Microfluidic electric parallel egg-laying assay and application to in-vivo toxicity screening of microplastics using C. elegans

AbstractEnvironmental pollutants like microplastics are posing health concerns on aquatic animals and the ecosystem. Microplastic toxicity studies using C. elegans as a model are evolving but methodologically hindered from obtaining statistically strong data sets, detecting toxicity effects based on microplastics uptake, and correlating physiological and behavioural effects at an individual-worm level. In this paper, we report a novel microfluidic electric egg-laying assay for phenotypical assessment of multiple worms in parallel. The effects of glucose and polystyrene microplastics at various concentrations on the worms’ electric egg-laying, length, diameter, and length contraction during exposure to electric signal were studied. The device contained eight parallel worm-dwelling microchannels called electric traps, with equivalent electrical fields, in which the worms were electrically stimulated for egg deposition and fluorescently imaged for assessment of neuronal and microplastic uptake expression. A new bidirectional stimulation technique was developed, and the device design was optimized to achieve a testing efficiency of 91.25%. Exposure of worms to 100mM glucose resulted in a significant reduction in their egg-laying and size. The effects of 1μm polystyrene microparticles at concentrations of 100 and 1000 mg/L on the electric egg-laying behaviour, size, and neurodegeneration of N2 and NW1229 (expressing GFP pan-neuronally) worms were also studied. Of the two concentrations, 1000 mg/L caused severe egg-laying deficiency and growth retardation as well as neurodegeneration. Additionally, using single-worm level phenotyping, we noticed intra-population variability in microplastics uptake and correlation with the above physiological and behavioural phenotypes, which was hidden in the population-averaged results. Taken together, these results suggest the appropriateness of our microfluidic assay for toxicological studies and for assessing the phenotypical heterogeneity in response to microplastics.

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Occurrence of Salmonella typhimurium resistance under sublethal/repeated exposure to cauliflower infusion and infection effects on Caernohabditis elegans host test organism

Food Science and Technology International ◽

10.1177/1082013219873500 ◽

2019 ◽

Vol 26 (2) ◽

pp. 151-159

Author(s):

Maria Sanz-Puig ◽

Alejandra Arana-Lozano ◽

M Consuelo Pina-Pérez ◽

Pablo Fernández ◽

Antonio Martínez ◽

...

Keyword(s):

Food Industry ◽

Research Work ◽

Test Organism ◽

Egg Laying ◽

Repeated Exposure ◽

Resistant Bacteria ◽

Bacterial Cells ◽

Natural Antimicrobial ◽

C Elegans

Resistant bacteria to antimicrobials are increasingly emerging in medical, food industry and livestock environments. The present research work assesses the capability of Salmonella enterica var Typhimurium to become adapted under the exposure to a natural cauliflower antimicrobial by-product infusion in consecutive repeated exposure cycles. Caenorhabditis elegans was proposed as in vivo host-test organism to compare possible changes in the virulent pattern of the different rounds treated S. enterica var Typhimurium and untreated bacterial cells. According to the obtained results, S. enterica var Typhimurium was able to generate resistance against a repeated exposure to cauliflower by-product infusion 5% (w/v), increasing the resistance with the number of exposed repetitions. Meanwhile, at the first exposure, cauliflower by-product infusion was effective in reducing S. enterica var Typhimurium (≈1 log10 cycle), and S. enterica var Typhimurium became resistant to this natural antimicrobial after the second and third treatment-round and was able to grow (≈1 log10 cycle). In spite of the increased resistance observed for repeatedly treated bacteria, the present study reveals no changes on C. elegans infection effects between resistant and untreated S. enterica var Typhimurium, according to phenotypic parameters evaluation (lifespan duration and egg-laying).

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In vitro and preliminary in vivo toxicity screening of high-surface-area TiO2–chondroitin-4-sulfate nanocomposites for bone regeneration application

Colloids and Surfaces B Biointerfaces ◽

10.1016/j.colsurfb.2015.02.027 ◽

2015 ◽

Vol 128 ◽

pp. 347-356 ◽

Cited By ~ 7

Author(s):

Kavitha Kandiah ◽

Rajendran Venkatachalam ◽

Chunyan Wang ◽

Suresh Valiyaveettil ◽

Kumaresan Ganesan

Keyword(s):

Bone Regeneration ◽

Surface Area ◽

High Surface ◽

High Surface Area ◽

Toxicity Screening ◽

In Vivo Toxicity

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Probing the relationship between BTBD9 and MEIS1 in C. elegans and mouse

Experimental Results ◽

10.1017/exp.2020.12 ◽

2020 ◽

Vol 1 ◽

Cited By ~ 1

Author(s):

Shangru Lyu ◽

Atbin Doroodchi ◽

Yi Sheng ◽

Mark P. DeAndrade ◽

Youfeng Yang ◽

...

