L4 Larval Stage Research Articles

Microbiota from Rhabditis regina may alter nematode entomopathogenicity.

Here we report the presence of the entomopathogenic nematode Rhabditis (Rhabditoides) regina affecting white grubs (Phyllophaga sp. and Anomala sp.) in Mexico and R. regina-associated bacteria. Bioassays were performed to test the entomopathogenic capacity of dauer and L2 and L3 (combined) larval stages. Furthermore, we determined the diversity of bacteria from laboratory nematodes cultivated for 2years (dauer and L2-L3 larvae) and from field nematodes (dauer and L2-L3 larvae) in addition to the virulence in Galleria mellonella larvae of some bacterial species from both laboratory and field nematodes. Dauer and non-dauer larvae of R. regina killed G. mellonella. Bacteria such as Serratia sp. (isolated from field nematodes) and Klebsiella sp. (isolated from larvae of laboratory and field nematodes) may explain R. regina entomopathogenic capabilities. Different bacteria were found in nematodes after subculturing in the laboratory suggesting that R. regina may acquire bacteria in different environments. However, there were some consistently found bacteria from laboratory and field nematodes such as Pseudochrobactrum sp., Comamonas sp., Alcaligenes sp., Klebsiella sp., Acinetobacter sp., and Leucobacter sp. that may constitute the nematode microbiome. Results showed thatsome bacteria contributing to entomopathogenicity may be lost in the laboratory representing a disadvantage when nematodes are cultivated to be used for biological control.

Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans.

Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts.

Circadian Activators Are Expressed Days before They Initiate Clock Function in Late Pacemaker Neurons fromDrosophila

Circadian pacemaker neurons in the Drosophila brain control daily rhythms in locomotor activity. These pacemaker neurons can be subdivided into early or late groups depending on whether rhythms in period (per) and timeless (tim) expression are initiated at the first instar (L1) larval stage or during metamorphosis, respectively. Because CLOCK-CYCLE (CLK-CYC) heterodimers initiate circadian oscillator function by activating per and tim transcription, a Clk-GFP transgene was used to mark when late pacemaker neurons begin to develop. We were surprised to see that CLK-GFP was already expressed in four of five clusters of late pacemaker neurons during the third instar (L3) larval stage. CLK-GFP is only detected in postmitotic neurons from L3 larvae, suggesting that these four late pacemaker neuron clusters are formed before the L3 larval stage. A GFP-cyc transgene was used to show that CYC, like CLK, is also expressed exclusively in pacemaker neurons from L3 larval brains, demonstrating that CLK-CYC is not sufficient to activate per and tim in late pacemaker neurons at the L3 larval stage. These results suggest that most late pacemaker neurons develop days before novel factors activate circadian oscillator function during metamorphosis.

Protective effects of novel organic selenium compounds against oxidative stress in the nematode Caenorhabditis elegans

Organic selenium compounds possess numerous biological properties, including antioxidant activity. Yet, the high toxicity of some of them, such as diphenyl diselenide (DPDS), is a limiting factor in their current usage. Accordingly, we tested four novel organic selenium compounds in the non-parasite nematode Caenorhabditis elegans and compared their efficacy to DPDS. The novel organic selenium compounds are β-selenoamines 1-phenyl-3-(p-tolylselanyl)propan-2-amine (C1) and 1-(2-methoxyphenylselanyl)-3-phenylpropan-2-amine (C2) and analogs of DPDS 1,2-bis(2-methoxyphenyl)diselenide (C3) and 1,2-bisp-tolyldiselenide (C4). Synchronized worms at the L4 larval stage were exposed for one hour in M9 buffer to these compounds. Oxidative stress conditions were induced by juglone (200μM) and heat shock (35°C). Moreover, we evaluated C. elegans behavior, GST-4::GFP (glutathione S-transferase) expression and the activity of acetylcholinesterase (AChE). All tested compounds efficiently restored viability in juglone stressed worms. However, DPDS, C2, C3 and C4 significantly decreased the defecation cycle time. Juglone-induced GST-4::GFP expression was not attenuated in worms pretreated with the novel compounds, except with C2. Finally, AChE activity was reduced by DPDS, C2, C3 and C4. To our knowledge, this is study firstly showed the effects of C1, C2, C3 and C4 selenium-derived compounds in C. elegans. Low toxic effects were noted, except for reduction in the defecation cycle, which is likely associated with AChE inhibition. The juglone-induced stress (reduced viability) was fully reversed by compounds to control animal levels. C2 was also efficient in reducing the juglone-induced GST-4::GFP expression, suggesting the latter may mediate the stress induced by this compound. Future studies could be profitably directed at addressing additional molecular mechanisms that mediate the protective effects of these novel organic selenium compounds.

