How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.

Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.

Symbiotic bacteria interact with their host through symbiotic cues. Here, we took advantage of the mutualism between Drosophila and Lactiplantibacillus plantarum (Lp) to investigate a novel mechanism of host-symbiont interaction. Using chemically defined diets, we found that association with Lp improves the growth of larvae-fed amino acid-imbalanced diets, even though Lp cannot produce the limiting amino acid. We show that in this context Lp supports its host's growth through a molecular dialogue that requires functional operons encoding ribosomal and transfer RNAs (r/tRNAs) in Lp and the general control nonderepressible 2 (GCN2) kinase in Drosophila's enterocytes. Our data indicate that Lp's r/tRNAs are packaged in extracellular vesicles and activate GCN2 in a subset of larval enterocytes, a mechanism necessary to remodel the intestinal transcriptome and ultimately to support anabolic growth. Based on our findings, we propose a novel beneficial molecular dialogue between host and microbes, which relies on a non-canonical role of GCN2 as a mediator of non-nutritional symbiotic cues encoded by r/tRNA operons.

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

Neural circuits detect environmental changes and drive behavior. The routes of information flow through dense neural networks are dynamic, but the mechanisms underlying this circuit flexibility are poorly understood. Here, we define a sensory context-dependent and neuropeptide-regulated switch in the composition of a C. elegans salt sensory circuit. The primary salt detectors, ASE sensory neurons, used BLI-4 endoprotease-dependent cleavage to release the insulin-like peptide INS-6 in response to large, but not small, changes in external salt stimuli. Insulins, signaling through the insulin receptor DAF-2, functionally switched the AWC olfactory sensory neuron into an interneuron in the salt circuit. Worms with disrupted insulin signaling had deficits in salt attraction, suggesting that peptidergic signaling potentiates responses to high salt stimuli, which may promote ion homeostasis. Our results indicate that sensory context and neuropeptide signaling modify neural networks and suggest general mechanisms for generating flexible behavioral outputs by modulating neural circuit composition.

We have used a paleogenetics approach to investigate the genetic landscape of coat color variation in ancient Eurasian dog and wolf populations. We amplified DNA fragments of two genes controlling coat color, Mc1r (Melanocortin 1 Receptor) and CBD103 (canine-β-defensin), in respectively 15 and 19 ancient canids (dogs and wolf morphotypes) from 14 different archeological sites, throughout Asia and Europe spanning from ca. 12 000 B.P. (end of Upper Palaeolithic) to ca. 4000 B.P. (Bronze Age). We provide evidence of a new variant (R301C) of the Melanocortin 1 receptor (Mc1r) and highlight the presence of the beta-defensin melanistic mutation (CDB103-K locus) on ancient DNA from dog-and wolf-morphotype specimens. We show that the dominant K(B) allele (CBD103), which causes melanism, and R301C (Mc1r), the variant that may cause light hair color, are present as early as the beginning of the Holocene, over 10,000 years ago. These results underline the genetic diversity of prehistoric dogs. This diversity may have partly stemmed not only from the wolf gene pool captured by domestication but also from mutations very likely linked to the relaxation of natural selection pressure occurring in-line with this process.

The rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions.

Nervous systems extract and process information from the environment to alter animal behavior and physiology. Despite progress in understanding how different stimuli are represented by changes in neuronal activity, less is known about how they affect broader neural network properties. We developed a framework for using graph-theoretic features of neural network activity to predict ecologically relevant stimulus properties, in particular stimulus identity. We used the transparent nematode, Caenorhabditis elegans, with its small nervous system to define neural network features associated with various chemosensory stimuli. We first immobilized animals using a microfluidic device and exposed their noses to chemical stimuli while monitoring changes in neural activity of more than 50 neurons in the head region. We found that graph-theoretic features, which capture patterns of interactions between neurons, are modulated by stimulus identity. Further, we show that a simple machine learning classifier trained using graph-theoretic features alone, or in combination with neural activity features, can accurately predict salt stimulus. Moreover, by focusing on putative causal interactions between neurons, the graph-theoretic features were almost twice as predictive as the neural activity features. These results reveal that stimulus identity modulates the broad, network-level organization of the nervous system, and that graph theory can be used to characterize these changes.