Keyword(s):

Restless Legs Syndrome ◽

Wheel Running ◽

Egg Laying ◽

Motor Deficit ◽

Double Knockout ◽

Genetic Studies ◽

C Elegans ◽

Increased Sensitivity ◽

The Relationship

AbstractRestless legs syndrome (RLS) is a neurological disorder characterized by an urge to move and uncomfortable sensations. Genetic studies have identified polymorphisms in up to 19 risk loci, including MEIS1 and BTBD9. Rodents deficient in either homolog show RLS-like phenotypes. However, whether MEIS1 and BTBD9 interact in vivo is unclear. Here, with C. elegans, we observed that the hyperactive egg-laying behavior caused by loss of BTBD9 homolog was counteracted by knockdown of MEIS1 homolog. This was further investigated in mutant mice with Btbd9, Meis1, or both knocked out. The double knockout mice showed an earlier onset of the motor deficit in a wheel running test but did not have increased sensitivity to heat stimuli as observed in single knock outs. Meis1 protein level was not influenced by Btbd9 deficiency, and Btbd9 transcription was not affected by Meis1 haploinsufficiency. Our results demonstrate that MEIS1 and BTBD9 do not regulate each other.

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The neural G protein Gαo tagged with GFP at an internal loop is functional in C. elegans

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab167 ◽

2021 ◽

Author(s):

Santosh Kumar ◽

Andrew C Olson ◽

Michael R Koelle

Keyword(s):

Genetic Analysis ◽

G Protein ◽

Molecular Mechanisms ◽

Fluorescent Protein ◽

Protein Complexes ◽

Egg Laying ◽

Biochemical Purification ◽

Internal Loop ◽

C Elegans

Abstract Gαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to complement the already well-developed genetic analysis of Gαo signaling.

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Acetylcholine Signaling Genes are Required for Cocaine-Stimulated Egg Laying in Caenorhabditis elegans

10.1101/2020.11.24.396333 ◽

2020 ◽

Author(s):

Soren Emerson ◽

Megan Hay ◽

Mark Smith ◽

David Blauch ◽

Nicole Snyder ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Nicotinic Acetylcholine Receptors ◽

Intracellular Signaling ◽

Acetylcholine Receptors ◽

Egg Laying ◽

Synthetic Enzymes ◽

C Elegans ◽

Degradative Enzymes ◽

Serotonin Reuptake Transporter

Despite the toxicity and addictive liability associated with cocaine abuse, its mode of action is not completely understood, and effective pharmacotherapeutic interventions remain elusive. The cholinergic effects of cocaine on acetylcholine receptors, synthetic enzymes, and degradative enzymes have been the focus of relatively little empirical investigation. Due to its genetic tractability and anatomical simplicity, the egg laying circuit of the hermaphroditic nematode, Caenorhabditis elegans, is a powerful model system to precisely examine the genetic and molecular targets of cocaine in vivo. Here, we report a novel cocaine-induced phenotype in Caenorhabditis elegans, cocaine-stimulated egg laying. In addition, we present the results of an in vivo candidate screen of synthetic enzymes, receptors, degradative enzymes, and downstream components of the intracellular signaling cascades of the main neurotransmitter systems that control Caenorhabditis elegans egg laying. Our results show that cocaine-stimulated egg laying is dependent on acetylcholine synthesis and synaptic release, functional nicotinic acetylcholine receptors, and the Caenorhabditis elegans acetylcholinesterases. Further, we show that cocaine-stimulated egg laying is not dependent on other neurotransmitters besides acetylcholine, including serotonin, dopamine, octopamine, and tyramine. Finally, our data show that cocaine-stimulated egg laying is increased in mutants for the C. elegans serotonin reuptake transporter as well as mutants for a 5-HT-gated chloride channel likely expressed in the locomotion circuit. Together, these results highlight serotonergic inhibition of egg laying behavior, functional connectivity between the egg laying and locomotion circuits in Caenorhabditis elegans, and possible discrete cholinergic and serotonergic effects of cocaine in the egg laying and locomotion circuits, respectively.

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Identified neurons in C. elegans coexpress vesicular transporters for acetylcholine and monoamines

AJP Cell Physiology ◽

10.1152/ajpcell.2001.280.6.c1616 ◽

2001 ◽

Vol 280 (6) ◽

pp. C1616-C1622 ◽

Cited By ~ 39

Author(s):

Janet S. Duerr ◽

Jennifer Gaskin ◽

James B. Rand

Keyword(s):

Motor Neurons ◽

Choline Acetyltransferase ◽

Egg Laying ◽

Vesicular Acetylcholine Transporter ◽

Vesicular Monoamine Transporter ◽

Monoamine Transporter ◽

C Elegans ◽

Vesicular Transporters ◽

The Individual

We have identified four neurons (VC4, VC5, HSNL, HSNR) in Caenorhabditis elegans adult hermaphrodites that express both the vesicular acetylcholine transporter and the vesicular monoamine transporter. All four of these cells are motor neurons that innervate the egg-laying muscles of the vulva. In addition, they all express choline acetyltransferase, the synthetic enzyme for acetylcholine. The distributions of the vesicular acetylcholine transporter and the vesicular monoamine transporter are not identical within the individual cells. In mutants deficient for either of these transporters, there is no apparent compensatory change in the expression of the remaining transporter. This is the first report of neurons that express two different vesicular neurotransmitter transporters in vivo.