FRAP Analysis of LET-23::GFP in the Vulval Epithelial Cells of Living Caenorhabditis elegans Larvae

The Caenorhabditis elegans (C. elegans) vulva is a well-established system to study organ development as the molecular mechanisms that govern its formation are conserved in animals. Of special interest is the EGFR/RAS/MAPK signaling pathway that is required for fate acquisition and morphogenesis of the vulva. let-23 encodes the sole homologue of the epidermal growth factor receptor (EGFR), is expressed at the plasma membrane of the vulval precursor cells (VPCs) and is activated by LIN-3 EGF at the end of the L3 larval stage to initiate vulva development. LET-23 activity can be modulated through altering its subcellular and plasma membrane localization. To study the trafficking of EGF receptor in a living organism, we created a functional LET-23::GFP translational reporter worm line (Haag et al., 2014) and quantified the mobile fraction of LET-23::GFP at the basolateral membrane of the VPCs by fluorescence recovery after photobleaching (FRAP). Here we describe the protocol for LET-23::GFP FRAP at the basolateral membrane of the VPCs and the data analysis using FIJI (ImageJ).

Tetraspanin (TSP-17) protects dopaminergic neurons against 6-OHDA-induced neurodegeneration in C. elegans.

Parkinson's disease (PD), the second most prevalent neurodegenerative disease after Alzheimer's disease, is linked to the gradual loss of dopaminergic neurons in the substantia nigra. Disease loci causing hereditary forms of PD are known, but most cases are attributable to a combination of genetic and environmental risk factors. Increased incidence of PD is associated with rural living and pesticide exposure, and dopaminergic neurodegeneration can be triggered by neurotoxins such as 6-hydroxydopamine (6-OHDA). In C. elegans, this drug is taken up by the presynaptic dopamine reuptake transporter (DAT-1) and causes selective death of the eight dopaminergic neurons of the adult hermaphrodite. Using a forward genetic approach to find genes that protect against 6-OHDA-mediated neurodegeneration, we identified tsp-17, which encodes a member of the tetraspanin family of membrane proteins. We show that TSP-17 is expressed in dopaminergic neurons and provide genetic, pharmacological and biochemical evidence that it inhibits DAT-1, thus leading to increased 6-OHDA uptake in tsp-17 loss-of-function mutants. TSP-17 also protects against toxicity conferred by excessive intracellular dopamine. We provide genetic and biochemical evidence that TSP-17 acts partly via the DOP-2 dopamine receptor to negatively regulate DAT-1. tsp-17 mutants also have subtle behavioral phenotypes, some of which are conferred by aberrant dopamine signaling. Incubating mutant worms in liquid medium leads to swimming-induced paralysis. In the L1 larval stage, this phenotype is linked to lethality and cannot be rescued by a dop-3 null mutant. In contrast, mild paralysis occurring in the L4 larval stage is suppressed by dop-3, suggesting defects in dopaminergic signaling. In summary, we show that TSP-17 protects against neurodegeneration and has a role in modulating behaviors linked to dopamine signaling.

Ethanol-induced differential gene expression and acetyl-CoA metabolism in a longevity model of the nematode Caenorhabditis elegans

Previous studies have shown that exposing adults of the soil-dwelling nematode Caenorhabditis elegans to concentrations of ethanol in the range of 100–400mM results in slowed locomotion, decreased fertility, and reduced longevity. On the contrary, lower concentrations of ethanol (0.86–68mM) have been shown to cause a two- to three-fold increase in the life span of animals in the stress resistant L1 larval stage in the absence of a food source. However, little is known about how gene and protein expression is altered by low concentrations of ethanol and the mechanism for the increased longevity. Therefore, we used biochemical assays and next generation mRNA sequencing to identify genes and biological pathways altered by ethanol. RNA-seq analysis of L1 larvae incubated in the presence of 17mM ethanol resulted in the significant differential expression of 649 genes, 274 of which were downregulated and 375 were upregulated. Many of the genes significantly altered were associated with the conversion of ethanol and triglycerides to acetyl-CoA and glucose, suggesting that ethanol is serving as an energy source in the increased longevity of the L1 larvae as well as a signal for fat utilization. We also asked if L1 larvae could sense ethanol and respond by directed movement. Although we found that L1 larvae can chemotax to benzaldehyde, we observed little or no chemotaxis to ethanol. Understanding how low concentrations of ethanol increase the lifespan of L1 larvae may provide insight into not only the longevity pathways in C. elegans, but also in those of higher organisms.

