronal cultures. Our results indicate that the iPSC-derived mDA neuronal culture platform can be used to investigate the spatiotemporal appearance of LBs, as well as their composition, architecture, and relationship with mitochondrial abnormalities.

Modeling Traumatic Brain Injury in Human Cerebral Organoids.

Traumatic brain injury (TBI) is a head injury that disrupts the normal brain structure and function. TBI has been extensively studied using various in vitro and in vivo models. Most of the studies have been done with rodent models, which may respond differently to TBI than human nerve cells. Taking advantage of the recent development of cerebral organoids (COs) derived from human induced pluripotent stem cells (iPSCs), which resemble the architecture of specific human brain regions, here, we adapted the controlled cortical impact (CCI) model to induce TBI in human COs as a novel in vitro platform. To adapt the CCI procedure into COs, we have developed a phantom brain matrix, matching the mechanical characteristics of the brain, altogether with an empty mouse skull as a platform to allow the use of the stereotactic CCI equipment on COs. After the CCI procedure, COs were histologically prepared to evaluate neurons and astrocyte populations using the microtubule-associated protein 2 (MAP2) and the glial fibrillary acidic protein (GFAP). Moreover, a marker of metabolic response, the neuron-specific enolase (NSE), and cellular death using cleaved caspase 3 were also analyzed. Our results show that human COs recapitulate the primary pathological changes of TBI, including metabolic alterations related to neuronal damage, neuronal loss, and astrogliosis. This novel approach using human COs to model TBI in vitro holds great potential and opens new alternatives for understanding brain abnormalities produced by TBI, and for the development and testing of new therapeutic approaches.

Protocol for controlled cortical impact in human cerebral organoids to model traumatic brain injury.

Modeling traumatic brain injury (TBI) has been a challenge. Rodent and cellular models have provided relevant contributions despite their limitations. Here, we present a protocol for a TBI model based on the controlled cortical impact (CCI) performed on human cerebral organoids (COs), self-assembled 3D cultures that recapitulate features of the human brain. Here, we generate COs from iPSCs obtained from reprogrammed fibroblasts. For complete details on the use and execution of this protocol, please refer to Ramirez et al. (2021).

Quantification of Bacteria Residing in Caenorhabditis elegans Intestine.

Quantification of intestinal colonization by pathogenic or commensal bacteria constitute a critical part of the analysis to understand host-microbe interactions during different time points of their interplay. Here we detail a method to isolate non-pathogenic and pathogenic bacteria from C. elegans intestines, and classify gut phenotypes induced by bacterial pathogens using fluorescently-tagged bacteria. Furthermore, these methods can be used to isolate and identify new culturable bacterial species from natural microbiomes of wild nematodes.

Intergenerational Pathogen-Induced Diapause in Caenorhabditis elegans Is Modulated by mir-243.

The interaction and communication between bacteria and their hosts modulate many aspects of animal physiology and behavior. Dauer entry as a response to chronic exposure to pathogenic bacteria in Caenorhabditis elegans is an example of a dramatic survival response. This response is dependent on the RNA interference (RNAi) machinery, suggesting the involvement of small RNAs (sRNAs) as effectors. Interestingly, dauer formation occurs after two generations of interaction with two unrelated moderately pathogenic bacteria. Therefore, we sought to discover the identity of C. elegans RNAs involved in pathogen-induced diapause. Using transcriptomics and differential expression analysis of coding and long and small noncoding RNAs, we found that mir-243-3p (the mature form of mir-243) is the only transcript continuously upregulated in animals exposed to both Pseudomonas aeruginosa and Salmonella enterica for two generations. Phenotypic analysis of mutants showed that mir-243 is required for dauer formation under pathogenesis but not under starvation. Moreover, DAF-16, a master regulator of defensive responses in the animal and required for dauer formation was found to be necessary for mir-243 expression. This work highlights the role of a small noncoding RNA in the intergenerational defensive response against pathogenic bacteria and interkingdom communication.IMPORTANCE Persistent infection of the bacterivore nematode C. elegans with bacteria such as P. aeruginosa and S. enterica makes the worm diapause or hibernate. By doing this, the worm closes its mouth, avoiding infection. This response takes two generations to be implemented. In this work, we looked for genes expressed upon infection that could mediate the worm diapause triggered by pathogens. We identify mir-243-3p as the only transcript commonly upregulated when animals feed on P. aeruginosa and S. enterica for two consecutive generations. Moreover, we demonstrate that mir-243-3p is required for pathogen-induced dauer formation, a new function that has not been previously described for this microRNA (miRNA). We also find that the transcriptional activators DAF-16, PQM-1, and CRH-2 are necessary for the expression of mir-243 under pathogenesis. Here we establish a relationship between a small RNA and a developmental change that ensures the survival of a percentage of the progeny.

Use of C. elegans Diapause to Study Transgenerational Responses to Pathogen Infection.

We present a protocol for the study of inter and transgenerational behavioral responses to pathogenesis in C. elegans. Transgenerational and intergenerational effects of microbes are best studied in model organisms with short life cycles, large progenies, and quantifiable cellular and behavioral outcomes. This chapter encompasses basic techniques used to study the consequences of bacterial infection in C. elegans, including worm growth, quantification of dauer larvae, and quantification of bacterial population dynamics within individual animals. Specific methods for studying transgenerational effects and their duration are also described.