Animals integrate changes in external and internal environments to generate behavior. While neural circuits detecting external cues have been mapped, less is known about how internal states like hunger are integrated into behavioral outputs. Here, we use the nematode C. elegans to examine how changes in internal nutritional status affect chemosensory behaviors. We show that acute food deprivation leads to a reversible decline in repellent, but not attractant, sensitivity. This behavioral change requires two conserved transcription factors MML-1 (MondoA) and HLH-30 (TFEB), both of which translocate from the intestinal nuclei to the cytoplasm during food deprivation. Next, we identify the insulin-like peptide INS-31 as a candidate ligand relaying food-status signals from the intestine to other tissues. Further, we show that neurons likely use the DAF-2 insulin receptor and AGE-1/PI-3 Kinase, but not DAF-16/FOXO to integrate these intestine-released peptides. Altogether, our study shows how internal food status signals are integrated by transcription factors and intestine-neuron signaling to generate flexible behaviors via the gut-brain axis.

Animals respond to predators by altering their behavior and physiological states, but the underlying signaling mechanisms are poorly understood. Using the interactions between Caenorhabditis elegans and its predator, Pristionchus pacificus, we show that neuronal perception by C. elegans of a predator-specific molecular signature induces instantaneous escape behavior and a prolonged reduction in oviposition. Chemical analysis revealed this predator-specific signature to consist of a class of sulfolipids, produced by a biochemical pathway required for developing predacious behavior and specifically induced by starvation. These sulfolipids are detected by four pairs of C. elegans amphid sensory neurons that act redundantly and recruit cyclic nucleotide-gated (CNG) or transient receptor potential (TRP) channels to drive both escape and reduced oviposition. Functional homology of the delineated signaling pathways and abolishment of predator-evoked C. elegans responses by the anti-anxiety drug sertraline suggests a likely conserved or convergent strategy for managing predator threats.

Animals possess conserved mechanisms to detect pathogens and to improve survival in their presence by altering their own behavior and physiology. Here, we utilize Caenorhabditis elegans as a model host to ask whether bacterial volatiles constitute microbe-associated molecular patterns. Using gas chromatography-mass spectrometry, we identify six prominent volatiles released by the bacterium Pseudomonas aeruginosa. We show that a specific volatile, 1-undecene, activates nematode odor sensory neurons inducing both flight and fight responses in worms. Using behavioral assays, we show that worms are repelled by 1-undecene and that this aversion response is driven by the detection of this volatile through AWB odor sensory neurons. Furthermore, we find that 1-undecene odor can induce immune effectors specific to P. aeruginosa via AWB neurons and that brief pre-exposure of worms to the odor enhances their survival upon subsequent bacterial infection. These results show that 1-undecene derived from P. aeruginosa serves as a pathogen-associated molecular pattern for the induction of protective responses in C. elegans.

Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.

Prey respond to predators by altering their behavior to optimize their own fitness and survival. Specifically, prey are known to avoid predator-occupied territories to reduce their risk of harm or injury to themselves and their progeny. We probe the interactions between Caenorhabditis elegans and its naturally cohabiting predator Pristionchus uniformis to reveal the pathways driving changes in prey behavior. While C. elegans prefers to lay its eggs on a bacteria food lawn, the presence of a predator inside a lawn induces C. elegans to lay more eggs away from that lawn. We confirm that this change in egg laying is in response to bites from predators, rather than to predatory secretions. Moreover, predator-exposed prey continue to lay their eggs away from the dense lawn even after the predator is removed, indicating a form of learning. Next, we find that mutants in dopamine synthesis significantly reduce egg laying behavior off the lawn in both predator-free and predator-inhabited lawns, which we can rescue by transgenic complementation or supplementation with exogenous dopamine. Moreover, we find that dopamine is likely released from multiple dopaminergic neurons and requires combinations of both D1- (DOP-1) and D2-like (DOP-2 and DOP-3) dopamine receptors to alter predator-induced egg laying behavior, whereas other combinations modify baseline levels of egg laying behavior. Together, we show that dopamine signaling can alter both predator-free and predator-induced foraging strategies, suggesting a role for this pathway in defensive behaviors.