Transcriptomics of selenium toxicity and deficiency in C. elegans (374.1)

Selenium (Se) is a trace element that is both essential and toxic. C. elegans expresses only 1 selenoprotein, and we have shown recently that Se is not essential but is toxic to C. elegans (PLOS ONE 8:e71525). To characterize Se regulation of the transcriptome, we conducted a microarray study in C. elegans cultured in axenic media with 0, 0.05, 0.1, 0.2 and 0.4 mM Se as selenite. Because worms cultured in 0.2 and 0.4 mM Se display delayed growth, all cultures were staged for harvest at the L4‐larval stage. Relative to 0.1 mM Se, this resulted in 2, 10, 6 and 2%, respectively, of the transcriptome being altered 蠅 2‐fold. C. elegans cultured in low Se (0 mM Se) exhibited no signs of deficiency, but a set of 222 genes changed 蠅 2‐fold; functional analysis revealed up‐regulation of genes related to lipid modification. Se toxicity (0.2 and 0.4 mM Se) altered the expression of 183 Se toxicity‐specific genes, after filtering against gene sets for sulfur and cadmium toxicity, that were significantly enriched in oxidoreductase genes, and significantly depleted in genes related to structural components of collagen and the cuticle. This study shows that while there is no phenotype associated with Se deficiency in C. elegans, low Se elicits a transcriptomic phenotype. In contrast, Se is toxic and elicits transcript changes supporting a hypothesis that derangement of cuticle thiols underlies altered development.Grant Funding Source: Supported by WIS 01435 and by UW Selenium Nutrition Research fund

Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm’s Excretory/secretory antigens.

In vivo effect of selected medicinal plants against gastrointestinal nematodes of sheep

Nematode resistance to anthelmintic drugs affects small ruminant production globally. This study evaluated in vivo effects of five plant extracts as alternative nematode treatments. Animals were divided into six groups. Each group was randomly assigned a treatment: Abamectin and Praziquantel (CAP) (positive Control), ethanol extracts of Ananas comosus (AC), Aloe ferox (AF), Allium sativum (AS), Lespedeza cuneata (LC) and Warburgia salutaris (WS). These were applied as an oral dose (100 mg kg(-1) BW), one dose per week per sheep for 42 days (Phase 1). From day 42 sheep were orally dosed for 3 consecutive days with the same treatments in the same groups (Phase 2). Rectal faecal samples were taken every 7 days up to day 63. Eggs per gram of faeces were counted in individual rectal samples, L3 larval stage was counted in faecal cultures, with four replicates per group. For plant extracts, EPG decreased (P < 0.001) with time and efficacy of plant increased (P < 0.001) with time. Ananas comosus and L. cuneata treatments had the highest efficacies of 58% and 61%, respectively, in Phase 1; and 77% and 81%, respectively, in Phase 2. Continuous treatment with these plants could further reduce nematode parasites and improve host health.

Patterning of sexually dimorphic neurogenesis in the Caenorhabditis elegans ventral cord by Hox and TALE homeodomain transcription factors

Reproduction in animals requires development of distinct neurons in each sex. In C. elegans, most ventral cord neurons (VCNs) are present in both sexes, with the exception of six hermaphrodite-specific neurons (VCs) and nine pairs of male-specific neurons (CAs and CPs) that arise from analogous precursor cells. How are the activities of sexual regulators and mediators of neuronal survival, division, and fate coordinated to generate sex-specificity in VCNs? To address this, we have developed a toolkit of VCN markers that allows us to examine sex-specific neurogenesis, asymmetric fates of daughters of a neuroblast division, and regional specification on the anteroposterior axis. Here, we describe the roles of the Hox transcription factors LIN-39 and MAB-5 in promoting survival, differentiation, and regionalization of VCNs. We also find that the TALE class homeodomain proteins CEH-20 and UNC-62 contribute to specification of neurotransmitter fate in males. Furthermore, we identify that VCN sex is determined during the L1 larval stage. These findings, combined with future analyses made possible by the suite of VCN markers described here, will elucidate how Hox-mediated cell fate decisions and sex determination intersect to influence development of neuronal sex differences.