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Neuropeptides encoded by nlp-49 modulate locomotion, arousal and egg-laying behaviours in Caenorhabditis elegans via the receptor SEB-3

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2017.0368 ◽

2018 ◽

Vol 373 (1758) ◽

pp. 20170368 ◽

Cited By ~ 9

Author(s):

Yee Lian Chew ◽

Laura J. Grundy ◽

André E. X. Brown ◽

Isabel Beets ◽

William R. Schafer

Keyword(s):

Caenorhabditis Elegans ◽

Egg Laying ◽

Electrical Synapse ◽

C Elegans ◽

Neuronal Communication ◽

Movement Dynamics ◽

Evolutionarily Conserved ◽

Genes Encoding ◽

High Content Analysis

Neuropeptide signalling has been implicated in a wide variety of biological processes in diverse organisms, from invertebrates to humans. The Caenorhabditis elegans genome has at least 154 neuropeptide precursor genes, encoding over 300 bioactive peptides. These neuromodulators are thought to largely signal beyond ‘wired’ chemical/electrical synapse connections, therefore creating a ‘wireless’ network for neuronal communication. Here, we investigated how behavioural states are affected by neuropeptide signalling through the G protein-coupled receptor SEB-3, which belongs to a bilaterian family of orphan secretin receptors. Using reverse pharmacology, we identified the neuropeptide NLP-49 as a ligand of this evolutionarily conserved neuropeptide receptor. Our findings demonstrate novel roles for NLP-49 and SEB-3 in locomotion, arousal and egg-laying. Specifically, high-content analysis of locomotor behaviour indicates that seb-3 and nlp-49 deletion mutants cause remarkably similar abnormalities in movement dynamics, which are reversed by overexpression of wild-type transgenes. Overexpression of NLP-49 in AVK interneurons leads to heightened locomotor arousal, an effect that is dependent on seb-3. Finally, seb-3 and nlp-49 mutants also show constitutive egg-laying in liquid medium and alter the temporal pattern of egg-laying in similar ways. Together, these results provide in vivo evidence that NLP-49 peptides act through SEB-3 to modulate behaviour, and highlight the importance of neuropeptide signalling in the control of behavioural states. This article is part of a discussion meeting issue ‘Connectome to behaviour: modelling C. elegans at cellular resolution’.

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The PAX gene egl-38 mediates developmental patterning in Caenorhabditis elegans

Development ◽

10.1242/dev.124.20.3919 ◽

1997 ◽

Vol 124 (20) ◽

pp. 3919-3928 ◽

Cited By ~ 5

Author(s):

H.M. Chamberlin ◽

R.E. Palmer ◽

A.P. Newman ◽

P.W. Sternberg ◽

D.L. Baillie ◽

...

Keyword(s):

Transcription Factors ◽

Genetic Analysis ◽

Molecular Genetic ◽

Egg Laying ◽

Paired Domain ◽

C Elegans ◽

Developmental Patterning ◽

Pax Family ◽

Insight Into

Mutations in the C. elegans gene egl-38 result in a discrete set of defects in developmental pattern formation. In the developing egg-laying system of egl-38 mutant hermaphrodites, the identity of four uterine cells is disrupted and they adopt the fate of their neighbor cells. Likewise, the identity of two rectal epithelial cells in the male tail is disrupted and one of these cells adopts the fate of its neighbor cell. Genetic analysis suggests that the egl-38 functions in the tail and the egg-laying system are partially separable, as different egl-38 mutations can preferentially disrupt the different functions. We have cloned egl-38 and shown that it is a member of the PAX family of genes, which encode transcription factors implicated in a variety of developmental patterning events. The predicted EGL-38 protein is most similar to the mammalian class of proteins that includes PAX2, PAX5 and PAX8. The sequence of egl-38 mutant DNA indicates that the tissue-preferential defects of egl-38 mutations result from substitutions in the DNA-binding paired domain of the EGL-38 protein. egl-38 thus provides the first molecular genetic insight into two specific patterning events that occur during C. elegans development and also provides the opportunity to investigate the in vivo functions of this class of PAX proteins with single cell resolution.

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