Insects living in nutritionally poor environments often establish long-term relationships with intracellular bacteria that supplement their diets and improve their adaptive and invasive powers. Even though these symbiotic associations have been extensively studied on physiological, ecological, and evolutionary levels, few studies have focused on the molecular dialogue between host and endosymbionts to identify genes and pathways involved in endosymbiosis control and dynamics throughout host development.

The survival of animals depends, among other things, on their ability to identify threats in their surrounding environment. Senses such as olfaction, vision and taste play an essential role in sampling their living environment, including microorganisms, some of which are potentially pathogenic. This study focuses on the mechanisms of detection of bacteria by the Drosophila gustatory system. We demonstrate that the peptidoglycan (PGN) that forms the cell wall of bacteria triggers an immediate feeding aversive response when detected by the gustatory system of adult flies. Although we identify ppk23+ and Gr66a+ gustatory neurons as necessary to transduce fly response to PGN, we demonstrate that they play very different roles in the process. Time-controlled functional inactivation and in vivo calcium imaging demonstrate that while ppk23+ neurons are required in the adult flies to directly transduce PGN signal, Gr66a+ neurons must be functional in larvae to allow future adults to become PGN sensitive. Furthermore, the ability of adult flies to respond to bacterial PGN is lost when they hatch from larvae reared under axenic conditions. Recolonization of germ-free larvae, but not adults, with a single bacterial species, Lactobacillus brevis, is sufficient to restore the ability of adults to respond to PGN. Our data demonstrate that the genetic and environmental characteristics of the larvae are essential to make the future adults competent to respond to certain sensory stimuli such as PGN.

Biodiversity has to be accurately evaluated to assess more precisely possible dam effects on fish populations, in particular on the most biodiverse rivers such as the Mekong River. To improve tools for fish biodiversity assessment, a methodological survey was performed in the surroundings of a recent hydropower dam in the Mekong basin, the Nam Theun 2 project. Results of two different approaches, experimental surface gillnets capture and environmental DNA metabarcoding assays based on 12S ribosomal RNA and cytochrome b, were compared during 3 years (2014-2016). Pitfalls and benefits were identified for each method but the combined use of both approaches indisputably allows describing more accurately fish diversity around the reservoir. Importantly, striking convergent results were observed for biodiversity reports. 75% of the fish species caught by gillnets (62/82) were shown by the metabarcoding study performed on DNA extracted from water samples. eDNA approach also revealed to be sensitive by detecting 30 supplementary species known as present before the dam construction but never caught by gillnets during 3 years. Furthermore, potential of the marker-genes study might be underestimated since it was not possible to assign some sequences at lower taxonomic levels. Although 121 sequences were generated for this study, a third of species in the area, that exhibits high endemism, are still unknown in DNA databases. Efforts to complete local reference libraries must continue to improve the taxonomic assignment quality when using the non-invasive and promising eDNA approach. These results are of broader interest because of increasing number of hydropower projects in the Mekong Basin. They reveal the crucial importance to sample tissues/DNA of species before dam projects, i.e. before the species could become endangered and difficult to catch, to obtain more precise biomonitoring in the future as we believe eDNA metabarcoding will rapidly be integrated as a standard tool in such studies.

The ability to evaluate variability in the environment is vital for making optimal behavioral decisions. Here we show that Caenorhabditis elegans evaluates variability in its food environment and modifies its future behavior accordingly. We derive a behavioral model that reveals a critical period over which information about the food environment is acquired and predicts future search behavior. We also identify a pair of high-threshold sensory neurons that encode variability in food concentration and the downstream dopamine-dependent circuit that generates appropriate search behavior upon removal from food. Further, we show that CREB is required in a subset of interneurons and determines the timescale over which the variability is integrated. Interestingly, the variability circuit is a subset of a larger circuit driving search behavior, showing that learning directly modifies the very same neurons driving behavior. Our study reveals how a neural circuit decodes environmental variability to generate contextually appropriate decisions.

5' AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl)-thioureido]-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl)-thioureido]-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.

Brominated flame retardants are suspected to act as disruptors of thyroid hormone signaling. This raises the concern that they might affect children's cognitive functions by influencing thyroid hormone signaling in the developing brain. We present here an in vitro analysis of the ability of the most common compounds, tetrabromobisphenol A (TBBPA) and BDE-209, to alter thyroid hormone response based on a model neural cell line and genome-wide analysis of gene expression.

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.