Absolute Quantification of a Steroid Hormone that Regulates Development in Caenorhabditis elegans

Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA increased 100-fold after carboxyl group derivatization with 2-picolylamine. High-resolution selected ion monitoring (HR-SIM) on a Q-Orbitrap mass spectrometer Q Exactive outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer TSQ Quantum Discovery. With a limit of quantification as low as 1 pg of DA, the HR-SIM method enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms, DA increases from 0.04 ± 0.02 ng/mg protein in the L1 larval stage to 1.21 ± 0.67 ng/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, four genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-β, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and most of them grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.

Structure of monomeric Na-GST-3, a glutathione S-transferase from the major human hookworm parasite Necator americanus.

Necator americanus is the major cause of human hookworm infection, which is a global cause of anemia in the developing world. Ongoing efforts to control hookworm infection include the identification of candidate vaccine antigens as well as potential therapeutic targets from the infective L3 larval stages and adult stages of the parasite. One promising family of proteins are the adult-stage-secreted cytosolic glutathione S-transferases (GSTs). Nematode GSTs facilitate the inactivation and degradation of a variety of electrophilic substrates (drugs) via the nucleophilic addition of reduced glutathione. Parasite GSTs also play significant roles in multi-drug resistance and the modulation of host immune defense mechanisms. Here, the structure of Na-GST-3, one of three GSTs secreted by adult-stage N. americanus, is reported. Unlike most GST structures, the Na-GST-3 crystal contains a monomer in the asymmetric unit. However, the monomer forms a prototypical GST dimer across the crystallographic twofold. A glutathione from the fermentation process is bound to the monomer. The overall binding cavity of Na-GST-3 is reminiscent of that of other N. americanus GSTs and is larger and capable of binding a wider array of ligands than GSTs from organisms that have other major detoxifying mechanisms. Furthermore, despite having low sequence identity to the host GST, Na-GST-3 has a greater tertiary-structure similarity to human sigma-class GST than was observed for the other N. americanus GSTs.

A microfluidic device for the continuous culture and analysis of Caenorhabditis elegans in a toxic aqueous environment

The nematode Caenorhabditis elegans (C. elegans) receives attention as a bioindicator, and the C. elegans condition has been recently analyzed using microfluidic devices equipped with an imaging system. To establish a method without an imaging system, we have proposed a novel microfluidic device with which to analyze the condition of C. elegans from the capacitance change using a pair of micro-electrodes. The device was designed to culture C. elegans, to expose C. elegans to an external stimulus, such as a chemical or toxicant, and to measure the capacitance change which indicates the condition of C. elegans. In this study, to demonstrate the capability of our device in a toxic aqueous environment, the device was applied to examine the effect of cadmium on C. elegans. Thirty L4 larval stage C. elegans were divided into three groups. One group was a control group and the other groups were exposed to cadmium solutions with concentrations of 5% and 10% LC50 for 24 h. The capacitance change and the body volume of C. elegans as a reference were measured four times and we confirmed the correlation between them. It shows that our device can analyze the condition of C. elegans without an imaging system.

NeuroChip: A Microfluidic Electrophysiological Device for Genetic and Chemical Biology Screening of Caenorhabditis elegans Adult and Larvae

Genetic and chemical biology screens of C. elegans have been of enormous benefit in providing fundamental insight into neural function and neuroactive drugs. Recently the exploitation of microfluidic devices has added greater power to this experimental approach providing more discrete and higher throughput phenotypic analysis of neural systems. Here we make a significant addition to this repertoire through the design of a semi-automated microfluidic device, NeuroChip, which has been optimised for selecting worms based on the electrophysiological features of the pharyngeal neural network. We demonstrate this device has the capability to sort mutant from wild-type worms based on high definition extracellular electrophysiological recordings. NeuroChip resolves discrete differences in excitatory, inhibitory and neuromodulatory components of the neural network from individual animals. Worms may be fed into the device consecutively from a reservoir and recovered unharmed. It combines microfluidics with integrated electrode recording for sequential trapping, restraining, recording, releasing and recovering of C. elegans. Thus mutant worms may be selected, recovered and propagated enabling mutagenesis screens based on an electrophysiological phenotype. Drugs may be rapidly applied during the recording thus permitting compound screening. For toxicology, this analysis can provide a precise description of sub-lethal effects on neural function. The chamber has been modified to accommodate L2 larval stages showing applicability for small size nematodes including parasitic species which otherwise are not tractable to this experimental approach. We also combine NeuroChip with optogenetics for targeted interrogation of the function of the neural circuit. NeuroChip thus adds a new tool for exploitation of C. elegans and has applications in neurogenetics, drug discovery and neurotoxicology.

Biological interactions between soil saprotrophic fungi and Ascaris suum eggs

The in vitro effect of saprotrophic soil fungi on the embryonic development of Ascaris suum was evaluated. The fungi tested were Aspergillus fumigatus, Aspergillus terreus, Penicillium citrinum, Penicillium expansum, Fusarium oxysporum and Trichothecium roseum, isolated from children's recreation areas in the city of Lodz (Poland). Each species was co-cultured with A. suum egg suspension (6×103eggs/ml) at 25±2°C for 60 days. Each day, 100 eggs were randomly collected and their developmental stage was classified macroscopically. Additionally, at days 4, 7, 14, 28, 42 and 60 of incubation, the viability and the percentage of eggs with morphological altered embryo/larva were determined in each sample. Microscopic examination revealed that exposure of eggs to the mycelium of examined fungi inhibited embryogenesis of A. suum. All control culture eggs reached L2 larval stage after 26 days of incubation, while the experimental cultures did so after 32–51 days, depending on the fungal species. Three species were found to exhibit very high inhibitory activity on A. suum egg development: A. terreus, P. expansum and F. oxysporum. Embryopathies and non-viable embryos/larvae were observed significantly more frequently in the eggs co-cultured with fungal species than in control cultures. The fungus-exposed eggs revealed morphological alternations in the early zygotic cleavage, blastula, gastrula and larval stages. After 60 days of incubation with mycelia of P. expansum, A. terreus and F. oxysporum, the mortality of the larvae reached 55.3–60.3%. P. expansum and F. oxysporum showed hyphal penetration and internal egg colonization of A. suum eggs.

Delayed accumulation of intestinal coliform bacteria enhances life span and stress resistance in Caenorhabditis elegans fed respiratory deficient E. coli

BackgroundStudies with the nematode model Caenorhabditis elegans have identified conserved biochemical pathways that act to modulate life span. Life span can also be influenced by the composition of the intestinal microbiome, and C. elegans life span can be dramatically influenced by its diet of Escherichia coli. Although C. elegans is typically fed the standard OP50 strain of E. coli, nematodes fed E. coli strains rendered respiratory deficient, either due to a lack coenzyme Q or the absence of ATP synthase, show significant life span extension. Here we explore the mechanisms accounting for the enhanced nematode life span in response to these diets.ResultsThe intestinal load of E. coli was monitored by determination of worm-associated colony forming units (cfu/worm or coliform counts) as a function of age. The presence of GFP-expressing E. coli in the worm intestine was also monitored by fluorescence microscopy. Worms fed the standard OP50 E. coli strain have high cfu and GFP-labeled bacteria in their guts at the L4 larval stage, and show saturated coliform counts by day five of adulthood. In contrast, nematodes fed diets of respiratory deficient E. coli lacking coenzyme Q lived significantly longer and failed to accumulate bacteria within the lumen at early ages. Animals fed bacteria deficient in complex V showed intermediate coliform numbers and were not quite as long-lived. The results indicate that respiratory deficient Q-less E. coli are effectively degraded in the early adult worm, either at the pharynx or within the intestine, and do not accumulate in the intestinal tract until day ten of adulthood.ConclusionsThe findings of this study suggest that the nematodes fed the respiratory deficient E. coli diet live longer because the delay in bacterial colonization of the gut subjects the worms to less stress compared to worms fed the OP50 E. coli diet. This work suggests that bacterial respiration can act as a virulence factor, influencing the ability of bacteria to colonize and subsequently harm the animal host. Respiratory deficient bacteria may pose a useful model for probing probiotic relationships within the gut microbiome in higher organisms.

Impact of Sublethal Levels of Environmental Pollutants Found in Sewage Sludge on a Novel Caenorhabditis elegans Model Biosensor

A transgenic strain of the model nematode Caenorhabditis elegans in which bioluminescence reports on relative, whole-organism ATP levels was used to test an environmentally-relevant mixture of pollutants extracted from processed sewage sludge. Changes in bioluminescence, following exposure to sewage sludge extract, were used to assess relative ATP levels and overall metabolic health. Reproductive function and longevity were also monitored. A short (up to 8 h) sublethal exposure of L4 larval stage worms to sewage sludge extract had a concentration-dependent, detrimental effect on energy status, with bioluminescence decreasing to 50–60% of the solvent control (1% DMSO). Following longer exposure (22–24 h), the energy status of the nematodes showed recovery as assessed by bioluminescence. Continuous exposure to sewage sludge extract from the L4 stage resulted in a shorter median lifespan relative to that of solvent or medium control animals, but only in the presence of 400–600 µM 5-fluoro-2′-deoxyuridine (FUdR), which was incorporated to inhibit reproduction. This indicated that FUdR increased lifespan, and that the effect was counteracted by SSE. Exposure to sewage sludge extract from the L1 stage led to slower growth and a delayed onset of egg laying. When L1 exposed nematodes reached the reproductive stage, no effect on egg laying rate or egg number in the uterus was observed. DMSO itself (1%) had a significant inhibitory effect on growth and development of C. elegans exposed from the L1 stage and on reproduction when exposed from the L4 stage. Results demonstrate subtle adverse effects on C. elegans of a complex mixture of environmental pollutants that are present, individually, in very low concentrations and indicate that our biosensor of energy status is a novel, sensitive, rapid, quantitative, whole-organism test system which is suitable for high throughput risk assessment of complex pollutant mixtures.

Specific Recognition of Biologically Active Amyloid-β Oligomers by a New Surface Plasmon Resonance-based Immunoassay and an in Vivo Assay in Caenorhabditis elegans

Soluble oligomers of the amyloid-β (Aβ) peptide play a key role in the pathogenesis of Alzheimer's disease, but their elusive nature makes their detection challenging. Here we describe a novel immunoassay based on surface plasmon resonance (SPR) that specifically recognizes biologically active Aβ oligomers. As a capturing agent, we immobilized on the sensor chip the monoclonal antibody 4G8, which targets a central hydrophobic region of Aβ. This SPR assay allows specific recognition of oligomeric intermediates that rapidly appear and disappear during the incubation of synthetic Aβ(1-42), discriminating them from monomers and higher order aggregates. The species recognized by SPR generate ionic currents in artificial lipid bilayers and inhibit the physiological pharyngeal contractions in Caenorhabditis elegans, a new method for testing the toxic potential of Aβ oligomers. With these assays we found that the formation of biologically relevant Aβ oligomers is inhibited by epigallocatechin gallate and increased by the A2V mutation, previously reported to induce early onset dementia. The SPR-based immunoassay provides new opportunities for detection of toxic Aβ oligomers in biological samples and could be adapted to study misfolding proteins in other neurodegenerative disorders.

Inhibition of ROS elevation and damage to mitochondrial function prevents lead-induced neurotoxic effects on structures and functions of AFD neurons in Caenorhabditis elegans

AbstractHere we investigated the possible roles of oxidative stress in the formation of decreased thermotaxis to cultivation temperature in lead (Pb)-exposed nematodes Caenorhabditis elagans. Exposure to Pb at the examined concentrations decreased thermotaxis behaviors, and induced severe deficits in the structural properties of AFD sensory neurons. Meanwhile, Pb exposure caused the induction of severe oxidative damage, reactive oxygen species (ROS) production, and mitochondrial dysfunction in young adults. Moreover, pre-treatment with the antioxidants dimethyl sulfoxide (DMSO), ascorbate and N-acetyl-L-cysteine (NAC), used to inhibit both the ROS elevation and the mitochondrial dysfunction caused by Pb exposure, at the L2-larval stage prevented the induction of oxidative damage and the formation of severe deficits in thermotaxis and structural properties of AFD sensory neurons in Pb-exposed young adults. Therefore, the formation of oxidative stress caused by Pb exposure may be due to both the induction of ROS elevation and damage to mitochondrial function, and oxidative stress may play a key role in inducing the neurotoxic effects on the structures and function of AFD sensory neurons in Pb-exposed nematodes.